非洲猪瘟病毒p54间接ELISA抗体检测方法的建立及试剂盒研制
Establishment of an indirect ELISA method and development of corresponding kit targeting African swine fever virus p54 antibodies
岳慧贤 1李其炫 1惠丽丽 1赵心馨 1陈腾 1张艳艳 1王述超 1缪发明 1张菲 1张守峰 1扈荣良1
作者信息
- 1. 中国农业科学院长春兽医研究所,吉林长春 130122
- 折叠
摘要
非洲猪瘟是对全球养猪业危害最严重的病毒性传染病,目前尚无有效疫苗.p54蛋白是非洲猪瘟病毒(ASFV)的重要结构蛋白之一,参与病毒的吸附和侵入,是非洲猪瘟血清学诊断的重要靶标之一.本研究采用无标签的重组p54蛋白作为包被抗原,建立了 ASFV p54间接ELISA抗体检测方法,优化了检测条件,组装成了试剂盒.根据对374份阴性猪血清的检测,确定了本试剂盒检测的临界值,并利用不同猪病原阳性质控血清进行了敏感性、特异性和稳定性研究.结果显示,本方法检测阴/阳性血清的临界值为0.250;敏感性血清质控品的最小检出限为1∶1 600倍,与ASFV阳性血清、猪伪狂犬病病毒阳性血清、猪繁殖与呼吸病毒综合征病毒阳性血清、猪圆环病毒2型阳性血清、猪细小病毒阳性血清和猪抗大肠杆菌阳性血清等6种特异性质控血清均无交叉反应;试制的3批试剂盒批间和批内变异系数均小于10％.试剂盒在2～8 ℃贮存9个月,特异性、敏感性保持不变.以上试验结果表明,本试剂盒具有良好的敏感性、特异性和稳定性,与OIE推荐的p72阻断ELISA抗体检测试剂盒具有较高符合率,为非洲猪瘟临床血清学诊断和抗体监测提供了良好的技术支持.
Abstract
The African swine fever virus(ASFV)is a highly contagious viral pathogen that poses a significant threat to the global pig industry.So far,no effective vaccine is available for the disease.The p54 protein,a prominent structural component of ASFV,is involved in virus adsorption and subsequent entry into cells during the invasion process.The protein also assumes a pivotal role in diagnostic techniques.In the present study,an optimized indirect enzyme-linked immunosorbent as-say(ELISA)was successfully developed,employing the non-tagged p54 protein as coating anti-gens.The cutoff value of the kit was determined by evaluating 374 pig ASFV negative serum sam-ples and preparation of the serum standard for the quality control of the kit.Furthermore,the sen-sitivity,specificity and repeatability of different pig pathogen positive quality control sera were e-valuated.The results showed that the established cutoff value of the kit was 0.250.The detection limit of the kit for control positive serum was up to a dilution ratio of 1:1 600.No cross-reactivity was detected for the antibodies to CSFV,PRRSV,PCV2,PRV,PPV,or E.coli Rosetta.Both the coefficients of intra-and inter-assay variability were found to be below 10％.The specificity and sensitivity remained unchanged when the kit was stored at 2-8 ℃ for 9 months.In conclusion,this antibody kit for ASFV exhibits a notable level of sensitivity,specificity,and stability,and has a high compliance rate with the OIE-recommended p72 blocking ELISA antibody detection kit,which provides strong technical support for the detection of ASFV antibodies and serological sur-veillance for African swine fever.
关键词
非洲猪瘟病毒/p54蛋白/间接ELISA/试剂盒Key words
African swine fever virus/p54 protein/indirect ELISA/kit引用本文复制引用
基金项目
非洲猪瘟病毒病原生态学、新型疫苗及抗病毒药物研究项目(U19A2039)
出版年
2024
NETL
NSTL
万方数据