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www.hytest.fi

Infectious Disease Reagents Influenza and Other Acute Respiratory Diseases Hepatitis A and B Torch Epstein Barr Virus Sexually Transmitted Diseases Malaria Tuberculosis Foodborne Pathogens Microbial and Plant Toxins Biodefense Veterinary


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INFECTIOUS DISEASE REAGENTS


tury. According to the Centers for Desease Control (CDC) and the World Health Organization (WHO) data among 20 leading causes of mortality throughout the world in 2002 at least 9 were infectious, with the first five positions kept by lower respiratory infections (6.8%), HIV/AIDS (4.9%), diarhheal diseases (3.2%), tuberculosis (2.7%) and malaria (2.2%). Thus it is still vitally important to modify and modernize therapy methods, to develop new vaccines and to find fast, precise and simple methods for the diagnostics of infectious diseases. This urgent need became all too clear after September 11, 2001. Antibodies are used extensively as diagnostic tools in a wide array of different analyses. Monoclonal and recombinant antibodies provide a never-ending source of molecules and can produce endless possibilities for novel genetic constructs. Antibodies are still very much in vogue and are now also being used in microarray analysis of the proteome using protein chips. Although PCR – which is targeted to DNA or RNA infectious agent identification – has become a method of choice in the infectious diseases diagnostics in the latest decades, the antibody-based methods major trends, however, over the past few decades have been increasing advances in assay specificity, detection technologies and sensitivity and are increasingly used in research, investigation of pathogenesis mechanisms and in routine clinical tests. This is especially true for serology analysis and for microbial toxin detection.

Introduction In spite of a considerable progress, which has been achieved in the field of diagnostics, therapy and prophylaxis during the recent decades, infectious diseases still keep the leading position among other human illnesses at the beginning of the 21st cen-

During the past 15 years Hytest has been involved in the development and production of highly purified viral and microbial antigens as well as monoclonal antibodies against pathogens. Taking into consideration the newly arisen global concern about bioterrorism and latest trends in most severe infectious diseases spreading, prompt supply of reagents for fast diagnosis is the major field of HyTest’s activity now. We have widened the product spectrum for infectious disease diagnostics and encourage contacts with our clients.

INFECTIOUS DISEASE REAGENTS

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Table of Contents

I INFLUENZA AND OTHER ACUTE RESPIRATORY DISEASES (ARDs)

II HEPATITIS A AND B

26

26 27

1. Influenza A antigens 2. Influenza A monoclonal antibodies 2.1. Influenza A Haemagglutinin (HA) monoclonal antibodies 2.1.1. Influenza A H1 and H3 monoclonal antibodies 2.1.2. Influenza A H1 and H3 immunodetection in ELISA 2.1.3. Influenza A quantitative sandwich immunoassay 2.1.4. Influenza A H1 and H3 immunodetection in Western blotting 2.1.5. Influenza A H5 and H7 monoclonal antibodies 2.2. Influenza A Matrix protein M2 monoclonal antibodies 2.3. Influenza A Nonstructural (NS) protein monoclonal antibodies 2.4. Influenza A Nucleoprotein (NP) monoclonal antibodies 2.4.1. Influenza A NP immunodetection in ELISA 2.4.2. Influenza A NP immunodetection in Western blotting 2.4.3. Influenza A NP quantitative sandwich immunoassay 3. Influenza B antigens 4. Influenza B monoclonal antibodies 4.1. Influenza B Nucleoprotein (NP) monoclonal antibodies 4.1.1. Influenza B NP immunodetection in ELISA 4.1.2. Influenza B NP quantitative sandwich immunoassay 4.1.3. Influenza B NP immunodetection in Western blotting 4.2. Influenza B Haemagglutinin (HA) monoclonal antibodies 4.2.1. Influenza B HA immunodetection in ELISA 4.2.2. Influenza B HA immunodetection in Western blotting 4.3. Influenza B Matrix protein M1 monoclonal antibodies 4.3.1. Influenza B M1 immunodetection in ELISA 4.3.2. Influenza B M1 immunodetection in Western blotting 5. Respiratory Syncytial Virus (RSV) 5.1. Respiratory Syncytial virus (RSV) antigen 5.2. Respiratory Syncytial virus (RSV) monoclonal antibodies 6. Adenovirus 6.1. Adenovirus antigen 6.2. Adenovirus monoclonal antibodies 7. Parainfluenza 7.1. Parainfluenza antigens 8. Klebsiella pneumoniae 9. Newcastle disease virus (NDV)

1. Hepatitis A 2. Hepatitis B

7 7 9 9 9 9 10 10 11 12 12 12 13 13 13 14 15 16 16 16 17 18 18 18 19 19 19 20 20 21 22 22 22 23 23 24 25

III TORCH

28

29

28 28 29 29

1. Toxoplasma gondii 2. Rubella virus 3. Cytomegalovirus (CMV) 4. Herpes simplex virus (HSV)

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Table of Contents IV EPSTEIN BARR VIRUS

30

V SEXUALLY TRANSMITTED DISEASES (STD)

31

31 31 32 32 33 34 34 35 35 36 36 37 37 38 39 39 40

VI MALARIA

41

VII TUBERCULOSIS

42

VIII FOODBORNE PATHOGENS

44

44 46 47 50 51 51

IX MICROBIAL AND PLANT TOXINS

52

52 52 52 53 53 53 54 54 55 55 56 57 57 57 58 58

1. Chlamydia trachomatis 2. Herpes simplex virus 3. Treponema pallidum (Syphilis) 4. Candida albicans 5. Human papilloma virus (HPV) 5.1. HPV, type 6, oncoprotein E7 monoclonal antibodies 5.1.1. Applications 5.2. HPV, type 11, oncoprotein E7 monoclonal antibodies 5.2.1. E7 HPV type 11 immunodetection in ELISA 5.2.2. E7 HPV type 11 immunodetection in Western blotting 5.3.HPV, type 16, oncoprotein E7 monoclonal antibodies 5.3.1. E7 HPV type 16 immunodetection in ELISA 5.3.2. E7 HPV type 16 immunodetection in Western blotting 5.4. HPV, type 18, oncoprotein E7 monoclonal antibodies 5.4.1. E7 HPV type 18 immunodetection in ELISA 5.4.2. E7 HPV type 18 immunodetection in Western blotting 5.5. Human papilloma virus (HPV) antigens

1. Gastroenteritis viruses: rotavirus and adenovirus 2. Salmonella 3. Listeria monocytogenes 4. Legionella pneumophila 5. Campylobacter jejuni 6. Astrovirus

1. Antibodies for the detection of Staphylococcus aureus enterotoxins 1.1. Antibodies for the detection of Staphylococcus aureus enterotoxin 1.2. Antibodies for the detection of Staphylococcus aureus enterotoxin A 1.3. Antibodies for the detection of Staphylococcus aureus enterotoxin B 1.4. Antibodies for the detection of Staphylococcus aureus enterotoxin G 1.5. Antibodies for the detection of Staphylococcus aureus enterotoxin I 2. Antibodies for the detection of Cholera toxin (CT) 3. Antibodies for the detection of Escherichia coli heat-labile enterotoxin 4. Antibodies for the detection of Clostridium botulinum toxoids 5. Antibodies for the detection of Diphtheria 6. Antibodies for the detection of Ricin RCA60 from Ricinus communis 7. Antibodies for the detection of HT-2 toxin 8. Antibodies for the detection Microcystin-LR 9. Antibodies for the detection Nodularin 10. Antibodies for the detection of Tetanus toxin 11. Antibodies for the detection of Aflatoxin from Aspergillus flavus

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Table of Contents

X BIODEFENSE ANTIBODIES

59

59 60 61 62 63 66 68 68 70

XI VETERINARY

71

71 71 71 72 72 72 73 73 73 73 74 74 75 75 75 75 75 75 76 76 76 76 77 77 77 78 78 79 79 79

XII MISCELLANEOUS

80

80 80 81 81 81 82 82 82

6

1. Antibodies for the detection of Bacillus anthracis 1.1. Antibodies for the detection of Bacillus anthracis Protective Antigen 1.2. Antibodies for the detection of Bacillus anthracis Lethal Factor 1.3. Antibodies for the detection of Bacillus anthracis Spore Antigen 2. Antibodies for the detection of Yersinia pestis 3. Antibodies for the detection of Francisella tularensis 4. Antibodies for the detection of Marburg and Ebola viruses 5. Antibodies for the detection of Vaccinia virus 6. Antibodies for the detection of Hemorrhagic fever with renal syndrome (HFRS)

1. Canine 1.1. Canine distemper virus (CDV) 1.2. Canine parvovirus (CPV) 1.3. Canine Adenovirus (CAV) 1.4. Rabies virus 1.5. Echinococcus granulosis 2. Bovine 2.1. Rotavirus 2.2. Bovine coronavirus 2.3. Brucella abortus (Brucellosis) 2.4. Alpha-1 – Acid Glycoprotein (AGP) 2.5. Foot-and-mouth disease (FMDV) 3. Equine 3.1. Burkholderia (Pseudomonas) mallei (Glanders) 4. Porcine 4.1. Transmissible Gastroenteritis (TGE) virus of Pigs 5. Piscine 5.1. Infectious Salmon Anemia virus 6. Avian 6.1. Newcastle disease virus (NDV) 6.2. Marek disease virus (MD) 6.3. Avian influenza 6.4. Infectious bursal disease virus (IBDV) 6.4.1. Immunodetection of VP2 and VP3 IBDV structure proteins in Western blotting 6.4.2. Direct ELISA 6.4.3. Histochemistry 6.4.4. Sandwich immunoassay for IBD virus detection 6.5. Infectious bronchitis virus (IBV) 6.5.1. Immunodetection of IBV nucleoprotein in Western blotting 6.5.2. Serological sandwich-type immunoassay

1. Borrelia burgdorferi (Borreliosis, Lyme Disease) 2. Tick-borne encephalitis virus (TBEV) 3. Cyclosporin A 4. Helicobacter pylori CagA 5. Hamster prion protein 6. FK 506 (Tacrolimus) 7. Allergen from Dermatotophagoides farinae 8. Streptavidin from Streptomyces avidinii INFECTIOUS DISEASE REAGENTS


I Influenza and Other Acute Respiratory Diseases (ARDs) Influenza viruses are unique in their ability to cause sudden, pervasive illnesses in all age groups of human population on a global scale. Three “pandemics” associated with “shift” of surface viral glycoproteins (HA, NA) have occurred in the past century. One of them, “Spanish flu”, in 1918 was responsible for more than 20 million deaths worldwide, primarily in young adults. Annual influenza epidemics associated with different virus types or subtypes caused excess morbidity and mortality especially in groups of high risk. In spite of special clinical signs of influenza (such as sudden fever (>38 °C), pronounced intoxication (headache, myalgias), dry cough, shortness of breath and sternal pain) common clinical picture varied in dependence of age, individual immunity condition, accompanied person pathology and pathogenicity of virus strain. Diagnosis during pre- and inter-epidemic periods became available as a result of laboratory test applications. Laboratory diagnosis for influenza is based on direct examination and paired serum specimens serological assay. Immunofluorescence (IF) testing performed directly on nasopharyngeal secretions. EIA tests are often more objective and more sensitive than IF for direct detection. These diagnoses are necessary for etiotropic chemotherapy prescription. Influenza virus types A and B belong to the family Orthomyxoviridae, containing eight segments of single-stranded RNA, generally spherical (30-100 nm in

diameter). The nucleoprotein (NP) antigen of influenza viruses is associated with viral RNA and determines the type of specificity (A, B or C). Two other important antigens are hemagglutinin (HA) and neuraminidase (NA). Both are glycoproteins and determine the subtypes. Technology of viral antigen production is similar for all the influenza viruses. The source is allantoic fluid of 10-12 days old embryonated hen eggs, inoculated with appropriate strain. Allantoic fluid is clarified by low speed centrifugation and the virus is pelleted by centrifugation at top speed for 1h in the SW 27 rotor (Beckman). Further virus purification is performed by two successive ultracentrifugations. After sucrose density gradient ultracentrifugation, the virus band is collected, diluted with STE buffer and finally pelleted by ultracentrifugation for 1h. To ensure the stability the viral stocks are stored at high protein concentration at -20 °C. Immunoreactivity is tested in in-house ELISA, using the panel of MAbs against influenza virus A and B. The viral antigen titer varies from 1:5 000 to 1:10 000. The infectivity testing on MDCK cultures is negative. Viral antigens of influenza viruses A and B have a very good performance in serology tests for specific IgG, IgM and IgA antibody detection, including EIA. We also recommend these viral antigens for polyclonal antibody production.

1. Influenza A antigens HyTest is offering following Influenza A antigens:

Influenza A (H1N1) virus, strain A/Taiwan/1/86 Influenza A (H1N1) virus, strain A/Beijing/262/95 Influenza A (H1N1) virus, strain A/New Caledonia/20/99 (See Fig. 1.) Influenza A (H1N1) virus, strain A/Solomon Islands/03/06

Influenza A (H3N2) virus, strain A/Shangdong/9/93 Influenza A (H3N2) virus, strain A/Panama/2007/99 (See Fig. 2.) Influenza A (H3N2) virus, strain A/Kiev/301/94 Influenza A (H3N2) virus, strain A/Wisconsin/67/05 Influenza A (H3N2) virus, strain A/Brisbane/10/07 INFECTIOUS DISEASE REAGENTS

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The source is allantoic fluid of 10-12 days old embryonated chicken eggs, inoculated with the appropriate influenza A strain. Purified viruses are inactivated with thimerosal and beta propiolactone treatment. Purity of all products is >90% and these antigens can be used for detection of antibodies to influenza A viruses in ELISA, HIT and Western blotting. Influenza A (H1N1) antigens do not have cross-reactivity in ELISA with panel of MAbs to HA of heter-

ological subtype of influenza A (H3N2) viruses, and Influenza A (H3N2) antigens do not have cross-reactivity in ELISA with panel of MAbs to HA of heterological subtype of influenza A (H1N1) viruses. Also these antigens are not cross-reacting with MAbs to HA of influenza B virus, MAbs to NP of influenza B virus and in hemagglutination inhibition test with antisera to different subtypes of influenza A and B viruses (See Table 1.).

Table 1. Control investigation of influenza A antigens in hemagglutination inhibition test.

Virus: A/Panama/2007/99 (H3N2) B/Tokio/53/99 A/New Caledonia/20/99 (H1N1) A/swine/1976/31 (Hsw1N1) A/St.Petersburg/186/00 (H3N2) A/Singapore/1/57 (H2N2)

Antibody titers in strain specific rabbit sera to: Influenza A virus Influenza B virus Ssw1 SH3 SH2 SB1 <10 320 <10 <10 <10 <10 <10 320 <10 <10 <10 <10 160 <10 <10 <10 <10 320 <10 <10 <10 <10 320 <10

SH1 <10 <10 640 <10 <10 <10

SH1: antiserum to strain A/New Caledonia/20/99 (H1N1) Ssw1: antiserum to strain A/swine/1976/31 (Hsw1N1) SH3: antiserum to strain A/St.Petersburg/186/00 (H3N2)

1,4

+

+

+

1,2

SH2: antiserum to strain A/ Singapore/1/57 (H2N2) SB1: antiserum to strain B/Tokio/53/99

MAb 3B3

2

MAb 1C6

1,8

MAb 4H7

+

0,8 0,6

OD 450

OD 450

1

+

+

+

MAb 12/5

1,6

MAb 1C6

1,4

+

MAb 4H7

+

MAb 3B3

1,2 1 0,8 0,6

0,4

+

0,4

5000

500

+

+

0,5

+ +

5

+

+

+

50

0

+

500

0,2 +

+

5000

+

+

+

0

+

0,2

50

5

0,5

Concentration of MAbs (ng/ml)

Concentration of MAbs (ng/ml) Figure 1. Control of specific activity and cross-reactivity of influenza A/New Caledonia/20/99 virus in ELISA with monoclonal antibodies to different influenza viruses. MAb 3B3 to HA of influenza A/Beijing/262/95 (H1N1) virus MAb 1C6 to NP of influenza A/chick/Pennsylvania/1370/83 (H5N1) virus MAb 4H7 to HA of influenza B/Panama/45/90 virus

Figure 2. Control of specific activity and cross-reactivity of influenza A/Panama/2007/99 virus in ELISA with monoclonal antibodies to different influenza viruses. MAb 12/5 to HA of influenza A/Panama/2007/99 (H3N2) virus MAb 1C6 to NP of influenza A/chick/Pennsylvania/1370/83 (H5N1) virus MAb 4H7 to HA of influenza B/Panama/45/90 virus MAb 3B3 to HA of influenza A/Beijing/262/95 (H1N1) virus

Ordering information: Product

Cat. #

Strain

Remarks

8IN73 8IN73-2 8IN73-3 8IN73-4 8IN74 8IN74-1 8IN74-2 8IN74-3 8IN74-4

A/Taiwan/1/86 A/Beijing/262/95 A/New Caledonia/20/99 A/Solomon Islands/03/06 A/Shangdong/9/93 A/Panama/2007/99 A/Kiev/301/94 A/Wisconsin/67/05 A/Brisbane/10/07

EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB

Influenza A (H1N1) virus Influenza A (H1N1) virus-2 Influenza A (H1N1) virus-3 Influenza A (H1N1) virus-4 Influenza A (H3N2) virus Influenza A (H3N2) virus-1 Influenza A (H3N2) virus-2 Influenza A (H3N2) virus-3 Influenza A (H3N2) virus-4

8

INFECTIOUS DISEASE REAGENTS


2. Influenza A monoclonal antibodies HyTest offers highly sensitive and specific monoclonal antibodies for detection of Influenza A virus. MAbs can be used in routine immunoassays (direct or indirect ELISA, sandwich immunodetection systems, Western blotting) and for specific detection of the most important Influenza A anti-

gens, such as Haemagglutnin (HA) and Nucleoprotein (NP) in different biological samples (nasal aspirates and swabs, cell lysates etc.). MAbs do not have cross-reactivity to Influenza B virus so they can be used for differentiation between Influenza A and B.

2.1. Influenza A Haemagglutinin (HA) monoclonal antibodies 2.1.1. Influenza A H1 and H3 monoclonal antibodies Anti-Influenza A virus H1 and H3 monoclonal antibodies Host Animal: Mice Balb/c Cell line used for fusion: Sp2/0 Immunogen: Purified influenza virus type A (H1N1) or (H3N2) Purification method: Protein A or protein G affinity chromatography

Hybridoma clones have been derived from hybridization of Sp2/0 myeloma with the spleen cells of Balb/c mice immunized with purified Influenza A viruses: A/New Caledonia/20/99 (strain H1N1) and

A/Shangdong/9/93 (strain H3N2). Haemagglutinin-specific antibodies selectively detect H1 or H3 haemagglutinins of Influenza A in ELISA and Western blotting.

2.1.2. Influenza A H1 and H3 immunodetection in ELISA Anti-haemagglutinin MAbs detect specific strain of Influenza A in direct and indirect ELISA. Titration curves of MAb InA4 (H1 specific) and MAb InA246 (H3 specific) are shown on Fig. 3.

0,8

Figure 3. Titration curves of MAbs specific to haemagglutinins H1 or H3 of Influenza A virus in indirect ELISA. A. MAb InA4 (H1 specific) B. MAb InA246 (H3 specific). Antigens: H1N1 – Influenza A/New Caledonia/20/99 -0.1 µg/well. H3N2 – Influenza A/Shangdong/9/93 - 0.1 µg/well 0,4

A

B

H1N1 0,6

H1N1

0,3

H3N2 A, 490nm

A, 490nm

H3N2 0,4

0,2

0

0,2

0,1

0,1

1

10

100

1000

10000

MAb concentration (ng/ml)

0

0,1

1

10

100

1000

10000

MAb concentration (ng/ml)

INFECTIOUS DISEASE REAGENTS

9


2.1.3. Influenza A quantitative sandwich immunoassay

1,0

0,8

OD 450

MAb C102 (Fig. 4) was obtained by use of avian influenza virus strain A (H1N1) as an immunogen and it is directed against relatively conservative H1 epitope. MAb crossreactivity pattern shows that it does not react with H3 and other hemagglutinins but interacts with H1 from human and avian influenza viruses, having indirect ELISA titers not less than 1:128 K. Thus MAb C102 may be used in EIA for subtype differentiation of isolates. MAb C102 can also be used for immunocytochemistry, haemagglutinin inhibition, ELISA and immunofluoresence.

0,6 0,4 0,2 0,0

MAb C102

1E-3

0,01

0,1

Concentration of MAb (µg/ml)

1

Figure 4. Specific activity of MAb C102 in ELISA with purified virus antigen A (H1N1).

10000000

H1N1

All pairs detect virus as well as recombinant haemagglutinin H1 and can be used in Influenza A H1Nxstrain immunodetection systems. Calibration curve for one of the pairs is shown on Fig. 5.

1000000

H3N2 Influenza B

100000 CPS

MAbs were tested in sandwich type fluoroimmunoassay as capture or detection MAbs. Pairs of MAbs were selected on their ability to detect specific strain of Influenza A with high specificity and sensitivity. Purified strains of Influenza A (H1N1 and H3N2) as well as recombinant H1 and H3 were used as antigens. For specific Influenza A H1 immunodetection following pairs are recommended (capture-detection): InA4 - InA88 InA4 - InA134 InA97 - InA134

10000

1000

100 0,1

1

10

100

1

2

1

MAbs detect haemagglutinin H1 or H3 in Western blotting after SDS-PAGE in reducing conditions. MAbs bind to HA1 chain of processed or non-processed haemagglutinin. Immunodetection of Influenza A haemagglutinins by specific antibodies is shown in Fig. 6.

97 kDa

97 kDa

66 kDa

66 kDa

45 kDa

45 kDa

Figure 6. Immunodetection of Influenza A viruses using anti-haemagglutinin monoclonal antibodies in Western blotting after PAGE in reducing conditions. Anti-mouse IgG conjugated with HRP was used for MAb haemagglutinin complex visualization. Antigens (1 μg/well): H1N1 – Influenza A/New Caledonia/20/99 H3N2 – Influenza A/Shangdong/9/93 Antibodies (5 μg/ml): 1: MAb InA4 – anti-Influenza A haemagglutinin H1 2: MAb InA246 – anti-Influenza A haemagglutinin H3

30 kDa

30 kDa

20 kDa

20 kDa

INFECTIOUS DISEASE REAGENTS

10000

100000

Figure 5. Calibration curve for Influenza A sandwich fluoroimmunoassay using anti haemagglutinin H1 antibodies. Capture: MAb InA97 – 1 µg/well. Detection (Eu-chelate labeled): MAb InA134 – 0.2 µg/well Incubation time 45 min. Antigens: H1N1 – Influenza A/New Caledonia/20/99 H3N2 – Influenza A/Shangdong/9/93 Influenza B – mixture of Influenza B viruses (strains B/Qingdao/102/91, B/Tokio/53/99, B/Victoria/504/00)

2.1.4. Influenza A H1 and H3 immunodetection in Western blotting

10

1000

Virus concentration (ng/ml)

H1N1

2

H3N2


2.1.5. Influenza A H5 and H7 monoclonal antibodies Avian influenza viruses occurring naturally among birds cause avian influenza infection. Usually â&#x20AC;&#x153;avian influenza virusâ&#x20AC;? refers to influenza A viruses found mainly in birds, but infections with these viruses can occur also in humans. Avian influenza was first identified over 100 years ago during an outbreak in Italy. Since then, the disease has cropped up at irregular intervals in all world regions. There are many different subtypes of type A influenza viruses and they differ because of changes

in certain proteins on the surface of the influenza A virus (hemagglutinin [HA] and neuraminidase [NA] proteins). Many different combinations of HA and NA proteins are possible and each combination represents a different subtype. Of the 16 different hemagglutinin types only strains within the H5 and H7 subtypes cause highly pathogenic avian influenza, which is highly contagious and rapidly fatal in susceptible avian species. When highly pathogenic influenza H5 viruses cause outbreaks, the mortality rate among poultry is usually between 90%- 100%.

Anti-Influenza A virus H5 and H7 monoclonal antibodies Host Animal: Mice Balb/c Cell line used for fusion: Sp2/0 Immunogen: Purified influenza virus type A (H5N1) or Influenza virus A/FPV (H7N1) Purification method: Protein A or protein G affinity chromatography

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3IH4 3AH1 3AH1 3AH1 3AH1 3AH1 3AH1 3HG3 3HG3 3H5N 3H5N 3H5N 3H5N 3H5N 3H5N 3H5N 3H5N 3H5N 3H5N 3H5N 3H5N 3H5N 3HI7 3HI7 3HI7 3HI7 3HI7 3HI7

C102 InA4 InA16 InA88 InA97 InA134 InA139 InA227 InA246 8D2 11A9 15A6 18D5 19C11 6C8 7E6 1C7 6B4 9B3 2D1 7D5 1B4 1H11 6B5 9A9 9F2 10C6 10H9

IgG1 IgG1 IgG2a IgG2a IgG1 IgG1 IgG1 IgG1 IgG2a IgG2a IgG2a IgG2a IgG2a IgG2a IgG1 IgG2a IgG2a IgG2a IgG2a IgG1 IgG2a IgG2a IgG2a IgG2b IgG2a IgG1 IgG2a IgG1

EIA, IF, HIT, IHC, H1 EIA (capture), WB EIA, WB EIA, WB EIA (capture), WB EIA (detection), WB EIA, WB EIA, WB EIA, WB EIA, HIT, Dot blot EIA, HIT, Dot blot EIA, HIT, Dot blot EIA, HIT, Dot blot EIA, HIT, Dot blot EIA, HIT EIA, HIT EIA, HIT EIA, HIT EIA, HIT EIA EIA EIA EIA, HIT, low C/r to H1 and H3 EIA, HIT, low C/r to H1 EIA, HIT EIA, HIT, low C/r to H1 and H10 EIA, HIT EIA, HIT, low C/r to H1

Anti-Influenza A virus haemagglutinin Anti-Influenza A virus haemagglutinin H1 Anti-Influenza A virus haemagglutinin H1 Anti-Influenza A virus haemagglutinin H1 Anti-Influenza A virus haemagglutinin H1 Anti-Influenza A virus haemagglutinin H1 Anti-Influenza A virus haemagglutinin H1 Anti-Influenza A virus haemagglutinin H3 Anti-Influenza A virus haemagglutinin H3 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H5 Anti-Influenza A virus haemagglutinin H7 Anti-Influenza A virus haemagglutinin H7 Anti-Influenza A virus haemagglutinin H7 Anti-Influenza A virus haemagglutinin H7 Anti-Influenza A virus haemagglutinin H7 Anti-Influenza A virus haemagglutinin H7

INFECTIOUS DISEASE REAGENTS

11


2.2. Influenza A Matrix protein M2 monoclonal antibodies Anti-Influenza A Matrix protein M2 monoclonal antibodies Host Animal: Mice Balb/c Cell line used for fusion: Sp2/0 Immunogen: Purified influenza virus type A Purification method: Protein A affinity chromatography

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Influenza A Matrix protein M2 Anti-Influenza A Matrix protein M2 Anti-Influenza A Matrix protein M2

3AM21 3AM21 3AM21

M2A10 M2D2 M2D4

IgG1 IgG2a IgG2b

Indirect EIA Indirect EIA Indirect EIA

2.3. Influenza A Nonstructural (NS) protein monoclonal antibodies MAbs were produced to non-structural antigens of Influenza A H5. MAbs can be used to detect antigen

in ELISA or others methods, in antibody screening in the competitive ELISA, etc.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Influenza A virus (NS protein) Anti-Influenza A virus (NS protein)

3NS8 3NS8

4A1 9F10

IgG2a IgG2b

EIA EIA

2.4. Influenza A Nucleoprotein (NP) monoclonal antibodies For the influenza virus type A determination we have a panel of MAbs against the nucleoprotein (NP). Influenza virus A (H1N1) was used as immunogen.

All MAbs detect NP of Influenza A with high specificity and have no cross reactivity to NP of Influenza B virus.

Anti-Influenza A virus (nucleoprotein) monoclonal antibodies Host Animal: Mice Balb/c Cell line used for fusion: Sp2/0 Immunogen: Purified influenza virus type A (H1N1) Purification method: Protein G affinity chromatography for MAb F8, Protein A affinity chromatography for other MAbs

12

INFECTIOUS DISEASE REAGENTS

3,0 2.5

OD 450

The investigation of F8 MAb specificity showed that it recognizes the conservative epitope expressed on the nucleoprotein, which is common for type A viruses with different antigenic structure and species origin. We investigated 25 strains of human and avian influenza virus A, isolated during different epidemics in the period from 1934 till 1993 and in all the cases specific reaction was observed. We investigated 265 samples of nasal washings from patients during influenza outbreaks in childrenâ&#x20AC;&#x2122;s communities by the method of direct immunofluorescense. Sensitivity and specificity of the influenza virus A detection reached 60% and 98.2% respectively.

2.0 1,5 1,0

A (H1N1) A (H3N2)

0,5 0,0

1E-3

0,01

0,1

1

Concentration of MAb F8 (Âľg/ml) Figure 7. Specific activity of MAb F8 in ELISA with purified virus antigens A (H1N1) and A (H3N2).


2.4.1. Influenza A NP immunodetection in ELISA Anti-NP MAbs equally detect different strains of Influenza A in ELISA. Titration curve of MAb InA108 is shown of Fig. 8. 0,800

2.4.3. Influenza A NP quantitative sandwich immu- noassay MAbs were tested in sandwich type immunoassay as capture or detection MAbs. Pairs of MAbs were selected on their ability do detect equally NP of H1N1 and H3N2 strains of Influenza A virus. The best pairs of anti-Influenza A NP MAbs are as follows (capturedetection):

A, 490nm

0,600

0,400

InA108 – InA245 InA180 – InA245

All pairs detect NP of Influenza A virus of different strains. Calibration curve for one of the pairs is shown on Fig. 10.

0,200

0,000 10

100

1 000

10 000

MAb concentr ation (ng/m l)

1000000

Figure 8. Titration curve of MAb InA108 specific to NP of Influenza A virus in indirect ELISA. Antigen: Influenza A/New Caledonia/20/99 (H1N1) -0.2 µg/well.

H1N1 H3N2 Influenza B

2.4.2. Influenza A NP immunodetection in Western blotting

CPS

100000

10000

MAbs InA108 and InA245 detect NP of Influenza A virus in Western Blotting after SDS-PAGE in reducing conditions. Immunodetection of Influenza A NP using anti-NP monoclonal antibody InA108 is shown on Fig. 9. Figure 9. Immunodetection of Influenza A viruses using antiNP monoclonal antibody 108 in Western blotting after PAGE in reducing conditions. Anti-mouse IgG conjugated with HRP was used for MAb NP complex visualization. Antigens (1 μg/well): 1: H1N1 – Influenza A/NewCaledonia/20/99 2: H3N2 – Influenza A/Shangdong/ 9/93 Antibody (5 μg/ml): MAb InA108 – anti-Influenza A nucleoprotein (NP).

1

2

97 kDa 66 kDa

45 kDa

1000 10

100

1 000

10 000

100 000

Virus concentration (ng/ml)

Figure 10. Calibration curve for Influenza A NP immunodetection in sandwich fluoroimmunoassay. Capture: MAb InA108 – 1 µg/well. Detection (Eu-labeled): MAb InA245 – 0.2 µg/well Incubation time 45 min. Antigens: H1N1 – Influenza A/New Caledonia/20/99 H3N2 – Influenza A/Shangdong/9/93 Influenza B – mixture of Influenza B viruses (strains B/Qingdao/102/91, B/Tokio/53/99, B/Victoria/504/00)

30 kDa

20 kDa

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3IN5 3IN5 3IN5 3IN5 3IN5

F8 InA108 InA180 InA224 InA245

IgG2a IgG1 IgG3 IgG1 IgG2b

EIA, IHC EIA (capture), WB EIA EIA (capture) EIA (detection), WB

Anti-Influenza A virus (nucleoprotein) Anti-Influenza A virus (nucleoprotein) Anti-Influenza A virus (nucleoprotein) Anti-Influenza A virus (nucleoprotein) Anti-Influenza A virus (nucleoprotein)

INFECTIOUS DISEASE REAGENTS

13


3. Influenza B antigens HyTest is offering following Influenza B antigens:

Influenza B virus, strain B/Qingdao/102/91 (See Fig. 11.) Influenza B virus, strain B/Tokio/53/99 (See Fig. 12.) Influenza B virus, strain B/Victoria/504/00 (See Fig. 13.) Influenza B virus, strain B/Malaysia/2506/04 Influenza B virus, strain B/Florida/07/04 Influenza B virus, strain B/Florida/04/06

The source is allantoic fluid of 10-12 days old embryonated chicken eggs, inoculated with the appropriate influenza B strain. Purified viruses are inactivated with thimerosal and beta propiolactone treatment. Purity of all products is >90% and these antigens can be used for detection of antibodies to influenza B viruses in ELISA, HIT and Western blotting.

Influenza B antigens do not have cross-reactivity in ELISA with panel of MAbs to HA of heterological subtype of influenza A (H3N2) viruses and MAbs to HA of influenza A (H1N1) viruses and in hemagglutination inhibition test with antisera to influenza A (H3N2) and influenza A (H1N1) viruses (See Table 2).

Figure 11. Electron microscopic image of influenza B virus. (Influenza B virus particles 100-120 nm in diameter, magnification 1x 110 000).

Table 2. Control investigation of influenza B antigens in hemagglutination inhibition test.

Antibodies titers in strain specific immune rabbit and rat sera to: Virus: B/Tokio/53/99 B/Victoria/504/00 A/New Caledonia/20/99 (H1N1) A/sw/1976/31 (Hsw1N1) A/St.Petersburg/186/00 (H3N2) SH1: SH3: Ssw1: SB1: SB2:

14

SH1 <10 <10 640 <10 <10

Influenza A virus Ssw1 <10 <10 <10 160 <10

antiserum to strain A/New Caledonia/20/99 (H1N1) antiserum to strain A/St.Petersburg/186/00 (H3N2) antiserum to strain A/sw/1976/31 (Hsw1N1) antiserum to strain B/Tokio/53/99 (B/Victoria/2/87 lineage) antiserum to strain B/Victoria/504/00 (B/Yamagata/16/88 lineage)

INFECTIOUS DISEASE REAGENTS

SH3 <10 <10 <10 <10 640

Influenza B virus SB1 SB2 320 <10 <10 320 <10 <10 <10 <10 <10 <10


MAb 2/3

1

MAb 12/5 MAb 4H7

+

OD 450

0,8 0,6 0,4

50

5

0,5

+

+

500

+

5000

+

+

0

+

0,2

0,05

1,4

+

Concentration of MAbs (ng/ml)

Figure 12. Control of specific activity and cross-reactivity of influenza B/Tokio/53/99 virus in ELISA with monoclonal antibodies to different influenza viruses. MAb 2/3 to NP of influenza B/Beijing/184/93 virus MAb 4H7 to HA of influenza B/Panama/45/90 virus (Yamagata/16/88 lineage) MAb 12/5 to HA of influenza A/Panama/2007/99 (H3N2) virus

+

1,2

MAb 2/3 MAb 12/5

0,8 0,6

+

+

D 450

1

MAb 4H7

0,4

+

0 5000

500

50

5

+

+

0,2

0,5

0,05

Concentration of MAbs (ng/ml)

Figure 13. Control of specific activity and cross-reactivity of influenza B/Victoria/504/00 virus in ELISA with monoclonal antibodies to different influenza viruses. MAb 2/3 to NP of influenza B/Beijing/184/93 virus MAb 4H7 to HA of influenza B/Panama/45/90 virus (Yamagata/16/88 lineage) MAb 12/5 to HA of influenza A/Panama/2007/99 (H3N2) virus

Ordering information:

Product

Cat. #

Strain

Remarks

Influenza B virus Influenza B virus-2 Influenza B virus-3 Influenza B virus-4 Influenza B virus-5 Influenza B virus-6

8IN75 8IN75-2 8IN75-3 8IN75-4 8IN75-5 8IN75-6

B/Qingdao/102/91 B/Tokio/53/99 B/Victoria/504/00 B/Malaysia/2506/04 B/Florida/07/04 B/Florida/04/06

EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB EIA, HIT, WB

4. Influenza B monoclonal antibodies HyTest offers a panel of monoclonal antibodies specific to nucleoprotein (NP), haemagglutinin (HA) and matrix protein M1 of Influenza B virus. MAbs work with high affinity and specificity in different immunoassays: direct or indirect ELISA, Sandwich immunodetection systems and in Western blotting. Anti-NP MAbs are highly specific to Influenza B nucleopro-tein and do not bind to NP of Influenza A virus or any other viral proteins. Low detection limit of our MAbs allows detection of Influenza B virus in different samples with low Influenza B titer. According to

high specificity and affinity they are recommended to be used in rapid Influenza B immunodetection systems. Anti-HA MAbs are specific to Influenza B haemagglutinin HA2 and detect equally different strains of Influenza B virus. Anti-matrix protein MAbs are highly sensitive to M1 matrix protein of Influenza B viruses and detect M1 of different Influenza B strains in EIA and Western blotting.

INFECTIOUS DISEASE REAGENTS

15


4.1. Influenza B Nucleoprotein (NP) monoclonal antibodies Anti-Influenza B virus monoclonal antibodies Host Animal: Mice SJL/J for MAb 2/3, mice Balb/c for other MAbs Cell line used for fusion: Px for MAb 2/3, Sp2/0 for other MAbs Immunogen: Purified influenza virus type B Purification method: Protein G or Protein A affinity chromatography

4.1.1. Influenza B NP immunodetection in ELISA All anti-NP MAbs detect different strains of Influenza B in direct and indirect ELISA. Titration curves of selected MAb are shown on Fig. 14.

2,500

Figure 14. Titration curves of MAb InB114 specific to NP of Influenza B virus in indirect (A) and direct (B) ELISA. A. MAb InB114 titration in indirect ELISA. Antigens: Influenza B: Influenza B/Tokyo/53/99 - 0.5 µg/well. Influenza A: mixture of two strains - A/Shangdong/9/93 and A/New Caledonia/20/99 - 0.5 µg/well B. MAb InB114 conjugated with Eu-chelate titration in direct ELISA. Antigen: Influenza B/Tokyo/53/99 - 0.2 µg/well.

A

100000

B

10000

1,500 Influenza B Influenza A 1,000

CPS

A, 490nm

2,000

B/Tokyo/53/99 1000

0,500

0,000 0,1

1

10

100

1000

100

10000

0,01

MAb concentration (ng/ml)

0,1

1

10

100

1000

InB114-Eu chelate concentration (ng/ml)

4.1.2. Influenza B NP quantitative sandwich immu- noassay 100000

10000

CPS

MAbs were tested in Sandwich type immunoassay as the capture or detection MAbs. Pairs of MAbs were selected on their ability to detect all tested strains of Influenza B with equal specificity and high sensitivity. Different strains of Influenza B (Influenza B/Leningrad/86/93, Influenza B/Tokyo/53/99, Influenza B/Victoria/504/00) as well as recombinant NP of Influenza B were used as antigens. For specific Influenza B NP immunodetection following pairs are recommended (capture-detection):

1000

100

InB12 – InB27 InB12 – InB64 InB36 – InB64

All pairs detect NP of influenza B and can be used in Influenza B immunodetection systems. Calibration curve for one pair is shown on Fig. 15.

16

INFECTIOUS DISEASE REAGENTS

10

100

1 000

10 000

100 000

Influenza B virus (ng/ml)

Figure 15. Calibration curve for Influenza B sandwich fluoroimmunoassay using anti NP antibodies. Capture: MAb InB36 – 1 µg/well. Detection (Eu-chelate labeled): MAb InB64 – 0.2 µg/well Incubation time 45 min. Antigen: Influenza B/Tokio/53/99.


MAbs are specific to different parts of NP molecule. For sensitive NP immunoassay MAbs that bind to diverse epitopes are recommended. Epitope specificity of all MAbs is shown in Table 3. Table 3. Epitope specificity of NP-specific MAbs.

Epitope

MAbs

Fragment 1: (1-80 a.a.r.)

InB12, InB36

Fragment 2: (120-200 a.a.r.)

InB27, InB64

Fragment 3: (240-320 a.a.r.)

InB204, InB210, 2/3

Fragment 4: (480-560a.a.r)

InB114, InB213

4.1.3. Influenza B NP immunodetection in Western blotting MAbs detect NP of Influenza B in Western blotting after SDS-PAGE in reducing and non-reducing conditions. Immunodetection of Influenza B NP by selected antibodies is shown on Fig. 16.

1

2

97 kDa 66 kDa 45 kDa

1,4

MAb 2/3

1,2

30 kDa

OD 450

1 0,8 0,6 0,4

20 kDa

0,2 0 5000

500

50

5

0,5

0,05

Concentration of MAb (ng/ml) Figure 17. Specific activity of MAb 2/3 in ELISA with purified virus antigen B/Beijing/184/93.

Figure 16. NP of Influenza B virus immunodetection using anti-NP monoclonal antibodies in Western blotting after PAGE in reducing conditions. Anti-mouse IgG conjugated with HRP was used for MAb NP complex visualization. Antigen: Influenza B/Tokio/53/99 - 1 μg/well Antibodies - 5 μg/ml 1: MAb InB27 2: MAb InB64

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18 3IF18

IB42 IB633 InB12 InB27 InB36 InB64 InB114 InB204 InB210 InB213 2/3 8-5 13-9 14-12 15-12

IgG2a IgG1 IgG2b IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG2a IgG2a IgG2a IgG2a IgG2a

EIA, WB WB EIA (capture), WB EIA, WB EIA, WB EIA (detection), WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB, IHC EIA, WB EIA, WB EIA, WB EIA, WB

Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein) Anti-Influenza Virus B (nucleoprotein)

INFECTIOUS DISEASE REAGENTS

17


4.2. Influenza B Haemagglutinin (HA) monoclonal antibodies Anti-Influenza B virus monoclonal antibodies Host animal: Mice Balb/c Cell line used for fusion: Sp2/0 Immunogen: Purified influenza B virus Purification method: Protein A affinity chromatography

4.2.1. Influenza B HA immunodetection in ELISA All of anti-haemagglutinin MAbs equally detect HA of different strains of Influenza B virus in direct and indirect ELISA. Titration curves of MAb InB190 is shown on Fig. 18.

4.2.2. Influenza B HA immunodetection in Western blotting All MAbs detect Influenza B haemagglutinin HA2 chain of different strains in Western Blotting after SDS PAGE in reducing conditions (Fig. 19). 1

1,000

2

3

A , 490nm

0,800

97 kDa

0,600 Influenza B/Tokyo/53/99

66 kDa

Influenza B/Leningrad/86/93

0,400

Influenza B/Victoria/504/00 Influenza A mixture

0,200

45 kDa

0,000 0,1

1

10

100

1000

10000

100000

MAb concentration (ng/ml)

Figure. 18. Titration curves of MAb InB190 specific to HA of Influenza B virus in indirect ELISA. A. MAb InB190 titration in indirect ELISA. Antigens - 0.5 µg/well: Influenza B/Tokyo/53/99 Influenza B/Leningrad/86/93 Influenza B/Victoria/504/00 Influenza A: mixture of two strains - A/Shangdong/9/93 and A/New Caledonia/20/99

30 kDa

20 kDa

Figure 19. Influenza B HA2 immunodetection after Western blotting. Anti-mouse IgG conjugated with HRP was used for MAb HA complex visualization. Antigens - 1 μg/well: 1 - Influenza B/Tokio/53/99 2 - Influenza B/Leningrad/86/93 3 - Influenza B/Victoria/504/00 Antibody: MAb InB190 – 3 μg/ml.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Influenza Virus B (haemagglutinin) Anti-Influenza Virus B (haemagglutinin)

3BH9 3BH9

InB18 InB190

IgG2a IgG2b

EIA, WB, HA2 EIA, WB, HA2

18

INFECTIOUS DISEASE REAGENTS


4.3. Influenza B Matrix protein M1 monoclonal antibodies Anti-Influenza B virus Matrix protein M1 monoclonal antibodies Host animal: Mice Balb/c Cell line used for fusion: Sp 2/0 Antigen: Purified Influenza B virus Purification method: Protein-A affinity chromatography

4.3.1. Influenza B M1 immunodetection in ELISA MAbs InB4 and InB15 detect matrix M1 protein of Influenza B virus in direct and indirect ELISA. Titration curve of MAb InB4 is shown on Fig. 20. 2,500

4.3.2. Influenza B M1 immunodetection in Western blotting All MAbs detect Influenza B matrix protein M1 in Western blotting after SDS PAGE in reducing conditions (Fig. 21). MAbs InB4 and InB15 equally detect M1 protein of different strains of Influenza B.

A , 490nm

2,000

1

2

1,500

97 kDa

1,000

66 kDa 0,500

45 kDa

0,000 1

10

100

1000

10000

100000

MAb concentration (ng/ml)

Figure 20. Titration curve of MAb InB4 specific to matrix protein M1 of Influenza B virus in indirect ELISA. A. MAb InB4 titration in indirect ELISA. Antigen: Influenza B/Tokyo/53/99 - 0.5 µg/well.

30 kDa

20 kDa

Figure 21. Influenza B matrix protein M1 immunodetection after Western blotting. Anti-mouse IgG conjugated with HRP was used for MAb M1 complex visualization. Antigen: Influenza B/Tokio/53/99 - 1 μg/well. MAbs: – 3 μg/ml. 1 - InB4 2 - InB15

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Influenza Virus B (Matrix protein M1) Anti-Influenza Virus B (Matrix protein M1)

3BM17 3BM17

InB4 InB15

IgG1 IgG1

EIA, WB EIA, WB

INFECTIOUS DISEASE REAGENTS

19


5. Respiratory Syncytial Virus (RSV) Respiratory syncytial virus is one of the most important respiratory pathogens in infants and children provoking considerable morbidity, which often requires bed care. Severe diseases caused by Respiratory Syncytial virus are most common among infants during the first six months of life and patients with immunodeficiency. Serious lesions of the low-

er respiratory tract induced by Respiratory Syncytial virus (bronchitis, bronchiolitis, pneumonia) are one of the important causes of mortality in infants. 6070% of infants less than six months of age fail to induce detectable antibody response to natural infection. Repeated infections with Respiratory Syncytial virus are common and result in neutralizing antibody formation.

5.1. Respiratory Syncytial virus (RSV) antigen Respiratory Syncytial virus (RSV), strain Long Source: MA-104 cells, inoculated with Respiratory Syncytial virus, strain Long. Purity: >90% Inactivation: Viruses are inactivated with thimerosal and beta propiolactone treatment.. Specificity: The identity of viral antigens, absence of contamination by other viruses (adenovirus, influenza A and B viruses and parainfluenza viruses) and immunoreactivity were checked in ELISA. See Fig. 23. Morphology: In investigation using electron microscopy: RS-virus particles 150 – 300 nm in diameter were observed. See Fig. 22. Applications: Detection of antibodies to Respiratory Syncytial virus in ELISA. Recommended concentration for ELISA is 2.5 – 5 μg/ml.

1,2

MAb 9C5

1

MAb 7F1

OD 450

0,8 0,6 0,4 0,2 0 5000

500

50

5

0,5

0,05

Concentration of MAbs (ng/ml) Figure 22. Electron microscopic image of Respiratory Syncytial virus (Virus particles 150-300 nm in diameter were observed, magnification 1x110 000).

Figure 23. Control of specific activity and cross-reactivity of Respiratory Syncytial virus in ELISA with monoclonal antibodies to different viruses MAb 9C5 to F-protein of Respiratory Syncytial virus. MAb 7F1 to hexon antigen of adenoviruses.

Ordering information: Product

Cat. #

Strain

Remarks

Respiratory Syncytial virus

8RSV79

Long

EIA

20

INFECTIOUS DISEASE REAGENTS


5.2. Respiratory Syncytial virus (RSV) monoclonal antibodies Anti-Respiratory Syncytial virus Host Animal: Mice SJL/J Cell line used for fusion: Px Immunogen: Purified Respiratory Syncytial virus Purification method: Protein G affinity chromatography

2,5

1,5 1,0 0,5 00,

1E-3

0,01

8C5, µg/ml

0,1

1

Figure 24. Specific activity of MAb 8C5 in ELISA with purified RSV antigen.

2,0

OD 450

MAbs 8C5 and 9C5 may be used in sandwich ELISA for RSV detection both with themselves and in a mixed combination, taking into account that 9C5 is especially suitable for conjugation. MAbs 8C5 and 9C5 have virus-neutralizing activity, 8C5 blocks RSVtarget cells binding, 9C5 hampers the virus penetration into the cell. MAb 8B10 is suitable for ELISA and could be used to detect incomplete virus assembly.

OD 450

2,0

We developed a panel of MAbs against RSV. In Western blot MAb 8C5 reacts with protein having Mr 90 K, that corresponds in mobility to protein G. MAb 9C5 specificity was determined by competitive ELISA with MAbs 131-2A and 92-11C (CDC, Atlanta): they are directed to the same F1a epitope, localized on Fprotein. MAb 8B10 in directed against nucleoprotein N. MAb 9C5 is highly reactive with the surface domains of both mature RSV virions and «empty» virion envelopes without formed inner nucleocapsid structures. MAb 8B10 is reacting well only with mature virions with completely assembled nucleocapsids. No cross-reactivity with influenza A and B viruses, adenovirus and parainfluenza type 1 and 2 viruses.

1,5 1,0 0,5 0,0 1E-4

1E-3

0,01 9C5, µg/ml

0,1

1

Figure 25. Specific activity of MAb 9C5 in ELISA with purified RSV antigen.

0,8 0,7

OD 450

0,6 0,5 0,4 0,3 0,2 0,1 0,0 1E-3

0,01

8B10, µg/ml

0,1

1

Figure 26. Specific activity of MAb 8B10 in ELISA with purified RSV antigen.

Ordering information: Product

Cat. #

MAb

Isotype

Anti-Respiratory Syncytial virus Anti-Respiratory Syncytial virus Anti-Respiratory Syncytial virus

3Res21 3Res21 3Res21

8C5 9C5 8B10

IgG2b IgG2b IgG1

Remarks G protein, EIA F protein, EIA Nucleoprotein, EIA

INFECTIOUS DISEASE REAGENTS

21


6. Adenovirus Adenoviruses are a large group (more than 80 types) of agents, which induce respiratory infections among human beings, animals and birds. Clinical pattern of adenoviral infection is characterized by pronounced pharyngitis, conjunctivitis, general intoxication and pulmonary lesions with high fever in children. Adenovirus types 3, 4, 7, 14 and 21 often spread in mil-

itary units and account for 72% of ARDs among recruits. A considerable part of these diseases results in hospitalization. Adenovirus types 3, 4, 7, 8 and 19 are known as causative agents of epidemic keratoconjunctivitis. Some types of adenoviruses provoke outbreaks of gastroenteritis with long (more than 2 months) carriage of viruses.

6.1. Adenovirus antigen Adenovirus, type 6, strain Tonsil 99 Source: HeLa cells, inoculated with Adenovirus, type 6, strain Tonsil 99 Purity: >90% Inactivation: Viruses are inactivated with thimerosal and beta propiolactone treatment.. Specificity: The identity of viral antigens, absence of contamination by other viruses (RSV, influenza A and B viruses and parainfluenza viruses) and immunoreactivity were checked in ELISA. See Fig. 28. Morphology: In investigation using electron microscopy - typical adenovirus particles 80 nm in diameter were observed. See Fig. 27. Applications: Detection of antibodies to Adenovirus in ELISA. Recommended concentration for ELISA is 2.5 - 5 Îźg/ml.

9C5

1,6

7F1

1,4

OD 450

1,2 1 0,8 0,6 0,4 0,2 0 5000

Figure 27. Electron microscopic image of Adenovirus, type 6 (Virus particles 80 nm in diameter were observed, magnification 1x110 000).

500

50

5

0,5

Figure 28. Control of specific activity and cross reactivity of adenovirus in ELISA with monoclonal antibodies to different viruses. MAb 9C5 to F-protein of RS-virus MAb 7F1 to hexon antigen of adenoviruses

Ordering information:

Product

Cat. #

Strain

Remarks

Adenovirus, type 6

8AV13

Tonsil 99

EIA

6.2. Adenovirus monoclonal antibodies Anti-Adenovirus monoclonal antibodies Host Animal: Mice Balb/c Cell line used for fusion: Sp2/0 Immunogen: Purified human and canine adenoviruses (type 1) Purification method: Protein A affinity chromatography

22

INFECTIOUS DISEASE REAGENTS

Adenovirus antibodies react with Hexon antigen of at least human, canine, bovine, monkey and rat adenoviruses. MAbs can be used in ELISA, immunodiffusion and immunohistochemistry. In sandwich ELISA we recommend using MAb 8C4 for capture and MAb 1E11 for detection.


Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Adenovirus hexon Anti-Adenovirus hexon Anti-Adenovirus hexon

3AV13 3AV13 3AV13

7C11 1E11 8C4

IgG2a + IgM IgG2a + IgM IgG2a

EIA, ID, IHC EIA (detection), ID, IHC EIA (capture), ID, IHC

7. Parainfluenza Parainfluenza virus types 1-3 are common agents of acute respiratory infections predominating among children less than 5 years old. They induce about 15% of acute respiratory infections. They are mostly

causative agents of severe croup, bronchitis, bronchiolitis and pneumonia (Parainfluenza virus type 3) in infants. Children can be infected with Parainfluenza virus several times during one year.

7.1. Parainfluenza antigens We produce extra purity grade parainfluenza virus type I (strain Sendai), parainfluenza type 2 (strain II-ALTB cc2056) and parainfluenza virus type 3 (strain 3v29) viral antigens for serology tests such as indirect EIA, HIT (hemagglutinin inhibition test), CFT (complement fixation test) and for the use as immunogens in polyclonal antibody production. The purification technology is in general similar to the one described earlier for influenza virus types A

and B, but parainfluenza virus types 2 and 3 were grown in a monolayer of cells MA-104. Viruses are inactivated with thimerosal. The quality control was made by electron microscopy, SDS-PAGE and protein concentration measurement by BCA (Pierce) assay. The identity of viral antigens, absence of contamination by other viruses and immunoreactivity were checked in indirect ELISA and HIT. Purity of the antigens is >90%.

Parainfluenza virus type 1, strain Sendai

Parainfluenza virus type 2, strain II ALTB cc 2056 (See Table 4 and Fig. 29.) In investigation using electron microscopy parainfluenza virus type 2 particles 150-300 nm in diameter were observed. Low level of cross-reactivity with parainfluenza virus type 3 was observed in ELISA.

Parainfluenza virus type 3, strain III v 2932 (See Table 4 and Fig. 30.)

MAb PIV21

0,8

MAb PIV23

+

MAb 1B5

0,4

5000

500

50

5

0,5

Concentration of MAbs (ng/ml)

+

+

+

0

+

+

0,2

+

OD 450

0,6

0,05

Figure 29. Control of specific activity and cross-reactivity of parainfluenza virus type 2 in ELISA with monoclonal antibodies to parainfluenza viruses. PIV21: MAb to F-protein of parainfluenza virus type 2 PIV23: MAb to F-protein of parainfluenza virus type 2 1B5: MAb to parainfluenza virus type 3

INFECTIOUS DISEASE REAGENTS

23


Table 4. Results of the control investigation of parainfluenza virus types 2 and 3 in hemagglutination inhibition test.

Parainfluenza viruses: PIV type 1 (Sendai strain) PIV type 2 PIV type 3

Antibody titers in strain specific immune rabbit sera to: S1 S2 S3 320 <20 <20 <20 320 <20 <20 <20 640

S1: antiserum to PIV type 1 (Sendai strain) S2: antiserum to PIV type 2 S3: antiserum to PIV type 3

+

+

1

0,6 0,4

+

OD 450

+

+

0,8

MAb PIV21 MAb PIV23 MAb 1B5

0

5000

500

50

5

+

+

0,2

0,5

0,05

Concentration of MAbs (ng/ml)

Figure 30. Control of specific activity and cross-reactivity of parainfluenza virus type 3 in ELISA with monoclonal antibodies to parainfluenza viruses. PIV21: MAb to F-protein of parainfluenza virus type 2 PIV23: MAb to F-protein of parainfluenza virus type 2 1B5: MAb to parainfluenza virus type 3

Ordering information:

Product

Cat. #

Strain

Remarks

Parainfluenza virus, type 1 Parainfluenza virus, type 2 Parainfluenza virus, type 3

8P76 8P76-2 8P76-3

Sendai II ALTB cc 2056 III v 2932

EIA, HIT EIA, HIT EIA, HIT

8. Klebsiella pneumoniae Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines. It is clinically the most important member of the Klebsiella genus of Enterobacteriaceae. It naturally occurs in the soil and about 30% of strains can fix nitrogen in anaerobic condition. Members of the Klebsiella genus typically express 2 types of antigens on their cell surface. The first, O antigen, is a lipopolysaccharide of which 9 varieties exist. The second is K antigen, a capsular polysaccharide with more than 80 varieties. Both contribute to pathogenicity and form the basis for subtyping.

K. pneumoniae can cause bacterial pneumonia, typically due to aspiration, though it is more commonly implicated in hospital-acquired urinary tract and wound infections, particularly in immunocompromised individuals (e.g. alcoholics). Klebsiella ranks second to E. coli for urinary tract infections in older persons. It is also an opportunistic pathogen for patients with chronic pulmonary disease, enteric pathogenicity, nasal mucosa atrophy, and rhinoscleroma. Monoclonal anti-Klebsiella pneumoniae antibodies were produced from hybridoma clones derived from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with lyophilized Klebsiella pneumoniae, strain 204.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Klebsiella pneumoniae 204 Anti-Klebsiella pneumoniae 204 Anti-Klebsiella pneumoniae 204

3KP4 3KP4 3KP4

KpE7 KpE10 KpH11

IgG2a IgG2a IgG2a

Indirect EIA Indirect EIA Indirect EIA

24

INFECTIOUS DISEASE REAGENTS


9. Newcastle disease virus (NDV) Newcastle disease (ND) is a highly contagious and sometimes fatal illness affecting many domestic and wild bird species. The causal agent, Newcastle disease virus (NDV), is a negative-sense single-stranded RNA virus. NDV affects the respiratory, nervous, and digestive systems. Clinical signs are extremely variable depending on the strain of virus, species and age of bird, concurrent disease, and preexisting immunity. NDV is so virulent that many birds die without showing any clinical signs.

Transmission occurs by exposure to foecal and other excretions from infected birds, and through contact with contaminated food, water, equipment and clothing. Virus-bearing material can be picked up on shoes and clothing and carried from an infected flock to a healthy one. Exposure of humans to infected birds (for example in poultry processing plants) can cause mild conjunctivitis and influenzalike symptoms, but NDV otherwise poses no hazard to human health. MAbs are negative with parainfluenza type 3 and avian influenza hemagglutinins.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3ND5 3ND5 3ND5 3ND5 3ND5 3ND5 3ND5

9F7 11F12 13H3 9C6 1C10 2H4 8H2

IgG1 IgG2a IgG2a IgG2a IgG2a IgM IgG2a

EIA (detection), WB, HIT EIA (detection), WB, HIT EIA, WB, HIT EIA, WB, HIT EIA (detection), WB, HIT EIA (capture), HIT EIA (capture)

Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus

INFECTIOUS DISEASE REAGENTS

25


II Hepatitis A and B Currently seven viruses, A, B, C, D, E, G and transfusion transmitted virus (TTV) are recognized in the hepatitis virus alphabet. Hepatitis G virus and TTV probably do not cause liver disease in humans. Hepatitis A and E usually cause a self-limiting hepatitis followed by complete recovery but occasionally cause fulminant hepatic failure. Hepatitis B and C are major public health problems worldwide due to their sequelae of chronic hepatitis, cirrhosis and primary liver cancer. Chronic hepatitis C is a particular health issue for Western Europe already, accounting for 40% of endstage cirrhosis and 30% of liver transplants. The contribution of hepatitis C to chronic liver disease is predicted to rise in the future. Vaccines can prevent hepatitis A and B. Interferon alpha is effective treatment in 25-30% of patients with chronic hepatitis B or C. The prospects for treating chronic hepatitis B have been improved by the introduction of reverse transcriptase inhibitors. Lamivudine is the first drug of this class to be licensed. The optimal use of these new drugs is currently being studied. The success rate for treating chronic hepatitis C can be raised to about 40% with combination therapy of interferon alpha and ribavirin. A large research effort to discover new antiviral agents against hepatitis C is already giving the prospect of more effective therapies in the next few years. Infections with the hepatitis B, C or D virus can all lead to chronic hepatitis. Serological and molecular methods are essential for diagnosis and for differentiation between the different forms of chronic virus hepatitis. In adults between 5% and 10% of all infections with the hepatitis B virus become chronic while the rate is as high as 80% with the hepatitis C virus. All forms of chronic hepatitis are frequently asymp-

tomatic for a long period of time. Complications are liver cirrhosis and hepatocellular carcinoma. During chronic hepatitis B infection in around 1% of the patients per year the virus is eliminated spontaneously while virus elimination occurs rarely in patients with chronic hepatitis C infection. In patients with chronic hepatitis C infection over a period of 30 years around 3% of the patients die due to chronic hepatitis C infection. Considerable evidence suggests that immune mechanisms are involved in the pathogenesis of both hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Both class I-restricted CD8+ T cell and class II-restricted CD4+ T cell responses to viral antigens are important mechanisms that may be responsible for the hepatocyte damage in hepatitis B and C. CD4+ T cell proliferative responses to hepatitis B core antigen (HBcAg) in terms of stimulation index are correlated with hepatitis activity. In terms of major histocompatibility complexes (MHC) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response, antigenic peptides derived from HBcAg, hepatitis B surface antigen (HBsAg), and polymerase have been demonstrated to be the targets for CTL recognition in hepatitis B patients. Multiple CTL epitopes within HBsAg, HBcAg and polymerase can be detected by sensitizing target cells with synthetic peptides. Likewise, multispecific, HCV-specific CTL responses can coexist with an extensive quasispecies of viral variants. The mechanisms of viral persistence in both hepatitis B and C remain to be clarified. The present situation of hepatitis infection being widely spread in the world, the necessity for rapid diagnostics is self evident.

1. Hepatitis A For the detection of Hepatitis A virus in ELISA and WB we offer MAb MK01, which can also be used in antibody capture assay. Ordering information:

Product

Cat. #

MAb

Isotype

Remarks

Anti-Hepatitis A Antigen

3HA18

MK01

IgG3

EIA, WB

26

INFECTIOUS DISEASE REAGENTS


2. Hepatitis B

MAbs HB11, HB12 and HB13 demonstrate a higher affinity to HBsAg and may be used as capture for sandwich ELISA. We recommend to use non-infectious recombinant HBsAg (ayw subtype, Cat. # 8HS7ay) and recombinant HBsAg (adw subtype, Cat. # 8HS7-2ad). These antigens are produced by yeast Saccharomyces cerevisiae, containing plasmid pCGA7. Both antigens could be used as a positive control in immunoassay and as immunogen for antibody production. The purity of the antigens more than 98% (SDS-PAGE). A supplementary product in this line is a MAb match pair against Hepatitis B virus core antigen (HBcAg). MAbs H3A4 and H6F5 recog-

nize 20 K and 40 K protein bands in WB and are suited for HBcAg detection in ELISA. Taking into consideration the growing requirements for the quantitative determination of HBsAg in EIA we are focused on the production of new MAbs. The example of this could be the newest matched pair Hs33-Hs41, which shows 50 pg/ml detection limit. (See Fig. 31) 1,0 0,8

OD 450

For the detection of Hepatitis B virus surface antigen (HBsAg) we offer a pair of MAbs, HB5 and HB6. These antibodies react equally well with subtypes ad and ay and recognize “a” common epitope with 10 9 M-1 binding constant. The detection limit in ELISA is 0.5 ng/ml when HB5 is used for capture and HB6 for labeling.

0,5 0,4 0,2 0,0 0,0

0,5

1,0

1,5

2,0

ng/ml

Figure 31. Calibration curve for sandwich ELISA using the best recommended pair Hs33 – Hs41. Coating: MAb Hs33, 5 μg/ml in 0.1 M carbonate buffer, detection: HRP-conjugated MAb Hs41, 1/20000 in PBS-AT, substrate: TMB. Sensitivity 50 pg/ml.

Ordering information:

Product

Cat. #

Purity

Source

Hepatitis B Virus Surface Antigen, ayw subtype Hepatitis B Virus Surface Antigen, adw subtype Hepatitis B Virus Surface Antigen, adr subtype

8HS7ay 8HS7-2ad 8HS7-3

>98% >98% >98%

Recombinant Recombinant Recombinant

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3HB12 3HB12 3HB12 3HB12 3HB12 3HB12 3HB12 3HB12 3HB12 3HB17 3HB17 3HBe24 3HBe24 3HBe24 3HBe24 3HBe24

Hs33 Hs41 HB5 HB6 HB11 HB12 HB13 B5 B1 H3A4 H6F5 AC5 HBe3 HBe5 HBe7 HBe8

IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG2a IgG1 IgG2a IgG2a IgG2b IgG2b IgG2b IgG1 IgG2b

EIA (capture) EIA (detection) EIA (capture) EIA (detection) EIA (detection) EIA (capture) EIA (capture) EIA (detection) EIA (capture) EIA, WB EIA, WB EIA EIA (detection) EIA (detection) EIA (capture) EIA (capture)

Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Surface Antigen Anti-Hepatitis B Virus Core Antigen Anti-Hepatitis B Virus Core Antigen Anti-Hepatitis B Virus “e” Antigen Anti-Hepatitis B Virus “e” Antigen Anti-Hepatitis B Virus “e” Antigen Anti-Hepatitis B Virus “e” Antigen Anti-Hepatitis B Virus “e” Antigen

INFECTIOUS DISEASE REAGENTS

27


III TORCH TORCH is an acronym for Toxoplasma, Rubella virus, Cytomegalovirus (CMV) and Herpes simplex virus (HSV). Serologic tests for detection of antibodies to these organisms are used for assessment of congenital infections. TORCH testing should reflect

detection of IgM rather than IgG class antibodies. Infants with congenital infection due to the TORCH agents may have low birth weight, prema-turity, purpura, jaundice, anemia, microcephaly, cerebral calcification, choriorenitis, cataracts, microphtalmia and pneumonia.

1. Toxoplasma gondii Prenatal protozoal infection with Toxoplasma gondii is associated with injury to the developing fetal nervous system. The severity of this condition is related to the stage of pregnancy during which the infection occurs; first trimester infections are associated with a greater degree of neurologic dysfunction. Clinical features include hydrocephalus, microcephaly, deafness, cerebral calcifications, seizures and psychomotor retardation. Signs of a systemic infection may also be present at birth, including fever, rash, and hepatosplenomegaly. We developed an optimal method of antigen preparation from T. gondii plasma membrane. We used

tachyzoites from mice, strain RH, as the source for antigen and then performed extraction by a detergent mixture and high-speed centrifugation. On the final stage the main emphasis has been made on the detergent removal from the preparation. It it possible to use the antigen for the detection of specific antibody in indirect ELISA and WB in the samples of sera and plasma. The antigen may be used in capture format, when labelled, to determine specific IgM. For the quality control of T. gondii antigen we use MAb TP3, directed against p30. MAb TP3 reacts with tachyzoites and purified p30 in ELISA, Western blotting and immunofluorescence.

2. Rubella virus An acute, usually benign, infectious disease caused by the Rubella virus is most often affecting children and nonimmune young adults, in which the virus enters the respiratory tract via droplet nuclei and spreads to the lymphatic system.

28

INFECTIOUS DISEASE REAGENTS

MAb 1C11 recognizes Rubella virus core-protein in Western blotting, whereas MAb 3D2 is evidently directed against E2 glycoprotein. MAb 3D2 is active in HIT. Recombinant Rubella virus antigen and MAb 1C11 as a HRP-conjugate may be used for the determination of Rubella-specific IgM in serum and plasma in capture assay.


3. Cytomegalovirus (CMV) Infection with Cytomegalovirus (CMV) is characterized by enlarged cells bearing intranuclear inclusions. Infection may be in almost any organ, but the salivary glands are the most common site in children, as are the lungs in adults. Especially acute character CMV acquires with immunocompromized patients and during transplantations.

We offer monoclonal antibody against Cytomegalovirus, MAb B2, which reacts with 65K protein of cytomegalovirus in Western blotting. MAb B2 is suitable for the CMV detection in ELISA, immunofluorescence and Western blotting, as well as for EIA construction for specific IgM by capture method.

4. Herpes simplex virus (HSV) MAb HSVA33 was obtained after mice immunization by HSV 2, strain BH. According to Western blotting data MAb HSVA33 reacts with 56-64 K protein band in purified HSV preparation under reduced condi-

tion and with a band 120-140 K in lyzates of the infected Vero cells under non-reduced condition, MAb HSVA33 is suited for HSV antigen detection in ELISA, Western blotting and immunofluorescence.

Ordering information: Product

Cat. #

Purity

Source

Toxoplasma gondii Antigen

8T68

N/A

RH Strain Tachyzoites

Ordering information: Product

Cat. #

MAb

Isotype Remarks

3Tx19 3R23 3R23 3R23 3R23 3CV14 3HS2

TP3 Ru5 Ru6 3D2 1C11 B2 HSVA33

IgG2a IgG2a IgG2a IgG2a IgG1 IgG2a IgG1

Anti-Toxoplasma gondii Anti-Rubella Virus Structural Glycoprotein Anti-Rubella Virus Structural Glycoprotein Anti-Rubella Virus Structural Glycoprotein Anti-Rubella Virus Structural Glycoprotein Anti-Cytomegalovirus Anti-Herpes Simplex Virus, Type 2

EIA, WB, IF, P30 Antigen, EIA, WB, Structural E1 Glycoprotein EIA, WB, Structural E1 Glycoprotein EIA, WB, Structural E2 Glycoprotein EIA, WB, Structural Core protein EIA, WB, p65 EIA, WB, Glycoprotein D

INFECTIOUS DISEASE REAGENTS

29


IV Epstein Barr virus Epstein-Barr virus (EBV) is a ubiquitous virus associated with a variety of different diseases and disorders. There are manifestations of Epstein-Barr virus-associated diseases or disorders within the liver, which involve a broad spectrum of histologic and clinical features, ranging from hepatitis through lymphoproliferative disorders to lymphoma. An important aspect of Epstein-Barr virus expression and infection is the biology of the Epstein-Barr virus. Documentation of infection can be performed using serology to detect the interaction of Epstein-Barr virus

with the immune system, and the detection of EBV proteins and use of molecular biologic techniques to identify the presence of EBV RNA and DNA sequences. Of particular utility are in situ hybridization, Southern blot analysis, and polymerase chain reaction as diagnostic methods to identify specific RNA or DNA sequences. Epstein-Barr virus-associated diseases and disorders including infectious mononucleosis, sporadic fatal infectious mononucleosis, X-linked proliferative disorder (Duncanâ&#x20AC;&#x2122;s disease), post-transplant lymphoproliferative disorders, lymphoma, and AIDS are described.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Epstein-Barr Virus Anti-Epstein-Barr Virus

3EB20 3EB20

1H1 4A8

IgG1 IgG2b

EIA, WB, IF, p120, p160 Capsid Antigen EIA, WB, IF, p120 Capsid Antigen

30

INFECTIOUS DISEASE REAGENTS


V Sexually transmitted diseases (STD) More than 25 diseases are spread primarily through sexual activity. The latest estimates indicate that there are 15 million new sexually transmitted disease cases in the United States each year. Approximately one-fourth of these new infections are in teenagers. Nearly two-thirds of all STD cases occur in people younger than 25 years.

While some sexually transmitted diseases, such as syphilis, have been brought to all-time lows, others, like genital herpes, gonorrhea, and chlamydia, continue to resurge and spread through the population. Not including HIV, the most common sexually transmitted diseases in the U.S. are chlamydia, gonorrhea, syphilis, genital herpes, human papillomavirus, hepatitis B, and trichomoniasis (Table 5.).

Table 5. Most common sexually transmitted diseases in the U.S.

Sexually transmitted disease

Incidence (estimated number of new cases every year)

Prevalence (estimated number of people currently infected)

Chlamydia

3 million

2 million

Gonorrhea

650,000

n.a.

Syphilis

70,000

n.a.

Herpes

1 million

45 million

Human papillomavirus (HPV)

5.5 million

20 million

Hepatitis B

120,000

417,000

Trichomoniasis

5 million

n.a.

1. Chlamydia trachomatis We have available mouse monoclonal antibody, which reacts with major outer membrane protein (MOMP) of Chlamydia trachomatis. Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Chlamydia trachomatis MOMP

3CT1

HT10

IgG2a

EIA, WB

2. Herpes simplex virus MAb HSVA33 was obtained after mice immunization by HSV 2, strain BH. According to Western blotting data MAb HSVA33 reacts with 56-64 K protein band in purified HSV preparation under reduced condi-

tion and with a band 120-140 K in lyzates of the infected Vero cells under non-reduced condition, MAb HSVA33 is suited for HSV antigen detection in ELISA, Western blotting and immunofluorescence.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Herpes Simplex Virus, Type 2

3HS2

HSVA33

IgG1

EIA, WB

INFECTIOUS DISEASE REAGENTS

31


3. Treponema pallidum (Syphilis) Recent studies indicate that the STDs that cause ulcerative diseases (herpes simplex virus type 2, Treponema pallidum and Haemophilus ducreyi) increase the risk of HIV transmission by at least two- to five-fold. Treponema pallidum is the causative agent of syphilis, which is the second most common cause of sexually transmitted ulcers in the United States. Unlike other STDs, syphilis has several defined clinical stages. Syphilis is characterized by multiple clinical stages and long periods of latent, asymptomatic

infection. The primary infection is localized, but organisms rapidly disseminate and cause manifestations throughout the body. Although effective therapies have been available since the introduction of penicillin, syphilis remains an important global health problem. We have developed a MAb which reacts with ultrasonicated lysates of Treponema pallidum and Treponema reiter.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Treponema pallidum

3T11

Tr33

IgG1

EIA, WB

4. Candida albicans Candidiasis, commonly called as yeast infection or thrush, is a fungal infection of any of the Candida species, of which Candida albicans is probably the most common. C. albicans is normally found in the mouth, gastrointestinal tract, vagina and the skin. In healthy individuals the presence of C. albicans is considered a normal part of the bowel flora. Usu-

ally, C. albicans is kept under control by the native bacteria and by the body’s immune defenses. If the “healthy” bacterial environment is compromised the C. albicans cells get an opportunity to proliferate and transform into a harmful fungus. Especially immunocompromised patients ranging from premature infants to AIDS patients are in risk.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti- Candida albicans

3CA4

CDA

IgG1

EIA, WB

32

INFECTIOUS DISEASE REAGENTS


5. Human papilloma virus Human papillomavirus (HPV) belongs to Papillomaviruses, a diverse group of DNA-based viruses that infect the skin and mucous membranes of humans and a variety of animals. Over 100 different human papillomavirus (HPV) types have been identified on the basis of difference in the virus genome nu- cleotide sequences (e.g. type 1, 2, 3 etc.). Today genital HPV infection is one of the most widespread sexually transmitted diseases. Approximately 20 million people around the world are currently infected with HPV. At least 50 percent of sexually active men and women acquire genital HPV infection at some point in their lives. By age 50, at least 80 percent of women will have acquired genital HPV infection. In accordance with WHO information, genital HPV infection was a reason of over 99% of cervical cancer cases, i.e. about 1.4 million women were affected worldwide and 239 000 of them died each year. All HPVs are transmitted by skin-to-skin contact. A group of about 30-40 HPVs is typically transmitted through sexual contact and infect the anogenital region. Some sexually transmitted HPVs, types 6 and 11, may cause genital warts. However, other HPV types which may infect the genitals do not cause any noticeable signs of infection. Persistent infection with a subset of about 13 socalled “high-risk” sexually transmitted HPVs, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 — different from the ones that cause warts — may lead to the development of cervical intraepithel-

ial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). These are precancerous lesions and can progress to invasive cancer. HPV infection is a necessary factor in the development of nearly all cases of cervical cancer. The HPV lifecycle begins from infection of epithelial tissues through micro-abrasions. At this point, the viral genome is transported to the nucleus and establishes itself at a copy number between 10-200 viral genomes per cell. A sophisticated transcriptional cascade then occurs as the host keratinocyte begins to divide and become increasingly differentiated in the upper layers of the epithelium. The viral oncogenes, E6 and E7, are thought to modify the cell cycle so as to make them amiable to the amplification of viral genome replication and consequent late gene expression. In the upper layers of the host epithelium, the late genes L1 and L2 are transcribed/ translated and serve as structural proteins which encapsidate the amplified viral genomes. HyTest offers a wide spectrum of monoclonal antibodies specific to oncoprotein E7 of “high-risk” HPV types 16 and 18 as well as of less oncogenic HPV type 6 and 11. MAbs can be used in routine immunoassays (direct or indirect ELISA, sandwich immunodetection systems, Western blotting). Some MAbs display high specificity to definite type of HPV while others can be used for determination of E7 proteins for all four types of viruses.

INFECTIOUS DISEASE REAGENTS

33


5.1. HPV, type 6, oncoprotein E7 monoclonal antibodies Host animal: Cell line used for fusion: Immunogen: Specificity: Purification method:

Mice Balb/c Sp2/0 Recombinant oncoprotein E7, type 6, conjugated with hsp70 Human papilloma virus, type 6 (for cross reactivity information see figure 33) Protein G affinity chromatography

5.1.1. Applications 2,5 2,208 2

OD450

MAb 706-C5 against E7 HPV type 6 can be used in routine immunoassays like HPV enzyme immunoaasay and HPD immunodetection in Western blotting. It should be noted that it is cross-reacting with HPV types 11, 16 and 18 (Fig. 32).

2,300

1,825 1,469

1,5

1

0,5 0,057 0

706-HSP70

711-HSP70

E7, type16

E7, type18

HSP70

Figure 32. Testing of MAb 706-C5 cross-reactivity in indirect ELISA. Coating: 1 mg/ml of each antigen; MAb 706-C5, 3 mg/ml

Figure 33. Results of the MAb mapping.

34

INFECTIOUS DISEASE REAGENTS


5.2. HPV, type 11, oncoprotein E7 monoclonal antibodies Host animal: Cell line used for fusion: Immunogen: Specificity: Purification method:

Mice Balb/c Sp2/0 Recombinant oncoprotein E7, type 11, conjugated with hsp70 Human papilloma virus, type 11 (for cross reactivity information see table 7) Protein G affinity chromatography

Table 6. MAbs main characteristics.

MAb

MAb isotype

Immunogen (hsp70 conjugated) HPV type

E7 oncoprotein fragment

Table 7. MAbs specificity. Study of cross-reactivity with HPV types 11, 16 and 18 Indirect ELISA, Coating: 5 mg/ml of each antigen; MAbs: 3 mg/ml

MAbs

Cross reactivity with E7, type 11

E7, type 16

E7, type 18

711-13

IgG1

11

Whole molecule

711-13

100%

100%

89%

711-45

IgG2a

11

Whole molecule

711-45

100%

38%

27%

711-66

IgG1

11

Whole molecule

711-66

100%

12%

12%

Figure 34. Results of the MAbs mapping.

5.2.1. E7 HPV type 11 immunodetection in ELISA

1,4 1,2 1

OD450

The best combination of monoclonal antibodies for E7 HPV type 11 sandwich ELISA were selected from several MAb combinations. The pairs were selected on the basis of MAb mapping data (maximal spatial determinant separation), sensitivity, specificity and kinetics characteristics.

1,6

0,8 0,6 0,4

Recommended pairs for sandwich ELISA are (capture - detection):

711-45 – 711-13 (see Fig. 33.) 711-66 – 711-13

0,2 0 0

2

4

6

8

10

12

[E7-11], ng/ml Figure 35. Calibration curves for E7 HPV type 11 sandwich immunoassays: 711-45 – 711-13. Coating: MAb 711-45, 5 µg/ml in 0.1 M, Carbonate buffer, pH 9.2 Detection: HRP-conjugted MAb 711-13, 1/20 000 Substrate: TMB

INFECTIOUS DISEASE REAGENTS

35


5.2.2. E7 HPV type 11 immunodetection in Western blotting

Dimer form

The results of MAb E7 HPV type 11 immunodetection in Western blotting after antigen SDS-gel electrophoresis and its transfer onto nitrocellulose membrane are presented on Fig. 36. Most of the tested MAbs recognize both monomer and dimer forms of HPV type 11.

Monomer form

Figure 36. Detection of E7 HPV type 11 (conjugated with hsp70) in Western blotting by different monoclonal antibodies after 12% SDS-PAAG electrophoresis. Strip 1: MAb 711-13, Strip 2: MAb 711-45, Strip 3: MAb 711-66, E7 HPV type 11 quantity: 15.0 Îźg/strip.

5.3. HPV, type 16, oncoprotein E7 monoclonal antibodies Host animal: Cell line used for fusion: Immunogen: Specificity: Purification method:

Mice Balb/c Sp2/0 Recombinant oncoprotein E7, type 16 (whole or fragments), conjugated with hsp70 Human papilloma virus, type 16 (for cross reactivity information see table 9) Protein G affinity chromatography

Table 8. MAbs main characteristics.

MAb

MAb isotype

Immunogen (hsp70 conjugated) HPV type

E7 oncoprotein fragment

716-281

IgG2b

16

Whole molecule

716-325

IgG2a

16

Whole molecule

Table 9. MAbs specificity. Study of cross-reactivity with HPV types 11, 16 and 18 Indirect ELISA, Coating: 5 mg/ml of each antigen; MAbs: 3 mg/ml

Cross reactivity with MAbs

E7, type 11

E7, type 16

E7, type 18

716-281

0%

100%

37%

0%

100%

0%

0%

100%

43%

716-332

IgG1

16

Whole molecule

716-325

716-A6**

IgG2a

16

Whole molecule

716-332

716-B2**

IgG1

16

Whole molecule

716-A6**

0%

100%

9%

0%

100%

83%

716-C4**

IgG1

16

Whole molecule

716-B2**

716-D1

IgG2a

16

Whole molecule

716-C4**

0%

100%

42%

0%

100%

114%

0%

100%

97%

716-E11

IgG1

16

Whole molecule

716-D1

716-F10

IgG1

16

Whole molecule

716-E11

ST1-A8**

IgG1

16

36-54 a.a.r. fragment

716-F10

0%

100%

100%

0%

13%

0%

ST1-A9**

IgG1

16

36-54 a.a.r. fragment

Com1-D9** *)

ST1-B7**

IgG1

16

36-54 a.a.r. fragment

Com2-D9** *)

0%

35%

100%

0%

0%

21%

ST1-B11**

IgG1

16

36-54 a.a.r. fragment

Com2-C11** *)

Com1-D9**

IgG1

16

CR1 fragment (common for all E7)

** MAb available only on special request.

Com2-D9**

IgG1

16

CR2 fragment (common for all E7)

Com2-C11**

IgG1

16

CR2 fragment (common for all E7)

Com2-D4**

IgG1

16

CR2 fragment (common for all E7)

** MAb available only on special request. 36

INFECTIOUS DISEASE REAGENTS

*) 100% reaction with corresponding peptide CR1 and CR2


5.3.1. E7 HPV type 16 immunodetection in ELISA The best combination of monoclonal antibodies for E7 HPV type 16 sandwich ELISA were selected from several MAb combinations. The pairs were selected

on the basis of MAb mapping data (maximal spatial determinant separation), sensitivity, specificity and kinetics characteristics.

Figure 37. Results of the MAbs mapping.

Recommended pairs for sandwich ELISA are (capture - detection): 716-D1 – 716-332 (see Fig. 38) 716-D1 – 716-E11 716-D1 – 716-F10 MAbs 716-D1, 716-281, 716-325 are equally suitable for capture of both HPV type 16 and 18.

5.3.2. E7 HPV type 16 immunodetection in Western blotting The results of MAb E7 HPV type 16 immunodetection in Western blotting after antigen SDS-gel electrophoresis and its transfer onto nitrocellulose membrane are presented on Fig. 39. Most of tested MAbs recognize both monomer and dimer (most common in physiological media) forms of HPV type 16.

1,8 1,6 1,4

Dimer form

OD450

1,2

(46 kDa)

1 0,8 0,6

Monomer form

0,4

(23 kDa)

0,2 0 0

1

2

3

4

5

[E7-16], ng/ml

Figure 38. Calibration curves for E7 HPV type 16 sandwich immunoassays: 716-D1 - 716-332. Coating: MAb 716-D1, 5 μg/ml, 0.1 M Carbonate buffer, pH 9.2 Detection: HRP-conjugated MAb 716-332, 1/50 000 Substrate: TMB

Figure 39. Detection of E7 HPV type 16 in Western blotting by different monoclonal antibodies after 15% SDS-PAAG electrophoresis. Strip 1: MAb 716-281, Strip 2: MAb 716-332, Strip 3: MAb 716-A6, Strip 4: MAb 716-B2, Strip 5: MAb 716-C4, Strip 6: MAb 716-D1, Strip 7: MAb 716-E11, Strip 8: MAb 716-F10; E7 HPV type 16 quantity: 15.0 μg/strip.

INFECTIOUS DISEASE REAGENTS

37


5.4. HPV, type 18, oncoprotein E7 monoclonal antibodies Host animal: Cell line used for fusion: Immunogen: Specificity: Purification method:

Mice Balb/c Sp2/0 Recombinant oncoprotein E7, types 18 (whole or fragments), conjugated with hsp70 Human papilloma virus, type 18 (for cross reactivity information see table 11) Protein G affinity chromatography

Table 10. MAbs main characteristics.

MAb

MAb isotype

Immunogen (hsp70 conjugated) E7 oncoprotein fragment

HPV type

718-15

IgG1

18

Whole molecule

718-67

IgG2a

18

Whole molecule

718-85**

IgG2b

18

Whole molecule

718-238

IgG2b

18

Whole molecule

718-A7**

IgG2a

18

Whole molecule

718-B5**

IgG1

18

Whole molecule

718-B6**

IgG1

18

Whole molecule

718-F9**

IgG2b

18

Whole molecule

718-G9**

IgG2a

18

Whole molecule

Figure 40. Results of the MAbs mapping.

38

INFECTIOUS DISEASE REAGENTS

Table 11. MAbs specificity. Study of cross-reactivity with HPV types 11, 16 and 18 Indirect ELISA, Coating: 5 mg/ml of each antigen; MAbs: 3 mg/ml

MAb

Cross reactivity with E7, type 11

E7, type 16

718-15

7%

43%

100%

718-67

0%

9%

100%

718-85**

0%

32%

100%

718-238

0%

8%

100%

718-B6**

5%

43%

100%

718-F9**

0%

21%

100%

718-G9**

0%

13%

100%

** MAb available only on special request.

E7, type 18


5.4.1. E7 HPV type 18 immunodetection in ELISA

2,5 2

OD450

The best combination of monoclonal antibodies for E7 HPV type 18 sandwich ELISA were selected from several MAb combinations. The pairs were selected on the basis of both MAb mapping data (maximal spatial determinant separation), sensitivity, specificity and kinetics characteristics.

3

1,5 1 0,5

Recommended pairs for sandwich ELISA are (capture - detection):

716-D1 – 718-238 (see Fig. 40) 718-15 – 718-85 718-67 – 718-238

0 0

1

2

3

4

5

[E7-18], ng/ml Figure 41. Calibration curves for E7 HPV type 18 sandwich immunoassays: 716-D1 – 718-238 Coating: MAb 716-D1 5 ug/ml, 0.1 M Carbonate buffer, pH 9.2 Detection: HRP-conjugated MAb 716-238, 1/20 000 Substrate: TMB

5.4.2. E7 HPV type 18 immunodetection in Western blotting The results of MAb E7 HPV type 18 immunodetection in Western blotting after antigen SDS-gel electrophoresis and its transfer onto nitrocellulose membrane are presented on Fig. 42. As can be seen, two tested MAbs, 716-281 and 716-D1, have ability to recognize E7 oncoprotein of both HPV type 16 and

18 that makes them suitable as a capture antibody for determination of both types of HPV. MAbs 718-67, 718-85 and 718-238 were found to be able to recognize both monomer and dimer forms of HPV type 18 and can be recommended for an ELISA application.

Dimer form (36-40 kDa)

Monomer form (18-20 kDa)

Figure 42. Detection of E7 HPV type 18 in Western blotting by different monoclonal antibodies after 15% SDS-PAAG electrophoresis: Strip 1: MAb 716-281, Strip 2: MAb 716-D1, Strip 3: MAb 718-15, Strip 4: MAb 718-67, Strip 5: MAb 718-85, Strip 6: MAb 718-238, Strip 7: MAb 718-A7; Strip 8: MAb 718-B5; Strip 9: MAb 718-B6; Strip 10: MAb 718-F9; Strip 11: MAb 718-G9; E7. HPV type 18 quantity: 15.0 μg/strip.

INFECTIOUS DISEASE REAGENTS

39


Ordering information: Product Anti-E7 HPV type 6 Anti-E7 HPV type 11 Anti-E7 HPV type 11 Anti-E7 HPV type 11 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 16 Anti-E7 HPV type 18 Anti-E7 HPV type 18 Anti-E7 HPV type 18 Anti-E7 HPV type 18 Anti-E7 HPV type 18 Anti-E7 HPV type 18 Anti-E7 HPV type 18 Anti-E7 HPV type 18 Anti-E7 HPV type 18

Cat. #

MAb

Isotype

Remarks

3HP6 3HP11 3HP11 3HP11 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP16 3HP18 3HP18 3HP18 3HP18 3HP18 3HP18 3HP18 3HP18 3HP18

706-C5 711-13 711-45 711-66 716-281 716-325 716-332 716-A6** 716-B2** 716-C4** 716-D1 716-F10 716-E11 ST1-A8** ST1-A9** ST1-B7** ST1-B11** Com1-D9** Com2-D9** Com2-C11** Com2-D4** 718-15 718-67 718-85** 718-238 718-A7** 718-B5** 718-B6** 718-F9** 718-G9**

IgG3 IgG1 IgG2a IgG3 IgG2b IgG2a IgG1 IgG2a IgG1 IgG1 IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG2a IgG2b IgG2b IgG2a IgG1 IgG1 IgG2b IgG2a

EIA, WB, C/r with types 11, 16 and 18 EIA, WB EIA, WB EIA, WB EIA (capture), WB EIA (detection), WB EIA (detection), WB, dimer and monomer WB, dimer and monomer WB WB, dimer and monomer EIA (capture), WB, dimer and monomer EIA (detection), WB, dimer and monomer EIA (detection), WB WB WB WB WB WB WB WB WB EIA (capture), WB EIA (capture), WB EIA (detection), WB EIA (detection), WB WB WB WB WB WB

** MAb available only on special request.

5.5. Human papilloma virus (HPV) antigens Ordering information: Product

Cat. #

Purity

Source

Human Papillomavirus L1 protein (HPVL1), type 16 Human Papillomavirus L1 protein (HPVL1), type 18

8HPV16 8HPV18

>90% >90%

Recombinant Recombinant

40

INFECTIOUS DISEASE REAGENTS


VI Malaria Malaria is a serious and sometimes fatal disease caused by a parasite. Forty-one percent of the worldâ&#x20AC;&#x2122;s population live in areas where malaria is transmitted (e.g., parts of Africa, Asia, the Middle East, Central and South America, Hispaniola, and Oceania). It is a leading cause of death and disease worldwide, especially in developing countries. Most deaths occur in young children. The World Health Organization estimates that each year 300-500 million cases of malaria occur and more than 1 million people die of malaria. Patients with malaria typically are very sick with high fevers, shaking chills, and flu-like illness. Four kinds of malaria parasites can infect humans: Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. Among these four malaria species, P. vivax and P. ovale can develop dormant liver stages that can reactivate after symptomless intervals of up to 2 (P. vivax) to 4 years (P. ovale). Infection with any of the malaria species can make a person feel very ill; infection with P. falciparum, if not promptly treated, may be fatal. The Plasmodium genus of protozoal parasites have a life cycle which is split between a vertebrate host and an insect vector. The Plasmodium species, with the exception of P. malariae (which may affect the higher primates) are exclusively parasites of man. The mos-

quito is always the vector and only female mosquitos are involved as the males do not feed on blood. The basic life cycle of the parasite is following: The gametocyte is the form that infects the mosquito and reproduces itself, as if it were both sexes. Inside a mosquito gametocytes pass into the salivary glands where they develop into a new form, the sporozoite. The sporozoite can be passed on to man when the mosquito bites. Sporozoites are more antigenic, and bear circumsporozoite polypeptide on their plasmalemma. The sporozoite travels with the blood to the liver where it enters the liver cells and some of the sporozoites divide (tachysporozoites) and become thousands of merozoites. The merozoites are released to the blood where they are taken up by the red blood corpuscles. Some of these turn into ring-formed trophozoites, which split again to form schizonts. Schizonts burst the red blood corpuscles at a certain moment, releasing the merozoites. This release coincides with the violent rises in temperature during the attacks seen in malaria.

Ordering information: Product

Cat. #

Mab

Isotype

Remarks

Anti-Plasmodium vivax merozoite surface protein 1 (MSP1)

3PLV5 3PLV5 3PLV5 3PLV5 3PLV5 3PLV2 3PLV2 3PLF3 3PLF3 3PLF3 3PLF1 3PLF1 3PLF1

PVM-1 PVM-2 PVM-3 PVM-4 PVM-5 PVC-1 PVC-2 PFS-1 PFS-2 PFS-3 PEM-1 PEM-2 PEM-3

IgG1 IgG2b IgG2b IgG2b IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG2a IgG1 IgG1

EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB

Anti-Plasmodium vivax merozoite surface protein 1 (MSP1) Anti-Plasmodium vivax merozoite surface protein 1 (MSP1) Anti-Plasmodium vivax merozoite surface protein 1 (MSP1) Anti-Plasmodium vivax merozoite surface protein 1 (MSP1) Anti-Plasmodium vivax circumsporozoite protein (CSP) Anti-Plasmodium vivax circumsporozoite protein (CSP) Anti-Plasmodium falciparum S-antigen (Sag) Anti-Plasmodium falciparum S-antigen (Sag) Anti-Plasmodium falciparum S-antigen (Sag) Anti-Plasmodium falciparum merozoite surface protein 1 (MSP1) Anti-Plasmodium falciparum merozoite surface protein 1 (MSP1) Anti-Plasmodium falciparum merozoite surface protein 1 (MSP1)

INFECTIOUS DISEASE REAGENTS

41


VII Tuberculosis Tuberculosis (TB) is a disease of global concern. About one third of the world population is infected with Mycobacterium tuberculosis. Every year, approximately 8 million people get the disease and 2 million die of TB. The currently available vaccine against TB is the attenuated strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), which has failed to provide consistent protection in different parts of the world. The commonly used diagnostic reagent for TB is the purified protein derivative (PPD) of M. tuberculosis, which is nonspecific because of the presence of antigens cross-reactive with BCG and environmental mycobacteria. Thus there is a need to identify M. tuberculosis antigens as candidates for new protective vaccines and specific diagnostic reagents against TB. Unfortunately, due to the great heterogeneity of the antibody response in tuberculosis patients no commercially available serological test has so far shown useful levels of sensitivity and specificity. It is therefore generally accepted that it will be necessary to include several antigens in a future serodiagnostic assay and the necessary improvements in sensitivity can be achieved by combining the best antigens available.

By using the techniques of recombinant DNA, synthetic peptides, antigen-specific antibodies and T cells etc., several major antigens of M. tuberculosis have been identified, e.g. heat shock protein (hsp) 60, hsp70, Ag85, ESAT-6 and CFP10 etc. These antigens have shown promise as new candidate vaccines and/or diagnostic reagents against TB. In addition, recent comparisons of the genome sequence of M. tuberculosis with BCG and other mycobacteria have unraveled M. tuberculosis specific regions and genes. Expression and immunological evaluation of these regions and genes can potentially identify most of the antigens of M. tuberculosis important for developing new vaccines and specific diagnostic reagents against TB. Moreover, advances in identification of proper adjuvant and delivery systems can potentially overcome the problem of poor immunogenicity/short-lived immunity associated with protein and peptide based vaccines. HyTest has started work over the development of a new line of TB diagnostic preparations. Our first products in this series are recombinant hsp70 and hsp65 and related monoclonal antibodies. We are offering also other M. tuberculosis related monoclonal antibodies, including the one specific to a number of antigens corresponding to dormancy regulon of M. tuberculosis.

Ordering information: Product

Cat. #

Purity

Source

M. tuberculosis 65 kDa Heat Shock Protein (HSP65) M. tuberculosis 70 kDa Heat Shock Protein (HSP70)

8HSP65 8HSP70

>95% >95%

Recombinant Recombinant

42

INFECTIOUS DISEASE REAGENTS


Ordering information: Product

Cat. #

MAb

Isotype Remarks

Anti-M. tuberculosis CFP10 Anti-M. tuberculosis CFP10 Anti-M. tuberculosis CFP10 Anti-M. tuberculosis ESAT6 Anti-M. tuberculosis ESAT6 Anti-M. tuberculosis, Heat Shock Protein 70 (HSP 70) Anti-M. tuberculosis, Heat Shock Protein 70 (HSP 70) Anti-M. tuberculosis, Heat Shock Protein 70 (HSP 70) Anti-M. tuberculosis, Rv1734 dormant protein from H37Rv strain Anti-M. tuberculosis, Rv2031 dormant protein from H37Rv strain Anti-M. tuberculosis, Rv2031 dormant protein from H37Rv strain Anti-M. tuberculosis, Rv2031 dormant protein from H37Rv strain Anti-M. tuberculosis, Rv2031 dormant protein from H37Rv strain Anti-M. tuberculosis, RV2623 Rec. Protein of Dormancy Anti-M. tuberculosis, RV2623 Rec. Protein of Dormancy Anti-M. tuberculosis, RV2623 Rec. Protein of Dormancy Anti-M. tuberculosis, RV2623 Rec. Protein of Dormancy Anti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain Anti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain Anti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain Anti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain Anti-M. tuberculosis, Rv2626 dormant protein from H37Rv strain Anti-M. tuberculosis, RV3134 Rec. Protein of Dormancy Anti-M. tuberculosis, RV3134 Rec. Protein of Dormancy Anti-M. tuberculosis, RV3134 Rec. Protein of Dormancy Anti-M. tuberculosis recombinant 16 kDa Ag Anti-M. tuberculosis recombinant 16 kDa Ag Anti-M. tuberculosis recombinant 16 kDa Ag Anti-M. tuberculosis recombinant 38 kDa Ag Anti-M. tuberculosis recombinant 38 kDa Ag Anti-M. tuberculosis recombinant 38 kDa Ag

3CFP1 3CFP1 3CFP1 3ES6 3ES6 3HSP70 3HSP70 3HSP70 3RV17 3RV20 3RV20 3RV20 3RV20 3RV26 3RV26 3RV26 3RV26 3RV66 3RV66 3RV66 3RV66 3RV66 3RV31 3RV31 3RV31 3MT16 3MT16 3MT16 3MT38 3MT38 3MT38

KFB16 KFB34 KFB42 RSB14 RSB34 TS489 TS29 TS31 17A9 31C11 31D7 31F11 31F12 A10 E1 E3 E5 26A8 26A11 26C5 26F6 26H6 B10 D5 G1 HTM61 HTM62 HTM63 HTM81 HTM82 HTM83

IgG1 IgG1 IgG2b IgG1 IgG2a IgG2b IgG1 IgG2a IgG2b IgG1 IgG1 IgG2a IgG2b IgG1 IgG3 IgG2a IgG2b IgG2a IgG2a IgG2a IgG2a IgG2b IgG3 IgG1 IgG1 IgG1 IgG2a IgG2a IgG1 IgG2b IgG1

EIA (capture) EIA (capture) EIA (detection) EIA EIA EIA (detection), WB EIA (capture), WB EIA (capture), WB EIA, WB Indirect EIA, WB Indirect EIA, WB Indirect EIA, WB Indirect EIA EIA, WB EIA, WB EIA, WB EIA, WB Indirect EIA, WB Indirect EIA, WB Indirect EIA, WB Indirect EIA, WB Indirect EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB EIA, WB

INFECTIOUS DISEASE REAGENTS

43


VIII Foodborne pathogens 1. Gastroenteritis viruses: rotavirus and adenovirus Acute gastroenteritis is among the most common illnesses of humankind, and its associated morbidity and mortality are greatest among those at the extremes of age, children and the elderly. In developing countries, gastroenteritis is a common cause of death in children < 5 years that can be linked to a wide variety of pathogens. In developed countries, while deaths from diarrhoea are less common, much illness leads to hospitalization or doctor visits. Much of the gastroenteritis in children is caused by viruses belonging to four distinct families rotaviruses, caliciviruses, astroviruses and adenoviruses. Other viruses, such as the toroviruses, picobirnaviruses, picornavirus (the Aichi virus), and enterovirus 22, may play a role as well. Viral gastroenteritis occurs with two epidemiologic patterns, diarrhoea that is endemic in children and outbreaks that affect people of all ages. Viral diarrhoea in children is caused by group A rotaviruses, enteric adenoviruses, astroviruses and the caliciviruses; the illness affects all children worldwide in the first few years of life regardless of their level of hygiene, quality of water, food or sanitation, or type of behaviour. For all but perhaps the caliciviruses, these infections provide immunity from severe disease upon reinfection. Epidemic viral diarrhoea is caused primarily by the Norwalk-like virus genus of the caliciviruses. These viruses affect people of all ages, are often transmitted by food or water contaminated with foeces, and are therefore subject to control by public health measures. The tremendous antigenic diversity of caliciviruses and shortlived immunity to infection permit repeated episodes throughout life. In the past decade, the molecular characterization of many of these gastroenteritis viruses has led to advances both in our understanding of the pathogens themselves and in development of a new generation of diagnostics. Application of these more sensitive methods to detect and characterize individual agents is just beginning, but has already opened up new avenues to reassess their disease burden, examine their molecular epidemiology, and consider new directions for their prevention and control through vaccination, improvements in food and water quality and sanitary practices. 44

INFECTIOUS DISEASE REAGENTS

Rotavirus (Reoviridae family) has 11 segments of double-stranded RNA. The virion has four major structural proteins, which form a capsid with three layers. The adenovirus (family Adenoviridae, genus Mastadenovirus) virion contains double-stranded DNA surrounded by a capsid 70-90 nm in diameter, which is comprised of 252 capsomers. The hexon capsomers share a cross-reacting group common antigen on their surface. Rotavirus and adenovirus antigens can be detected in stool specimens by EIA or electron microscopy. We developed a representative panel of murine MAbs against rotavirus. Purified bovine rotavirus, cell culture adapted strain MR was used as an immunogen. Further on we chose one MAb 3C10, being of the greatest diagnostic potential. Western blot showed that MAb 3C10 recognized p42-major inner-capsid antigen. MAb 3C10 demonstrates broad cross-reactivity and interact equally well with monkey rotavirus (SA-11), porcine rotavirus (PP) and with numerous human rotaviruses (field strains). Specific antibody titer is 10 -6 in ELISA. In combination with polyclonal bovine antibody to rotavirus as capture and labeled MAb 3C10 form a perfect sandwich for the quantitative virus determination in clinical specimens (see Fig. 43). We used a purified human adenovirus type 2 as an immunogen for the MAb panel generation. After selection, 2 MAbs were chosen: 1E11 and 7C11, directed against genus-specific hexon antigen, which is present in all human serotypes, and being of greater affinity to adenovirus. Traditional sandwich ELISA with the use of a homologous pair of MAb 1E11/1E11, as well as a combination of MAb 7C11 (capture) and 1E11 (detection) provides for the detection limit of viral antigens 1ng/ml. (See Fig. 44).


The new MAb 8C4 having the same specificity showed even better performance as capture antibody in sandwich ELISA with MAb 1E11-HRP conjugate.

observed. FITC-labelled MAb 7C11 provided for the adenovirus detection with the sensitivity and specificity 71.4 and 100% respectively at testing of 20 clinical specimens of nasal washings (see Fig. 45).

We tested 100 faecal specimes and 100 nasal washings using in-house ELISA with MAb 7C11 and 1E11, compared to adenovirus Antigen EIA (Biotrin International) and a complete coinsidence of results was

The fermentation conditions for bulk production of rota- and adenovirus antigens as positive controls have been optimised for ELISA kits production.

0,1

1

10

100

1000

10000

7C11

Ab (poly) 5 Âľg/ml, 3C10 0.25 Âľg/ml

Figure 43. ELISA calibration curve for Rotavirus antigen determination.

1E11

Figure 44. ELISA calibration curve for adenovirus antigen determination.

A

Negative

B

Negative

Positive

Positive

Figure 45. Determination of Adenovirus antigen in biologic materials. Comparison in-house ELISA versus BIOTRIN ELISA. A: Nasal washing, B: Stool specimens.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3R10 3AV13 3AV13 3AV13

3C10 7C11 1E11 8C4

IgG2a IgG2a + IgM IgG2a + IgM IgG2a

EIA, IHC, WB, P42 Antigen EIA, ID, IHC EIA (detection), ID, IHC EIA (capture), ID, IHC

Anti-Rotavirus Group Specific Antigen Anti-Adenovirus hexon Anti-Adenovirus hexon Anti-Adenovirus hexon

Ordering information: Product

Cat. #

Source

Remarks

Adenovirus, type 6

8AV13

Tonsil 99

EIA

INFECTIOUS DISEASE REAGENTS

45


2. Salmonella Genus Salmonella (Enterobacteriaceae family) is an important enteric pathogen that is responsible for a variety of diseases, including gastroenteritis and enteric fever. Human infection is primarily acquired by ingestion of contaminated poultry or meat. Water that has been contaminated by faeces or urine from humans or animals may also serve as a vehicle of transmission. Ninety-five percent of the salmonella serotypes isolated from humans belong to Subgroup 1 containing serogroup A, B, C1, C2, D and E. For immunoserologic identification one should be able to detect Vi capsular antigen of S. typhi and group-specific O-antigens. For production of monoclonal antibodies anti-Salmonella O-antigens the following heat-inactivated (100 °C, 60 min) microor-

ganisms were used for immunization: S. paratyphi A (group A), S. typhimurium (group B) and S. enteriditis (group D). Finally we produced a panel of MAbs, being of a different cross-reactivity spectrum in respect to Salmonella spp. (see Table 12). Thus for serogroup typing of Salmonella spp. we offer the following MAbs: 5D12A: 10D9H: 10B10G: 1E6:

Group A, B, C1, C2, D, E1, E2 (pan-Salmonella) Group A, B, D, E1, E2 Group A Group B

Immunogen serogroup

Mouse IgG subisotype

S. paratyphi A

S. typhimurium

S. choleraesuis

S. newport

S. enteriditis

S. anatum

S. selandia

E. coli 055:B5

E. coli K12

Klebsiella pneum.

10B10G 5D12A 1E6 10D9H

A A B D

IgG1 IgG1 IgG1 IgG1

A + + +

B + + +

C1 + -

C2 + -

D + +

E1 + ±

E2 + ±

-

-

-

To prove cross-reactivity we determined binding -1 constants (Ka, M ) for MAbs with lipopolysaccharides (LPS) of A, B, D and E groups (Table 13). Table 13: Binding constants (Ka, M1).

MAb 10B10G 10D9H 5D12A

LPS A 2.0 x 107 2.1 x 10 9 1.0 x 10 9

LPS B

LPS D

1.1 x 10 9 6.5 x 10 8 1.0 x 107 1.0 x 107

LPS E 1.0 x 10 6 1.0 x 1010

MAbs may be used for Salmonella spp. detection in dot-blot, ELISA and immunofluorescence. For production of monoclonal antibody anti-S. typhimurium lipopolysaccharides of S. typhimurium have been used as immunogen. Obtained MAb 1E6 is specif-

46

INFECTIOUS DISEASE REAGENTS

Tentative LPS antigenic determinant

Immunogen MAb

Table 12. O-Antigen specificity of the anti-Salmonella MAbs.

0-2 core 0-4 0-12

ic for LPS of S. typhimurium. Later on it was shown that Eu-labelled MAb 1E6 has a very broad crossreactivity range recognizing E. coli 1243 and Listeria monocytogenes (ATCC 7644) species as well, being a potential positive control antibody for a variety of assays. For production of anti-Salmonella virchow MAbs hybridoma clones deriving from hybridization of Sp2/0 myeloma cells with spleen cells of Balb/c mice immunized with lyophilized Salmonella virchow, serotype C has been used. MAbs SvB3 and SvE2 crossreact with S. typhimurium. MAbs SvA1 and SvB3 bind LPS. MAb SvE2 binds a protein of approximately 3840 kDa in Western blotting.


Ordering information: Product

Cat. #

MAb

Isotype Remarks

3SO22 3SO22 3SO22 3S9 3SV4 3SV4 3SV4

10B10G 5D12A 10D9H 1E6 SvA1 SvB3 SvE2

IgG1 IgG1 IgG1 IgG1 IgG2b IgG2a IgG2a

Anti-Salmonella O-Antigens Anti-Salmonella O-Antigens Anti-Salmonella O-Antigens Anti-Salmonella typhimurium Anti-Salmonella virchow Anti-Salmonella virchow Anti-Salmonella virchow

A-group, C/r Data Available Pan Salmonella, C/r Data Available A, B and D-group, C/r Data Available LPS of Salmonella typhimurium LPS LPS, C/r with S. typhimurium 38-40 kDa in WB, C/r with S. typhimurium

3. Listeria monocytogenes The genus Listeria comprises six species: L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii and L. grayi. Listeria monocytogenes, the most commonly isolated pathogenic member, is associated with a wide spectrum of human and animal diseases. In the smear from the original tissue, L. monocytogenes may appear as gram-positive coccobacilli that may be confused with Streptococcus agalactiae (group B), enterococci, or Corynebacterium spp. Listeria is differentiated from streptococci by a positive catalase test.

L. monocytogenes is the only species of the genus Listeria that has been clearly documented as a pathogen for humans. The forms of disease caused by this organism are myriad and age-related. The most common clinical manifestations are meningitis and septicemia. An electrophoresis picture of L. monocytogenes can be seen in Fig. 46.

Figure 46. 12% SDS-PAGE (Laemmli, silver stain)

94 67 43

Lane 1: Positive control (KPL) without β-mercaptoethanol Lane 2: L. monocytogenes cell suspension without β-mercatoethanol Lane 3: L. monocytogenes OM fraction without β-mercaptoethanol Lane 4: L. monocytogenes OM fraction with β-mercaptoethanol Lane 5: L. monocytogenes cell suspension with β-mercaptoethanol Lane 6: Positive control (KPL) with β-mercaptoethanol Lane 7: Molecular mass standards from top to bottom: 94K, 67K, 43K, 30K, 20K, 14K

30

20 14 1

2

3

4

5

6

7

The antibodies are working in indirect ELISA and they are specific and show high immunoreactivity (end-point dilution is about 1 – 3 ng/ml) against outer membrane (OM) fraction of L. monocytogenes. The best calibration curves were obtained with antibodies LZG7 and LZF7 (Fig. 47).

The best pair for sandwich ELISA was selected among the antibodies by testing all possible pairs. The best pair according to the results was LZH1 – LZF7 (capture – detection) (Fig. 48, 49 and 50).

INFECTIOUS DISEASE REAGENTS

47


0,8

0,25

0,7

LZH1 - LZF7-POD 1/10 000 LZH1 - LZF7-POD 1/20 000 LZH1 - LZF7-POD 1/50 000 LZH1 - LZF7-POD 1/100 000

0,20

+

0,6

+

0,4

+

0,3

10

1

0,05

0 0,0001

0,001

0,01

0,1

0,10

0,1

++

+ +

+

+

+

+ +

+

+

+

0,2

0,15

OD 450

+

0,5

0,00

LZG5

LZG7

0,1

LZH1

+

LZF7

+

MAbs, µg/ml

10

1

OM fraction L. monocytogenes, ng/ml

LZA2

Figure 47. Calibration curves of anti-L. monocytogenes antibodies in indirect ELISA.

Figure 48. Calibration curves for L. monocytogenes detection at different conjugate dilutions using pair LZH1 – LZF7.

0,6 1,6 2,5 µg/ml 5,0 µg/ml 10,0 µg/ml

1,4

0,4

1,0

OD 450

OD 450

1,2

2,5 µg/ml 5,0 µg/ml 10,0 µg/ml

0,5

0,8 0,6

0,3

0,2

0,4 0,1 0,2 0,0

0,0 0,1

1

10

100

1

10

100

1000

10 000

Cell/ml

OM fraction L. monocytogenes, ng/ml

Figure 49. Calibration curves for L. monocytogenes detection with different MAb coating concentrations using pair LZH1 – LZF7.

1,4 1,6 1,2

1/2000 1/5000 1/10000

1,4 1/2000 1/5000 1/10000

1,0

0,8

OD 450

OD 450

1,2

1,0

0,8 0,6

0,6 0,4

0,4 0,2

0,2

0,0

0,0 0,1

1

10

100

OM fraction L. monocytogenes, ng/ml

1

10

100

1000

10000

Cell/ml

Figure 50. Calibration curves for L. monocytogenes detection at different conjugate dilutions and 2.5 µg/ml coating concentration using pair LZH1 – LZF7.

Using pair LZH1 – LZF7 the detection limit 0.3 ng/ ml for L. monocytogenes OM fraction and 100 cells per well for L. monocytogenes cell suspension have been achieved.

48

INFECTIOUS DISEASE REAGENTS

All antibodies recognize L. monocytogenes whole cell lysate and OM fraction in Western blotting. The antibodies detect 23 kDa protein band (Fig. 51).


L. monocytogenes cell suspension L. monocytogenes OM fraction

94 67 43 30

Lanes 1 and 16: Molecular weight markers Lanes 2 and 9: MAb LZF7 Lanes 3 and 10: MAb LZG5 Lanes 4 and 11: MAb LZG7 Lanes 5 and 12: MAb LZH1 Lanes 6 and 13: MAb LZA2 Lanes 7 and 14: Anti-Legionella pneumophila antibodies Lanes 8 and 15: Control (anti-mouse IgG, hrp-conjugated)

20 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 51. Western blotting of L. monocytogenes with different antibodies.

All antibodies recognize specifically L. monocytogenes OM fraction, L. monocytogenes cell suspension and Listeria-genus specific positive control in dot-blot EIA (Fig. 52). LZF7, hrp-conjugate, 1/10000 1 Line 1: L. monocytogenes OM fraction in concentrations 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.3 μg/ml, 3.1 μg/ml 2 μl/point, i.e. 100 ng, 50 ng, 25 ng, 12.5 ng, 6.3 ng per dot.

2 3 LZG7, hrp-conjugate, 1/1000 1 2

Line 2: L. monocytogenes cell suspension from initial stock with step 1/10 2 μl/point, i.e. 2 x 10 6 cells, 2 x 10 5 cells, 2 x 104 cells etc. per dot.

3

LZH1, hrp-conjugate, 1/3000 1 2 3

Line 3: Listeria-genus specific positive control was reconstituted according to manufacture’s instruction and spotted from initial stock with 10-fold serial dilutions 2 μl/point. (KPL, Cat # 50-90-90).

Figure 52. Dot-blot EIA with conjugated anti-L. monocytogenes antibodies.

INFECTIOUS DISEASE REAGENTS

49


The detection limits for L. monocytogenes OM fractions were 6 - 12 ng for conjugated LZF7 (diluted 1/10000), 25 ng for conjugated LZG7 (diluted 1/1000) and 6 ng for conjugated LZH1 (diluted 1/3000). Using L. monocytogenes cells the detection limit for all conjugates was 2000 cells/dot (the fourth dilution dot). Using Listeria-genus specific positive control from KPL only the initial concentration was detected with conjugated LZF7. With conjugated LZG7 the second dilution (1/10) was detected and conjugated LZH1 detected also the third dilution (1/1000).

Cross-reactivity:

Anti-L. monocytogenes antibodies have been tested negative for cross-reactivity (ELISA) with the following species: E. coli, Salmonella, Y. pestis, F. tularensis, L. pneumophila and B. abortus. MAbs LZH1, LZF7 and LZG5 have been tested for cross-reactivity with L. innocua and L. ivanovii. The antibodies are cross-reacting with these species.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3L1 3L1 3L1 3L1 3L1

LZF7 LZG5 LZG7 LZH1 LZA2

IgG2a IgG2a IgG2a IgG1 IgM

EIA (detection), WB EIA, WB EIA, WB EIA (capture), WB EIA, WB

Anti-Listeria monocytogenes Anti-Listeria monocytogenes Anti-Listeria monocytogenes Anti-Listeria monocytogenes Anti-Listeria monocytogenes

4. Legionella pneumophila Legionella pneumophila is the bacterium associated with Legionnaires’ disease and Pontiac fever. Respiratory transmission of this organism can lead to infection, which is usually characterized by a gradual onset of flu-like symptoms. Patients may experience fever, chills, and a dry cough as part of the early symptoms. Patients can develop severe pneumonia, which is not responsive to penicillins or aminoglycosides. Legionnaires’ disease also has the potential to spread into other organ-systems of the body such as the gastrointestinal tract and the central nervous system. Accordingly, patients with advanced infections may experience diarrhea, nausea, disorientation, and confusion. Pontiac fever is also caused by L. pneumophila but does not produce the severity of the symptoms found in Legionnaires’ disease. The flu-like symptoms are still seen in Pontiac fever patients but pneumonia does not develop and

infection does not spread beyond the lungs. Most L. pneumophila infections are easily treated with erythromycin. MAbs react with LPS of Legionella pneumophila serogroup 1 and could be used in sandwich ELISA (2F10 for capture, 5F4 for detection), WB and dotblot as well as in lateral flow devices with MAb 5F4colloidal gold conjugate. MAbs do not cross-react with Pseudomonas fluorescens, Pseudomonas cepacia, Pseudomonas aerugenose, Bordetella pertusis, Leptospira interrogens (Australia), Leptospira interrognes (Pomona), Leptospira interrognes (Icterogemorrhagia), Toxoplasma gondii, Hemophilus influenza (type B), Brucella abortus, Bacillus anthracis, Yersinia pseudotuberculosis, Salmonella typhi, Escherichia coli K88, Francisella tularensis.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Legionella pneumophila LPS Anti-Legionella pneumophila LPS

3L15 3L15

2F10 5F4

IgG3 IgG3

EIA (capture) EIA (detection)

50

INFECTIOUS DISEASE REAGENTS


5. Campylobacter jejuni Campylobacters are bacteria that are a major cause of diarrheal illness in humans and are generally regarded as the most common bacterial cause of gastroenteritis worldwide. In developed and developing countries, they cause more cases of diarrhea than, for example, foodborne Salmonella bacteria. In developing countries, Campylobacter infections in children under the age of two years are especially frequent, sometimes resulting in death. In almost all developed countries, the incidence of human campylobacter infections has been steadily increasing for several years. The reasons for this are unknown.

We used Campylobacter jejuni intact cell suspension as immunogen and succeeded in developing 3 monoclonal antibodies E10, H3 and H2, which recognize C. jejuni in indirect ELISA and show no cross-reactivity against Salmonella spp and E. coli. MAbs H3, H2 and E10 recognize 38 kDa C. jejuni antigen in Western blotting. MAbs H2 and H3 could be used to capture C. jejuni in double antibody sandwich ELISA using MAb E10 as a conjugate in the presense of nonidet NP40.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Campylobacter jejuni Anti-Campylobacter jejuni

3CJ2 3CJ2

E10 H3

IgG2b IgG2b

EIA (detection), WB EIA (capture), WB

Anti-Campylobacter jejuni

3CJ2

H2

IgG1

EIA (capture), WB

6. Astrovirus Astroviruses are small, nonenveloped and singlestranded RNA viruses. In humans they are transmitted primarily through the foecal-oral route (including food- and waterborne transmission) and occasionally by aerosols. Numerous studies indicate that human astrovirus serotype 1 is the most predominant

serotype worldwide. It has been postulated that the incidence of astrovirus-associated gastroenteritis has been underestimated and that astrovirus infections may be one of the common infections of childhood.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Astrovirus

3AS6

ASV

IgG3

EIA, WB

INFECTIOUS DISEASE REAGENTS

51


IX Microbial and plant toxins 1. Antibodies for the detection of Staphylococcus aureus enterotoxins Staphylococcal enterotoxins (S.enterotoxins) represent a group of proteins, which are secreted by Staphylococcus aureus and cause food poisoning. Their molecular masses range between 27-30 kDa. At present, seven S. enterotoxins are known, namely A, B, C1, C2, C3, D and E. Their amino acid sequences have been determined and it was shown that all are single-chain polypeptides containing one disulfide bond formed by two half-cystines located in the

middle of the polypeptide chain, which form the socalled cysteine loop. S. enterotoxins are known to be most potent T cell mitogens. T cell activation accompanied by induction of interleukin 2 and interferon is conditioned by highâ&#x20AC;&#x201C;affinity interaction of S. enterotoxins with class II major histocompatibility complex (MHC) molecules and subsequent presentation of the complex formed to a variable region of the subunit of the T cell receptor.

1.1. Antibodies for the detection of Staphylococcus aureus enterotoxin Specificity of MAbs was established in indirect ELISA using standard staphylococcal enterotoxins A, B, C1, C2, D, E from Serva (Germany) at appropriate dilution of ascitic fluids.

Percentage of cross-reactivity is shown in Table 14. At 0.1 nM MAb S5 inhibits enterotoxins induced stimulation of T-lymphocytes in vitro.

Table 14: Percentage of cross-reactivity with enterotoxins of S. aureus

MAb

A

B

C1

C2

D

E

S1

21

0

0

0

100

16

S2

8

0

0

0

100

0

S5

2

100

7

76

29

0

1.2. Antibodies for the detection of Staphylococcus aureus enterotoxin A We used S. aureus enterotoxin A (SEA) as an immunogen to produce the MAbs C4, E8, E11, F12, G10, H5 and H10. All MAbs are working in Western blotting and EIA applications. In Table 15 are the recommended pairs for sandwich immunoassay and the detection limits with these pairs.

52

INFECTIOUS DISEASE REAGENTS

Table 15: Recommended pairs for sandwich immunoassay and the detection limits with these pairs

Capture MAb

Detection MAb

Detection limit ng/ml

H5

C4

< 0.1

H10

C4

0.1

E8

C4

0.1

G10

C4

0.1

F12

H5

0.1-0.2

C4

H5

0.1-0.2

E11

C4

0.1-0.2

C4

E8

0.1-0.2


1.3. Antibodies for the detection of Staphylococcus aureus enterotoxin B

3,5 3,0

Fig. 53 shows the calibration curve for SEB antigen quantification using double antibody sandwich ELISA with MAbs S222 and S643. The detection limit for the immunoassay is 50 pg/ml.

2,5

OD 450

MAbs S222 and S643 recognize two different epitopes of SEB molecule. There is no cross-reactivity with S. aureus enterotoxins A, C1, C2, C3, D and E.

2,0 1,5 mean

1,0 0,5 0,0 0

2

4

6

ng/ml

8

10

12

14

Figure 53. Calibration curve for double antibody sandwich with MAb S222 used for capture and MAb S643 as an HRP conjugate for SEB antigen detection.

1.4. Antibodies for the detection of Staphylococcus aureus enterotoxin G

1.5. Antibodies for the detection of Staphylococcus aureus enterotoxin I

We used S. aureus enterotoxin G (SEG) as an immunogen to produce the MAbs SEG-59 and SEG16. Both MAbs are working in Western blotting and EIA applications. Recommended pair for sandwich immunoassay is (capture - detection):

We used S. aureus enterotoxin I (SEI) as an immunogen to produce the MAbs SEI-17A and SEI-68. Both MAbs are working in Western blotting and EIA applications. Recommended pair for sandwich immunoassay is (capture - detection):

SEG-59 - SEG-16

SEI-17A - SEI-68

The detection limit for the immunoassay is 0.1 ng/ml.

The detection limit for the immunoassay is 0.1 ng/ml.

Ordering information: Product

Cat.#

MAb

Subclass

Application

Anti-S. aureus enterotoxin

2S3 2S3 2S3 2S7 2S7 2S7 2S7 2S7 2S7 2S7 2S4 2S4 2S6 2S6 2S5 2S5

S1 S2 S5 C4 E8 E11 F12 G10 H5 H10 S222 S643 SEG-59 SEG-16 SEI-17A SEI-68

Ig2a Ig2a Ig2a IgG1 IgG1 IgG2a IgG1 IgG1 IgG1 IgG2b IgG1 IgG1 IgG1 IgG2a IgG2a IgG1

A, D and E enterotoxin, EIA A and D enterotoxin, EIA A, B, C1, C2 and D enterotocin, EIA EIA (capture, detection), WB EIA (capture, detection), WB EIA (capture), WB EIA (capture), WB EIA (capture), WB EIA (capture, detection), WB EIA (capture), WB EIA (capture), N/cr with A, C, D and E enterotoxin EIA (detection), N/cr with A, C, D and E enterotoxin EIA (capture), WB EIA (detection), WB EIA (capture), WB EIA (detection), WB

Anti-S. aureus enterotoxin Anti-S. aureus enterotoxin Anti-S. aureus enterotoxin A Anti-S. aureus enterotoxin A Anti-S. aureus enterotoxin A Anti-S. aureus enterotoxin A Anti-S. aureus enterotoxin A Anti-S. aureus enterotoxin A Anti-S. aureus enterotoxin A Anti-S. aureus enterotoxin B Anti-S. aureus enterotoxin B Anti-S. aureus enterotoxin G Anti-S. aureus enterotoxin G Anti-S. aureus enterotoxin I Anti-S. aureus enterotoxin I

INFECTIOUS DISEASE REAGENTS

53


2. Antibodies for the detection of Cholera toxin (CT) Cholera is an acute intestinal infection caused by ingestion of food or water contaminated with the bacterium Vibrio cholerae. The bacterium may also live in the environment in brackish rivers and coastal waters. In an epidemic, the source of the contamination is usually the foeces of an infected person. The disease can spread rapidly in areas with inadequate treatment of sewage and drinking water. The infection is often mild or without symptoms, but sometimes it can be severe (approximately one in 20 cases). It has a short incubation period, from less than one day to five days, and produces an enterotoxin that in severe cases causes painless and watery diarrhoea that can quickly lead to severe de-

hydration and death even within hours if treatment is not promptly given. Vomiting and leg cramps can also occur. Cholera toxin and the closely related E. coli heatlabile enterotoxin are 85 kDa hexameric assemblies consisting of a single 240 residue A subunit and five identical 103 residue B subunits. The B subunits assemble into a regular pentamer containing a central pore, through which the C-terminal tail of the A subunit extends to hold the AB 5 holotoxin together. All MAbs react with B subunit of cholera toxin. MAb 3D11 reacts also with E.coli heat-labile enterotoxin. Specific antibody titer in indirect ELISA is 10 -6.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

2C4 2C4 2C4 2C4 2C4

3D11 B8 E6 E10 G9

IgG1 IgG1 IgG2a IgG2a IgG1

EIA, B-subunit of CT EIA (capture), B-subunit of CT EIA (capture), B-subunit of CT EIA (detection), WB, B-subunit of CT EIA (detection), B-subunit of CT

Anti-Cholera toxin Anti-Cholera toxin Anti-Cholera toxin Anti-Cholera toxin Anti-Cholera toxin

3. Antibodies for the detection of Escherichia coli heat-labile enterotoxin We have used recombinant A- and B-chains of E. coli heat-labile enterotoxin as immunogens for MAb

development. 19 clones were produced and tested for reactivity in ELISA and WB against LTA, LTB and Cholera toxin.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

2LTA2 2LTA2 2LTA2 2LTB2 2LTB2 2LTB2

AB3 AE4 AE7 BB2 BB12 BG12

IgG2b IgG2b IgG2b IgG2b IgG2b IgG1

EIA (capture), C/r with Cholera toxin EIA (detection), No C/r with Cholera toxin WB, C/r with Cholera toxin EIA, WB EIA, WB EIA, WB

Anti-E. coli heat-labile enterotoxin A-chain Anti-E. coli heat-labile enterotoxin A-chain Anti-E. coli heat-labile enterotoxin A-chain Anti-E. coli heat-labile enterotoxin B-chain Anti-E. coli heat-labile enterotoxin B-chain Anti-E. coli heat-labile enterotoxin B-chain

54

INFECTIOUS DISEASE REAGENTS


4. Antibodies for the detection of Clostridium botulinum toxoids Clostridium botulinum is an anaerobic, Gram-positive, spore-forming rod that produces a potent neurotoxin. The spores are heat-resistant and can survive in foods that are incorrectly or minimally processed. Foodborne disease caused by C. botulinum is referred to as botulism. Seven types (A, B, C, D, E, F and G) of botulism are recognized, based on the antigenic specificity of the toxin produced by each strain. Only types A, B, E and F cause illness in humans. Types C and D cause most cases of botulism in animals. Toxin is called a neurotoxin because it affects the nervous system. Symptoms appear 12

to 48 hours after eating a food, which contains the toxin. The symptoms include double vision, droopy eyelids, trouble with speaking and swallowing and difficulty with breathing. Without treatment death may result from suffocation because the nerves can no longer stimulate breathing. We have used formaldehyde inactivated C. botulinum toxins A, B, D and E to generate monoclonal antibodies. New MAbs 2A33 and 24A29 were produced against two synthetic peptides, a.a.r. 869-887 and a.a.r. 1177-1195, of Clostridium botulinum toxin A heavy chain. These new MAbs recognize natural and non-inactivated toxin A.

Ordering information: Product

Cat. #

MAb

Isotype Remarks

3Cb19 3Cb19 3Cb20 3Cb20 3Cb21 3Cb21 3Cb21 3Cb23 3Cb24 3Cb24

2A33 24A29 KBA211 KBA468 KBB18 KBB27 KBB36 KB21 KBE169 KBE42

IgGM IgGM IgG1 IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG1

Anti-C. botulinum toxin A Anti-C. botulinum toxin A Anti-C. botulinum A Toxoid Anti-C. botulinum A Toxoid Anti-C. botulinum B Toxoid Anti-C. botulinum B Toxoid Anti-C. botulinum B Toxoid Anti-C. botulinum D Toxoid Anti-C. botulinum E Toxoid Anti-C. botulinum E Toxoid

a.a.r. 869-887, C. botulinum toxin A heavy chain a.a.r. 1177-1195, C. botulinum toxin A heavy chain EIA (capture), C/r data Available EIA (detection), C/r data Available EIA (detection), C/r data Available EIA (capture), C/r data Available EIA (capture), C/r data Available EIA, IF, C/r data available EIA (capture), WB, C/r data Available EIA (detection), WB, C/r data Available

Ordering information: Product

Cat. #

Host animal

Polyclonal Anti-C. botulinum D Toxoid

3Cb23/2

goat

Remarks EIA

5. Antibodies for the detection of Diphtheria Diphtheria is an acute disease caused by a bacterium Corynebacterium diphtheriae, which produces a toxin that is carried in the bloodstream. Diphtheria is passed from person to person by droplet transmission, usually by breathing in diphtheria bacteria after an infected person has coughed, sneezed or even laughed. Diphtheria is a very contagious and potentially life-threatening infection. It usually attacks the throat and nose causing breathing problems but in more serious cases it can attack the nerves and also cause heart failure, paralysis and even death. Because of widespread immunization, diphtheria is very rare. However, some people are not adequate-

ly vaccinated, and cases still occur. People carrying diphtheria germs are contagious for up to 4 weeks without antibiotic therapy, even if they themselves do not develop symptoms. MAbs 1H2 and 3B6 react with different determinants of Diphtheria toxin and anatoxin. They do not react with free A or B subunits of Diphtheria toxin. Antibodies can be used for detection of Diphtheria toxin by different immunochemical technique. We recommend to use MAb 3B6 for capture and MAb 1H2 for labeling.

INFECTIOUS DISEASE REAGENTS

55


MAb 8A4 reacts with free A subunit of Diphtheria toxin. It does not react with the whole Diphtheria toxin and free B subunit of Diphtheria toxin. MAb 7F2 react with the epitope exposed on free A subunit and on whole Diphtheria toxin molecule. It does not react with free B subunit of Diphtheria toxin. Antibodies can be used for detection of Diphtheria toxin A subunit by different immunochemical technique. We recommend to use MAb 8A4 for capture and MAb 7F2 for labeling. MAb DiB4 reacts with free A subunit and with whole Diphtheria toxin and does not re-

act with free B subunit (WB results). On the contrary, MAb DiD1 reacts in WB with the epitope of free B subunit and on whole Diphtheria toxin molecule and do not react with free A subunit. Antibodies can be used for detection of Diphtheria toxin by different immunochemical technique. We recommend using the following pairs (capture- detection):

DiH1 - DiB4 DiH1 - DiD1

Ordering information: Product

Cat. #

MAb

Isotype Remarks

2DT13 2DT13 2DT13 2DT13 2DT13 2DT14 2DT14

1H2 3B6 DiD1 DiB4 DiH1 8A4 7F2

IgG1 IgG1 IgG2b IgG2b IgG1 IgG2a IgG1

Anti-Diphtheria Toxin Anti-Diphtheria Toxin Anti-Diphtheria Toxin Anti-Diphtheria Toxin Anti-Diphtheria Toxin Anti-Diphtheria Toxin, A-subunit Anti-Diphtheria Toxin, A-subunit

EIA (detection), N/cr with Free A- and B-subunits EIA (capture), N/cr with Free A- and B-subunits EIA, WB EIA, WB EIA N/cr with Whole Toxin and Free B-subunit, EIA (capture) N/cr with Free B-subunit, EIA (detection), Whole Toxin

6. Antibodies for the detection of Ricin RCA60 from Ricinus communis Ricin toxin is a protein derived from the beans of the castor plant (Ricinus communis). The naturally occurring toxin is fairly easily removed from the bean pulp waste, which remains after castor oil extraction. The toxin is stable in powder and aerosolized form, can be disseminated as an aerosol, an injection, or as a food or water contaminant, and has no known treatment or vaccine. Ricin poisoning cannot be spread from person to person through casual contact. Symptoms of ricin poisoning depend on the dose received and route of exposure and they usu-

ally begin within six hours of ingestion exposure and within eight hours of inhalation exposure. MAbs CP23, CP37, CP75 and RA999 recognize the A-chain of RCA60. MAb RB999 recognizes the Bchain of RCA60 and also the RCA120. The antibodies are working in ELISA. All MAbs (except CP23) are working also in Western blotting. The matched pair RA999 (capture) and RB999 (detection) has a detection limit of 100 pg/ml for RCA60 and shows no crossreactivity for RCA120.

Ordering information: Product

Cat. #

MAb

Isotype Remarks

2R1 2R1 2R1 2R1 2R1

CP23 CP37 CP75 RA999 RB999

IgM IgG2a IgG1 IgG1 IgG1

Anti-Ricin, RCA60 from Ricinus communis Anti-Ricin, RCA60 from Ricinus communis Anti-Ricin, RCA60 from Ricinus communis Anti-Ricin, RCA60 from Ricinus communis Anti-Ricin, RCA60 from Ricinus communis

56

INFECTIOUS DISEASE REAGENTS

A-chain, EIA A-chain, EIA, WB A-chain, EIA, WB A-chain, EIA (capture), WB, C/r with RCA120 B-chain, EIA (detection), WB


7. Antibodies for the detection of HT-2 toxin HT-2 toxin is mycotoxin of the group trichothecenes type A produced by fungi of the Fusarium genus, i.e. Fusarium sporotrichioides, F.poae, F.equiseti, and F.acuminatum which are commonly found in various cereal crops (wheat, maize, barley, oats, and rye) and processed grains (malt, beer and bread). HT-2 toxin (together with related T-2-toxin) often occurs in infected cereals. The fungi producing trichothcenes are soil fungi and are important plant pathogens which grow on the crop in the field. The trichothecenes are in general very stable compounds, both during storage/milling and processing/cooking of food, and they do not degrade at high temperatures.

Both HT-2 and T-2 toxins have a multiple toxic actions, including inhibition of DNA and RNA synthesis and of initiation phase of protein synthesis, affecting the permeability of cell membranes, causing apoptosis in living tissues (e.g. thymic and splenic lymphocytes). Monoclonal mouse anti-HT-2 toxin antibody were produced from ascitic fluid of tumour bearing BALB/c mice, inoculated with corresponding hybridoma clones using HT-2 toxin conjugated with HAS as an immunogen. All MAbs recognize HT-2 toxin and C6B4 and C6D4 also react with T2-toxin.

Ordering information: Product

Cat. #

MAb

Isotype

2HT2 2HT2 2HT2 2HT2

C6B4 C6D4 C6E6 C6F1

IgG1 C/r with T2-toxin IgG1 C/r with T2-toxin IgG1 â&#x20AC;Ż IgG1 â&#x20AC;Ż

Anti-HT-2 Toxin Anti-HT-2 Toxin Anti-HT-2 Toxin Anti-HT-2 Toxin

Remarks

8. Antibodies for the detection of Microcystin-LR Microcystin-LR is a cyanobacterial toxin and a potent inhibitor of protein phosphatase types 1 and 2A (has no effect on protein kinase). It is a tumor promoter rather than a carcinogen. Among more than 80 microcystins identified to date, only few occur frequently and in high concentrations but Microcystin-LR is

among the most frequent and most toxic microcystin congeners. Frequently occurring cyanobacterial genera that contain these toxins are Microcystis, Planktothrix and Anabaena. Microcystins usually occur within the cells; substantial amounts are released to the surrounding water only in situations of cell rupture.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Microcystin-LR Anti-Microcystin-LR

2MC2 2MC2

C64A1 C64C12

IgG1 IgG1

Indirect EIA Indirect EIA

9. Antibodies for the detection of Nodularin Nodularin is cyanobacterial toxins and it inhibits protein phosphatases 1 and 2A with the same potency as does microcystin-LR. Nodularin might be a tumor

promoter in rat liver. Nodularin itself is a new liver carcinogen.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Nodularin Anti-Nodularin Anti-Nodularin

2ND3 2ND3 2ND3

C66H3 C66B6 C66A2

IgG1 IgG1 IgG1

Indirect EIA Indirect EIA Indirect EIA

INFECTIOUS DISEASE REAGENTS

57


10. Antibodies for the detection of Tetanus toxin inhibitory impulses by interfering with the release of neurotransmitters. This leads to unopposed muscle contraction and spasm. Seizures may occur, and the autonomic nervous system may also be affected.

Tetanus toxin (tetanospasmin, TeNT) is the neurotoxin produced by the vegetative spore of Clostridium tetani in anaerobic conditions, causing tetanus â&#x20AC;&#x201C; a medical condition that is characterized by a prolonged contraction of skeletal muscle fibers, which can eventually cause respiratory failure.

The peptide tetanospasmin has a molecular weight of 150kDa. It is made up of two parts: a 100 kDa heavy or B-chain and a 50 kDa light or A-chain. The chains are connected by a disulfide bond. The B-chain binds to disialogangliosides (GD2 and GD1b) on the neurone membrane. The A-chain, a zinc endopeptidase, attacks the vesicle-associated membrane protein. The chains are non-toxic after separation.

Tetanospasmin is one of the most potent toxins known: the estimated minimum human lethal dose is 2.5 nanograms per kilogram of body weight, or 175 nanograms in a 70 kg human. The toxin acts at several sites within the central nervous system, including peripheral nerve terminals, the spinal cord, and brain, and within the sympathetic nervous system. The clinical manifestations of tetanus are caused when tetanus toxin blocks

Using supernatant from C. tetani we managed to produce three monoclonal antibodies specific to tetanus toxin having toxin neutralization activity (in vivo assay) and suitable for EIA and WB applications.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Tetanus Toxin Anti-Tetanus Toxin Anti-Tetanus Toxin

2TE8 2TE8 2TE8

TetE3 TetG2 TetH6

IgG1 IgG2a IgG1

EIA, WB EIA, WB EIA, WB

11. Antibodies for the detection of Aflatoxin from Aspergillus flavus Aspergillus flavus is a plant, animal, and human pathogen that produces the carcinogen, aflatoxin. Aflatoxins are potent toxic, carcinogenic, mutagenic and immunosuppressive agents, produced as secondary metabolites by the fungus Aspergillus flavus and A. parasiticus on variety of food products. Among 18 different types of aflatoxins identified, major members are aflatoxin B1, B2, G1 and G2. Aflatoxins M1 and M2 are major metabolites of aflatoxin

B1 and B2 respectively, found in milk of animals that have consumed feed contaminated with aflatoxins. We used aflatoxin from Aspergillus flavus conjugated to BSA as immunogen to produce monoclonal antibody against aflatoxin. MAb ATB recognizes free aflatoxins B1 and B2. No cross-reactivity with G1, G2 and M1 was observed.

Ordering information: Product

Cat.#

MAb

Isotype

Remarks

Anti-Aflatoxin

3Af27

ATB

IgG1

EIA

58

INFECTIOUS DISEASE REAGENTS


X Biodefense antibodies 1. Antibodies for the detection of Bacillus anthracis According to JAMA Consensus Statement, of the numerous biological agents that may be used as weapons, the Working Group on Civilian Biodefense has identified a limited number of organisms that could cause disease and deaths in sufficient numbers to cripple a city or region. Anthrax is one of the most serious of these diseases. For centuries, anthrax has caused disease in animals and, uncommonly, serious illness in humans throughout the world. Research on anthrax as a biological weapon began more than 80 years ago. Today, at least 17 nations are believed to have offensive biological weapons programs; it is uncertain how many are working with anthrax. In humans, 3 types of anthrax infection occur: inhalational, cutaneous, and gastrointestinal. Naturally occurring inhalational anthrax is now a rare cause of human disease. Historically, wool sorters at industrial mills were at highest risk. Cutaneous anthrax is the most common naturally occurring form, with an estimated 2000 cases reported annually, disease typically follows exposure to anthrax-infected animals, whereas gastrointestinal anthrax is uncommonly reported. However, gastrointestinal outbreaks have been reported in Africa and Asia. B. anthracis derives from the Greek word for coal, anthrakis, because the disease causes black, coallike skin lesions. B. anthracis is an aerobic, grampositive, spore-forming, nonmotile Bacillus species. The nonflagellated vegetative cell is large (1-8 μm in length, 1-1.5 μm in breadth). Spore size is approximately 1 μm. Spores grow readily on all ordinary laboratory media at 37 °C, with a “jointed bamboorod” cellular appearance and a unique “curledhair” colonial appearance, and display no hemolysis on sheep agar. This cellular and colonial morphology theoretically should make its identification by an experienced microbiologist straightforward, although few practicing microbiologists outside the veterinary community have seen anthrax colonies other than in textbooks. Anthrax spores germinate when they enter an environment rich in amino acids, nucleosides, and glucose, such as that are found in the blood or tissues of an animal or human host. The

Figure 54. Gram Stain of Bacillus anthracis.

rapidly multiplying vegetative anthrax bacilli, on the contrary, will only form spores after local nutrients are exhausted, such as when anthrax-infected body fluids are exposed to ambient air. Full virulence requires the presence of both an antiphagocytic capsule and 3 toxin components (ie, protective antigen, lethal factor, and edema factor). Vegetative bacteria have poor survival outside of an animal or human host; colony counts decline to undetectable within 24 hours following inoculation into water. This contrasts with the environmentally hardy properties of the B. anthracis spore, which can survive for decades. For diagnostics, identification and investigation of pathogenesis mechanisms of B. anthracis HyTest offers a wide spectrum of immunochemical reagents. We have produced 13 monoclonal antibodies to Protective Antigen (PA), which according to WB recognize both 83K and truncated 63K forms of PA. Traditional sandwich ELISA with the use of the match pair BAP0105 (capture) – BAP0106 (HRP conjugate) easily provides for the detection limit 100 pg/ml. For the detection of the lethal factor we offer match pair BAL0105 and BAL0106. HyTest has started the process of production of immunogenic preparations from B. anthracis spores. As a result of the rabbits immunization and a special exhaustion procedure we now have at our disposal IgG fraction of rabbit serum to spore antigen of B. anthracis which interact only with anthrax spores and do not cross-react with spores of other bacilli: B. cereus, B. turingiensis, B. subtilis, B. megaterium and others. According to WB they recognize S-layer proteins (92-94K) and work in dot-blot ELISA, immunofluorescence as well as in sandwich-ELISA with MAb when used as capture. INFECTIOUS DISEASE REAGENTS

59


1.1. Antibodies for the detection of Bacillus anthracis Protective Antigen Bacillus anthracis produces a three-component toxin, which contributes to the symptoms and lethality of anthrax. The anthrax toxin consists of three proteins, termed Protective Antigen (PA), Edema Factor (EF) and Lethal Factor (LF). The components are non-toxic individually until they combine to each other and enter cells. Full size PA molecule (83K) triggers this process via binding to the cell surface ATR Immunogen: Immunoreactivity: Applications:

receptor. After binding PA is cleaved enzymatically, yielding 63K fragment, which combine to form a ring-shaped heptamer. The heptamer captures EF and LF and is transported to endosome. Monoclonal antibodies against PA are useful for anthrax diagnostics, toxin neutralization and analyses of how it enters cells.

Purified B. anthracis Protective Antigen, 83K. MAbs C3, BAP0101, BAP0102, BAP0103, BAP0104, BAP0105, BAP0106 are directed against different PA epitopes. They recognize both 83K and truncated 63K forms of PA. There is no cross-reactivity with B. anthracis LF, spore and vegetative microbe surface antigens. Western blotting, ELISA. Fig. 55 shows the detection of crude B. anthracis Protective Antigen preparation in Western blotting using BAP0101, BAP0103, BAP0105 and BAP0106. Fig. 56 shows the calibration curve for PA quantification using double antibody sandwich ELISA with MAbs BAP0105 and BAP0106. The detection limit for the immunoassay is 50 pg/ml.

kDa

2,0

1,5

OD 450

94 67 43

1,0 PA 0,5

30 0,0

20 1

2

3

4

0

5

10

20

30

40

50

60

ng/ml

Figure 55. Detection of B. anthracis Protective Antigen in Western blotting using MAbs BAP0101 (1); BAP0103 (2); BAP0105 (3); BAP0106 (4); molecular mass standards (5). 10% SDS-PAGE was run under non-reducing conditions.

Figure 56. Calibration curve in ELISA for B. anthracis Protective Antigen determination using MAb BAP0105 for capture and MAb BAP0106 as a HRP-conjugate.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3BA16 3BA16 3BA16 3BA16 3BA16 3BA16 3BA16

C3 BAP0101 BAP0102 BAP0103 BAP0104 BAP0105 BAP0106

IgG1 IgG2b IgG2b IgG2b IgG2b IgG1 IgG1

EIA (capture), WB EIA, WB EIA (detection), WB EIA (detection), WB EIA, WB EIA (capture), WB EIA (detection), WB

Anti-B. anthracis Protective Antigen Anti-B. anthracis Protective Antigen Anti-B. anthracis Protective Antigen Anti-B. anthracis Protective Antigen Anti-B. anthracis Protective Antigen Anti-B. anthracis Protective Antigen Anti-B. anthracis Protective Antigen

60

INFECTIOUS DISEASE REAGENTS


1.2. Antibodies for the detection of Bacillus anthracis Lethal Factor B. anthracis full virulence requires the presence of both an anti-phagocyte capsule and 3 toxin collaborating components: Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). Up to three copies of EF or LF or a combination of the two bind to the Protective Antigen heptamer on the outer cell surface and are delivered to an endosome. Mild acidImmunogen: Immunoreactivity: Applications:

ity in the endosome compartment causes EF and LF transport into the cytosol. The exact mechanism of the LF action is not known. It was shown that LF is a protease and cleaves enzymes belonging to MAPKK family. Anti-LF monoclonals could be useful for anthrax toxin neutralization and LF intracellular localization.

Purified B. anthracis Lethal Factor. MAbs BAL0105 and BAL0106 recognize two different epitopes of LF molecule with high affinity. There is no cross-reactivity with B.anthracis PA, spore and vegetative microbe surface antigens. Western blotting, ELISA. Fig. 57 shows the detection of crude B. anthracis Lethal Factor preparation in Western blotting using MAbs BAL0105 and BAL0106. Fig. 58 shows the calibration curve for LF quantification using double antibody sandwich ELISA with MAbs BAL0105 and BAL0106. The detection limit for the immunoassay is 5 ng/ml.

kDa

94 67

2,5

2,0

OD 450 nm

43

30

1,5

1,0 stand. curve

0,5

0,0

1

2

20

0

100

200

300

400

500

[c] Ag ng/ml

Figure 57. Detection of B. anthracis Lethal Factor in Western blotting using MAbs BAL0105 (1); BAL0106 (2). 10% SDS-PAGE was run under non-reducing conditions.

Figure 58. Calibration curve in ELISA for Lethal Factor determination using MAb BAL0106 for capture and MAb BAL0105 as an HRP-conjugate

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3BA17 3BA17 3BA17 3BA17

BAL0105 BAL0106 LFA58 LFA27

IgG1 IgG1 IgG1 IgG2a

EIA (detection), WB EIA (capture), WB EIA (capture), WB EIA (detection), WB

Anti-B. anthracis Lethal Factor Anti-B. anthracis Lethal Factor Anti-B. anthracis Lethal Factor Anti-B. anthracis Lethal Factor

INFECTIOUS DISEASE REAGENTS

61


1.3. Antibodies for the detection of Bacillus anthracis Spore Antigen B. anthracis is an aerobic, gram-positive, sporeforming, nonmotile Bacillus species. Spore size is approximately 1 Âľm. Anthrax spores germinate when they enter an environment rich in amino acids, nucleosides and glucose. The rapidly multiplying vegetative anthrax bacilli will only form spores after local nutrients are exhausted, such as when anthrax-infected body fluids are exposed to ambi-

Immunogen: Immunoreactivity: Applications:

ent air. Spores can survive for decades in an environment. Current methods for anthrax spore identification are based on microscopy, germination and spore destruction PCR/DNA-probes. The use of immunochemical assays was limited in connection with poor antigens expositions and cross-reactivity with thousands of similar but harmless spore-forming microorganisms that colonize air, water and soil.

B. anthracis spore extract made by chaotropic agents and detergent treatment. MAbs SA26 and SA27 recognize S-layer protein (92K). There is no cross-reactivity with PA, LF and B. anthracis vegetative form. They do not bind to spores and vegetative forms of B. thuringiensis, B. subtilis, B. cereus, B. brevis, B. megaterium. Western blotting, ELISA. Fig. 59 shows the detection of crude B.anthracis spore preparation in Western blotting using MAbs SA26 and SA27. Fig. 60 shows the calibration curve for B. anthracis spore quantification using double antibody sandwich ELISA with MAbs SA26 and SA27. The detection limit for the immunoassay is 3 x 104 spores/ml.

1

2

3 B C D E F

3,0 2,5

OD 450

2,0 1,5 1,0 0,5 0,0 10

1000

100

thousand spore/ml Figure 59. Detection of B. anthracis spore in Western blotting using MAbs SA26 (1); SA27 (2); Molecular mass standards are 94, 67, 43, 30, 20 K from top to bottom (3). 10% SDS-PAGE was run under reducing conditions.

Figure 60. Calibration curves for B. Anthracis spore detection using MAb SA26 for capture and SA27 as an HRP conjugate. The spores were suspended in PBST (B), 0.05% SDS (C), 0.5M Urea (D), 0.4% Formaldehyde (E), 1% Formaldehyde (F).

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-B. anthracis Spore Antigen Anti-B. anthracis Spore Antigen

3BA19 3BA19

SA26 SA27

IgG2a IgG2a

EIA (capture), WB EIA (detection), WB

Ordering information: Product

Cat. #

Host animal

Remarks

Polyclonal B. anthracis Spore Antigen

3BA18

rabbit

EIA, WB

62

INFECTIOUS DISEASE REAGENTS


2. Antibodies for the detection of Yersinia pestis In AD 541, the first recorded plague pandemic began in Egypt and swept across Europe with attributable population losses of between 50% and 60% in North Africa, Europe, and central and southern Asia. The second plague pandemic, also known as the ”black death or great pestilence”, began in 1346 and eventually killed 20 to 30 million people in Europe, one third of the European population. The pandemic lasted more than 130 years and had major political, cultural, and religious ramifications. The third pandemic began in China in 1855, spread to all inhabited continents, and ultimately killed more than 12 million people in India and China alone. Small outbreaks of plague continue to occur throughout the world. Advances in living conditions, public health, and antibiotic therapy make future pandemics improbable. However, plague outbreaks following use of a biological weapon are a plausible threat. In World War II, a secret branch of the Japanese army, Unit 731, is reported to have dropped plague-infected fleas over populated areas of China, thereby causing outbreaks of plague. In the ensuing years, the biological weapons programs of the United States and the Soviet Union developed techniques to aerosolize plague directly, eliminating dependence on the unpredictable flea vector. In 1970, the World Health Organization (WHO) reported that, in a worst-case scenario, if 50 kg of Y. pestis were released as an aerosol over a city of 5 million, pneumonic plague could occur in as many as 150,000 persons, 36,000 of whom would be expected to die. Human plague most commonly occurs when plague-infected fleas bite humans who then develop bubonic plague. As a prelude to human epidemics, rats frequently die in large numbers, precipitating the movement of the flea population from its natural rat reservoir to humans. Although

most persons infected by this route develop bubonic plague, a small minority will develop sepsis with no bubo, a form of plague termed ”primary septicemic plague”. Neither bubonic nor septicemic plague spreads directly from person to person. A small percentage of patients with bubonic or septicemic plague develop secondary pneumonic plague and can then spread the disease by respiratory droplet. Persons contracting the disease by this route develop primary pneumonic plague. Plague remains an enzootic infection of rats, ground squirrels, prairie dogs, and other rodents on every populated continent except Australia. Worldwide, on average in the last 50 years, 1700 cases have been reported annually. In the United States, 390 cases of plague were reported from 1947 to 1996, 84% of which were bubonic, 13% septicemic, and 2% pneumonic. Concomitant case fatality rates were 14%, 22%, and 57%, respectively. Y. pestis is a nonmotile, gram-negative bacillus, sometimes coccobacillus, that shows bipolar (also termed safety pin) staining with Wright, Giemsa, or Wayson stain. (Fig. 59)

Figure 61. Peripheral Blood Smear From Patient With Septicemic Plague.

INFECTIOUS DISEASE REAGENTS

63


Y. pestis is a lactose nonfermenter, urease and indole negative, and a member of the Enterobacteriaceae family. It grows optimally at 28 째C on blood agar or MacConkey agar, typically requiring 48 hours for observable growth, but colonies are initially much smaller than other Enterobacteriaceae and may be overlooked. Y. pestis has a number of virulence factors that enable it to survive in humans by facilitating use of host nutrients, causing damage to host cells, and subverting phagocytosis and other host defense mechanisms. Capsular F1 antigen is a main immunochemical component of Y. pestis surface. Its synthesis is determined by pFra plasmid and induced at 37 째C. F1 is aggregated on the outer membrane of the Y. pestis microbe as an olygomeric protein, forming a granular layer and gradually diffusing into the environment. Capsular polymer consists of multiple similar hydrophobic protein subunits, aggregating at physiological conditions. F1 subunit of the antigen is originally synthesized from 170 aminoacids and has a molecular mass 17.6 kD, then, after the elimination of the signal peptide during the secretion onto the surface, a protein with molecular mass 15.6 kD and isoelectric point 4.1 is formed. We used highly purified F1 antigen for hybridoma development. 1

2

3

4

5

To confirm capsular antigen identity Ouchterlony double immunodiffusion in agarose gel and Western blotting analysis were performed with F1-specific rabbit polyclonal antibodies. (Fig. 63 and 64) 2

1

3

5

4 Figure 63. Ouchterlony immunodiffusion plate. Well 1: commercial F1, 1 mg/ml positive control; Wells 2, 3, 4: samples of F1: batch 7 non diluted; batches 7 and 8 diluted 4-fold; Well 5: LPS EV 76 negative control.

6 94 67

1

2

3

43 30 20 14

Figure 62. Electrophoretic analysis of Y. pestis F1 antigens Lane 1: Cytochrome C 12.5 kD Lane 2 F1 reference Lane 3: F1 batch 8 Lane 4: F1 batch 7 Lane 5: F1 reference Lane 6: Molecular mass standards

64

INFECTIOUS DISEASE REAGENTS

Figure 64. Immunoblotting of capsular F1 antigen with rabbit antisera. Lane 1: F1 batch 7 Lane 2: F1 batch 8 Lane 3: F1 commercial preparation

Electrophoresis and immunoblotting data testify to the contents of F1 in the immunogen preparations at not less than 95%. For the detection of Y. pestis we produced MAb YPF19, which is characterized by high affinity towards F1 capsular antigen. This antibody was checked in different formats: sandwich ELISA, hem-and-latex agglutination, lateral flow device as a colloidal gold conjugate, Western blotting and immunofluorescence. Data confirm high specificity and sensitivity of the assays on the base of these MAb. There is no cross-reactivity with Y. pseudotuberculosis and Y. enterocolitica.


V antigen of Yersinia pestis is a multifunctional protein that has been implicated as a protective antigen, a virulence factor, and a regulatory protein. Some studies suggest that V antigen is also a virulence factor that reduces local expression of the host cytokines tumor necrosis factor alpha and gamma interferon in response to Yersinia infection, thus allowing the bacterium to become established. V antigen

also has a role in the regulation of the low-calcium response. To produce MAbs Va13, Va22, Va48, Va52 and Va68 we used Y. pestis, V antigen as an immunogen. All these MAbs are working in ELISA and Western blotting. Recommended pair for sandwich immunoassay is (capture - detection): Va13 - Va48

Anti-Yersinia pestis V antigen Immunogen: Y. pestis full-size recombinant V antigen. Immunoreactivity: MAbs Va13 and Va22 are not cross-reacting with B. anthracis protective antigen and spore, E. coli or Y. pestis F1 antigen. Applications: Western blotting, ELISA

Anti-Yersinia pestis capsular F1 antigen Immunogen: Purified Y. pestis strain EV76 F1 capsular antigen. Immunoreactivity: MAb YPF19 recognize Y. pestis F1 capsular antigen. There is no cross-reactivity with Y. pseudotuberculosis and Y. enterocolitica. Applications: Western blotting, ELISA. Fig. 65 shows the detection of purified Y. pestis F1 antigen preparation in Western blotting using MAb YPF19. Fig. 66 shows the calibration curve for Y.pestis F1 antigen quantification using double antibody sandwich ELISA with MAb YPF19. The detection limit for the immunoassay is 0.5 ng/ml.

2,0

OD 492

1,5

1,0

0,5

con. 20 t con. 40 t

0,0

1

2

3

0

4

2

4

6

8

10

12

14

ng/ml

Figure 65. Detection of Y. pestis F1 capsular antigen in Western blotting using MAb YPF19 (2). Molecular mass standards are 67, 43, 30, 20, 14 K control and track 3 shows F1 antigen staining with Amido black. 10% SDSPAGE was run under reducing conditions.

Figure 66. Calibration curve for double antibody sandwich with MAb YPF19 used for capture and as an HRP conjugate for Y. pestis F1 antigen detection. The conjugate working dilutions were 1/20000 (black squares) and 1/40000 (red squares).

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3YPV8 3YPV8 3YPV8 3YPV8 3YPV8 3YP8

Va13 Va22 Va48 Va52 Va68 YPF19

IgG1 IgG1 IgG1 IgG1 IgG1 IgG1

EIA (capture), WB EIA, WB EIA (detection), WB EIA, WB EIA, WB EIA (capture, detection), WB, IHC

Anti-Yersinia pestis V Antigen Anti-Yersinia pestis V Antigen Anti-Yersinia pestis V Antigen Anti-Yersinia pestis V Antigen Anti-Yersinia pestis V Antigen Anti-Yersinia pestis capsular F1 Antigen

INFECTIOUS DISEASE REAGENTS

65


3. Antibodies for the detection of Francisella tularensis Tularemia was first described as a plague-like disease of rodents in 1911 and, shortly thereafter, was recognized as a potentially severe and fatal illness in humans. Tularemiaâ&#x20AC;&#x2122;s epidemic potential became apparent in the 1930s and 1940s, when large waterborne outbreaks occurred in Europe and the Soviet Union and epizootic-associated cases occurred in the United States. As well, F. tularensis quickly gained notoriety as a virulent laboratory hazard. Public health concerns impelled substantial early investigations into tularemiaâ&#x20AC;&#x2122;s ecology, microbiology, pathogenicity, and prevention. Tularemia occurs throughout much of North America and Eurasia. In the United States, human cases have been reported from every state except Hawaii; however, most cases occur in south-central and western states (especially Missouri, Arkansas, Oklahoma, South Dakota, and Montana). In Eurasia, the disease is also widely endemic, although the greatest numbers of human cases are reported from northern and central Europe, especially Scandinavian countries and those of the former Soviet Union. Tularemia is almost entirely a rural disease, although urban and suburban exposures occasionally do occur. Throughout its range, F. tularensis is found in widely diverse animal hosts and habitats and can be recovered from contaminated water, soil, and vegetation. A variety of small mammals, including voles, mice, water rats, squirrels, rabbits, and hares, are natural reservoirs of infection. They acquire infection through bites by ticks, flies, and mosquitoes, and by contact with contaminated environments. Although enzootic cycles of F. tularensis typically occur without notice, epizootics with sometimes extensive dieoffs of animal hosts may herald outbreaks of tularemia in humans.

66

INFECTIOUS DISEASE REAGENTS

Humans become infected with F. tularensis by various modes, including bites by infective arthropods handling infectious animal tissues or fluids, direct contact with or ingestion of contaminated water, food or soil, and inhalation of infective aerosols. Persons of all ages and both sexes appear to be equally susceptible to tularemia. Certain activities, such as hunting, trapping, butchering, and farming, are most likely to expose adult men. Laboratory workers are especially vulnerable to infection, either by accidentally inoculating themselves or by inhaling aerosolized organisms. Ordinary exposures during examination of an open culture plate can cause infection. Although F. tularensis is highly infectious and pathogenic, its transmission from person to person has not been documented. Francisella tularensis is a small, nonmotile, aerobic, gram-negative coccobacillus. It has a thin lipopolysaccharide-containing envelope and is a hardy nonspore-forming organism that survives for weeks at low temperatures in water, moist soil, hay, straw, and decaying animal carcasses. Francisella tularensis has been divided into 2 major subspecies (biovars) by virulence testing, biochemical reactions, and epidemiological features. Francisella tularensis biovar tularensis (type A) may be highly virulent in humans and animals, produces acid from glycerol, demonstrates citrulline ureidase activity, and is the most common biovar isolated in North America. Francisella tularensis biovar palaearctica (type B) is relatively avirulent, does not produce acid from glycerol, and does not demonstrate citrulline ureidase activity. In Europe and Asia, all human tularemia is thought to be caused by the milder type B strains, although recent studies there have identified naturally occurring F. tularensis related to F. tularensis biovar tularensis. A few rapidly growing strains of F. tularensis have been recovered from the blood of immunocompromised patients not showing seroreactivity to F. tularensis.


Lipopolysaccaride (LPS) is a main species-specific antigen of Francisella tularensis. According to the data available LPS of tularemia microbe differs from LPS of other gram-negative bacteria. LPS of tularemia microbe havenâ&#x20AC;&#x2122;t yet revealed the properties of classical endotoxin, which may be connected with non-typical structure of lipid A â&#x20AC;&#x201C; a toxic component of LPS. We used LPS purified from f vaccine strain 15 of Francisella tularensis for hybridoma development. Fig. 67 shows the results of electrophoresis in 15% SDS-PAGE and silver stained by the method of Tsai and Frasch .

1

Balb/c mice were repeatedly immunized with ultrasonic disintegrate of F. tularensis strain 15 cells. Primary MAbs screening was done using highly purified LPS as capture antigen in indirect ELISA. Two MAbs T14 and FB11 were established, both MAbs are suitable for F. tularensis detection in sandwich ELISA (detection limit 100 pg/ml)(Fig. 68), immunofluorescence and lateral flow devices. Antibodies are not cross-reacting with the following microorganisms: F. novicida, B. abortus, B. suis, B. melitensis, B. ovis, B. neotomae, Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, S. typhimurium, V. cholera, E. coli.

2

0,30 Mean 0,25

Max Min

OD 492

0,20

0,15

0,10

0,05 1

0,5

0,25

0,125

0

ng/ml Figure 67. Electrophoresis of F. tularensis strain 15. Lane 1: Ra-lipopolysaccaride Salmonella Minnesota SF 1112 Lane 2: LPS15-lipopolysaccaride F. tularensis 15. LPS F. tularensis is presented on the gel as a brown spot in the region 5-10 kDa.

Figure 68. Determination of the detection limit for F. tularensis LPS in sandwich ELISA using MAb T14 as capture and HRP conjugate.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-F. tularensis LPS Anti-F. tularensis LPS

3FT6 3FT6

T14 FB11

IgG3 IgG2a

EIA (capture, detection) IF EIA, IF

INFECTIOUS DISEASE REAGENTS

67


4. Antibodies for the detection of Marburg and Ebola viruses Marburg virus (MBG) is a representative of Filoviridae family of RNA containing viruses. MBG is an exceptionally dangerous pathogen, which induces a severe contagious and highly lethal (53-88%) febrile disease with hemorrhagic syndrome. The name â&#x20AC;&#x153;thread virusesâ&#x20AC;? is based on their morphology. MBG made its appearance in 1967 in the form of frightening nosocomial outbreak, initially among polio vaccine pro-

duction workers in Germany in contact with Ugandan green monkeys and their kidney tissues. The pathoand immuno-genesis of MBG fever have so far been little studied, no specific agents or treatment methods have been developed. We used purified formaldehyde inactivated Marburg virus as an immunogen to produce MAbs FM213, FM11, FM32 and FM44. All MAbs react with MBG in Western blotting and indirect ELISA.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3M1 3M1 3M1 3M1

FM213 FM11 FM32 FM44

IgG1 IgG1 IgG1 IgG1

EIA, WB EIA, WB EIA, WB EIA, WB

Anti-Marburg virus Anti-Marburg virus Anti-Marburg virus Anti-Marburg virus

Ebola haemorrhagic fever (EHF) is one of the most virulent viral diseases known to humankind, causing death in 50-90% of all clinically ill cases. Several different species of Ebola virus have been identified. The Ebola virus is transmitted by direct contact with the blood, body fluids and tissues of infected persons. Transmission of the Ebola virus has also occurred by handling ill or dead infected chimpanzees.

Ebola virus is one of at least 18 known viruses capable of causing the viral hemorrhagic fever syndrome. Although agents of the viral hemorrhagic fever syndrome constitute a geographically diverse group of viruses, to date, all are RNA viruses, all are considered zoonoses, all damage the microvasculature resulting in increased vascular permeability, and all are members of 1 of 4 families: Arenaviridae, Bunyaviridae, Flaviviridae, and Filoviridae.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Ebola virus, Zaire strain Anti-Ebola virus, Zaire strain Anti-Ebola virus, Zaire strain

3E1 3E1 3E1

FE18 FE25 FE37

IgG2a IgG2a IgG2a

EIA, WB EIA EIA, WB

5. Antibodies for the detection of Vaccinia virus Vaccinia virus is a double-stranded DNA orthopoxvirus. Orthopoxviruses are the largest viruses found. The viruses are about 200 nm in diameter, 250-300 nm long and 250 nm high. Smallpox (variola) is a viral disease characterized by a skin rash and a high death rate. Smallpox is highly contagious from one person to another. It may spread extremely rapidly e.g. from bed sheets and clothing and from saliva droplets. In the past smallpox was found all over the world causing illness and death where ever it occurred. It was primarily a disease of children and young adults. Vaccinia virus can be used as an ef-

68

INFECTIOUS DISEASE REAGENTS

fective immunizing agent against human smallpox. In 1980 WHO declared that the world is free of smallpox and the vaccinations were stopped. The vaccine is, however, still used for the people working in laboratories directly involved with smallpox, monkeypox, vaccinia and other closely related orthopoxviruses. Even it has been successfully eradicated; smallpox may still pose a threat to humanity. We used Vaccinum variola vivim siccum (live dry smallpox vaccine) as an immunogen to produce monoclonal antibodies against vaccinia. Pep-


tone was used as cryoprotector and stabilizer. For screening purposes vaccinia virus preparation used as immunogen was dialyzed against PBS to remove peptone. 10% SDS-PAGE of vaccinia virus preparation before and after the dialysis under non-reducing and reducing conditions are presented on Fig. 69. As a reference on Fig. 68 there are 10% SDS-PAGE images of different highly purified orthopoxviruses. According to Ichihashi et al: 1988, Virology, Vol.163, 133-144, major polypeptides of vaccinia virus have

the following molecular masses: 27, 34, 57 and 61 kDa, as is shown on the picture. The same proteins are found in the immunogen preparation. The antibodies are working in ELISA and stain 27 kDa protein band at the concentration 10 Îźl/ml in Western blotting (see Fig. 71). Cross-reaction against cowpox virus, ectromelia virus, avipox virus and smallpox virus in ELISA and Western blotting are currently being determined. Neutralization was not observed against live vaccinia virus in cell culture. 1

kDa

2

3

M

4

kDa

216 132

94

78

61 57

67

45,7

34

43

32,5

27 30

18,4

20 1 2 3 4 5 Figure 69. SDS-PAGE of vaccinia antigen used as immunogen and for screening. Lane 1: Vaccinia antigen before dialysis, non-reducing conditions Lane 2: Vaccinia antigen before dialysis, reducing conditions Lane 3: Molecular mass standards (from top to bottom: 94, 67, 43, 30, 20 kDa) Lane 4: Vaccinia antigen after dialysis, non-reducing conditions Lane 5: Vaccinia antigen after dialysis, reducing conditions

Figure 70. SDS-PAGE analysis of orthopoxviruses. Lane 1: Vaccinia virus, Elstree Lane 2: Ectromelia virus K-1, CAM Lane 3: Ectromelia virus K-1, cell culture Lane M: Molecular mass standards Lane 4: Cowpox virus, Grishak

kDa 94 67 43 30 27 20 14

Figure 71. 10% SDS-PAGE (lanes 1-3 non-reducing conditions, lanes 4-6 reducing conditions), Western blotting. Lane 1: Molecular mass standards (from top to bottom: 94, 67, 43, 30, 20, 14 kDa) Lane 2: Anti-Vaccinia, clone TV43, ascites, dilution 1:1000 Lane 3: Anti-Vaccinia, clone TV46, ascites, dilution 1:1000 Lane 4: Anti-Vaccinia, clone TV43, ascites, dilution 1:1000 Lane 5: Anti-Vaccinia, clone TV46, ascites, dilution 1:1000 Lane 6: Molecular mass standards (from top to bottom: 94, 67, 43, 30, 20, 14 kDa)

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Vaccinia virus

3V1

TV43

IgG2a

EIA, WB

Anti-Vaccinia virus

3V1

TV46

IgG2a

EIA, WB

INFECTIOUS DISEASE REAGENTS

69


6. Antibodies for the detection of Hemorrhagic fever with renal syndrome (HFRS) Hemorrhagic fever with renal syndrome (HFRS) is a group of clinically similar illnesses caused by hantaviruses from the family Bunyaviridae. HFRS includes diseases such as Korean hemorrhagic fever, epidemic hemorrhagic fever and nephropathis epidemica. The viruses that cause HFRS include Hantaan, Dobrava-Belgrade, Seoul, and Puumala strains. Symptoms of HFRS usually develop within 1 to 2 weeks after exposure to infectious material, but in rare cases, they may take up to 8 weeks to develop. Initial symptoms begin suddenly and include intense headaches, back and abdominal pain, fever, chills, nausea, and blurred vision. The severity of the disease varies depending upon the virus causing the infection: Hantaan and Dobrava virus infections usually cause severe symptoms, while Seoul and Puumala virus infections are usually more moderate. Complete recovery can takes weeks or months. Hantaviruses are carried and transmitted by rodents and the occurrence of the disease in endemic areas is at its highest when rodent densities are highest (May-June and October-November). The disease is

worldwide but is most prevalent in Korea and neighboring provinces of China. People can become infected with these viruses and develop HFRS after exposure to aerosolized urine, droppings, or saliva of infected rodents or after exposure to dust from their nests. In addition, individuals who work with live rodents can be exposed to hantaviruses through rodent bites from infected animals. Transmission from one human to another may occur, but is extremely rare. Like other members of the bunyavirus family, hantaviruses have spherical virions with diameters of 90100 nm enveloped with glycoproteins G1 (aka Gn) and G2 (Gc) and contain no matrix proteins. For production of HFRS virus (Puumala strain) specific monoclonal antibodies a lysate of virus-infected cells (MAb PG10) and synthetic peptide conjugated with hsp70 (MAb G2D11) have been used as immunogens. All MAbs are specific to HFRS virus, Puumala strain without cross reactivity with Dobrava, Hanta and Seoul strains. MAb G2D11 was proved to be specific to glycoprotein G2 of the viral envelope.

Ordering information: Product

Cat.#

MAb

Isotype

Remarks

Anti-HFRS

3CCH5

PG10

IgG2a

Anti-HFRS

3CCH5

G2D11

IgG1

Reacts with Puumala strain, N/cr with Dobrava, Hanta and Seoul strains, virus neutralizing activity Reacts with Puumala strain, G2 protein, WB

Anti-HFRS

3CCH5

Puu-H6

IgG1

Anti-HFRS

3CCH5

Puu-D21

IgG1

Anti-HFRS

3CCH5

Dob-A4

IgG2a

Anti-HFRS

3CCH5

Dob-F1

IgG2b

Anti-HFRS Anti-HFRS

3CCH5 3CCH5

HANT-E9 HANT-B5

IgG1 IgG1

70

INFECTIOUS DISEASE REAGENTS

EIA, fluorescent immunoassay, reacts with Puumala, Dobrava, Hanta and Seoul strains EIA, fluorescent immunoassay, reacts with Puumala, Dobrava, Hanta and Seoul strains EIA, fluorescent immunoassay, reacts with Puumala, Dobrava, Hanta and Seoul strains EIA, fluorescent immunoassay, reacts with Puumala, Dobrava, Hanta and Seoul strains EIA, fluorescent immunoassay, reacts with Hanta strain EIA, fluorescent immunoassay, reacts with Hanta strain


XI Veterinary 1. Canine 1.1. Canine distemper virus (CDV) Canine distemper is a contagious, incurable, often fatal, multisystemic viral disease that affects the respiratory, gastrointestinal, and central nervous systems. Distemper is caused by the canine distemper virus (CDV). It occurs among domestic dogs and many other carnivores, including raccoons, skunks, and foxes. CDV is fairly common in wildlife. Infected dogs shed the virus through bodily secretions and excretions, especially respiratory secretions. The primary mode of transmission is airborne viral particles that dogs breathe in. The development of a vaccine in the early 1960s led to a dramatic reduction

in the number of infected domestic dogs. It tends to occur now only as sporadic outbreaks. For the production of MAb to CDV, splenocytes of the Balb/c mice recovered after acute infection by CDV envelope protein (Onderstepoort) were used. MAbs 8-1 and 5-4 against CDV could be used in ELISA with the native CDV and purified CDV, in immunofluorescence with infected Vero cells and in immunohistochemistry. These MAbs may be used for the detection of CDV in clinical samples (blood, saliva, tears, feces) of animals.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Canine distemper virus Anti-Canine distemper virus

3CD10 3CD10

8-1 5-4

IgG2a IgG2a

EIA (detection), PLA EIA (capture), PLA

1.2. Canine parvovirus (CPV) Canine parvovirus (CPV) is a highly contagious and serious disease caused by a virus that attacks the gastrointestinal tract of puppies, dogs, and wild canids. Parvoviral infection must be considered as a possible diagnosis in any young dog with vomiting and/or diarrhea. Puppies and dogs usually become infected when they ingest virus that is passed in the feces of an infected dog. There is a 3-7 day incubation period before the puppy seems obviously ill. Canine parvovirus causes lethargy; loss of appetite; fever; vomiting; and severe, often bloody, diarrhea. Vomiting and diarrhea can cause rapid dehydration, and most deaths from parvovirus occur within 48 to

72 hours following onset of clinical signs. Virus is shed for the first two weeks or less after infection in the stool of an infected dog. MAbs 5G7 and 8H7 are directed against CPV. These MAb are working in ELISA with the native CPV and purified CPV, in immunodiffusion, hemagglutinin inhibition, immunohistochemistry and immunofluorescence. These MAb may be used for the detection of CPV in clinical samples (foeces). MAbs 5G7, 8H7 are cross-reacting with Mink enteritis virus and FPLV (Feline panleucoperia).

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Parvovirus, canine Anti-Parvovirus, canine

3PV16 3PV16

5G7 8H7

IgG2a IgG2a

EIA (capture), WB, ID, HIT EIA (detection), WB, ID, HIT

INFECTIOUS DISEASE REAGENTS

71


1.3. Canine Adenovirus (CAV) MAbs 8C4, 1E11 and 7C11 were produced by immunization with human adenovirus type 1 and boosting with canine hepatitis virus. These MAbs are working in ELISA with the native CAV and purified CAV in im-

munodiffusion, immunohistochemistry and immunofluorescence. These MAbs may be used for the detection of Canine adenovirus infections in clinical samples (blood, nasal swabs) of animals.

Ordering information: Product

Cat. #

MAb

Isotype

Anti-Adenovirus hexon Anti-Adenovirus hexon Anti-Adenovirus hexon

3AV13 3AV13 3AV13

8C4 7C11 1E11

IgG2a EIA (capture), ID, IHC IgG2a+IgM EIA, ID, IHC IgG2a+IgM EIA (detection), ID, IHC

Remarks

1.4. Rabies virus Rabies is a disease caused by a virus found in the saliva of infected animals and is transmitted to pets and humans by bites, or possibly by contamination of an open cut. It infects the central nervous system, causing encephalopathy and ultimately death. The time between exposure to the virus and the onset of symptoms can range from about two weeks to several months. The rabies virus can be found in animal saliva days before any obvious symptoms develop. However, all animals that have the virus will develop symptoms and eventually die of the disease. Most animals can be infected by the virus and can transmit the disease to humans. Infected bats, raccoons, foxes, skunks, dogs or cats provide the greatest risk to humans. For immunization we used vaccine strain of the rabies virus. Finally we got quite a number of

MAbs, which were characterized by the specificity to the glycoprotein (GP) and nucleoprotein (NP) of the rabies virus. MAbs 4G4 and 5B12 are specific to glycoprotein. MAb 1C5 against GP is of special interest. This MAb is very good in ELISA with the native RV and the purified GP and in immunofluorescence with intact infected BHK cells. Hybridoma cells, when inoculated into mice, protect them from RV infection and the MAb 1C5 demonstrates a therapeutic effect on the infected mice. MAb 1C5 has a virus-neutralizing activity in respect to the majority of standard RV and rabies-relative viruses: CVS, Lagos bat, Mokola, Flury lep, Duvenhage and the field isolates. This antibody may be used for the detection of rabies virus and for creation of new vaccine anti-rabies preparations.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Rabies virus Anti-Rabies virus Anti-Rabies virus

3R7 3R7 3R7

1C5 4G4 5B12

IgG2a IgG2a IgG2a

EIA, IHC EIA EIA, WB

1.5. Echinococcus granulosis Echinococcus granulosis, also called the Hydatid worm, is a cyclophyllid cestode that parasitizes the small intestine of canids as an adult, but which has important intermediate hosts such as livestock and humans, where it causes hydatid disease. In canids, E. granulosis causes a typical tapeworm infection.

Adult worms mature in the intestine of the dog (definitive host) and the eggs are released in the foeces. By an accidental ingestion in humans, oncospheres hatch in the duodenum, penetrate the intestine and are carried via the bloodstream to various organs. Hydatid cysts form in organs like liver, lungs and brain.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Echinococcus granulosis

3EG3

EHG

IgG1

EIA, WB

72

INFECTIOUS DISEASE REAGENTS


2. Bovine 2.1. Rotavirus The incubation period for rotavirus disease is approximately 2 days. The disease is characterized by vomiting and watery diarrhea for 3 - 8 days, and fever and abdominal pain occur frequently. As with all viruses, some rotavirus infections cause few or no symptoms, especially in adults. Rotavirus spreads by fecal-oral transmission.

We used purified bovine rotavirus as an immunogen and MAb 3C10 is specific for mammalian group A rota viruses. Antibody is cross-reacting with monkey rotavirus (SA-11), porcine rotavirus (PP) and with numerous human rotaviruses. Antibody is reacting in Western blotting with p42 major inner capsid antigen and it is working also in ELISA and immunohistochemistry.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Rotavirus Group Specific Antigen

3R10

3C10

IgG2a

P42 Antigen, EIA, IHC, WB

2.2. Bovine coronavirus MAb 5A4 was produced by immunization with bovine coronavirus. MAb recognizes bovine coronavirus surface antigen (peplomer).

Bovine coronavirus infects neonatal calves and presents as an acute diarrhea. It frequently leads to death. Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Bovine coronavirus

3BCV1

5A4

IgG1

EIA, HIT

2.3. Brucella abortus (Brucellosis) Brucellosis is an infectious disease caused by the bacteria of the genus Brucella. Cows are the source of Brucella abortus but other species of Brucella can be contracted from other animals and can also cause brucellosis. The disease may be either subclinical, acute and subacute, relapsing, or chronic. The incubation period may be weeks to months. It may be mild or an explosive, toxic illness. Symptoms are non-specific and few localizing physical signs develop. Diagnosis is usually from blood or bone marrow cultures, or a rise in anti-brucella antibodies of 4 fold or greater.

We used two antigens for anti-B. abortus MAb production: crude cell lysate and purified LPS. Respectively 2 panels of MAb were obtained: BA35, BA37, BA39 and Bx85, Bx87, Bx88. Selection of the optimal pair for B. abortus detection in sandwich-ELISA showed that MAb Bx85 is suitable for capture and MAb Bx88 works well and specifically as HRP-conjugate. Detection limit when using purified LPS as a standard is lower than 100 pg/ml, sensitivity is improved at sample boiling before the analysis.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3BR11 3BR11 3BR11 3BR11 3BR11 3BR11

BA35 Bx85 Bx88 BrH1 BrF11 BrG11

IgG2a IgG1 IgG1 IgG2a IgG1 IgG2a

EIA, WB EIA (capture), LPS Brucella abortus EIA (detection), LPS Brucella abortus EIA, WB EIA, WB EIA, WB

Anti-Brucella abortus Anti-Brucella abortus Anti-Brucella abortus Anti-Brucella abortus Anti-Brucella abortus Anti-Brucella abortus

INFECTIOUS DISEASE REAGENTS

73


2.4. Alpha-1 – Acid Glycoprotein (AGP) The Alpha-1 – Acid Glycoprotein (AGP) level in 152 Holsteins from 1 year to 12 years is 283 μg/ml. There are no differences between male and female or between breeds. The upper limit of normal AGP in healthy bovine is 450 μg/ml. The serum value of AGP in a calf immediately following birth is 300 to 1750 μg/ml, but this drops to normal by the end of the third weekn following birth. Levels of AGP that remains

high longer than the 3-d week are indicative of a background health problem that will adversely affect growth and productivity. AGP is elevated in stressed cattle that are clinically normal. In particular the levels are elevated with hepatitis, pericarditis, arthritis, mastitis and pneumonia. With successful treatment or stress removal levels return to normal rapidly.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Alpha-1-Acid Glycoprotein, Bovine (Orosomucoid)

5AG1

GPB2

IgG1

EIA, WB

2.5. Foot-and-mouth disease (FMDV) MAbs against virulent serotype O1 of foot-andmouth disease (FMDV) were screened in different serological reactions like ELISA and gel precipitation, in which both antibodies were active. Antibodies can be used to detect FMDV in the field isolates for differentiating type “O” from other types, for virus purification and for antibody screening in competitive ELISA, etc.   MAbs against the non-structural protein (NSP) of foot-and-mouth disease can be used for example

for antigen purification and for antibody screening in the competitive ELISA, etc. Combination of an ELISA test, that detects antibodies directed against FMDV viral non-structural proteins (NSPs) and a liquid phase blocking competitive ELISA for the detection of antibodies against the viral structural proteins (SPs strain specific), can be used in analysis of field samples allowed for a clear differentiation between infected and uninfected animals, with high specificity and sensitivity, regardless of the animal’s vaccination status.

Ordering information: Product

Cat. #

MAb

Isotype Remarks

3FM2 3FM2 3FM2 3FM2

2D2 3G8 1G2 1H4

IgG2a IgG2a IgG2a IgG2a

Anti-Foot-and-mouth disease Anti-Foot-and-mouth disease Anti-Foot-and-mouth disease Anti-Foot-and-mouth disease

74

INFECTIOUS DISEASE REAGENTS

virulent serotype O1, EIA, ID virulent serotype O1, EIA, ID non-structural proteins, EIA non-structural proteins, EIA


3. Equine 3.1. Burkholderia (Pseudomonas) mallei (Glanders) Burkholderia (Pseudomonas) mallei is a gram-negative, non-sporing bacilli and it is the etiologic agent of glanders. Glanders is a highly contagious disease of solipeds and it is characterized by nodular lesions of the lungs and other organs, as well as ulcerative lesions of the skin and mucous membranes of the nasal cavity and respiratory passages. Ingestion of the pathogen, present in secretions from infected animals, constitutes the major route of infection

in glanders. Carnivores are susceptible to disease if they consume glandered meat. Humans also are susceptible to infection with glanders, which is an important occupational disease of veterinarians, farriers, and other animal workers. We have used cell extract of Pseudomonas mallei to develope a monoclonal antibody for detection of Pseudomonas mallei. MAb 3D11 MAb reacts with LPS of Burkholderia mallei in ELISA and Western blotting.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Pseudomonas mallei LPS

3PM15

3D11

IgG1

EIA, WB

4. Porcine 4.1. Transmissible Gastroenteritis (TGE) virus of Pigs Transmissible gastroenteritis (TGE) is an acute highly contagious disease of pigs caused by virus from the Coronaviridae family. It is a common viral disease of the small intestine that causes vomiting and profuse diarrhea in pigs of all ages. There is high morbidity and mortality in piglets.

MAb 1E11 is specific to peplomer of TGE virus. MAb can be used in Immunometric detection of the TGE virus in stool and cell culture fluids. The same MAb can be used as capture and detection MAb. MAb can also be used in hemagglutinin inhibition.

Ordering information: Product

Cat. #

MAb

Isotype Remarks

Anti-Transmissible Gastroenteritis Pig Virus

3TG1

1E11

IgG1

EIA (capture, detection), HIT

5. Piscine 5.1. Infectious Salmon Anemia virus Infectious salmon anemia virus (ISAV) is the causative agent of ISA, which is a highly infectious disease of farmed Atlantic salmon in the Northern hemisphere. This virus is a member of the family Orthomyxoviridae, genus Isavirus. We have used two synthetic peptides of Infectious Salmon Anemia Vi-

rus from putative haemagglutinin sequence, conjugated to KLH to generate monoclonal antibodies suitable for EIA ans WB. All MAbs recognize peptides of putative haemagglutinin of Infectious Salmon Anemia Virus (8-23 a.a.r. for MAbs 16C7 and 16F4 and 296-312 a.a.r. for MAbs 18D8 and 18F9).

Ordering information: Product

Cat. # MAb

Isotype Remarks

3SA1 3SA1 3SA1 3SA1

IgG1 IgG2a IgG2b IgG1

Anti-Infectious Salmon Anemia Virus, Putative Haemagglutinin Anti-Infectious Salmon Anemia Virus, Putative Haemagglutinin Anti-Infectious Salmon Anemia Virus, Putative Haemagglutinin Anti-Infectious Salmon Anemia Virus, Putative Haemagglutinin

16C7 16F4 18D8 18F9

8-23 a.a.r., EIA 8-23 a.a.r., EIA 296-312 a.a.r., EIA 296-312 a.a.r., EIA

INFECTIOUS DISEASE REAGENTS

75


6. Avian 6.1. Newcastle disease virus (NDV) Newcastle disease (ND) is a highly contagious and sometimes fatal illness affecting many domestic and wild bird species. The causal agent, Newcastle disease virus (NDV), is a negative-sense single-stranded RNA virus. NDV affects the respiratory, nervous, and digestive systems. Clinical signs are extremely variable depending on the strain of virus, species and age of bird, concurrent disease, and pre-existing immunity. NDV is so virulent that many birds die without showing any clinical signs. Ordering information:

Transmission occurs by exposure to foecal and other excretions from infected birds, and through contact with contaminated food, water, equipment and clothing. Virus-bearing material can be picked up on shoes and clothing and carried from an infected flock to a healthy one. Exposure of humans to infected birds (for example in poultry processing plants) can cause mild conjunctivitis and influenzalike symptoms, but NDV otherwise poses no hazard to human health. MAbs are negative with parainfluenza type 3 and avian influenza hemagglutinins.

Product

Cat. #

MAb

Isotype Remarks

3ND5 3ND5 3ND5 3ND5 3ND5 3ND5 3ND5

9F7 11F12 13H3 9C6 1C10 2H4 8H2

IgG1 IgG2a IgG2a IgG2a IgG2a IgM IgG2a

Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus Anti-Newcastle disease virus

EIA (detection), WB, HIT EIA (detection), WB, HIT EIA, WB, HIT EIA, WB, HIT EIA (detection), WB, HIT EIA (capture), HIT EIA (capture)

6.2. Marek disease virus (MDV) Marek disease (MD) is a highly contagious, lymphoproliferative disease of chickens. It is caused by the MD virus (MDV), an oncogenic avian herpesvirus. It is the most serious chronic concern to the poultry industry. Chickens are exposed at an early age to cellfree MDV through inhalation of contaminated dust. MDV-infected lymphocytes in the peripheral blood distribute the virus to other tissues. In susceptible chickens second round of cytolytic infection occurs after about 2 weeks. After 3 weeks the chronic in-

flammation of the peripheral nerves is often seen and changes in lymphoid cells may induce malignant transformation in this cell type. The disease is characterized by presence of T cell lymphoma as well as infiltration of nerves and organs by lymphocytes. Infected birds can suffer from asymmetric paralysis of one or more limbs, difficulty of breathing, dilation of the crop, depression and paralysis. Death occurs usually in a large number of birds (up to 80 percent).

Ordering information: Product

Cat. #

MAb

Isotype Remarks

Anti-Marek disease virus

3MD8 3MD8 3MD8 3MD8 3MD8

14C8 1G6 5C2 3G2 3H9

IgG3 IgG2a IgM IgM IgG2b

Anti-Marek disease virus Anti-Marek disease virus Anti-Marek disease virus Anti-Marek disease virus

EIA EIA EIA EIA EIA

6.3. Avian influenza Of the 16 different influenza hemagglutinin types only strains within the H5 and H7 subtypes cause highly pathogenic avian influenza, which is highly conta76

INFECTIOUS DISEASE REAGENTS

gious and rapidly fatal in susceptible avian species. Find more information about antibodies against H5 and H7 from page 11.


6.4. Infectious bursal disease virus (IBDV)

The genome of IBDV consists of two segments (A and B) of double-stranded RNA, which are packaged in a nonenveloped icosahedral shell about 60 nm in diameter. The segment A of the IBDV genome has at least two partially overlapping open reading frames (ORF). The large continuous ORF encodes the precursor protein which is subsequently processed into the mature viral proteins: pVP2, VP3 and VP4. During virus maturation in a host cell, pVP2 (48 kDa), also known as VPX, turns into 37 kDa outer capsid structure protein by host proteases cleavage. VP2 carries the determinants responsible for the causing of antigenic variation as well as for virus tropism to the cell. VP3 (32 kDa) is the inner capsid structure protein, while VP4 (28 kDa) is the nonstructural protein, which is involved in processing of the precursor. For detection and identification of the IBD virus in field isolates and in vitro studies along with different types of reverse transcription-polymerase chain reactions, many methods are based on antigenantibody interaction principle. Among them: histochemistry, virus neutralization, sandwich (antigencapture) immunoassay, dot- and western blotting. Methods utilizing high affinity monoclonal antibodies are much more sensitive and have lower nonspecific reactions than those which utilize polyclonal antisera. Pairs of high affinity MAbs could also be used in one-step diagnostic kits based on membrane chromatography. HyTest offers a panel of MAbs, which could be sucessfully used in dot- and Western blotting analsis of VP2 and VP3 virus proteins, for immunohistochemical analysis in diagnostics and for peroxidase immunostaining of infected cell cultures. Several pairs of MAbs could be utilized for the designing of sandwich immunoassays to be used for virus titration; for live and inactivated vaccine preparation as well as in diagnostics.

6.4.1. Immunodetection of VP2 and VP3 IBDV structure proteins in Western blotting Two structure proteins of IBDV capsid, VP2 (about 37 kDa) and VP3 (32 kDa) are the major proteins of interest in Gumboro disease investigation, as they carry neutralizing epitopes and play important role in virus evolution (4, 5, 10, 11, 12). HyTest offers monoclonal antibodies selectively recognizing VP2 or VP3 proteins of IBDV. MAbs IBDV67 and IBDV92 are VP2specific, MAbs IBDV9, IBDV99, IBDV105 are VP3specific. 250 kDa 130 kDa 72 kDa 55 kDa

VP2 36 kDa

VP3

28 kDa

Figure 72. Immunodetection of major structure proteins of IBD virus capsid in Western blotting by different monoclonal antibodies after Tricine-SDS-PAGE in reducing conditions. Lanes: 1- MAb IBDV9, 2- MAb IBDV67, 3- MAb IBDV92, 4- MAb IBDV99, 5- MAb IBDV105, 6- molecular mass markers.

6.4.2. Direct ELISA All MAbs recognize IBD virus in direct ELISA. 2.5

2 IBDV92 1.5

OD 490

Infectious bursal disease (IBD) is an acute, highly contagious avian viral infection, which is disseminated worldwide and brings major economic losses in the poultry industry. Infectious bursal disease was described by Cosgrove in 1962, and since the first outbreaks occurred in the area of Gumboro, Delaware, “Gumboro disease” became a synonym for this disease.

1

0.5

0 0.0001

0.001

0.01

0.1

1

10

MAb concentration, ng/ml

Figure 73. Titration curve for MAb IBDV92. Antigen: purified IBD virus, 0.5 µg/well.

INFECTIOUS DISEASE REAGENTS

77


6.4.3. Histochemistry

A

1000000

IBDV9 - IBDV105

100000

10000

cps

To determine if the virus is able to infect cells in vitro or in virus neutralization tests in cell culture, immunostaining of infected cells is usually used. We recommend our MAbs, conjugated with HRP for rapid and very specific detection of IBD virus in cell culture experiments (Fig. 74). B

1000

100 0,1

1

10

100

1000

10000

Purified IBDV concentration, ng/ml

A. Early stage of virus infection. After blocking cells were incubated 1 hour with MAb IBDV 9, conjugated with HRP, 1:500 dilution in PBST. After washing, 3,3`-diaminobenzidine HRP substrate was added. B. Totally infected cell culture. After blocking cells were incubated 1 hour with non-conjugated MAb IBDV105 in PBST (10 μg/ml). Polyclonal antimouse IgG conjugated with HRP was used for MAb-IBDV complex visualization. 3-amino-9-ethilcarbozol was used as HRP substrate.

6.4.4. Sandwich immunoassay for IBD virus detection All MAbs were tested in sandwich type immunoassay as capture or detection antibodies. Best pairs were tested with different strains of IBDV (MB, D 78, GM 97, S 706, LC75) see Table 16. For IBDV immunoassay development following two-site MAb combinations are recommended (capture-detection): IBDV9 - IBDV105 (Fig. 75) IBDV99 - IBDV105 (Fig. 76) IBDV67 - IBDV92 (Fig. 77)

IBDV99 - IBDV105

100000

10000

1000

100 0,1

1

10

100

1000

10000

Purified IBDV concentration, ng/ml Figure 76. Calibration curve for assay IBDV99- IBDV105. Capture MAb: IBDV99, 1 µg/well. Detection MAb (Eu-chelate labeled): IBDV 105, 0.4 µg/well. Incubation time – 30 min.

1000000

IBDV9IBDV105

IBDV99IBDV105

IBDV67IBDV92

MB

-

+

++

S 706

+++

+++

++

GM 97

-

-

+

D 78

-

-

+

LC75

+++

+++

+++

+++ very high response, ++ high responce; + low responce; - no interaction.

IBDV67 - IBDV92

100000

cps

IBDV strain

INFECTIOUS DISEASE REAGENTS

1000000

10000000

Table 16. Detection of different IBDV strains by three pairs.

78

10000000

cps

Figure 74. Immunodetection of IBD virus in Vero cells. Vero cells were cultured in 96-well plate, than infected with S706 IBDV strain and fixed with ethanol-acetone mixture. Sites of non-specific binding were blocked with 5% dry milk in PBS containing 0,1% Tween 20 (PBST).

Figure 75. Calibration curve for the assay IBDV9-IBDV105. Capture MAb: IBDV9, 1 µg/well. Detection MAb (Eu-chelate labeled): IBDV105, 0.4 µg/well. Incubation time – 30 min.

10000

1000

100 0,1

1

10

100

1000

Purified IBDV concentration, ng/ml Figure 77. Calibration curve for assay IBDV67- IBDV92. Capture MAb: IBDV67, 1 µg/well. Detection MAb (Eu-chelate labeled): IBDV92, 0.4 µg/well. Incubation time – 30 min.

10000


6.5. Infectious bursal disease virus (IBDV) Avian infectious bronchitis (IB) is a highly contagious upper-respiratory disease in chickens. It was first observed in 1930 and it is disseminated worldwide costing the poultry industry billions of dollars annually. The causative agent of IB, infectious bronchitis virus (IBV), belongs to the Group 3 of Coronavirus genus of Coronaviridae family. IBV is enveloped, lipid-containing positive-sense RNA virus with non-segmented, single-stranded genome, polyadenylated at 3â&#x20AC;&#x2122;-terminus. Like other coronaviruses the IBV virus particle contains three major protein structures: the spike, membrane, and the nucleocapsid. The nucleocapsid (N-protein) of IBV is a phosphoprotein of 409 amino acids and molecular mass about 50 kDa. N-protein forms a protective shell that packages the viral genomic RNA and its phosphorylation is thought to control the RNA binding activity, replication and transcription. Nucleocapsid protein is highly con6.5.1. Immunodetection of IBV nucleoprotein in Western blotting

served protein across various IBV strains within major antigenic groups of Coronaviruses. It has immunogenic properties and N-specific antibodies crossreact between different strains of the group. We developed the monoclonal antibody specific to the N-protein of infectious bronchitis virus. This antibody recognizes the N-protein in Western blotting assay with excellent specificity. It could be used as the capture antibody in sandwich-immunoassays with polyclonal antibodies as the detection. It also could be utilized as the capture antibody in whole virus based serology assays to increase sensitivity and specificity in case of low purity of the antigen. 6.5.2. Serological sandwich-type immunoassay MAb IB95 could be used as capture antibody in serology assay to detect anti-IBV antibodies in vaccinated chickens when semi-purified IB virus is used as an antigen. MAb presents nucleoprotein and provide better sensitivity and low non-specific reaction comparing direct surface virus adsorption; less amount of the antigen is needed (Fig. 79). 3

250 kDa 130 kDa 95 kDa 72 kDa

2.5

1 2

55 kDa

2

OD 450

N-protein

36 kDa

1.5

28 kDa

1

0.5

17 kDa 0

11 kDa

1

Figure 78. Immunodetection of IBV nucleoprotein in Western blotting by MAb IB95 after Tricine-SDS-PAGE in reducing conditions. N-protein specificity was confirmed by mass spectrometry. Molecular mass markers are marked at the right side of the picture.

0.1

0.01

0.001

serum dilution

0.0001

0.00001

Figure 79. Titration curve of serum sample from IBV-immunized chicken in two different immunoassays. 1: Sandwich type assay with MAb IB95 as the capture antibody and semipurified IB virus as the antigen. 2: Direct sorption of semi-purified IB virus onto the plate surface. In both cases semi-purified virus was diluted 1000-fold (about 1 Âľg/ml of total protein).

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3BD5 3BD5 3BD5 3BD5 3BD5 3BN1

IBDV9 IBDV67 IBDV92 IBDV99 IBDV105 IB95

IgG2a IgG1 IgG1 IgG1 IgG3 IgG2a

EIA (capture), WB, IHC, Inner capsid structure protein VP3 EIA (capture), WB, IHC, Outer capsid structure protein VP2 EIA (capture, detection), WB, IHC, Outer capsid structure protein VP2 EIA (capture, detection), WB, IHC, Inner capsid structure protein VP3 EIA (capture, detection), WB, IHC, Inner capsid structure protein VP3 EIA (capture), WB

Anti-IBDV Anti-IBDV Anti-IBDV Anti-IBDV Anti-IBDV Anti-IBV

INFECTIOUS DISEASE REAGENTS

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XII Miscellaneous 1. Borrelia burgdorferi (Borreliosis, Lyme Disease) Lyme borreliosis is an infection caused by a bite from a tick infected with the bacterium Borrelia burgdorferia. It is an inflammatory disease characterized by a skin rash, joint inflammation and flu-like symptoms. Tick bite results in a skin lesion and is followed by heart and nervous system involvement and later on by arthritis. Late involvement of eye, nervous system, joints, and skin can also occur. The only sign that enables a reliable clinical diagnosis of borreliosis is erythema migrans. Microbial or serological confirmation of borrelial infection is needed for all manifestations of the disease except for typical early skin lesions. Treatment with antibiotics is beneficial for all stages of Lyme borreliosis, but is most successful early in the course of the illness.

Two different partially purified B. burgdorferi strains, sensu stricto and garinii, were used as immunogens to generate MAb panels. MAbs Bss42, and Bss98 are specific for B. burgdorferi sensu stricto, while MAbs Bg14 and Bg64 are directed against B. burgdorferi garinii. End-point titer in indirect ELISA for all the MAbs is 7.2 x 10 6 reflecting high antibody avidity towards corresponding antigens. B. burgdorferi US isolated strain 39/40 is well recognized by MAbs Bss42 and Bss98 in indirect ELISA and WB with a single band in the region 30K (outer surface protein A, OspA, 31kD), which is a highly specific protein for B. burgdorferi spp. MAbs Bg14, and Bg64 give no reactivity with B. burgdorferi strains Colic and Sidn.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3BS25 3BS25 3BB24 3BB24

Bss42 Bss98 Bg14 Bg64

IgG2a IgG2b IgG1 IgG1

EIA, WB, IF EIA, WB, IF EIA, WB, IF EIA, WB, IF

Anti-Borrelia burgdorferi sensu stricto Anti-Borrelia burgdorferi sensu stricto Anti-Borrelia burgdorferi garinii Anti-Borrelia burgdorferi garinii

2. Tick-borne encephalitis virus (TBEV) Tick-borne encephalitis (TBE) is a human viral infectious disease involving the central nervous system. It most often manifests as meningitis, encephalitis, or meningoencephalitis. It is most commonly recognized as a neurological disorder but mild fever can also occur.

TBE is caused by tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus in the family Flaviviridae. It is usually transmitted by the bite of several species of infected ticks, including Ixodes scapularis, Ixodes ricinus and Ixodes persulcatus.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Tick born encephalitis virus (TBEV)

3TBE1

TBE-F2

IgG2a

EIA (peptide)

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INFECTIOUS DISEASE REAGENTS


3. Cyclosporin A Cyclosporin A (CsA) is a fungal metabolite from Tolypocladium inflatum, an undeca - cyclic peptide, it is a potent immunosuppressive agent, it inhibits primarily T lymphocytes and interleukin 2 production. CsC-BSA was used as immunogen, MAb CSZ22

has according to Biacore the following Kd: 4.45 x 1010, 5.93 x 10-8 and 1.54 x 10-9M for CsC-BSA, CsA and CsC-Biotin respectively. In competitive assay MAb CSZ22 are well correlated with Sandimmun-kit at CsA determination in blood.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Cyclosporin A

3C13

CSZ22

IgG1

EIA

4. Helicobacter pylori CagA Helicobacter pylori is a Gram-negative, microaerophilic bacterium that can inhabit various areas of the stomach, particularly the antrum. It causes a chronic low-level inflammation of the stomach lining and is strongly linked to the development of duodenal and gastric ulcers and stomach cancer. Over 80% of individuals infected with the bacterium are asymptomatic. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is

associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like cellular morphological change termed the “hummingbird phenotype.”, which is characterized by dramatic cell elongation.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Helicobacter pylori CagA-protein Anti-Helicobacter pylori CagA-protein Anti-Helicobacter pylori CagA-protein

3HE70 3HE70 3HE70

HP-317 HP-387 HP-1811

IgG IgG IgG

EIA (capture) EIA (detection) EIA (capture), WB, IP

5. Hamster prion protein Prions are infectious agents composed of protein. They propagate by transmitting a mis-folded protein state; the protein does not itself self-replicate and the process is dependent on the presence of the polypeptide in the host organism. The mis-folded form of

the prion protein has been implicated in a number of diseases in a variety of mammals, including BSE (“mad cow disease”) and Creutzfeldt-Jakob disease in humans. All prion diseases affect the structure of the brain or other neural tissue, and all are currently untreatable and are always fatal.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-Prion protein, hamster Anti-Prion protein, hamster Anti-Prion protein, hamster

3PP3 3PP3 3PP3

PrPA5 PrPB7 PrPH8

IgM IgG1 IgG1

N-terminal, WB, indirect EIA, N/cr wtih human PrP C-terminal, WB, indirect EIA, N/cr wtih human PrP C-terminal, WB, indirect EIA, N/cr wtih human PrP

INFECTIOUS DISEASE REAGENTS

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6. FK 506 (Tacrolimus) FK-506, a macrolide antibiotic obtained from Streptomyces isukubaensis, strain No 9993, was isolated from the soil of Tsuluba, northern Japan. Its structure has been determined chemically and by X-ray crystallography. FK-506 has been shown to have strong immunosuppressive activity against a mixed lymphocyte reaction (MLR) and promises to be useful in organ transplantation. Its mechanism is considered to be a suppression of both interleukin 2(IL-2) and in-

terleukin-2 receptor expression on T cells. Its activity has been reported to be 100 times more potent, than that of cyclosporin A (CsA). We used FK506-BSA conjugate as immunogen and for screening. MAb FK1 interacts with free FK506, conjugate, but doesn’t recognize protein-carrier. It works in competitive ELISA and provides for the detection limit ca 1 ng/ml for free FK-506.

Ordering information: Product

Cat. #

MAb

Isotype

Remarks

Anti-FK 506 (Tacrolimus)

4FK42

FK1

IgM

EIA

7. Allergen from Dermatotophagoides farinae In the early 1920’s it was discovered that house dust causes allergic reactions and later during that decade the presence of mites in house dusts was known. Today we know that dust mites (Dermatotophagoides farinae) are one of the most common sources of sensitisation in all parts of the world. A connection between dust mite allergy and asthma has also been reported by many researchers. Mites’ faeces seem to be the major source of allergenic exposure. They are about the size of a pollen grain and

could therefore very easily become airborne and penetrate the lung alveolus. So far the only effective way to get a permanent reduction of house-dustmite allergens seems to be lowering of air humidity and temperature together with efficient cleaning. We used recombinant 15K major allergen from excretions of house dust mite Dermatophagoides farinae to produce MAb Df10. MAb could be used in EIA and WB.

Ordering information: Product

Cat. #

MAb

Isotype Remarks

Anti-15 K allergen of House Dust Mite Dermatophagoides farinae

3K15

Df10

IgG1

EIA, WB

8. Streptavidin from Streptomyces avidinii MAb panel S8E4, S8C12, S10D4 and S3E11, was generated against streptavidin from Steptomyces avidinii. MAbs are suitable for IHC and EIA. Ordering information: Product

Cat. #

MAb

Isotype

Remarks

3ST10 3ST10 3ST10 3ST10

S8E4 S8C12 S10D4 S3E11

IgG1 IgG1 IgG1 IgG1

EIA, WB, IHC EIA, WB, IHC EIA, WB, IHC EIA, WB, IHC

Anti-Streptavidin from Streptomyces avidinii Anti-Streptavidin from Streptomyces avidinii Anti-Streptavidin from Streptomyces avidinii Anti-Streptavidin from Streptomyces avidinii

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INFECTIOUS DISEASE REAGENTS


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