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Abstract：
African swine fever (ASF), caused by African swine fever virus (ASFV), is a fatal infectious disease of pigs and causes great socioeconomic losses globally. The reliable diagnostic method is critical for prevention and control of the disease. In this study, an improved Luciferase immunosorbent assay (LISA) for detecting ASF was developed using the cell lysates containing ASFV p35 protein fused with a reporter Nano-luciferase (p35-Luc protein). The improved method avoids the complicate procedures of immobilizing the serum samples with protein G in the normal LISA method, and replaced by directly coating the serum samples with carbonate buffer, therefore reduces the productive cost and simplifies the operation procedures. The p35-Luc LISA exhibited high specificity for anti-ASFV sera while no cross-reactions with the sera against other swine viruses. The detection limit of the p35-Luc LISA was shown to be at least four times higher than that of the p35 based indirect ELISA established in our lab. The receiver operating characteristic (ROC) analysis showed the 96.36% relative specificity and 96.97% relative sensitivity of the p35-Luc LISA with the cutoff values of 3.55 as compared to the commercial Ingezim p72-ELISA kit. Furthermore, a total of 248 serum samples were tested by both the p35-Luc LISA and commercial Ingezim p72-ELISA kit, and there was a high degree of agreement (97.6%, kappa = 0.9753) in the performance of the two assays. Collectively, the improved LISA based on the p35-Luc protein could be used as a rapid, ultrasensitive, cost-effective and reliable diagnostic tool for serological survey of ASF in pig farms.
摘要：
非洲猪瘟(ASF)由非洲猪瘟病毒（ASFV）引起，是一种致命的猪传染病，在全球范围内造成巨大的社会经济损失。可靠的诊断方法对于疾病的预防和控制至关重要。在这项研究中,使用含有与报道纳米荧光素酶（p35-Luc蛋白）融合的ASFVp35蛋白的细胞裂解物，开发了一种用于检测ASF的改进的荧光素酶免疫吸附测定（LISA）。改进后的方法避免了常规LISA法用蛋白G固定血清样品的复杂程序，并用碳酸盐缓冲液直接涂覆血清样品，因此降低了生产成本，简化了操作程序。p35-LucLISA对抗ASFV血清表现出高特异性，而与血清对其他猪病毒没有交叉反应。p35-LucLISA的检测限比我们实验室建立的基于p35的间接ELISA的检测限高至少四倍。与商业Ingezimp72-ELISA试剂盒相比，接受者工作特征（ROC）分析显示p35-LucLISA的96.36％相对特异性和96.97％相对灵敏度，截止值为3.55。此外，通过p35-LucLISA和商业Ingezimp72-ELISA试剂盒共检测了248份血清样品，并且有很高的一致性(97.6%，kappa=0.9753）在两个测定的性能中。总的来说，基于p35-Luc蛋白的改进LISA可以作为一种快速、超灵敏,用于猪场ASF血清学调查的经济有效且可靠的诊断工具。
