应用HTS-ELISA筛选方法制备抗黄曲霉毒素M1单抗

Using HTS-ELISA Method To Make Anti-Aflatoxin M1 Monoclonal Antibody

摘要:

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0.01 μg/L，线性范围为0.1-10 μg/L，竞争性抑制抗体反应50%的抑制浓度IC50为0.82 μg/L，添加0.25-5 μg/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。

Abstract:

Abstract: [objective] To prepare high-affinity anti-aflatoxin M1 monoclonal antibodies by High Throughput Screening ELISA (HTS-ELISA) [Methods] Balb/C mice were immunized by aflatoxin M1-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 5.5x10-10 mol/L. The cross-reactivity of the monoclonal antibody clone against aflatoxin M1, aflatoxin M2, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol 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 - 10μg/L aflatoxin M1. The binding inhibition IC50 of the anti-aflatoxin M1 monoclonal antibody was 0.82μg/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 high-affinity 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.