Hand Book Of ELISA
1. ELISA History
2. Basic Principles of ELISA
3. Common Terms used in ELISA
4. Types of ELISA a. Direct ELISA
b. Indirect ELISA
c. Dot ELISA
d. Sandwich ELISA
e. Competitive ELISA
5. Advantages of ELISA
6. Disadvantages of ELISA
8. Application of ELISA
9. Precautions – during ELISA Procedure 10. Trouble Shooting TIPS.
ELISA- ENZYME LINKED IMMUNOSORBENT ASSAY ELISA : Enzyme-linked immunosorbent assay (or) Enzyme immunoassay (EIA) It is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. ELISA, or enzyme-linked immunosorbent assay, are quantitative immunological procedures in which the Ag- Ab reaction is monitored by enzyme measurements. (or) An enzyme linked immunosorbent assay (ELISA) is a test performed in an immunology laboratory to determine levels of protein in a biological sample.
Why known as ......? Enzyme Linked Immunosorbent Assay : 1. Antigen of interest is absorbed on to plastic surface (‘sorbent’). 2. Antigen is recognized by specific antibody (‘immuno’). 3. This antibody is recognized by second antibody (‘immuno’) which has enzyme attached (‘enzyme-linked’). 4. Substrate reacts with enzyme to produce product, usually colored.
HISTORY : Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960 The term ELISA was first used by Engvall & Perlma in 1971.
What is an ELISA test ? : An enzyme-linked immunosorbent assay, or ELISA, test is a type of medical diagnostic test used to detect whether a certain antibody or antigen is present in a patient. It can be useful for a range of different purposes relating to immunology, such as disease testing and virus testing. For example: An ELISA HIV test may be administered to determine whether a patient has been infected with HIV antibodies. ELISA tests are sometimes also used in testing for illegal drug use. An ELISA test can also help detect allergic reactions in food products like nuts or dairy items.
What is the use of an ELISA test? : ELISA tests are widely utilized to detect substances that have antigenic properties, primarily proteins. The substances detected by ELISA tests include hormones, bacterial antigens and antibodies.
How does an ELISA test work? : There are variations of the ELISA test, but the most basic type consists of an antibody attached to a solid surface. This antibody has affinity for the substance of interest For example: human chorionic gonadotropin (HCG), the commonly measured protein which indicates pregnancy. A mixture of purified HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only HCG with linked enzyme will bind. The more HCG which is present in the test sample, the less enzyme linked HCG will bind.
BASIC PRINCIPLE OF ELISA : BASIC PRINCIPLE OF ELISA use an enzyme to detect the binding of antigen (Ag) antibody (Ab). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag : Ab binding. An ELISA can be used to detect either the presence of Antigens or antibodies in a sample depending how the test is designed.
ELISA Qualitative/Quantitative : Qualitative determines antigen or antibody is present or absent. Quantitative determines the quantity of the antibody Titer the highest dilution of the specimen usually serum which gives a positive reaction in the test
Common terms used in ELISA : Antigen : A substance that when introduced into the body stimulates the production of an antibody. An antigen is an organic compoundâ€Śâ€Ś.. protein, polysaccharide or glycolipid Antigens include Toxins, Bacteria, Foreign blood cells, Microorganisms Allergens. Viruses Etc.
Small part of an antigen that interacts with an antibody.
Any given antigen may have several epitopes.
Each epitope is recognized by a different antibody.
Antibodies : Antibodies (also known as immunoglobulins, abbreviated Ig) are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. (OR) proteins produced by the immune system which help defend against antigens
Antibodies are produced by a kind of white blood cell called a plasma cell. They are typically made of basic structural units—each with two large heavy chains and two small light chains
Enzyme substrate : In biochemistry, a substrate is a molecule upon which an enzyme acts. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzymesubstrate complex is formed initially the substrate should be colorless after degradation by the enzyme it should be strongly colored or fluorescent. Substrates are critical for the detection and visualization steps of an ELISA. The choice of substrate depends on a number of factors, including the degree of sensitivity required, different instrumentation and filter requirements, safety issues, and choice of enzyme. Most immunoassays use antibodies/proteins conjugated to enzymes in order to generate signal through the catalytic properties of the enzyme. Horseradish peroxidase (HRP) and alkaline phosphatase (AP). are the two widely used enzymes employed in ELISA assay
Microtiter plate : A Microtiter plate or microplate is a flat plate with multiple "wells" used as small test tubes. A very common usage is in the enzyme-linked immunosorbent assay (ELISA)
ELISA Plate 96 Wells : ELISA Plate 96 Wells Made from clear polystyrene, these ELISA plate is high grade for optic transmission. Arranged in the standard 8 x12 well configuration, available as flat bottom wells. The ELISA plate fits all standard optical readers. Marked on one side alphabetically Numerically on the other side
Importance : Incubation & washing Incubation step- During the test performance incubation time and mentioned temperature is must required For the proper binding between antigen and antibody and also binding with conjugate and color development of substrate. Washing- For the removal of any unbound Antibody/Antigen proper washing and tapping is required other wise we get the incorrect result. So incubation & washing is much important for good results
Coating Solution: Antigen or antibody are diluted in coating solution to immobilize them to the microplate. Commonly used coating solutions are: 50 mM sodium carbonate, pH 9.6; 20 mM TrisHCl, pH 8.5; or 10 mM PBS, pH 7.2. A protein concentration of 1-10 µg/ml is usually sufficient. •
Primary/Secondary Antibody Solution: Primary/secondary antibody should be diluted in 1X blocking solution to help prevent non-specific binding. A concentration of 0.1-1.0 µg/ml is usually sufficient. • Wash Solution: Typically 0.1 M Phosphate-buffered saline or Trisbuffered saline (pH 7.4) with a detergent such as Tween 20
Types of ELISA : Direct ELISA Indirect ELISA Dot ELISA Sandwich ELISA Compétitive ELISA
Direct ELISA : The direct detection method originated in the 1940s when Coons and colleagues labeled antibodies with a fluorescent tag to mark tissue antigens. The direct ELISA uses the method of directly labeling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample.
Advantages of Direct ELISA : Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated.
Disadvantages of Direct ELISA : Immuno reactivity of the primary antibody may be reduced as a result of labeling. Labeling of every primary antibody is time-consuming and expensive. No flexibility in choice of primary antibody label from one experiment to another. Little signal amplification
INDIRECT ELISA : The indirect, two-step method uses a labeled secondary antibody for detection. First, a primary antibody is incubated with the antigen. This is followed by incubation with a labeled secondary antibody that recognizes the primary antibody. Then secondary antibody will bind any antibody produced by a member of the donor's species (for example, an antibody produced in a mouse that will bind any rabbit antibody). This secondary antibody often has an enzyme attached to it, which has a negligible effect on the binding properties of the antibody. A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. The color change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. This can be helpful in a clinical testing, and in R&D. The higher the concentration of the primary antibody that was present in the serum, the stronger the color change. Often a spectrometer is used to give quantitative values for color strength
Advantages of Indirect ELISA : A wide variety of labeled secondary antibodies are available commercially. Since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. Immuno-reactivity of the primary antibody is not affected by labeling. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Different visualization markers can be used with the same primary antibody.
Disadvantages of Indirect Detection : Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal. An extra incubation step is required in the procedure.
DOT ELISA : In this type ,we use nitrocellulose membrane instead of polysterene plate. The Substrate used is Bromocreso indoylpyrophosphate. This gives blue colour. This is useful in immuno blot technique.
DISADVANTAGE: Quantification is not possible.
Sandwich ELISA : Sandwich ELISA is used to determine the antigen concentration in unknown samples The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to antibody, since at least two antibodies act in the sandwich. Sandwich assays are restricted to the quantification of multivalent antigens such as proteins or polysaccharides. Sandwich ELISAs for quantification of antigens are especially valuable when the concentration of antigens is low and/or they are contained in high concentrations of contaminating protein.
Sensitivity of the sandwich ELISA is dependent on four factors : The number of molecules of the first antibody that are bound to the solid phase. The avidity of the first antibody for the antigen. The avidity of the second antibody for the antigen. The specific activity of the second antibody.
Applications of sandwich ELISA : To detect and quantitate large molecules with multiple antigenic sites, usually a protein, in a sample. To test recognition or binding between antigen and antibody, where polyclonals are usually used. To make an ELISA when a pair of monoclonal antibody is not immediately available.
Competitive ELISA : Unlabeled antibody is incubated in the presence of its antigen (Sample) These bound antibody/antigen complexes are then added to an antigen coated well. The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.") The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
Advantages of ELISA : ELISA tests are generally highly sensitive and specific and compare to radioimmune assay (RIA) tests. They have the added advantages of not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus). Reagents are relatively cheap & have a long shelf life ELISA is highly specific and sensitive No radiation hazards occur during labelling or disposal of waste. Easy to perform and quick procedures Equipment can be inexpensive and widely available. ELISA can be used to a variety of infections.
Disadvantage of ELISA : It requires skilled laboratory technicians and specialized laboratory equipment. Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes. Enzyme activity may be affected by plasma constituents. Kits are commercially available, but not cheap Very specific to a particular antigen. Wonâ€™t recognize any other antigen False positives/negatives possible, especially with mutated/altered antigen
PRECAUTIONS : Negative control with strong signal : The excessive background signal can be caused by inadequate rinsing of plates, Reagents not sufficiently diluted, Inadequate blocking of plates or non-specific binding of enzyme conjugate. The appearance of color in negative control wells may also indicate cross-reactivity of secondary antibody with components in the antigen sample.
Positive control with no signal : Microtiter plates not coated properly. Reagents applied in wrong order or step omitted. Secondary antibody not matched to the species of primary antibody. Enzyme conjugates defective or inhibited by contaminant.
Detector antibody not compatible with capture antibody (for sandwich assays).
ELISA with weak signal : Wash buffer not adequately drained after every wash step. Inadequate incubation times. Detection reagents too dilute. Perform checkerboard titrations. Enzyme conjugate defective or inhibited by contaminant. Substrate defective or contaminated. Micro well plates poorly coated. Loss of capture antibody during blocking/washing. Decrease or eliminate use of Tween-20.
APPLICATIONS : Enzyme Linked Immunosorbant Assays (ELISA) have been used for decades in research and development, disease diagnosis and other critical fields in sciences. Detecting infections sexually-transmitted agents like HIV, syphilis and chlamydia hepatitis B and C Toxoplasma gondii Detecting allergens in food and house dust Measuring "rheumatoid factors" and other autoantibody in autoimmune diseases like Lupus erythematosus. Measuring toxins in contaminated food Screening donated blood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-HIV antibodies) hepatitis C (presence of antibodies) hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, T3 and T4 (for thyroid function) ELISA is used to detect many bacterial and viral antigens, including human immunodeficiency virus (HIV), malaria, cholera, measles, and mumps The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
Application on ELISA Qualitative Method Semi Quantitative Method Quantitative Method
1. Quantitative Method: The interpretation of result are expressed in Positive / Negative using the Cut-off value Cut-off Value = (X) * MNC + (Y) * MPC + F MPC – Mean of Positive Control MNC – Mean of Negative Control F – is the Factor provided along the kit. Eg., HIV ,HbSAg , HCV
2. Semi Quantitative Method The extinction values of the samples exceeding those of the Cut off Calibrator value are considered to be Positive and those below are considered to be Negative. For eg, dsDNA, Leptospira
3. Quantitative Method A standard curve from the Multi Standards is produced by point-to-point plotting of the measured extinction values and the concentration is calculated against the absorbance. In Quantitative method usually 6 – 7 standard is used for Plotting a point-to-point curve. Eg, Hormones-Thyroid, Fertility, Tumour Markers etc.
Precautions to be taken during ELISA performance General
check that the kit has not expired do not open the protection bag of microplate before it has reached room temperature and in case of a used microplate check if the bag was sealed properly (risk of condensation)
do not pipette cold reagents or calibrators (reduced activity)
store substrate in dark bottle
dissolve any crystals in the wash buffer before diluting it
dilute the wash buffer using distilled water (never use tap water!)
calibrate pipettes regularly use pipette of appropriate accuracy for the task (e.g. do not use a 1 ml pipette for pipetting 10 µl!)
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do not pipette volumes < 5 µl (10 µl is better)
pipette tips are for single-use only
use correct pipette tips do not cross use pipette tips between different samples or reagents (cross contamination!)
do not touch pipette tips without gloves
Dilution of patient samples
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ensure both sera and sample buffer are thoroughly mixed before diluting use correct sample buffer Note! Phospholipids (yellow), IgM (green) never use water for serum dilution! after adding of serum to buffer mix by vortexing (e.g. 5 sec) mix diluted serum again directly before use
the controls and calibrators are pre-diluted and ready for use!
Pipette samples and reagents as rapidly as possible
max. 10 minutes for a complete microplate
pre-dilute samples and reagents
the use of stepper or multi-channel pipette recommended
During transfer of liquids into microplates avoid:
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touching the bottom of the wells with pipette tip production of air bubbles splashing liquids from one well to another
ensure correct incubation times 30 min sample incubation 30 min conjugate incubation 15 min substrate incubation!
start timer on addition of sample/reagent to the first well
ensure correct incubation temperature (+18ºC to +25ºC ) temperature too high ⇒ increased enzyme activity temperature too low ⇒ decreased enzyme activity
if incubation must be performed at a high or low temperature (e.g. when room temp. deviates substantially from +20ºC) the substrate incubation time can be adjusted accordingly
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300 µl wash buffer per well 3 cycles 30-60 sec soak time
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check suitability of system check correct programming 400 µl (overflow modus) 3 cycles 30-60 sec soak time check that buffer does not overflow wells
clean washer regularly Note! Wrong washing influences results enormously
do not leave washed wells standing for long period of time
(risk of drying out!)
Note! Remove all traces of buffer by banging the microplate upside down onto a stack of absorbent paper after each incubation step
already 10 µL wash buffer rest remaining in the well can reduce activity by approx. 40%
TROUBLESHOOTING TIPS – ELISA Positive results in negative control Contamination of reagents/samples May be contamination of reagents or samples, or cross contamination from splashing between wells. Use fresh reagents and pipette carefully. Insufficient washing of plates Ensure wells area washed adequately by filling the wells with wash buffer. Ensure all residual antibody solutions are removed before washing. To much antibody used leading to non-specific binding Check the recommended amount of antibody suggested. Try using less antibody. High background across entire plate Conjugate to strong or left on too long Check dilution of conjugate, use it at the recommended dilution. Stop the reaction using stop buffer as soon as the plate has developed enough for absorbance readings.
Substrate solution or stop solution is not fresh Use fresh substrate solution. Stop solution should be clear (if it has gone yellow, this is a sign of contamination and it should be replaced). Reaction not stopped Colour will keep developing if the substrate reaction is not stopped. Plate left too long before reading on the plate reader Colour will keep developing (though at a slower rate if stop solution has been added) Contaminants from laboratory glassware Ensure reagents are fresh and prepared in clean glassware. Sterilize glassware Substrate incubation carried out in the light Substrate incubation should be carried out in the dark. Incubation temperature too high Antibodies will have optimum binding activity at the correct temperature. Ensure the incubations are carried out at the correct temperature and that incubators are set at the correct temperature and working. Incubation temperature may require some optimization. Insufficient antibody Check the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results Substrate solutions not fresh or combined incorrectly Prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed at the correct concentration. Incubation time not long enough Ensure you are incubating the antibody for the recommended amount of time, if an incubation time is suggested. The incubation time may require increasing for optimization of results. Incubation temperature too low Antibodies will have optimum binding activity at the correct temperature. Ensure the incubations are carried out at the correct temperature and that incubators are set at the
correct temperature and working. Incubation temperature may require some optimization. Ensure all reagents are at room temperature before proceeding. Stop solution not added Addition of stop solution increases the intensity of colour reaction and stabilizes the final colour reaction High absorbance values for samples and/or positive control - absorbance does not go down as the sample is diluted down the plate). The concentration of samples or positive control is too high and out of range for the sensitivity of the assay. Re-assess the assay you are using OR reduce the concentration of samples and control by dilution before adding to the plate. Take the dilution into account when calculating the resulting concentrations. Inconsistent absorbances across the plate Plates stacked during incubations Stacking of plates does not allow even distribution of temperature across the wells of the plates. Avoid stacking. Pipetting inconsistent Ensure pipettes are working correctly and are calibrated. Ensure pipette tips are pushed on far enough to create a good seal. Take particular care when diluting down the plate and watch to make sure the pipette tips are all picking up and releasing the correct amount of liquid. This will greatly affect consistency of results between duplicates. Wells allowed to dry out Ensure lids are left on the plates at all times when incubating. Place a humidifying water tray (bottled clean/sterile water) in the bottom of the incubator. Inadequate washing This will lead to some wells not being washed as well as others, leaving different amounts of unbound antibody behind which will give inconsistent results. Bottom of the plate is dirty affecting absorbance readings. Clean the bottom of the plate carefully before re-reading the plate Colour developing slowly Plates are not at the correct temperature Ensure plates are at room temperature and that the reagents are at room temperature before use
Conjugate too weak Prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed, at the correct concentration. Contamination of solutions Presence of contaminants, such as sodium azide and peroxidise can affect the substrate reaction. Avoid using reagents containing these preservatives.