黄曲霉毒素B1检测试剂盒的制备及应用

Preparation and application of aflatoxin B1 detection kit

中文关键词:  黄曲霉毒素 B1  单克隆抗体  检测  试剂盒  酶联免疫吸附法  

英文关键词:aflatoxin B1  monoclonal antibodies  detection kit  enzyme-linked immunosorbent assay

中文摘要:

      目的 制备高效价黄曲霉毒素B1 (aflatoxin B1, AFB1)单克隆抗体检测试剂盒, 快速准确检测食品中的AFB1。方法 通过碳二亚胺法, 将AFB1分别与牛血清白蛋白(bovine albumin, BSA)偶联合成免疫抗原、与鸡卵白蛋白(ovalbumin, OVA)偶联制备检测抗原; 通过免疫小鼠、细胞融合、杂交瘤细胞筛选, 体内诱生腹水、分离纯化等过程制备AFB1单克隆抗体, 并开发可用于快速检测AFB1含量的试剂盒。结果 制备的单克隆抗体效价高达1:27 w, 对柄曲霉素有弱交叉反应, 反应率为13%, 与其他2种化合物未见交叉反应; 对加标样品的批内、批间试验相对标准偏差分别为2.6%~3.6%和5.0%~8.0%; 检测回收率达到89.82%~103.64%; 该试剂盒检测灵敏度半数抑制浓度值为650 pg/mL, 检测范围为156~5000 pg/mL, 检出限为100 pg/mL; 以不同产地的玉米面粉为检测样品进行AFB1含量检测, 各地检测结果分别为9.756、2.483、3.995、39.080和7.831 μg/kg。结论 该研究制备出高效价AFB1单克隆抗体, 该抗体不仅可以用于试剂盒检测实验方法的开发, 同时为开发基于AFB1单克隆抗体和胶体金法的快速检测试纸条奠定基础。

英文摘要:

      Objective To prepare a high-titer monoclonal antibody detection kit for aflatoxin B1 (AFB1) and rapidly and accurately detect AFB1 in food. Methods By carbodiimide method, AFB1 was conjugated with bovine albumin (BSA) to form immune antigen, and with chicken ovalbumin (OVA) to prepare detection antigen. AFB1 monoclonal antibody was prepared by immunizing mice, cell fusion, screening of hybridoma cells, inducing ascites in vivo, isolation and purification, and a kit for rapid detection of AFB1 was developed. Results The prepared monoclonal antibody had a titer of up to 1:27 w and showed weak cross-reactivity to Shentuqumycin, with a reaction rate of 13%. No cross-reactivity was observed with the other 2 compounds. The relative standard deviation of the intra- and inter-batch tests for the spiked samples was 2.6%–3.6% and 5.0%–8.0%, respectively. The recoveries were 89.82%–103.64%. The detection sensitivity (median inhibitory concentration value) was 650 pg/mL, with a detection range of 156–5000 pg/mL with the limit of detection of 100 pg/mL. When AFB1 content was detected in corn flour from different origins, the detection results were 9.756, 2.483, 3.995, 39.080 and 7.831 μg/kg. Conclusion This research has prepared high-titer monoclonal antibodies against AFB1. These antibodies can not only be utilized for the development of test kit detection experimental methods but also lay the foundation for the development of rapid test strips based on AFB1 monoclonal antibodies and the colloidal gold method.