抗青霉素单克隆抗体的制备及初步应用.doc

1 抗青霉素单克隆抗体的制备及初步应用 Preparation and primary application of monoclonal antibody against benzylpenicillin [Abstract] AIM: To develop monoclonal antibody(mAb) against benzylpenicillin and to establish a Sandwich ELISA method for relative quantitative analysis of the allergen which induced penicillin monly in clinic. METHODS: Penicillin, asa kind of hapten, was conjugated with carrier protEin to plete antigen and then was used to immunize BALB/c mice. Hybridoma cells secrecting mAb against penicillin were developed. Ascites from the immunized mice were purified by caprylic acid 拟 ammonium sulfate precipitation. The specificity of the purified antibodies was detected and the suitable antibodies were used for establishing Sandwich ELISA. RESULTS: Nine hybridoma cells stably secrecting mAb were prepared through cell fusion, screening 2 and cloning, and five of these purified mAb had relative high affinity. The double 拟 antibody Sandwich ELISA for relative quantitative analysis of benzylpenicilloyl protEIn was established with a sensitivity of 870U/ average recovery rate was % and the average intra 拟 and inter 拟 coefficient of varitation (CV) was % and % respectively. The method was applied for relative quantitative analysis of benzylpenicilloyl protein from Group A us Preparation. CONCLUSION: Nine hybridoma cell strains stably secreting mAb against benzylpenicillin were obtained. The double 拟 antibody Sandwich ELISA for relative quantitative analysis of benzylpenicilloyl protein was established. [Keywords]benzylpenicillin;benzylpenicilloyl;mAb;double 拟 antibody Sandwich ELISA [摘要] 目的: 制备抗青霉素的单克隆抗体(mAb) 并建立双抗体夹心 ELIS A 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 3 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, 用辛酸拟硫酸铵法纯化, 并对纯化的 mA b 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 mA b 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 %, 批内变异系数平均为 %, 批间变异系数平均为 %, 可用于 A 群链球菌制剂中