A型肉毒神经毒素单克隆抗体的制备与化学发光酶免疫检测技术的应用  

Preparation of Monoclonal Antibodies against Type A Botulinum Neurotoxin and Application of Chemiluminescent Enzyme Immunoassay Technology

作　　者：刘英 张慧云 高有为 李晨昊 李恺 王云天 朱衍志 张若晖 LIU Ying;ZHANG Huiyun;GAO Youwei;LI Chenhao;LI Kai;WANG Yuntian;ZHU Yanzhi;ZHANG Ruohui(Lanzhou Biotechnology Development Co.,Ltd.Medical Aesthetics Research and Development Center,Lanzhou 730046,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：《药物生物技术》2024年第3期245-249,共5页Pharmaceutical Biotechnology

摘　　要：从A型肉毒神经毒素杂交瘤细胞中提取单抗基因并在哺乳动物系统中表达纯化,制备A型肉毒神经毒素单克隆抗体。应用化学发光酶免疫分析技术(CLEIA),建立A型肉毒神经毒素快速检测方法。通过cDNA末端快速扩增技术(RACE)检测杂交瘤细胞序列,以含有单抗基因的质粒为模板进行亚克隆,分别构建轻重链的表达质粒并转到JM108克隆菌株中,后通过质粒大抽试剂盒提取转染级质粒,再将质粒转染到哺乳动物细胞CHO中进行表达,最后亲和层析Protein G柱纯化抗体。利用双抗体夹心原理,优化出CLEIA法最佳检测方案。建立A型肉毒神经毒素CLEIA检测方法,并对该分析方法的检测限、专属性、耐用性逐一进行验证。制备了A型肉毒神经毒素单克隆抗体且建立了A型肉毒神经毒素快速检出方法,检测限为0.156 ng/mL;A型肉毒神经毒素与其他型别无交叉反应且其在不同溶剂介质中检测限不受影响,显示出良好的特异性和耐用性。应用该方法证明了100 U衡力^(Ⓡ)注射用A型肉毒毒素成品在0.02 mol/L,pH=7.8磷酸缓冲液中复溶检测效果最佳。本研究制备的A型肉毒神经毒素单克隆抗体和建立的A型肉毒神经毒素CLEIA检测方法具有良好的实用性,为A型肉毒神经毒素的临床检测及质控分析提供另一种思路和方法。Monoclonal antibodies against type A botulinum toxin were prepared by extracting monoclonal antibody genes from hybridoma cells of type A botulinum toxin and expressing and purifying them in mammalian systems.In order to establish a rapid detection method for type A botulinum toxin using chemiluminescence enzyme immunoassay(CLEIA)technology,hybridoma cell sequences were detected using rapid amplification of cDNA ends(RACE)technology,plasmids containing monoclonal antibody genes were used as templates for subcloning,and expression plasmids for light and heavy chains were constructed and transferred to the JM108 clone strain.The transfected plasmids were extracted using a plasmid extraction kit,and then transfected into mammalian cell CHO for expression.Finally,the antibodies were purified by affinity chromatography using a Protein G column.Then using the double antibody sandwich principle,the optimal detection scheme for the CLEIA method was optimized,a detection method for type A botulinum toxin CLEIA was established and the detection limit,specificity,and durability of this analytical method were verified one by one.This study prepared monoclonal antibodies against type A botulinum toxin and established a rapid detection method for type A botulinum toxin with a detection limit of 0.156 ng/mL;Type A botulinum toxin has no cross reactivity with other types and its detection limit is not affected in different solvent media,demonstrating good specificity and durability.This method has been applied to prove 100 U balance forceⓇbest redissolution detection effect of type A botulinum toxin for injection is achieved under 0.02 mol/L,pH=7.8 phosphate buffer conditions.The monoclonal antibody against type A botulinum toxin prepared in this study and the established CLEIA detection method for type A botulinum toxin have good practicality,providing another approach and method for clinical detection and quality control analysis of type A botulinum toxin.

关 键 词：A型肉毒神经毒素 A型肉毒神经毒素单克隆抗体 化学发光酶免疫分析技术 CDNA末端快速扩增技术 双抗体夹心法 

分 类 号：R392[医药卫生—免疫学]