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为了建立捕获法ELISA检测基孔肯雅病毒IgM抗体的方法,本研究分离基孔肯雅病毒结构蛋白编码基因CE3-E2-6K-E1基因元件,通过昆虫细胞杆状病毒表达体系,制备病毒样颗粒,免疫昆明鼠制备多克隆抗体,纯化后用辣根过氧化物酶标记,建立一种以羊抗人IgM抗体、VLPs抗原和酶标多克隆抗体为基础的MacELISA检测基孔肯雅病毒IgM抗体的方法,通过商业化质控血清,实验室确诊基孔肯雅热、肾综合症出血热患者和健康人群血清标本对所建立方法进行优化和初步评价,并与德国欧蒙公司商业化CHIKV IgM抗体检测试剂盒IIFA-IgM进行比较研究。所建立方法特异性为99%(99/100),重复性检测板内变异性系数低于10%,板间变异系数低于15%,检测限明显低于IIFA-IgM商业化试剂盒(P<0.0001)。结果显示,本方法属常规ELISA方法,操作简单,具有较高的敏感性、特异性,可用于基孔肯雅病毒IgM抗体实验室检测,为该病的诊断和流行病学调查提供新的手段。
In order to establish a method for the detection of IgM antibodies to capture Chikungunya virus by ELISA, we isolated the CE3-E2-6K-E1 gene of Chikungunya virus structural protein gene from Baculovirus expression system and prepared virus-like The polyclonal antibody was prepared from the immunized Kunming mice and purified by horseradish peroxidase (HRP). The detection of Chikungunya virus (IgM) by MacELISA was based on goat anti-human IgM antibody, VLPs antigen and enzyme-labeled polyclonal antibody Antibody method to optimize and preliminarily evaluate the established method by commercial quality control serum, laboratory confirmed chikungunya, hemorrhagic fever with renal syndrome and healthy population, and with the commercialization of CHIMV IgM antibody test kit IIFA-IgM comparative study. The specificity of the established method was 99% (99/100). The coefficient of variation of reproducibility was less than 10% and the coefficient of variation (CV) was less than 15%. The detection limit was significantly lower than that of IIFA-IgM commercial kit <0.0001). The results show that the present method belongs to conventional ELISA method is simple, has high sensitivity, specificity, may be used Chikungunya virus IgM antibody laboratory testing, to provide new means for the diagnosis and epidemiology of the disease .