Chinese Academy of Inspection and Quarantine, Beijing 100176, China.
Chongqing Animal Disease Control Center, Chongqing 401120, China.
J AOAC Int. 2022 Sep 6;105(5):1428-1436. doi: 10.1093/jaoacint/qsac050.
African swine fever virus (ASFV) is the etiologic agent of African swine fever (ASF), a disease of highly contagious and significant threat to pork production. At present, the sensitive detection methods are the keys to the disease control.
Full-length p72 is produced by a eukaryotic system, and its monoclonal antibody (mAb) 34C10 is subsequently recovered. A blocking ELISA kit for detection of ASFV antibody is developed based on p72 trimers and 34C10.
Full-length p72 is expressed and is used as an immunogen to prepare a panel of monoclonal antibodies. The mAb 34C10 is verified by immunofluorescence and tested by ELISAs with positive serums. The constant affinity of 34C10 is then confirmed. A blocking ELISA kit is further developed and is compared with two commercial kits.
The mAb 34C10 is specifically bound to p72 protein, and it exhibits a blocking effect to positive serum. The immunofluorescence assay experiment shows that 34C10 could bind to p72 expressed by baculoviruses, and the binding affinity of 34C10 is found to be as high as 1.85 × 1011 L/mol. The blocking ELISA kit shows high coincidence with a commercial ELISA kit. The sensitivity between these two kits is 97.6% (95%, CI: 90.65-99.58), and the specificity between them is 100% (95%, CI: 98.34-100).
The blocking ELISA developed in this study may have great potential for diagnosis of ASF. The structure of the antigen p72 is found to be a key factor for the performance of the kit.
For the first time, the eukaryotic expressed full-length p72 protein is used to recover the monoclonal antibody, and it is coated as antigen during the development of the blocking ELISA kit. This study sheds new light on the development of the blocking ELISA kits, especially for the development of a diagnostic kit for the contagious virus with bio-safety problems.
非洲猪瘟病毒（ASFV）是非洲猪瘟（ASF）的病原体，是一种高度传染性的疾病，对猪肉生产构成重大威胁。目前，敏感的检测方法是疾病控制的关键。
全长 p72 由真核系统表达，随后回收其单克隆抗体（mAb）34C10。基于 p72 三聚体和 34C10 开发了用于检测 ASFV 抗体的阻断 ELISA 试剂盒。
表达全长 p72 作为免疫原制备单克隆抗体。通过免疫荧光和阳性血清 ELISA 验证 mAb 34C10。进一步证实了 34C10 的恒定亲和力。然后开发了一种阻断 ELISA 试剂盒，并与两种商业试剂盒进行了比较。
mAb 34C10 特异性结合 p72 蛋白，并对阳性血清表现出阻断作用。免疫荧光实验表明，34C10 可与杆状病毒表达的 p72 结合，34C10 的结合亲和力高达 1.85×1011 L/mol。阻断 ELISA 试剂盒与商业 ELISA 试剂盒高度吻合。两种试剂盒的灵敏度为 97.6%（95%CI：90.65-99.58），特异性为 100%（95%CI：98.34-100）。
本研究开发的阻断 ELISA 试剂盒可能对 ASF 的诊断具有很大的潜力。抗原 p72 的结构是试剂盒性能的关键因素。
首次使用真核表达的全长 p72 蛋白回收单克隆抗体，并将其作为包被抗原用于阻断 ELISA 试剂盒的开发。本研究为阻断 ELISA 试剂盒的开发，特别是为具有生物安全问题的传染性病毒的诊断试剂盒的开发提供了新的思路。
