摘要
【目的】确立基于高通量酶联免疫(HTS-ELISA)筛选方法制备高亲和力抗黄曲霉毒素M1单克隆抗体的方法体系。【方法】采用AFM1-BSA(Aflatoxin M1-bovine serum albumin)免疫Balb/C小鼠,利用HTS-ELISA方法筛选分泌抗黄曲霉毒素M1单抗的杂交瘤细胞株,并分析抗体的特性。【结果】筛选到14株具有分泌高活性抗黄曲霉毒素M1单克隆抗体的杂交瘤细胞株,纯化的最佳抗体的亲和力为5.5×10-10 mol/L。与黄曲霉毒素M1及其结构类似物黄曲霉毒素M2、B1、B2、G1、G2以及其他物质脱氧雪腐镰刀菌烯醇和BSA的交叉反应率分别为100%、4.5%、21.5%、1.0%、16.6%、1.0%、0%、0%。间接竞争ELISA检测最低检测限可达0.01μg/L,线性范围为0.1-10μg/L,竞争性抑制抗体反应50%的抑制浓度IC50为0.82μg/L,添加0.25-5μg/L黄曲霉毒素的牛奶间接竞争ELISA检测回收率在60.3%-152.8%之间。【结论】HTS-ELISA方法可以制备具有高亲和力的抗黄曲霉毒素M1单克隆抗体,可为黄曲霉毒素M1免疫检测体系的建立提供优质抗体材料。
[ objective] To prepare high-affinity anti-aflatoxin M1 monoelonal antibodies by High Throughput Screening ELISA （HTS-ELISA） [Methods] Balb/C mice were immunized by aflatoxin Ml-bovine serum albumin conjugate, and screen secret anti-aflatoxin M1 monoclonal antibody hybridoma by HTS-ELISA. The antibody was characterized. [ Results] Fourteen hybridoma cell lines which could secret high activity anti-aflatoxin M1 monoclonal antibodies were obtained. The affinity of the purified monoclonal antibody was 5.5 ×10^-10 mol/L. The cross-reactivity of the monoclonal antibody clone against aflatoxin M1, aflatoxin M2, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivatenol and BSA was 100% , 4.5% , 21.5% , 1.0% , 16.6% , 1.0% , 0% , 0% , respectively. The sensitivity of the anti-AFM1 monoclonal antibody binding to aflatoxin M1 was 0.01 μg/L and the linear range for developed indirect competitive ELISA was 0. 1 - 10 μg/L aflatoxin M1. The binding inhibition IC50 of the anti-aflatoxin M1 monoclonal antibody was 0.82 μg/ L. Assays of milk samples mixed with AFM1 ranging in concentration from 0.25 to 5.0 μg/L gave mean indirect competitive ELISA recovery of 60.3% - 152.8% . [ Conclusion] HTS-ELISA can be used for the preparation of the highaffinity anti-aflatoxin M1 monoclonal antibodies. The anti-aflatoxin M1 monoclonal antibody could be provided as the high quality material in the system of aflatoxin M1 immune detection.
出处
《微生物学报》
CAS
CSCD
北大核心
2010年第10期1406-1411,共6页
Acta Microbiologica Sinica
基金
黑龙江省教育厅项目(1151hz025)
国家自然科学基金(30771799)~~
作者简介
作者简介：裴世春（1966-），男（朝鲜族），吉林省通化市人，教授，博士，从事食品中真菌毒素的检测研究。E-mail：peisc2002cn@yahoo·.com．cn
