Institute of Animal Inspection and Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100176, People's Republic of China.
School of Agroforestry and Medicine, Open University of China, Beijing, 100039, China.
Appl Microbiol Biotechnol. 2021 Jun;105(11):4751-4759. doi: 10.1007/s00253-021-11339-2. Epub 2021 May 29.
Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and "sandwiched" by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78-100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.
感染非洲猪瘟病毒（ASFV）会导致一种急性、高致死性的出血性疾病，该疾病已导致中国遭受巨大的经济损失。为了准确检测 ASFV 的抗原，我们使用针对 ASFV p72 的两种单克隆抗体（mAb）M-5 和 M-6，建立了一种快速、免洗、一步夹心型免疫测定法，该方法基于扩增发光接近均相测定联免疫吸附测定（AlphaLISA）。样品中的 ASFV p72 被生物素化 mAb M-5 通过链霉亲和素连接到供体珠表面捕获，并被涂覆在受体珠上的 mAb M-6“夹在中间”。功效和敏感性试验表明，AlphaLISA 可以检测到≥0.78ng/ml 的纯化 p72，线性范围为 0.78-100ng/ml。AlphaLISA 对 ASFV 具有特异性，与其他常见的致病性猪病毒没有交叉反应。与 RealPCR ASFV DNA 测试和 ASFV 抗原检测试剂盒相比，在 60 份猪血清样本中评估的 AlphaLISA 的敏感性分别为 93%和 100%。特异性分别为 100%和 91.7%。本研究为敏感、高效地检测猪血清中的 ASFV 提供了一种良好的实验室诊断工具。 关键点： • MAb M-5 和 M-6 识别 ASFV p72 的各种表位。 • 建立的 ASFV p72 AlphaLISA 显示出良好的特异性、高灵敏度和满意的相关系数。
