水体中微囊藻毒素的高灵敏快速免疫检测技术的研究

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1、江南大学博士学位论文水体中微囊藻毒素的高灵敏快速免疫检测技术的研究姓名：赵晓联申请学位级别：博士专业：食品科学指导教师：汤坚20070301江南大学博士学位论文AbstractTworapidandsensitivedetectionmethodsforMicrocystin-LR(MC-LRlweredevelopedbyusingandgoldimmunoehromatographicassay(CGICA)andenzyme-linkedimmunoassayfELISA)．nesensitiveELISAkitsandrapidCGICTimmunostripsforanaly

2、zingMC-LRwithChineseindependentintellectualpropertyrightweredeveloped．Inthispaper,theimmuno-antigen(MC-LR-KLH)ofMC-LRwaspreparedaccordingtothemethodofactiveester,andthedetectionantigen(MC·-LR--BSA)ofMC·-LRwaspreparedaccordingtothemethodofEDCcatalyze．ThenNewZealandwhilerabbitswereimmunizedfor20we

3、ekswiththeimmtmo-antigenbyusingtheroutinemethod．PolyclonalantibodyofMC．LRwasobtainedafterprecipitationwithsaturatedammoniumsulphurinat．AndmonoclonalantibodyagainstMC．LRwasobtainedthroughBalb／emiceimmunizing，cellfusion,hybridomascreening,cellcuitivatingandpurification．AnindirectcompetitiveELISAwa

4、sdevelopedusingthepreparedpolyclonalantibody,MC-LR·BSA．andotherreagentsforELISA．Theconditionsofcoatingandimmunoreaction,colorreactionandproportionofreagentswereoptimized，andthesensitivity,linearrange，specificity,reproducibilityanderrorelementswereinvestigated，ThelltheELlSAkitsagainstMC—LRwerepre

5、pared，andtlleirstabilityandrecoveringratewereassessed．11他kitswerecomparedwithsimilarkitsandothersanalysismethods．ArapidCGICAtestforMC-LRwasdevelopedbyusingmonoclonalantibodyagainstMC．LRMC．LR-BSAandotherreagents．7n忙conditionsofimmersion,linearrange．etc．wereoptimized，andthesensitivity,specificity,

6、reproducibilityandreactiontimewereinvestigated．RapidCGICTimmunostripsagainstMC—LRwereinvented,andtheirstabilityrecoveringratewerestudiedchiefly．mexperimentalresultswerevalidatedbvuseOfHPLC．TheELISAkitagainstMC-LRwaspreparedusingpolyclonalantibody,itssensibilitywasO．05pg／L．1inearrangewasO．1ug／L-1

7、0．g／L，fittinglinearequationwasY=-202355x．0．4119．correlationode伍cient∥=0．9987，IC50ValHeWas0．65pg／L，crossreaetivitywithMC．ItRwaslowerthan30％．erOSSreactivitywithMC·LW,MC-LFwerealllowerthan1％．reproducibilityinsamestripswas0．86—7