摘要
目的非洲猪瘟缺乏有效的疫苗,防控有赖于及时准确的诊断和清除病原,随着低致病力毒株的出现和持续流行,检测抗体成为感染监测的重要手段。方法本研究在表达纯化非洲猪瘟病毒P72蛋白并筛选获得单克隆抗体的基础上,将羧基磁珠与重组P72蛋白进行偶联反应形成免疫磁珠,建立了磁微粒化学发光抗体检测技术(P72-MPCLIA)。结果在对220份临床样品的检测结果分析后确定其阈值阻断率为50.00%,敏感性检测表明P72-MPCLIA检测为阳性的最大稀释倍数为1:256,特异性试验与其他生猪疫病阳性血清无交叉反应性,重复性检测批内、批间变异系数分别为1.68%~4.70%和1.90%~4.72%。在对66份临床样品的检测中,与世界动物卫生组织推荐的确诊技术间接免疫荧光抗体检测方法和商品化间接ELISA试剂盒,相符率分别为96.97%和100%。结论P72-MPCLIA特异性高、敏感性好,且检测时间缩短至30min以内,可实现自动化操作,为非洲猪瘟的抗体检测技术提供了新的技术手段。
Objective African swine fever(ASF)lacks an effective vaccine;therefore,its prevention and control depend heavily on timely and accurate diagnosis and rapid removal of infection sources.With the emergence and continued circulation of low-virulence African swine fever virus(ASFV)strains,serological detection has become an essential tool for monitoring ASFV infection.Methods In this study,the P72 protein of ASFV was expressed and purified,and monoclonal antibodies(mAbs)targeting P72 were screened.Carboxyl-functionalized magnetic beads were then conjugated with the recombinant P72 protein to generate immunomagnetic beads,enabling the establishment of a magnetic-particle chemiluminescence immunoassay(P72-MPCLIA).Results Based on the analysis of 220 clinical samples,the cutoff blocking rate was determined to be 50.00%.Sensitivity evaluation showed that P72-MPCLIA remained positive at a maximum serum dilution of 1:256.Specificity testing confirmed the absence of cross-reactivity with sera positive for other major swine pathogens.The assay demonstrated excellent repeatability,with intra-assay and inter-assay coefficients of variation ranging from 1.68%–4.70%and 1.90%–4.72%,respectively.Among 66 clinical samples tested,the concordance rates of P72-MPCLIA with the World Organization for Animal Health(WOAH)–recommended indirect immunofluorescence assay(IFA)and with a commercial indirect ELISA kit were 96.97%and 100%,respectively.Conclusion P72-MPCLIA exhibits high specificity and sensitivity,reduces assay time to within 30 minutes,and is compatible with automated platforms,providing a novel and efficient approach for ASFV antibody detection.
作者
汪葆玥
任雪建
胡冬梅
吕园园
马静
李秀梅
顾小雪
刘玉良
倪建强
周智
李琦
WANG Baoyue;REN Xuejian;HU Dongmei;LYU Yuanyuan;MA Jing;LI Xiumei;GU Xiaoxue;LIU Yuliang;NI Jianqiang;ZHOU Zhi;LI Qi(China Animal Disease Control Center,Beijing 102618,China;Hexu(Zhengzhou)Biotechnology Co.,Ltd.,Zhengzhou 450048,China)
出处
《病毒学报》
北大核心
2026年第1期135-145,共11页
Chinese Journal of Virology
基金
国家重点研发计划项目(项目号:2023YFD1800504),题目:猪重要疫病感染和免疫效果评价的高通量监测技术研究。
关键词
非洲猪瘟病毒
磁微粒化学发光检测方法
化学发光免疫分析法
间接免疫荧光试验
African swine fever virus
Magnetic particle chemiluminescence assay
Chemiluminescence immunoassay
Indirect immunofluorescent assay
