ELISA实验英文介绍

ELISA（Enzyme-LinkedImmunoSorbantAssay）

ELISA实验和ELISA试剂盒的原理介绍（英文原版）

ThepurposeofanELISAistodetermineifaparticularproteinispresentinasampleandifso,howmuch.Therearetwomainvariationsonthismethod:youcandeterminehowmuchantibodyisinasample,oryoucandeterminehowmuchproteinisboundbyanantibody.Thedistinctioniswhetheryouaretryingtoquantifyanantibodyorsomeotherprotein.Inthisexample,wewilluseanELISAtodeterminehowmuchofaparticularantibodyispresentinanindividualsblood.

ELISAsareperformedin96-wellplateswhichpermitshighthroughputresults.Thebottomofeachwelliscoatedwithaproteintowhichwillbindtheantibodyyouwanttomeasure.Wholebloodisallowedtoclotandthecellsarecentrifugedouttoobtaintheclearserumwithantibodies（calledprimaryantibodies）.Theserumisincubatedinawell,andeachwellcontainsadifferentserum（seefigurebelow）.Apositivecontrolserumandanegativecontrolserumwouldbeincludedamongthe96samplesbeingtested.

Aftersometime,theserumisremovedandweaklyadherentantibodiesarewashedoffwithaseriesofbufferrinses.Todetecttheboundantibodies,asecondaryantibodyisaddedtoeachwell.Thesecondaryantibodywouldbindtoallhumanantibodiesandistypicallyproducedinarodent.Attachedtothesecondaryantibodyisanenzymesuchasperoxidaseoralkalinephosphatase.Theseenzymescanmetabolizecolorlesssubstrates（sometimescalledchromagens）intocoloredproducts.Afteranincubationperiod,thesecondaryantibodysolutionisremovedandlooselyadherentonesarewashedoffasbefore.Thefinalstepistheadditiontheenzymesubstrateandtheproductionofcoloredproductinwellswithsecondaryantibodiesbound.

Whentheenzymereactioniscomplete,theentireplateisplacedintoaplatereaderandtheopticaldensity（i.e.theamountofcoloredproduct）isdeterminedforeachwell.Theamountofcolorproducedisproportionaltotheamountofprimaryantibodyboundtotheproteinsonthebottomofthewells.

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