间接ELISA测定人血IgM型抗-A抗体方法的建立及应用

Establishment and application of an indirect ELISA for determining IgM anti-A antibodies in human plasma

目的建立间接酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测方法,快速定量检测人血IgM抗-A抗体。方法通过制备A型红细胞膜蛋白作为捕获抗原,采用棋盘滴定实验筛选出最佳抗原抗体工作浓度,建立人血IgM型抗-A抗体定量检测方法。结果所构建的间接ELISA定量检测方法检测下限为0.02μg/mL;批内变异系数及批间变异系数均小于10%;通过ROC曲线分析,当临界D(450)为0.259时,ROC曲线下面积最大,为0.947,检测灵敏度为87.7%,特异性为100%。采用本研究自建的ELISA检测57例B型血浆标本的IgM型抗-A抗体浓度为(1.71±1.95)μg/mL,定量结果与经典半定量效价评分呈正相关(r=0.71,P<0.01)。结论成功建立了人血IgM型抗-A抗体定量检测方法。

Objective To develop an indirect enzyme linked immunosorbent assay （ELISA） for rapid and quantitative detection of IgM anti-A antibodies in human plasma. Methods An in-house indirect ELISA was developed using the ghost protein of group A erythrocyte as coating antigen. The optimal combination of antigens and antibodies was selected by checkerboard titration. The methodological performance was evaluated, and the plasma concentrations of IgM anti-A antibodies of clinical samples were determined. Results The detection limit of the in-house indirect ELISA was 0.02 μg/mL. The coefficient of variation for inner-batch and inter-batch were both less than 10%. According to the receiver operating characteristic （ROC） curve analysis, the largest area under the curve accounted for 0.947 when the cut-off value at D（450） was 0.259, and the detection sensitivity and specificity were 87.7% and 100% respectively. The plasma level of IgM anti-A antibodies in 57 samples of type B plasma was 1.71±1.95 μg/mL. The quantitative results were positively correlated to the titer scores from the classical semi-quantitative detection of IgM anti-A antibodies （r=0.71, P〈0.01）. Conclusion An indirect ELISA is developed successfully for quantitative detection of IgM antiA antibodies.