盐酸西布曲明人工抗原的合成与鉴定

摘要

In order to establish an immunoanalytical method for the detection of sibutramine hydrochloride,the hapten of N,N-didesmethyl sibutramine hydrochloride（M2） was synthesized from 4-chlorophenylacetonitrile and 1,3-dibromopropane and then conjugated to the carrier protein of bovine serum albumin（BSA） or ovalbumin（OVA） by active ester method,glutaraldehyde method or mixed anhydride method to prepare immunogens and coating antigens.The results indicated that the coupling ratios of M2-BSA and M2-OVA were 24.6：1 and 16.2：1 as indicated by UV spectroscopic analysis.The antisera displayed high-level affinity to each coating antigen with a titer of more than 1：8000 and an IC50 of 0.42 μg/mL.Therefore,M2 hapten and carrier proteins were successfully conjugated.The active ester method revealed the smallest damage on the reactive groups of the hapten and the highest specificity.These results suggested that the artificial antigens could be used for the preparation of monoclonal antibodies.%目的：建立盐酸西布曲明的免疫分析方法。方法：4-氯苯乙腈和1,3-二溴丙烷为原料合成与盐酸西布曲明具有相同母核结构的小分子双去甲基西布曲明（M2）,以双去甲基西布曲明为半抗原,并分别通过活泼酯法、戊二醛法和混合酸酐法将半抗原与牛血清白蛋白（BSA）和卵清蛋白（OVA）偶联制备免疫原（M2-BSA）和包被抗原（M2-OVA）。结果：紫外光谱扫描证明半抗原M2与载体蛋白偶联比为24.6：1（M2-BSA）和16.2：1（M2-OVA）,抗血清ELISA效价均达到1：8000以上,IC50=0.42μg/mL。结论：半抗原M2与载体蛋白均已成功偶联,其中活泼酯法对半抗原活性基团的影响最小,合成人工抗原的特异性最强。