Enzyme-linkedImmunosorbentAssays(ELISAs)combinethespecificityofantibodieswiththesensitivityofsimpleenzymeassays,byusingantibodiesorantigenscoupledtoaneasily-assayedenzymethatpossessesahighturnovernumber.ELISAscanprovideausefulmeasurementofantigenorantibodyconcentration.

Oneofthemostusefuloftheimmunoassaysisthetwo-antibody"sandwich"ELISA.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.

1. ThesensitivityoftheSandwichELISAisdependentonfourfactors:
2. Thenumberofmoleculesofthefirstantibodythatareboundtothesolidphase.
3. TheAvidityofthefirstantibodyfortheantigen.
4. Theavidityofthesecondantibodyfortheantigen.
5. Thespecificactivityofthesecondantibody.

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

GeneralProtocolfortheSandwichELISAmethod:

1. Beforetheassay,bothantibodypreparationsshouldbepurifiedandonemustbelabeled.
2. Formostapplications,apolyvinylchloride(PVC)microtiterplateisbest;however,consultmanufacturerguidelinestodeterminethemostappropriatetypeofplateforproteinbinding.
3. Bindtheunlabeledantibodytothebottomofeachwellbyaddingapproximately50mLofantibodysolutiontoeachwell(20mg/mLinPBS).PVCwillbindapproximately100ng/well(300ng/cm2).Theamountofantibodyusedwilldependontheindividualassay,butifmaximalbindingisrequired,useatleast1mg/well.thisiswellabovethecapacityofthewell,butthebindingwilloccurmorerapidly,andthebindingsolutioncanbesavedandusedagain.
4. Incubatetheplateovernightat4?Ctoallowcompletebinding.
5. WashthewellstwicewithPBS.A500mLsquirtbottleisconvenient.Theantibodysolutionwashescanberemovedbyflickingtheplateoverasuitablecontainer.
6. Theremainingsitesforproteinbindingonthemicrotiterplatemustbesaturatedbyincubatingwithblockingbuffer.Fillthewellstothetopwith3%BSA/PBSwith0.02%sodiumazide.Incubatefor2hrstoovernightinahumidatmosphereatroomtemperature.(Note:SodiumazideisaninhibitororhorserADIshperoxidase.Donotincludesodiumazideinbuffersorwashsolutions,ifanHRP-labeledantibodywillbeusedfordetection.)
7. WashwellstwicewithPBS.
8. Add50mLoftheantigensolutiontothewells(theantigensolutionshouldbetitrated).Alldilutionsshouldbedoneintheblockingbuffer(3%BSA/PBSwith0.02%sodiumazide).Incubateforatleast2hrsatroomtemperatureinahumidatmosphere.
9. WashtheplatefourtimeswithPBS.
10. Addthelabeledsecondantibody.Theamounttobeaddedcanbedeterminedinpreliminaryexperiments.Foraccuratequantitation,thesecondantibodyshouldbeusedinexcess.Alldilutionsshouldbedoneintheblockingbuffer.
11. Incubatefor2hrsormoreatroomtemperatureinahumidatmosphere.
12. WashwithseveralchangesofPBS.
13. Addsubstrateasindicatedbymanufacturer.Aftersuggestedincubationtimehaselapsed,opticaldensitiesattargetwavelengthscanbemeasuredonanELISAreader.

Note:Someenzymesubstratesareconsideredhazardous,duetopotentialcarcinogenicity.HandlewithcareandrefertoMaterialSafetyDataSheetsforproperhandlingprecautions.

Forquantitativeresults,comparesignalofunknownsamplesagainstthoseofastandardcurve.Standardsmustberunwitheachassaytoensureaccuracy.

TroubleshootingELISAAssays:

• Interpretthecontrolresults.
• Ifthenegativecontrolsaregivingpositiveresults,theremaybecontaminationofthesubstratesolution,orcontaminationoftheenzyme-labeledantibody,orofthecontrolsthemselves.
• Ifnocolorhasdevelopedforthepositivecontrolsorforthesamples,checkallreagentsfordating,concentration,andstorageconditions.Checktheintegrityoftheantibodyreagent.
• Ifverylittlecolorhasdevelopedforthepositivecontrolsandthetestsamples,checkthedilutionoftheenzyme-labeledantibody,andtheconcentrationofthesubstrate.
• Ifcolorhasdevelopedforthetestsamplesbutnotthepositiveornegativecontrols,checkthesourceofthepositivecontrols,theirexpirationdateandtheirstorage.Havetheybeenstoredinadiluteform,sothattheantigenmayhaveadheredtothesurfaceofthestoragevessel?
• Ifcolorcanbeseen,buttheabsorbanceisnotashighasexpected,checkthewavelengthsetting.
• Whenrerunninganassaywhiletroubleshooting,changeonlyonefactoratatime.

相关仪器试剂设备

• 酶标仪
• 洗板机
• 化学发光检测仪
• ELISA检测试剂盒
• ELISPOT检测
• ELISA试剂盒
• 抗体
• 酶标板

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。