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夹心ELISA

蚂蚁淘 /发布时间:1545760195 /浏览量:219

Enzyme-linkedImmunosorbentAssays(ELISAs)combinethespecificityofantibodieswiththesensitivityofsimpleenzymeassays,byusingantibodiesorantigenscoupledtoaneasily-assayedenzyme.ELISAscanprovideausefulmeasurementofantigenorantibodyconcentration.Therearetwomainvariationsonthismethod:TheELISAcanbeusedtodetectthepresenceofantigensthatarerecognizedbyanantibodyoritcanbeusedtotestforantibodiesthatrecognizeanantigen.AnELISAisafive-stepprocedure:

coatthemicrotiterplatewellswithantigen;
blockallunboundsitestopreventfalsepositiveresults;
addantibodytothewells;
addanti-mouseIgGconjugatedtoanenzyme;
reactionofasubstratewiththeenzymetoproduceacoloredproduct,thusindicatingapositivereaction.

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.

ThesensitivityoftheSandwichELISAisdependentonfourfactors:
Thenumberofmoleculesofthefirstantibodythatareboundtothesolidphase.
TheAvidityofthefirstantibodyfortheantigen.
Theavidityofthesecondantibodyfortheantigen.
Thespecificactivityofthesecondantibody.

Theamountofthecaptureantibodythatisboundtothesolidphasecanbeadjustedeasilybydilutionorconcentrationoftheantibodysolution.Theavidityoftheantibodiesfortheantigencanonlybealteredbysubstitutionwithotherantibodies.Thespecificactivityofthesecondantibodyisdeterminedbythenumberandtypeoflabeledmoietiesitcontains.Antibodiescanbelabeledconvenientlywithiodine,enzymes,orbiotin.

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