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目的:建立检测马白细胞介素-18(equine interleu-kin-18,EIL-18)的双抗体夹心ELISA,为马传染病的免疫学研究提供新方法。方法:将识别不同表位的EIL-18的2株单克隆抗体(mAb)B1G1和S6A3纯化后,利用生物素标记试剂盒对B1G1株mAb进行标记,以重组马白细胞介素-18(rEIL-18)为捕获抗原进行ELISA检测来建立EIL-18双抗体夹心ELISA。结果:经过方阵滴定试验确定捕获抗体的最佳浓度为4mg/L,检测抗体的工作效价为1∶2000。建立的方法可检测马血清中的EIL-18,与猪、牛和羊血清中的IL-18不发生交叉性反应,此方法检测敏感度达到15ng/L,特异性良好。结论:成功建立了EIL-18的双抗体夹心ELISA,为马细胞免疫学研究提供了方法,也为下一步EIL-18ELISA试剂盒的商品化开发奠定了坚实基础。
OBJECTIVE: To establish a double antibody sandwich ELISA for the detection of equine interleukin-18 (EIL-18) in horses and to provide a new method for the immunological study of infectious diseases of horses. Methods: The two monoclonal antibodies (mAb) B1G1 and S6A3 of EIL-18 with different epitopes were purified and labeled with the biotin labeling kit to identify the mAb of recombinant strain B1G1. Recombinant horse IL-18 (rEIL- 18) EIL-18 Diabody Sandwich ELISA was established by ELISA for capture antigen. Results: The optimum concentration of capture antibody was 4 mg / L after square titration test, and the titer of antibody was 1: 2000. The established method can detect EIL-18 in horse serum, and does not cross-react with IL-18 in the serum of pigs, cattle and sheep, and the detection sensitivity is 15ng / L and the specificity is good. Conclusion: The double antibody sandwich ELISA of EIL-18 has been successfully established, which provides a method for the study of equine immunology and laid a solid foundation for the commercialization of EIL-18 ELISA kit.