B型肉毒毒素单克隆抗体的制备及其检测试剂盒的研制  

Preparation of monoclonal antibody against botulinum toxin B and establishment of a sandwich ELISA for botulinum toxin B detection

作　　者：潘宪云 彭林峰 张甲菊 康玺泰 张正雷 罗广 席仲兴 PAN Xian-yun;PENG Lin-feng;ZHANG Jia-ju;KANG Xi-tai;ZHANG Zheng-lei;LUO Guang;XI Zhong-xing(The Department of Diagnostic Products,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司诊断用品室、甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2023年第1期18-25,共8页Progress In Microbiology and Immunology

摘　　要：目的 建立B型肉毒毒素(botulinum toxin B, BoNT/B)快速检测方法,研制B型肉毒毒素酶联免疫吸附测定(ELISA)检测试剂盒。方法 以B型肉毒类毒素为抗原免疫小鼠,用杂交瘤技术获得鼠源单克隆抗体(单抗),体内法扩大制备单抗。间接ELISA测定杂交瘤细胞株分泌单抗的稳定性、腹水效价以及单抗的亚型、理化性质和抗原识别表位。过碘酸钠法对抗原表位差异较大的抗体进行HRP标记,筛选最佳包被抗体和标记抗体,并确定包被抗体包被浓度和标记抗体工作浓度。将其配制成试剂盒,对试剂盒的线性、定量限、准确度和重复性等进行研究。结果 筛选、鉴定8株能稳定分泌B型肉毒毒素特异性单抗的杂交瘤细胞株。8株单抗腹水效价1∶32 000~1∶512 000,重链为IgG类、轻链为κ型,均耐碱、耐热,不耐酸,其中4C11、4D7、7F9和8D2 4株单抗抗原表位差异较大。以4D7为包被抗体、8D2为标记抗体配制试剂盒,B型肉毒毒素滴度在2~32 LD_(50)/mL范围内呈现良好的线性(r=0.996),定量限为0.96 LD_(50)/mL,试剂盒2~8℃有效期为12个月,与A、C、D、E和F型肉毒毒素均无交叉反应。B型肉毒毒素3、6、12、24 LD_(50)/mL的回收率均在85%~115%之间,重复性CV≤15%。结论 筛选得到8株鼠源单抗,建立了B型肉毒毒素快速ELISA检测方法,研制了检测周期短且高灵敏度的B型肉毒毒素ELISA检测试剂盒。Objective To prepare monoclonal antibody against botulinum toxin type B(BoNT/B) and establish a rapid detection method for BoNT/B. Methods Mice were immunized with BoNT/B, and monoclonal antibodies(MAbs) against BoNT/B were obtained by hybridoma technology. Large amounts of MAbs were prepared in vivo. Indirect enzyme-linked immunosorbent assay(ELISA) was used to determine the stability of monoclonal antibodies secreted by hybridoma cell lines. Antibody subtypes, titers, physicochemical properties and identification of antigenic epitopes were also detected by indirect ELISA. Antibodies with different epitopes were labeled with horseradish peroxidase(HRP) using sodium periodate method. Antibody pairs screening was conducted, and the best suitable concentration of coating antibody and labeled antibody were optimized. Validation tests including linearity and range, limit of quantitation, accuracy and precision of the method were conducted. Results Eight hybridoma cell lines that can stably secrete antibodies against BoNT/B were screened and identified. The titers of ascites antibody produced by the eight hybridoma cells ranged from 1∶32 000 to 1∶512 000. The detection results of antibodies showed that the heavy chain was IgG type, the light chain was κ type, and all of MAbs were alkali-resistant and heat-resistant, but not acid-resistant. Among them, four antibodies 4C11, 4D7, 7F9 and 8D2 have different antigenic epitopes. The kit was prepared using 4D7 as the coating antibody and 8D2 as labeled antibody, and the linear range was 2-32 LD_(50)/mL with a correlation coefficient r=0.996. The limit of quantitation was 0.96 LD_(50)/mL. The kit can be stored at 2~8 ℃ for 12 months, and it showed no cross reaction with botulinum toxin A, C, D, E and F. The recovery of BoNT/B(3, 6, 12, 24 LD_(50)/mL) ranged from 85% to 115%, and the precision of this method was less than 15%. Conclusion Eight MAbs against BoNT/B were generated, and a kit for rapid detection of BoNT/B was developed.

关 键 词：B型肉毒毒素 杂交瘤 单克隆抗体 酶联免疫吸附测定 定量限 试剂盒 抗原识别表位 

分 类 号：R392[医药卫生—免疫学]