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目的本研究合成并鉴定了AFB1人工抗原,制备AFB1的单克隆抗体(AFB1mAb)。方法采用NHS法将AFB1分别偶联于载体蛋白BSA和OVA上,分别合成W人工抗原AFB1-BSA和AFB1-OVA,紫外分光光度法和SDS-PAGE进行鉴定;AFB1-BSA免疫BALB/C小鼠,通过间接ELISA和阻断ELISA法选择细胞融合备用鼠;用杂交瘤技术制备AFB1mAb,并对其效价、亲和力、敏感性、特异性、亚型进行鉴定;体内诱生腹水法大量制备单抗。结果 UV图谱和SDS-PAGE图表明结半抗原AFB1和载体BSA及OVA偶联成功;筛选出2H5-F6、2H5-C9、2H9-C3三株杂交瘤细胞;鉴定单抗亚型均为IgG1;细胞上清效价1∶2.0×102～1∶1.28×103,2H5-F6的腹水效价1∶1.28×106,AFB1mAb亲和常数Ka为2.65×1010 L/moL,对AFB1的IC50为2.58 ng/mL;与AFB2的交叉反应率为1.61%,与其他类药物无交叉反应。结论通过试验获得高效价、敏感、特异的AFB1mAb,可用于各种食品中AFB1残留的快速免疫学检测试验。
Objective In this study, AFB1 artificial antigen was synthesized and identified, and AFB1 monoclonal antibody (AFB1 mAb) was prepared. METHODS: AFB1 was coupled to carrier protein BSA and OVA respectively by NHS method. The artificial antigen AFB1-BSA and AFB1-OVA were synthesized by UV-Vis spectrophotometry and were identified by UV-Vis spectrophotometry and SDS-PAGE respectively. BALB / C mice were immunized with AFB1-BSA AFB1 mAb was selected by hybridoma technique and the titer, affinity, sensitivity, specificity and subtype of AFB1 mAb were determined by indirect ELISA and blocking ELISA. Massive monoclonal antibodies . Results The UV spectrum and SDS-PAGE showed that the coupling of AFB1 with BSA and OVA was successful. Three hybridoma cells, 2H5-F6, 2H5-C9 and 2H9-C3, were screened out. Cell supernatants titer of 1: 2.0 × 102 ~ 1: 1.28 × 103,2H5-F6 ascites titer 1:1.28 × 106, AFB1mAb affinity constant Ka was 2.65 × 1010 L / moL, the IC50 of AFB1 was 2.58 ng / mL; the cross-reactivity rate with AFB2 was 1.61%, no cross reaction with other drugs. Conclusion The high titer, sensitive and specific AFB1 mAb obtained from the experiment can be used for the rapid immunological detection of AFB1 residues in various foods.