竞争ELISA法检测人抗脊髓灰质炎病毒(Ⅲ型)中和抗体  

Development of A Competitive ELISA for Detecting Neutralizing Antibody against Poliovirus (Type Ⅲ)

作　　者：张冰洁 赵卫忠 高菁霞[1] 朱文勇[1] 李卫东[1] 廖国阳[1] ZHANG Bing-jie;ZHAO Wei-zhong;GAO Jing-xia;ZHU Wen-yong;LI Wei-dong;LIAO Guo-yang(Institute of Medical Biology,Chinese Academy of Medical Science and Peking Union Medical College,Kunming Yunnan 650018,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650018

出　　处：《昆明医科大学学报》2020年第9期7-11,共5页Journal of Kunming Medical University

基　　金：中国医学科学院医学与健康科技创新工程基金资助项目(2016-I2M-3-026):云南省创新团队基金资助项目(2015HC027)。

摘　　要：目的建立竞争ELISA检测方法,用于检测人群中抗脊髓灰质炎病毒Ⅲ型中和抗体的监测,旨在消灭脊灰后时代中替代中和试验,提供方便、快速和准确的检测方法。方法在前期研究的双抗体夹心ELISA方法的基础上,用纯化后的SabinⅢ型的D抗原作为包被抗原,然后将待测人血清和兔多克隆抗脊灰抗体共同孵育,形成竞争性结合,使用方阵滴定法确定各项抗体、抗原的包被条件,建立抗脊灰中和抗体竞争性ELISA检测方法,与中和试验进行结果的一致性评价。结果该方法仅对脊灰病毒Ⅲ型D抗原血清检测为阳性,与脊灰病毒Ⅰ、Ⅱ型血清无交叉反应,比中和试验敏感性更高。批内批间变异系数分别为3.29%~4.51%和5.26%~6.11%,具有良好的重复性。与中和试验比较,阳性符合率为100%,阴性符合率为88.9%。结论使用本实验建立的竞争ELISA方法用于检测人群抗脊灰中和抗体能达到快速、准确的评价目的并且可避免活病毒的使用。Objective To construct a competitive ELISA method to test neutralizing antibody against poliovirus(type Ⅲ) in the crowd, which is more convenient, fast and accurate compared with traditional poliovirus neutralization test.Method Based on the double-antibody sandwich method previously established in our laboratory,the purified Sabin poliovirus(type Ⅲ) D antigen was used as the coating antigen, and rabbit anti-poliovirus polyclonal antibody as a competitive antibody against human serum after the immunization. Incubating both together can form a competitive binding. The coating conditions of various antibodies and antigens were determined by square matrix titration and the experimental conditions were further optimized.The consistency of the results with the traditional poliovirus neutralization test was evaluated.Results The experimental conditions of the competitive ELISA method were determined by repeated experiments: the antigen coating concentration was 99.6 DU/m L, and the working concentration of tested serum was 1:16. The coincidence rate of this method and the traditional poliovirus neutralization test was 100%, which effectively reduced the interference of false negative results,did not react with the poliovirus C antigen,and had the good specificity between types,which could meet the detection requirements. The study also established the good quality control standards beneficial to the study of clinical samples. Conclusion The competitive ELISA method established in this study can be used for the rapid and accurate detection of the neutralizing antibody against poliovirus(type Ⅲ) in the crowd,and also can avoid the use of live viruses.

关 键 词：竞争ELISA 脊髓灰质炎病毒 D抗原 中和抗体 血清监测 

分 类 号：R373.2[医药卫生—病原生物学]