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, 730046, China.
Appl Microbiol Biotechnol. 2024 Oct 8;108(1):482. doi: 10.1007/s00253-024-13321-0.
Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 10 50% tissue culture infective dose per mL (TCID/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread.
猪流行性腹泻（PED）是一种由猪流行性腹泻病毒（PEDV）引起的传染性肠道疾病，由于其发病迅速、传播迅速以及仔猪死亡率高，给全球养猪业造成了巨大的经济损失。在本研究中，我们使用保守且具有抗原性的 PEDV N 蛋白作为免疫原，制备了针对 PEDV 核衣壳（N）蛋白的兔多克隆抗体和单克隆抗体 6C12。建立了双抗体夹心定量酶联免疫吸附试验（DAS-qELISA），使用兔多克隆抗体作为捕获抗体，辣根过氧化物酶（HRP）标记的 6C12 作为检测抗体，用于检测 PEDV。使用 DAS-qELISA，重组 PEDV N 蛋白和病毒滴度检测的下限分别约为 0.05ng/mL 和 10 50%组织培养感染剂量/mL（TCID/mL）。与猪繁殖与呼吸综合征病毒（PRRSV）、猪轮状病毒（PoRV）、猪伪狂犬病病毒（PRV）、猪德尔塔冠状病毒（PDCoV）或猪圆环病毒（PCV）无交叉反应性。验证了 DAS-qELISA 的重现性，批内和批间重复的变异系数（CV）小于 10%，表明重现性良好。使用 DAS-qELISA 检测 PEDV 感染仔猪的肛拭子样本时，与逆转录-聚合酶链反应（RT-PCR）的符合率为 92.55%，kappa 值为 0.85，与 PEDV 抗原检测试剂盒的符合率为 94.29%，kappa 值为 0.88，表明该方法具有可靠性。这些发现为 PEDV 的基础和实际研究提供了基本的物质支持，并为临床应用提供了重要的诊断工具。 关键点： • 制备了一种新的抗 PEDV N 蛋白单克隆抗体株 • 建立了一种更敏感的双抗体夹心定量 ELISA • DAS-qELISA 可用于控制 PEDV 的传播。
