State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730000, China.
Appl Microbiol Biotechnol. 2024 May 29;108(1):350. doi: 10.1007/s00253-024-13146-x.
The African swine fever virus (ASFV) has the ability to infect pigs and cause a highly contagious acute fever that can result in a mortality rate as high as 100%. Due to the viral epidemic, the pig industry worldwide has suffered significant financial setbacks. The absence of a proven vaccine for ASFV necessitates the development of a sensitive and reliable serological diagnostic method, enabling laboratories to effectively and expeditiously detect ASFV infection. In this study, four strains of monoclonal antibodies (mAbs) against p72, namely, 5A1, 4C4, 8A9, and 5E10, were generated through recombinant expression of p72, the main capsid protein of ASFV, and immunized mice with it. Epitope localization was performed by truncated overlapping polypeptides. The results indicate that 5A1 and 4C4 recognized the amino acid 20-39 aa, 8A9 and 5E10 are recognized at 263-282 aa, which is consistent with the reported 265-280 aa epitopes. Conserved analysis revealed 20-39 aa is a high conservation of the epitopes in the ASFV genotypes. Moreover, a blocking ELISA assay for detection ASFV antibody based on 4C4 monoclonal antibody was developed and assessed. The receiver-operating characteristic (ROC) was performed to identify the best threshold value using 87 negative and 67 positive samples. The established test exhibited an area under the curve (AUC) of 0.9997, with a 95% confidence interval ranging from 99.87 to 100%. Furthermore, the test achieved a diagnostic sensitivity of 100% (with a 95% confidence interval of 95.72 to 100%) and a specificity of 98.51% (with a 95% confidence interval of 92.02 to 99.92%) when the threshold was set at 41.97%. The inter- and intra-batch coefficient of variation were below 10%, demonstrating the exceptional repeatability of the method. This method can detect the positive standard serum at a dilution as high as 1:512. Subsequently, an exceptional blocking ELISA assay was established with high diagnostic sensitivity and specificity, providing a novel tool for detecting ASFV antibodies. KEY POINTS: • Four strains of ASFV monoclonal antibodies against p72 were prepared and their epitopes were identified. • Blocking ELISA method was established based on monoclonal antibody 4C4 with an identified conservative epitope. • The established blocking ELISA method has a good effect on the detection of ASFV antibody.
非洲猪瘟病毒（ASFV）能够感染猪并引发高度传染性的急性发热，死亡率高达 100%。由于这种病毒性疫情，全球的养猪业遭受了重大的经济挫折。由于没有经过验证的 ASFV 疫苗，因此需要开发一种敏感可靠的血清学诊断方法，使实验室能够有效地快速检测到 ASFV 感染。在这项研究中，通过重组表达 ASFV 的主要衣壳蛋白 p72，生成了针对 p72 的四株单克隆抗体（mAb）5A1、4C4、8A9 和 5E10，并对其进行免疫小鼠。通过截断重叠多肽进行表位定位。结果表明，5A1 和 4C4 识别氨基酸 20-39 aa，8A9 和 5E10 识别 263-282 aa，与报道的 265-280 aa 表位一致。保守性分析表明，20-39 aa 是 ASFV 基因型中表位的高度保守区。此外，基于 4C4 单克隆抗体建立了一种用于检测 ASFV 抗体的阻断 ELISA 检测方法，并对其进行了评估。使用 87 个阴性和 67 个阳性样本进行接收者操作特性（ROC）分析以确定最佳阈值。建立的测试获得了 0.9997 的曲线下面积（AUC），95%置信区间范围为 99.87 至 100%。此外，当阈值设置为 41.97 时，该测试的诊断灵敏度为 100%（95%置信区间为 95.72%至 100%），特异性为 98.51%（95%置信区间为 92.02%至 99.92%）。批内和批间变异系数均低于 10%，表明该方法具有出色的重复性。该方法可检测到高达 1:512 的稀释度的阳性标准血清。随后，建立了一种具有高诊断灵敏度和特异性的阻断 ELISA 检测方法，为检测 ASFV 抗体提供了一种新工具。 关键点： • 制备了四株针对 p72 的 ASFV 单克隆抗体，并鉴定了其表位。 • 基于鉴定出的保守表位，建立了基于单克隆抗体 4C4 的阻断 ELISA 检测方法。 • 建立的阻断 ELISA 检测方法对 ASFV 抗体的检测效果良好。
