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目的 为了建立非洲猪瘟病毒（African Swine fever virus,ASFV）pB602L蛋白抗体间接ELISA检测方法。方法本研究利用大肠杆菌表达系统表达了His-pB602L重组蛋白，并以纯化的His-pB602L重组蛋白为包被抗原，以表达pB602L的重组狂犬病病毒免疫后的小鼠阳性血清和ASFV猪阳性血清为一抗，辣根过氧化物酶（HRP）标记的葡萄球菌A蛋白（SPA）作为广谱二抗，采用棋盘滴定法优化各反应条件，建立针对pB602L蛋白的间接ELISA抗体检测方法，并对其特异性、敏感性和重复性进行评价。结果 ASFV pB602L蛋白间接ELISA抗体检测方法的最优反应条件为：抗原包被浓度为4μg/mL,4℃包被16 h～18 h,5%脱脂奶粉37℃封闭0.5 h，待检血清孵育0.5 h,SPA-HRP 1:10000倍稀释后37℃孵育0.5 h。该方法仅对ASFV阳性血清检测结果为阳性，与猪瘟病毒、猪蓝耳病毒、猪细小病毒、猪伪狂犬病毒、猪乙型脑炎病毒和猪圆环病毒2型阳性血清均无交叉反应，具有良好的特异性；该方法检测稀释度为1∶6400的猪ASFV临床阳性血清时仍呈现阳性，检测分别稀释3200倍和1600倍的ASFVⅠ型和Ⅱ型阳性血清均为阳性；批内重复和批间重复变异系数均小于10%。结论 本研究建立的ASFV pB602L蛋白间接ELISA抗体检测方法不仅可用于ASF的临床诊断和流行病学调查，亦能够为ASF疫苗研发过程中免疫动物的特异性抗体评价提供技术支持。
Abstract：Objective To establish an indirect enzyme-linked immunosorbent assay(ELISA) for the detection of antibodies against the African swine fever virus(ASFV) pB602L protein. Methods In this study, a recombinant His-tagged pB602L protein was expressed using an Escherichia coli expression system and purified for use as the coating antigen. Positive mouse sera obtained after immunization with a recombinant rabies virus expressing pB602L, as well as ASFV-positive swine sera, were used as primary antibodies. Horseradish peroxidase(HRP)-conjugated staphylococcal protein A(SPA) was employed as a broad-spectrum secondary antibody. Reaction conditions were optimized by checkerboard titration to establish an indirect ELISA specific for the pB602L protein. The specificity, sensitivity, and reproducibility of the assay were subsequently evaluated.Results The optimal assay conditions were determined as follows: antigen coating concentration of 4 μg/mL with incubation at 4°C for 16–18 h; blocking with 5% skim milk at 37 ℃ for 0.5 h, serum incubation for 0.5 h; and incubation with SPA-HRP diluted 1∶10,000 at 37 ℃ for 0.5 h. The established assay yielded positive results exclusively for ASFV-positive sera and showed no cross-reactivity with positive sera against classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine pseudorabies virus, Japanese encephalitis virus, or porcine circovirus type 2, demonstrating high specificity. The assay was able to detect ASFV-positive clinical swine sera at a dilution of 1∶6,400, and successfully identified ASFV genotype I and genotype II positive sera at dilutions of 1∶3,200 and 1∶1,600, respectively. Both intra-assay and inter-assay coefficients of variation were less than 10%, indicating good reproducibility.Conclusion The indirect ELISA based on the ASFV pB602L protein established in this study is suitable for the clinical diagnosis and epidemiological surveillance of African swine fever. In addition, it provides a useful tool for the evaluation of specific antibody responses in immunized animals during ASF vaccine development.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250354
中图分类号:S852.651
引用信息:
[1]张向阳,矫健,黄培,等.非洲猪瘟病毒pB602L蛋白间接ELISA抗体检测方法的建立[J].病毒学报,2026,42(01):126-134.DOI:10.13242/j.cnki.bingduxuebao.250354.
基金信息:
“十四五”国家重点研发计划青年科学家项目（项目号：2021YFF0703600）,题目：实验动物病原快速检测新技术研究; 吉林省科技厅（高层次人才培养项目）（项目号：20250601001RC）,题目：人兽共患病毒新型检测技术研究~~
2025-11-05
2025
2025-12-16
2026-01-04
2026
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2026-01-05
2026-01-05
2026-01-05