结核分枝杆菌特异性γ-干扰素化学发光免疫检测体系的建立

Establishment of a system of chemiluminescence immunoassay for specific Mycobacterium tuberculosis interferon-γ

目的建立一种用于便携式检测结核分枝杆菌特异性γ-干扰素的化学发光免疫检测体系,为下一步研制便携式检测试剂盒奠定基础。方法利用生物素-链霉亲和素系统偶联磁珠和抗体,利用酰胺键结合方式偶联吖啶酯和标记抗体。采用正交实验法,优化结核分枝杆菌特异性γ-干扰素检测试剂盒所需的吖啶酯标记检测抗体及磁珠偶联包被抗体的方法、缓冲液配方、封闭液配方等条件。最后将建立的检测体系与商品化酶联免疫吸附试验(ELISA)参照试剂盒进行比较。结果建立的检测体系与商品化ELISA参照试剂盒阳性符合率为99.20%,阴性符合率为98.91%,总符合率为99.10%,Kappa值为0.982,最低检出限0.66 pg/mL。结论建立的便携式结核分枝杆菌特异性γ-干扰素化学发光免疫检测体系与ELISA参照试剂盒相比具有类似的检测能力,可用于临床辅助诊断。

Objective To establish a chemiluminescence immunoassay which can portably detect the specific Mycobacterium tuberculosis interferon-γ,so as to lay a foundation for the development of portable detection kit in the next step.Methods A biotin streptavidin system was used to couple magnetic beads and antibodies,and an amide bond was used to couple acridine ester and label antibodies.Using orthogonal experimental method to optimize the conditions such as the method,buffer solution formula,and blocking solution formula for acridine ester labeling detection antibodies and magnetic bead coupling coated antibodies required for the specific Mycobacterium tuberculosis interferon-γdetection kit.Finally,the established detection system was compared with the commercial ELISA reference kit.Results When the established detection system was compared with commercial ELISA reference kit,the positive coincidence rate was 99.20%,the negative coincidence rate was 98.91%,the total coincidence rate was 99.10%,Kappa value was 0.982,and the minimum detection limit was 0.66 pg/mL.Conclusion The established portable chemiluminescence immunoassay system for specific Mycobacterium tuberculosis interferon-γcompared with the ELISA reference kit has the same detection ability and could be used for clinical auxiliary diagnosis.