摘要
猴痘病毒(MPXV)是一种能引起人兽共患病的正痘病毒属(Orthopoxvirus)成员,在人群和野生动物中具有较高的感染率,对人类公共卫生安全造成重大威胁。为了建立快速、准确的MPXV感染抗体ELISA检测方法,对人群和动物中MPXV感染情况进行流行病学监测,本研究构建了MPXV-A27L蛋白的原核表达系统,以其作为包被抗原建立了MPXV间接ELISA抗体检测方法,并优化了一系列反应条件,确定了检测结果判定标准,评估了建立方法的特异性、重复性和敏感性。结果显示,成功表达了MPXV-A27L蛋白,确定了ELISA最佳反应条件:抗原包被浓度为0.25μg/mL;1%BSA封闭1 h;待检血清1∶200稀释,37℃孵育30 min;酶标二抗1∶20000稀释,孵育30 min;底物显色10 min。待检血清样品OD_(450)值>0.316时判定为阳性,OD_(450)值<0.262时判定为阴性,OD_(450)值介于0.262~0.316时则判为可疑。该检测方法与山羊痘病毒属的山羊痘病毒(GTPV)、绵羊痘病毒(SPPV)和牛结节性皮肤病病毒(LSDV)阳性血清不发生交叉反应;批内变异系数为2.75%~7.45%,批间变异系数为6.55%~9.59%;该方法可检测到血清最大稀释度为1∶104。结果表明,本研究建立的MPXV间接ELISA抗体检测方法具有良好的特异性、重复性和敏感性,为MPXV的临床检测和流行病学调查提供了依据,对于猴痘疫情的防控具有重要作用。
Monkeypox virus(MPXV),a member of the Orthopoxvirus(OPXV)genus that causes zoonotic illness,has a high prevalence in human population as well as wildlife,and poses a major threat to human public health safety.To develop a rapid and accurate ELISA for MPXV infection and to facilitate epidemiological surveillance of MPXV infection in the human population and animals,this study constructed a prokaryotic expression system for the MPXV-A27L protein,which was used as the coating antigen to establish an MPXV indirect ELISA antibody method,and optimized a series of reaction conditions to determine the criteria for adjudication of the results and to evaluate the specificity,reproducibility,and sensitivity of the method.The results showed that MPXV-A27L protein was successfully expressed,and the optimal reaction conditions for the indirect ELISA were determined as follows:the antigen coating concentration was 0.25μg/mL;blocking was performed with 1%BSA for 1 h;serum to be examined was diluted 1∶200 and incubated at 37℃for 30 min;the secondary antibodies for the microplate were diluted 1∶20000 and reacted for 30 min;substrate was developed for 10 min.The serum samples were considered positive when the OD_(450) value was greater than 0.316,considered negative when the OD_(450) value was less than 0.262,and considered suspicious when the OD_(450) value was between 0.262 and 0.316.The indirect ELISA did not show cross reaction with positive sera of goatpox virus(GTPV),sheeppox virus(SPPV)or lumpy skin disease virus(LSDV),which belong to the Capripoxvirus genus.Intra assay coefficients of variation ranged from 2.75%to 7.45%,and inter assay coefficients of variation ranged from 6.55%to 9.59%.This indirect ELISA detected serum up to a maximum dilution of 1∶104.Thus,the MPXV indirect ELISA antibody assay developed in this study has good specificity,reproducibility,and sensitivity,provides a basis for clinical detection and epidemiological investigation of MPXV,and greatly aids the prevention and control of monkeypox outbreaks.
作者
朱俊达
王爽
任书凝
张子卉
李雅睿
刘国瑞
吴文学
王永强
彭辰
ZHU Jun-da;WANG Shuang;REN Shu-ning;ZHANG Zi-hui;LI Ya-rui;LIU Guo-rui;WU Wen-xue;WANG Yong-qiang;PENG Chen(College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)
出处
《中国兽医杂志》
CAS
北大核心
2022年第10期1-7,共7页
Chinese Journal of Veterinary Medicine
基金
“十四五”国家重点研发计划(2021YFD800700)
国家自然科学基金面上项目(32172822)。
