PlaqueReductionAssay
Normalcellscantakeupreactivedyesduringthemetabolicprocess,but whentheyareinfectedwithviruses,thecellswilllosetheirabilitytotake upthedyeandthusformcolorlessplaques.Afterthevirusisneutralizedby specificantibodiesandtheninfectsthecells,thenumberofcellplaques formeddecreasesaccordingly.Therefore,theantibodytiterforneutralizing theviruscanbecalculatedaccordingtothenumberofcellplaques.The plaquereductionassaymeasurestheplaque-formingefficiencyofavirusin thepresenceofdifferentconcentrationsofatestarticle.Thetimerequired fortheplaquetobecomevisibledependsonthekineticsofvirus replication,whichcanvaryfrom24hourstoseveralweeks.Sinceplaques needtobeatleast1mmwideforaccuratescoring(especiallywiththe nakedeye),thisanalysisisusuallyperformedon24or6-wellsplates,asany smallerholescanaffectreadabilityandresolution.Theplaquereduction neutralizationtest(PRNT)isavariantofplaquereductionassayandis consideredthegoldstandardfordetectingneutralizingantibodiesto certainviruses(ie,dengueviruses)
ThevirusPRNTassaywasfirstdescribedinthe1950sandlaterappliedto thedenguevirus(DENV).ThebasicdesignofPRNTistogenerate virus-antibodyinteractionsintesttubesormicrotiterplatesandthen
measuretheeffectofantibodiesonvirusinfectivitybyspreadingthe mixtureonvirus-sensitivecells.Thecellsarecoveredwithasemi-solid mediumtolimitthetransmissionoftheprogenyvirus.Everyvirusthat causesaproductiveinfectionproducesalocalinfectionarea(plaque). Plaquesarecountedandcomparedwiththeinitialconcentrationofthe virustodeterminethepercentagereductionintotalviralinfectivity.In PRNT,theserumsampletobetestedusuallyundergoesaserialdilution beforemixedwithastandardamountofvirusThevirusconcentration remainsunchangedsothatwhenvirus-sensitivecellsareaddedand coveredwithsemi-solidmedia,individualplaquescanbeidentifiedand counted.Inthisway,thePRNTendpointtiterofeachserumsampleatany selectedpercentreductionofvirusactivitycanbecalculated.Forexample, thevirusisfirstdilutedtoanappropriateconcentrationsothatevery0.2 mlviruscontains80~100PFU(plaque-formingunits).Then,itismixed withthesameamountofserumwithdifferentdilutions,incubatingat37°C for1to2h.Finally,thePFUatdifferentserumdilutionsiscalculated,and theserumdilutionthatreducesplaqueby50%istheneutralizationtiterof theserum.
Assayapplication:
1.Detectantibodiesfromtheserumtobetestedordetectvirusesfromthe diseasedmaterialstodiagnoseviralinfectiousdiseases.
2.Useantitoxinserumtocheckthetoxinsinthepathologicalmaterialor identifythetypeofbacteriatoxins.
3.Determineantiviralserumorantitoxintiters.
4.Identifyandtypenewlyisolatedviruses.
CreativeDiagnosticsprovidesantiviraltestingandcustomizedearly detectionsolutionsforotherlifesciencecompaniesworkinginthefieldof antiviralandinfectiousdiseasediagnosisWeofferdifferentopportunities tomeettheneedsofourclients,includingawiderangeofanti-virustesting options(eg,plaquereductionassays),aswellasopportunitiestoestablish cooperativeefforts.
OurFeatures
Plaquereductionassayisapowerfulandwell-establishedtest, becauseithasbeendevelopedforalongtimeandhasbeenthoroughly optimizedbyourexperiencedexperts.
Forvirusesthatonlyhaveamildcytopathyortakealongtimeto formplaques,weuseimmunostainingofinfectedfocitoprovidefaster resultsinasmallerformatwithoutsacrificingaccuracy.
Weusestandardserumsasthebenchmarkforeachresultandallow comparisonsacrosslaboratoriesandovertime.
OurAvailability
Denguevirus(DENV)
HepatitisBvirus(HBV)
HepatitisCvirus(HCV)
Herpessimplexvirus(HSV)
Humancytomegalovirus(HCMV)
Humanimmunodeficiencyvirus(HIV)
Humanrhinovirus(HRV)
Influenzavirus(IFV)
Respiratorysyncytialvirus(RSV)
RossRivervirus(RRV)
Semlikiforestvirus(SFV)
Sindbisvirus(SINV)
Vacciniavirus(VACV)
Zikavirus(ZIKA)
(Othervirusesormicroorganismsmaybeavailableonrequest)
Wecombineinfectionandanalyticalexpertisetoprovideourclientswith themostpowerfulportfolioofantiviralandantimicrobialinvitrotesting services.Facinganincreasingdemandfornewantiviralandantimicrobial compoundsforthetreatmentofinfectiousdiseases,CreativeDiagnostics cantestthesecompoundsinvitrotodeterminetheirpotentialefficacyin vivomodels