高特异性三唑仑代谢物单克隆抗体的制备

目的建立分泌抗三唑仑代谢物α-羟基三唑仑单克隆抗体的杂交瘤细胞株,制备高特异性的三唑仑代谢物单克隆抗体,为三唑仑及其代谢物免疫分析方法的开发奠定基础。方法在三唑仑分子的6位苯环对位上引入活性氨基基团,然后通过缩合反应分别与匙孔血蓝蛋白(KLH)和牛血清白蛋白(BSA)相偶联形成完全抗原。以三唑仑-KLH免疫Balb/c小鼠,通过细胞融合,筛选等杂交瘤技术建立稳定的分泌特异性单克隆抗体的杂交瘤细胞株。纯化后的单克隆抗体,分别用SDS-PAGE电泳法、间接ELISA法和胶体金免疫层析法对其纯度、效价及灵敏度和特异性进行测定。结果获得3株能稳定分泌三唑仑代谢物单克隆抗体的杂交瘤细胞株,分别命名为2G4,4B2和5H6。2G4和4B2抗体只与三唑仑代谢物α-羟基三唑仑有反应,灵敏度分别为500ng/mL和750ng/mL。与其他参试物无交叉反应。因5H6抗体为IgM,考虑到纯化难度和实际应用的限制,暂未做深入研究。结论本研究制备的2G4和4B2单克隆抗体仅识别三唑仑代谢物α-羟基三唑仑,具有高度特异性和灵敏度。

Preparation and characterization of monoclonal antibodies against metabolite of triazolam

Objective To prepare monoclonal antibodies against α-hydroxytriazolamm,the main metabolite of triazolam and identify their immunological characteristics,in order to promote the developing of immunoassay methods of triazolam and its metabolites further.Methods By introducing an active amino group to benzene ring at triazolam molecule,the hapten triazolam was conjugated to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) by EDC method.Immnunized the Balb/c mice with triazolam-KLH,the spleen cells fused with SP2/0 cell to form the hybridoma.The hybridoma cell lines which could secret mAbs against α-hydroxytriazolamm were acquired after several round screening.The purified monoclonal antibodies were tested for purity,titration,sensitivity and specificity by SDS-PAGE,indirect ELISA and colloidal gold immunochromatography.Results Three hybridoma cell lines were obtained and named as 2G4,4B2 and 5H6,respectively.The monoclonal antibodies secreted by 2G4 and 4B2 react with α-hydroxytriazolam,the main metabolites of triazolam.The sensitivity was 500ng/mL and 750ng/mL.It was showed there is no any cross reaction to other benzodiazepines.Conclusion The monoclonal antibodies secreted by hydrodoma cell lines 2G4 and 4B2 recognize the triazolam metabolite α-hydroxytriazolamm with high sensitivity and specificity.