Enzyme-linkedImmunosorbentAssays(ELISAs)combinethespecificityofantibodieswiththesensitivityofsimpleenzymeassays,byusingantibodiesorantigenscoupledtoaneasily-assayedenzyme.ELISAscanprovideausefulmeasurementofantigenorantibodyconcentration.Therearetwomainvariationsonthismethod:TheELISAcanbeusedtodetectthepresenceofantigensthatarerecognizedbyanantibodyoritcanbeusedtotestforantibodiesthatrecognizeanantigen.AnELISAisafive-stepprocedure: Theamountofthecaptureantibodythatisboundtothesolidphasecanbeadjustedeasilybydilutionorconcentrationoftheantibodysolution.Theavidityoftheantibodiesfortheantigencanonlybealteredbysubstitutionwithotherantibodies.Thespecificactivityofthesecondantibodyisdeterminedbythenumberandtypeoflabeledmoietiesitcontains.Antibodiescanbelabeledconvenientlywithiodine,enzymes,orbiotin. [1][2]下一页
TherearemanydifferenttypesofELISAs.OneofthemostcommontypesofELISAis"sandwichELISA."Thisassayisusedtodeterminetheantigenconcentrationinunknownsamples.ThisELISAisfastandaccurate,andifapurifiedantigenstandardisavailable,theassaycandeterminetheabsoluteamountsofantigeninunknownsamples.ThesandwichELISArequirestwoantibodiesthatbindtoepitopesthatdonotoverlapontheantigen.Thiscanbeaccomplishedwitheithertwomonoclonalantibodiesthatrecognizediscretesitesoronebatchofaffinity-purifiedpolyclonalantibodies.Toutilizethisassay,oneantibody(the"capture"antibody)ispurifiedandboundtoasolidphase.Antigenisthenaddedandallowedtocomplexwiththeboundantibody.Unboundproductsarethenremovedwithawash,andalabeledsecondantibody(the"detection"antibody)isallowedtobindtotheantigen,thuscompletingthe"sandwich".Theassayisthenquantitatedbymeasuringtheamountoflabeledsecondantibodyboundtothematrix,throughtheuseofacolorimetricsubstrate.Amajoradvantageofthistechniqueisthattheantigendoesnotneedtobepurifiedpriortouse,alsothattheseassaysareveryspecific.However,onedisadvantageisthatnotallantibodiescanbeused.Monoclonalantibodycombinationsmustbequalifiedat"matchedpairs",meaningthattheycanrecognizeseparateepitopesontheantigen.UnlikeWesternblots,whichusepreciptatingsubstrates,ELISAproceduresutilizesubstratesthatproducesolubleproducts.Ideallytheenzymesubstratesshouldbestable,safeandinexpensive.Popularenzymesarethosewhichconvertacolorlesssubstratetoacoloredproduct,e.g.p-nitrophenylphosphate(pNPP)whichisconvertedtotheyellowp-nitrophenolbyalkalinephosphatase.Substratesusedwithperoxidaseinclude2,2?-azo-bis(3-ethylbenzthiazoline-6-sulfonicacid)(ABTS),o-phenylenediamine(OPD)and3,3?,5?tetramethylbenzidinebase(TMB),whichyieldgreenorangeandbluecolors,respectively.Atableofcommonly-usedenzyme-substratecombinationsisincludedinAppendixA.
