黄芪甲苷单克隆抗体的制备及鉴定

Prepration and Identification of Monoclonal Antibody against Astragaloside IV

人工合成黄芪甲苷（Astragaloside IV，简称AST）的免疫抗原AST-BSA（黄芪甲苷-牛血清白蛋白）和包被抗原AST．OVA（黄芪甲苷-卵清白蛋白），以AST-BSA为免疫原，分5次免疫Balb／C小鼠，取免疫脾细胞与骨髓瘤细胞进行融合，HAT筛选，有限稀释法克隆。建立分泌单克隆抗体的杂交瘤细胞株。采用间接ELISA和间接竞争ELISA（酶联免疫吸附测定）法分别检测抗体效价和特异性。结果免疫小鼠血清抗体效价达1：27000，获得两株稳定分泌抗AST单克隆抗体的细胞株，分别命名为1D5和3D11，以3D11细胞株制备的单抗检测AST显示在0．01—1μg／mL呈线性关系，检测范围达50—300ng／mL，制备的单抗可用于AST含量的检测。该方法有望用于AST免疫检测试剂的研制开发。

In this study,astragaloside IV （AST） immune antigen AST-BSA （astragaloside and bovine serum albumin） and the envelope antigen AST-OVA （astragaloside and egg albumin） were synthesized. Using AST-BSA as immunogen, Balh/C mice were immunized five times. The immunized splenocytes were isolated and fused with myeloma cells, SP2/0. After HAT screening, hybfidoma cell line that secretes McAb was obtained by limited dilution cloning. Indirect ELISA and indirect competitive ELISA （enzyme-linked immunosorbent assay） were used to detect the antibody titer and specificity. Results showed that the serum antibody titer of immunized mice was 1： 27000. Two cell lines which secret monoclonal antibodies against AST,named 1 D5 and 3D11 cells were obtained. Using 3D11 cells, the expression of AST was detected to have linear relationship within the range of 0. 01- 1 μg/mL with detection range of 50 - 300 ng/mL. The monoclonal antibody can be used to detect the content of AST. The method can be used for research and development of AST immune detection reagent.