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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。
Objective To establish a hybridoma cell line secreting anti-triazolam monoclonal antibody α-hydroxy triazolam to prepare a highly specific monoclonal antibody to triazolam metabolites for the method of immunoassay of triazolam and its metabolites Development laid the foundation. Methods The active amino groups were introduced into the para position of the 6-position of the triazolam molecule and then coupled with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) respectively to form the complete antigen through the condensation reaction. Balb / c mice were immunized with triazolam-KLH and hybridoma cell lines secreting monoclonal antibodies were established by hybridization, screening and other hybridoma techniques. Purified monoclonal antibodies were detected by SDS-PAGE electrophoresis, indirect ELISA and colloidal gold immunochromatography, respectively, to determine their purity, potency and sensitivity and specificity. Results Three hybridoma cell lines that could stably secrete monoclonal antibodies to triazolam metabolites were obtained and named as 2G4, 4B2 and 5H6.2G4 and 4B2 antibodies, respectively, reacted with the triazolam metabolite α-hydroxy triazolam, The sensitivity was 500 ng / mL and 750 ng / mL, respectively. No cross reaction with other test substances. Due to the 5H6 antibody is IgM, taking into account the difficulty of purification and practical limitations, yet to do in-depth study. Conclusions The 2G4 and 4B2 monoclonal antibodies prepared in this study only recognize α-hydroxy-triazolam, a triazolam metabolite, with high specificity and sensitivity.