奶牛孕酮單抗克隆抗體的制備、鑒定及ELISA方法的建立.pdf

1、黑龍江八一農(nóng)墾大學(xué)碩士學(xué)位論文奶牛孕酮單抗克隆抗體的制備、鑒定及ELISA方法的建立姓名：曾文淵申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：動(dòng)物營(yíng)養(yǎng)與飼料科學(xué)指導(dǎo)教師：張洪友20060401AbstractPr02estefonewasakindofbebinItreaectedtI】econdjtjonofovaryaccurateJySothaLwemonjtoredPro鼯steronejnthedairycowaSamethodwitchcould

2、preventand廿eateddisorderofpmpagationdisease1fwemonitored，weshouldpreparetheantigenwithdicyclohexylcarbodiimide，someantj巾rOgesteronemonoclonalantibodieswiththehy嘶domatec|lIl0109y，thendevelopavalidsensitivemethodofEn珂me—li

3、nkedimmunosorbantassayf研t11edetection0fPmgegceroneThecourseofexperimentsaIldtheresu】tsasfol】ows：ProgeSteronewaSah印tenTopr印areantgen，11慢一hydroxyprogesteronehemisuccinatewaSrespectjvelyconjugatedtocarriefproteinsBsA鋤dOVAby

4、dicyclohexylcarbodiimideP4BsAwasusedfort11eimmunizationofmiceP4oVAwasusedasmeco鰣ngantigenfortheELlSABotha11ti邸nswereaccreditedbvSDS—PAGE，、^michwassucceeded6～8weeksmalemousewereimmunizedwithf珥BSAfor4times。everytimewasonem

5、OntllWecou】dgetthespleenonthe廿lird出【yanertheforIllimmunizaIionThespleencellswerefusedwimSP2／omyelomacellsusingPEG，th即96cellcultureboardswereculturedon37℃，5％C02(2～3da”halfmediumwaschangedl，andselectedwj廿1HATandHTmediumTwo

6、hybridomacelllinesof8G6afld11AllwereobtainedusingP4一OVAascoatingantigenbyindirectELlSA，鋤dclonedby1imiteddiIutionmethodforthreetimeswhosetitersofcunivatemediumaIldasciteswere1：16000、1：8000，and1：512000、l：216000。respectiveJ

7、y，Thesubl)，pesofthemwereIgGIandIgG2IrespectiVelyAnalyzedbypmhibitiVeEusA，antipmgestemnemonoclonalaIltibodiesshowed900dsensitivityThe娟nityof8G6wasbetterthall1lAllImmunoaSsaymethodofPro驛stemnewasestabHshedini廿allywnhamif葉O

8、geStemnemonoclonala11tibody8G6incowbvindirectELISA：協(xié)eoptimaIdensityofP4OVAcoatingwas218105“∥ml，Tecoatingtimewas2hat37℃，t1en4℃ovemi曲也Theoptimalblockingbu髓rwas10％90atse咖theblockingtimewas2hat37℃Theoptimaldensityof8G6was139

9、10pg／ml，Incub撕onwascontinuedforlhwith100肛lofl，lOoogoat觚ti哪ouseilTlmun0910bulin(GAMl曲coupledtoHRPOPD，H202subs仃atewasaddedThecolorreactionwasteI孫inated啦er10minbyadditionof501of2MH2S04andODswerereadat490『帥ThewashingwasPBSTT

10、heoptimaIscopeofPmgestemnestandardwasO1～lOOng／mLThjsimmunoassayproVidedaa11al”jcaJmethodforthefouIldationofthecljnicaldetec石ona11dbeuseinpreparationofimmunoassaykita11dtheresearchofbiosensortoProgeste∞neKeyword：Progestel