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

作　　者：宋敏 范娅涵 王世春 张强[1] 徐婷 蒋天伦 赵树铭 SONG Min, FAN Yahan, WANG Shichun, ZHANG Qiang, XU Ting, JIANG Tianlun, ZHAO Shuming Department of Blood Transfusion, First Affiliated Hospital, Army Medical University(Third Military Medical University ）, Chongqing, 400038, Chi)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院输血科,重庆400038

出　　处：《第三军医大学学报》2018年第7期556-560,共5页Journal of Third Military Medical University

基　　金：全军"十二五"计划专项课题(ASW11J007-05);第三军医大学西南医院院管课题(SWH2014SX02);国家自然科学基金面上项目(81570167)~~

摘　　要：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.

关 键 词：ABO血型 血影 血型抗体 定量 ELISA 

分 类 号：R392-33[医药卫生—免疫学] R392.11[医药卫生—基础医学]