外周血中抗EV71 VP1特异性抗体分泌细胞检测方法的建立  

Development of a Method for Detecting VP1-Antigen Specific Antibody Secreting Cells in Peripheral Blood

作　　者：赵培培 陈建国 ZHAO Pei-pei;CHEN Jian-guo(Department of Clinical Laboratory,Wuxi Huishan District People's Hospital,Wuxi,Jiangsu,214000,China)

机构地区：[1]无锡市惠山区人民医院检验科,江苏无锡214000

出　　处：《中国血液流变学杂志》2023年第1期115-119,共5页Chinese Journal of Hemorheology

摘　　要：目的应用酶联免疫斑点(ELISPOT)技术,建立一种检测外周血中抗EV71VP1特异性抗体分泌细胞的方法,并评价该方法在检测EV71感染诊断中的应用价值。方法无菌分离外周血中单个核细胞,建立一系列特异性ELISPOT检测外周血VP1抗原特异性抗体分泌细胞的参数:包括抗原最佳包被浓度、反应体系中细胞浓度以及HRP标记抗体工作浓度的确定,与ELISA比较,检测方法的敏感性和特异性。结果当抗原包被浓度为20μg/mL时,不同细胞浓度反应曲线最稳定;细胞浓度为5×10^(6)/mL时,不同包被浓度形成的斑点数适量,易于计数;HRP标记抗体释稀度为1:10000时,背景最浅容易识别。ELISPOT敏感性为90.0%,特异性96.7%。与ELISA检测抗EV71抗体方法比较,ELISPOT优于ELISA。结论该方法有较高的特异性和灵敏度,有望成为诊断EV71感染的一种理想方法。Objective To establish a method for detecting VP1-antigen specific antibody-secreting cells(ASCs)in peripheral blood with enzyme linked immunospot assay,and avaluated its clinical value for EV71 infection diagnosis.Methods Peripheral blood mononuclear cells were isolated.A series of parameters for detecting VP1 specific ASCs was established,including optimal concentration of coated antigen,optimal concentration of cells,and performance concentration of HRP-Igs.The sensitivity and specificity of the method was compared with ELISA.Results When antigen concentration was 20μg/mL,the reaction curve was the most stable compared with the others.The amount of spots in cell with concentration of 5 ×10^(6)/mL were in moderate level and easy to be recognized.The detected spots were the clearest and the background was the lightest when performance concentration of HRP-Igs were 1:10000.The sensitivity and specificity of ELISPOT were 90.0%and 96.7%,respectively.In comparison with ELISA,ELISPOT was better than ELISA.Conclusion This research indicates that ELISPOT assay with high sensitivity and specificity,will be an idea diagnostic method for EV71 infection.

关 键 词：肠道病毒71型 VP1蛋白 外周血 酶联免疫斑点 

分 类 号：R446.6[医药卫生—诊断学]