Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing 210014, China.
Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014, China.
Viruses. 2023 Jan 4;15(1):154. doi: 10.3390/v15010154.
African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs of all breeds and ages, caused by African swine fever virus (ASFV). Due to the absence of a safe and efficacious vaccine, accurate laboratory diagnosis is critical for the control of ASF prevention. The p30 protein is immunogenic and stimulates a high level of antibody response to ASFV infection. We developed a panel of 4 monoclonal antibodies (mAbs) against p30 protein, and mAb-2B4 showed the highest percent of inhibition (PI) of 70% in the solid phase blocking ELISA (bELISA). Epitope mapping revealed the mAb-2B4 recognized the epitope of aa 12-18 of p30, which is conserved among various ASFV genotypes. Subsequently, a competitive enzyme-linked immunosorbent assay (cELISA) was established using HRP-labeled mAb-2B4. The cutoff for discrimination between 98 negative sera and 40 positive sera against ASFV was determined by plotting a receiver operating characteristic (ROC) curve. It yielded the area under the curve (AUC) of 0.998, and a diagnostic specificity of 97.96% and a sensitivity of 97.5% were achieved when the cutoff value was determined at 37.1%. Furthermore, the results showed an excellent repeatability of the established cELISA and no cross-reaction to antisera against six other pig pathogens. Additionally, the cELISA detected a titer of 1:256 in the positive standard serum. Overall, mAb-2B4 showed a conserved epitope and high ability to be inhibited by positive sera in ASFV antibody detection. The cELISA based on HRP-labeled mAb-2B4 offers an alternative to other assays for a broader diagnostic coverage of ASFV infection.
非洲猪瘟（ASF）是一种高度传染性的出血性病毒性疾病，可感染所有品种和年龄段的家猪和野猪，由非洲猪瘟病毒（ASFV）引起。由于缺乏安全有效的疫苗，因此准确的实验室诊断对于控制 ASF 的预防至关重要。p30 蛋白具有免疫原性，可刺激针对 ASFV 感染的高水平抗体反应。我们开发了针对 p30 蛋白的 4 种单克隆抗体（mAb），mAb-2B4 在固相阻断 ELISA（bELISA）中的抑制百分率（PI）最高，为 70％。表位作图显示，mAb-2B4 识别 p30 的 aa12-18 表位，该表位在各种 ASFV 基因型中均保守。随后，使用 HRP 标记的 mAb-2B4 建立了竞争酶联免疫吸附试验（cELISA）。通过绘制接收者操作特征（ROC）曲线确定了区分 98 份阴性血清和 40 份抗 ASFV 阳性血清的截断值。曲线下面积（AUC）为 0.998，当截断值确定为 37.1％时，获得了 97.96％的诊断特异性和 97.5％的敏感性。此外，结果表明，建立的 cELISA 具有出色的重复性，与针对其他六种猪病原体的抗血清无交叉反应。此外，cELISA 在阳性标准血清中检测到 1：256 的效价。总体而言，mAb-2B4 显示出针对 ASFV 抗体检测中阳性血清的保守表位和高抑制能力。基于 HRP 标记的 mAb-2B4 的 cELISA 为更广泛地诊断 ASFV 感染提供了替代其他检测方法的选择。
