摘要
为制备特异性强的副溶血性弧菌的单克隆抗体,解决单克隆抗体对免疫学检测产品研发的制约,以副溶血性弧菌(Vibrio parahaemolyticus)ATCC 17802标准菌株免疫Balb/c小鼠,经细胞融合、间接酶联免疫吸附测定法筛选,获得稳定分泌抗副溶血性弧菌(ATCC 17802)菌株的单克隆杂交瘤细胞株3F7D7E8C4,通过体内诱生腹水大量制备抗体,用亚类试剂盒测定抗体亚类为Ig G1;采用辛酸硫酸铵沉淀法以及亲和层析柱对腹水进行纯化,聚丙烯酰胺凝胶电泳实验鉴定单克隆抗体的纯度。制备得到腹水抗体效价为1∶16 000,纯化后抗体效价为1∶8 000,抗体敏感性IC_(50)达到10~6 CFU/m L。纯化后的抗体与12株副溶血性弧菌均能特异性结合,与其他9种非副溶血性弧菌的食源性致病菌均无交叉反应。
This study aimed to prepare a highly specific monoclonal antibody(mAb)against Vibrio parahaemolyticus for the purpose of solving the constraints on the development of immunological assays for this pathogenic microorganism.We injected Balb/c mice with V.parahaemolyticus ATCC17802.The hybridoma cell line3F7D7E8C4,which could stably secret mAb against ATCC17802,was obtained after cell fusion and screening by indirect ELISA.By using a commercial kit,the mAb was identified to belong to the IgG1subclass.The antibody titer of the ascites was1:16000.After purification with saturated ammonium sulfate precipitation and protein G affinity chromatography and purity analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),the titer of the antibody was1:8000and the sensitivity(IC50)was106CFU/mL.The purified antibody could specifically bind to12V.parahaemolyticus strains,and had no cross-reaction with9non-V.parahaemolyticus foodborne pathogens.
作者
谢曼曼
李建武
王广彬
曾海娟
翟绪昭
丁承超
刘武康
刘箐
XIE Manman;LI Jianwu;WANG Guangbin;ZENG Haijuan;ZHAI Xuzhao;DING Chengchao;LIU Wukang;LIU Qing(School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China;Xuzhou Lüjian-Dairy Beverage Co. Ltd., Xuzhou 221006, China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2017年第8期63-68,共6页
Food Science
基金
上海市科委"科技创新行动计划"长三角科技联合攻关领域项目(15395810900)
徐州绿健乳品饮料有限公司"乳制品生产体系致病菌快速检测"项目(3A15308006)
上海理工大学研究生创新基金项目
关键词
副溶血性弧菌
单克隆抗体
酶联免疫吸附测定
Vibrio parahaemolyticus
monoclonal antibody (mAb)
enzyme linked immunosorbent assay (ELISA)
