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目的制备抗黄曲霉毒素B1(AFB1)的重链IgG2b亚型单克隆抗体,并建立黄曲霉毒素B1的间接竞争ELISA检测方法。方法采用AFB1-BSA偶联物为免疫抗原,AFB1-STI偶联物为检测抗原,利用间接竞争ELISA筛选分泌抗AFB1单抗的细胞株,并对抗体进行鉴定。结果筛选到1株能分泌特异性抗AFB1单克隆抗体的杂交瘤细胞株,其染色体数目为(90±10)条;所分泌的单抗亚类重链为IgG2b,轻链为λ;间接ELISA检测该株杂交瘤细胞分泌上清效价在1∶12800～1∶25600,纯化腹水效价为1∶105～1∶106。传30代及液氮中保存6个月,抗体效价稳定;建立的间接竞争ELISA检测方法的最低检出限为0.001ng/ml,校正曲线的线性范围为0.005～5ng/ml,IC50为0.01ng/ml;与AFB1的结构类似物AFM1和化学结构有差异的脱氧雪腐镰刀菌烯醇的交叉反应率分别为1.4%和<1%。结论所制备的抗黄曲霉毒素B1单克隆抗体具有高度特异性和稳定性,可应用于间接竞争ELISA检测方法。
Objective To prepare a monoclonal antibody against heavy chain IgG2b of aflatoxin B1 (AFB1) and to establish an indirect competitive ELISA for detection of aflatoxin B1. Methods AFB1-BSA conjugate was used as antigen and AFB1-STI conjugate was used as antigen. Antibody against AFB1 was screened by indirect competitive ELISA. Results A hybridoma cell line secreting specific anti-AFB1 monoclonal antibody was screened. The number of chromosomes was (90 ± 10). The secreted mAb subtype heavy chain was IgG2b and the light chain was λ. The indirect ELISA The titer of the secreted supernatant of the hybridoma cell line was 1:12800 to 1:25600, and the titer of purified ascites was 1:105 to 1:106. The 30th generation and liquid nitrogen were stored for 6 months, the titer of the antibody was stable. The minimum detectable limit of indirect ELISA was 0.001ng / ml. The linear range of the calibration curve was 0.005 ~ 5ng / ml and the IC50 was 0.01 ng / ml. The cross-reactivity with AFM1, a structural analog of AFB1, and deoxynivalenol with different chemical structures were 1.4% and <1%, respectively. Conclusion The monoclonal antibody against aflatoxin B1 has high specificity and stability and can be used in the indirect competitive ELISA assay.