A型肉毒神经毒素双抗体夹心ELISA的建立及验证  

Establishment and verification of sandwich ELISA with double antibody to botulinum neurotoxin type A

作　　者：刘英 朱衍志 陆俭 周易 马亚峰 张政 朱俐 张荣 张若晖 LIU Ying;ZHU Yanzhi;LU Jian;ZHOU Yi;MA Yafeng;ZHANG Zheng;ZHU Li;ZHANC Rong;ZHANG Ruohui(Medical Beauty Research and Development Center,Lanzhou Biotechnology Development Co.,Lid.,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物技术开发有限公司医美研发中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2024年第1期40-47,共8页Progress In Microbiology and Immunology

摘　　要：目的 建立A型肉毒神经毒素双抗体夹心ELISA快速定量检测方法,并进行验证及初步应用。方法 通过杂交瘤细胞培养技术制备鼠抗A型肉毒神经毒素单克隆抗体,用ELISA及其他免疫学方法测定抗体效价、亚型及理化性质。优化出最佳试验方案,建立定量检测A型肉毒神经毒素的双抗体夹心ELISA,并对该方法的线性及范围、定量限、准确性、精密度(重复性、中间精密度)、专属性和耐用性进行验证。对本公司相关产品进行测试。结果 制备的A型肉毒神经毒素单克隆抗体纯度为88.15%,效价为1∶102 400,重链亚型为IgG1类,轻链为Kappa类;可与A型肉毒神经毒素的重链特异性结合。建立的A型肉毒神经毒素双抗体夹心ELISA在5.000~80.000 ng/mL范围内线性良好(r>0.99);定量限为2.500 ng/mL;准确度介于80%~115%;CV<15%;该方法与破伤风毒素、B、C、D、E和F型肉毒甘油毒素均无交叉;且A型肉毒神经毒素产品复溶后在2~8℃储存3 d,检测结果不受影响。应用该方法检测本公司开发的新型A型肉毒神经毒素产品,结果符合预期。结论 制备了鼠抗A型肉毒神经毒素单克隆抗体,建立了特异性好且灵敏度较高的A型肉毒神经毒素双抗体夹心ELISA,为A型肉毒神经毒素产品质量控制提供参考价值。Objective To establish a rapid quantitative detection method for botulinum toxin type A double antibody sandwich ELISA,and to verify and preliminarily apply it. Method Murine monoclonal antibodies against botulinum toxin type A were prepared by hybridoma cell culture technique. The titer, subtypes, and physicochemical properties of antibody were determined by ELISA and other immunological methods. Optimize the optimal experimental protocol, a double antibody sandwich ELISA for the quantitative detection of botulinum toxin type A was established. The linearity, range, limit of quantitation, accuracy, precision(repeatability, intermediate precision),specificity, and durability of the method were verified. Related products of our company were tested. Results The purity of the monoclonal antibody against botulinum toxin type A prepared was 88.15%,the titer was 1∶102 400. The heavy chain subtype was IgG1 class, and the light chain was Kappa class;it could bind specifically to the heavy chain of botulinum toxin type A. The established double antibody sandwich ELISA for botulinum toxin type A showed good linearity in the range of 5.000-80.000 ng/mL(r>0.99);the limit of quantitation was 2.500 ng/mL;the accuracy ranged from 80% to 115%;CV<15%;there was no crossover with tetanus toxin, B,C,D,E,and F type botulinum toxin;the results were not affected when the botulinum toxin type A products were stored at 2-8 ℃ for 3 days after dissolution. The method was applied to detect the new botulinum toxin type A product developed by our company, and the results were in line with expectations. Conclusion A mouse monoclonal antibody against botulinum toxin type A was prepared, and a sandwich ELISA with good specificity and high sensitivity for type A botulinum toxin dual antibodies was established, which provided reference value for the quality control of botulinum toxin type A products.

关 键 词：A型肉毒神经毒素 单克隆抗体 杂交瘤细胞 双抗体夹心ELISA 

分 类 号：R392-33[医药卫生—免疫学]