抗青霉素单克隆抗体的制备及初步应用.docx

抗青霉素单克隆抗体的制备及初步应用抗青霉素单克隆抗体的制备及初步应用抗青霉素单克隆抗体的制备及初步应用PreparationandprimaryapplicationofmonoclonalantibodyagainstbenzylpenicillinAIM:Todevelopmonoclonalantibody(mAb)againstbenzylpenicillinandtoestablishaSandwichELISAmethodforrelativequantitativeanalysisoftheallergenwhichinducedpenicillinallergycommonlyinclinic.METHODS:Penicillin,asakindofhapten,wasconjugatedwithcarrierproteintoformcompleteantigenandthenwasusedtoimmunizeBALB/cmice.HybridomacellssecrectingmAbagainstpenicillinweredeveloped.Ascitesfromtheimmunizedmicewerepurifiedbycaprylicacid?ammoniumsulfateprecipitation.ThespecificityofthepurifiedantibodieswasdetectedandthesuitableantibodieswereusedforestablishingSandwichELISA.RESULTS:NinehybridomacellsstablysecrectingmAbwerepreparedthroughcellfusion,screeningandcloning,andfiveofthesepurifiedmAbhadrelativehighaffinity.Thedouble?antibodySandwichELISAforrelativequantitativeanalysisofbenzylpenicilloylproteinwasestablishedwithasensitivityof870U/L.Theaveragerecoveryratewas107.81%andtheaverageintra?andinter?coefficientofvaritation(CV)was6.7%and9.3%respectively.ThemethodwasappliedforrelativequantitativeanalysisofbenzylpenicilloylproteinfromGroupAStreptococcusPreparation.CONCLUSION:NinehybridomacellstrainsstablysecretingmAbagainstbenzylpenicillinwereobtained.Thedouble?antibodySandwichELISAforrelativequantitativeanalysisofbenzylpenicilloylproteinwasestablished.benzylpenicillin;benzylpenicilloyl;mAb;double?antibodySandwichELISA目的:制备抗青霉素的单克隆抗体(mAb)并建立双抗体夹心ELISA检测方法,对临床上引起青霉素过敏反应的过敏原青霉噻唑蛋白进行研究。方法:将半抗原青霉素和载体蛋白偶联后免疫BALB/c小鼠,应用杂交瘤技术建立稳定分泌抗青霉素mAb的杂交瘤细胞株。常规制备腹水,用辛酸?硫酸铵法纯化,并对纯化的mAb进行特异性鉴定。通过对不同抗体组合的分析和条件的优化,建立检测过敏原的双抗体夹心ELISA方法。结果:经细胞融合、筛选及克隆化,共获得9株稳定分泌抗青霉素mAb的杂交瘤细胞株,其中5株亲和力较高。建立了双抗体夹心ELISA相对定量检测方法,该方法灵敏度达到870U/L,平均回收率为107.81%,批内变异系数平均为6.7%,批间变异系数平均为9.3%,可用于A群链球菌制剂中青霉噻唑蛋白的检测。结论:成功地制备了抗青霉素的mAb,并建立了相对定量检测青霉噻唑蛋白的.双抗体夹心ELISA法。青霉素;青霉噻唑基;单克隆抗体;双抗体夹心ELISA青霉素因其高效、低毒的特点在临床上被广泛应用,但其结构中的β?内酰胺环很不稳定,在溶液状态尤其是碱性条件下易开环与蛋白、多肽的氨基发生亲核反应生成稳定的青霉噻唑蛋白,形成临床主要的过敏原。青霉素类抗生素的过敏反应发生率居各种药物之首,青霉素不纯物0.01μg即可使青霉素高度敏感者产生严