The present study describes the development of a truncated recombinant peste des petits ruminants virus (PPRV) nucleoprotein (rPPRV-NPN) and its polyclonal antibodies-based immuno-diagnostic assay, Avidin-Biotin (AB) recombinant nucleoprotein competitive ELISA (ABrC-ELISA) for the detection of PPRV antibodies in the sheep and goats. The PPRV N-terminal immunogenic region (1–266 aa) of nucleoprotein (NPN) coding sequence was amplified and cloned into the pETite vector. The rPPRV-NPN with a molecular weight of ∼ 30 kDa was expressed in E. coli, purified, and characterized by SDS-PAGE and immunoblot using standard PPRV specific sera. The Ni-NTA affinity-purified rPPRV-NPN as coating antigen and its hyperimmune serum as competitive antibodies raised in guinea pigs were evaluated as diagnostic reagents in ABrC-ELISA using the known standard panel of sera. The threshold (cut-off) Percentage Inhibition (PI) value was determined as 45 (mean ± 3 SD) based on the reactivity of the known sheep and goats sera to PPRV antibodies [negative (n = 140) and positive (n = 98)] and the assay had a sensitivity of 97 % (95 % Confidence Interval (CI): 91.3–99.4 %) and specificity of 100 % (95 % CI: 97.4–100 %) with an excellent Area under curve (AUC) of 0.997 (95 % CI: 0.99–1.0). On evaluation of diagnostic performance of the assay using the sheep and goats sera (n = 391) from vaccinated, infected, and non-vaccinated animals, the ABrC-ELISA showed the relative diagnostic sensitivity of 95.88 % (95 % CI: 92.56–98.01 %) & 98.77 % (95 % CI: 96.43–99.74 %) and diagnostic specificity of 97.97 % (95 % CI: 94.19–99.58 %) & 90.54 % (95 % CI: 84.64–94.73 %) against indigenous PPR competitive ELISA kit & IDvet Screen® PPR Competition kit, respectively. The study showed that ABrC-ELISA is rapid, sensitive, and specific and can be a better alternative assay for the detection of the PPRV antibodies in the sera of small ruminants for serosurveillance / seromonitoring of PPR not only at the eradication and post-eradication phases in the disease-controlled endemic countries but also in the PPR non-endemic countries.

本研究描述了一种截短重组小反刍兽疫病毒 (PPRV) 核蛋白 (rPPRV-NPN) 及其基于多克隆抗体的免疫诊断测定法，抗生物素蛋白-生物素 (AB) 重组核蛋白竞争 ELISA (ABrC-ELISA)用于检测绵羊和山羊体内的 PPRV 抗体。将核蛋白 (NPN) 编码序列的 PPRV N 端免疫原性区域 (1-266aa) 扩增并克隆到 pETite 载体中。分子量约为 30 kDa 的 rPPRV-NPN 在大肠杆菌中表达, 纯化, 并使用标准 PPRV 特异性血清通过 SDS-PAGE 和免疫印迹进行表征。Ni-NTA 亲和纯化的 rPPRV-NPN 作为包被抗原，其超免疫血清作为在豚鼠中培养的竞争性抗体，使用已知的标准血清组在 ABrC-ELISA 中作为诊断试剂进行了评估。根据已知绵羊和山羊血清对 PPRV 抗体的反应性 [阴性 (n = 140) 和阳性 (n = 98 )]，该测定的灵敏度为 97 %（95 % 置信区间 (CI)：91.3–99.4 %），特异性为 100 %（95 % CI：97.4–100 %），曲线下面积 (AUC) 为0.997（95% CI：0.99–1.0）。关于使用来自接种疫苗、感染疫苗的绵羊和山羊血清 (n = 391) 评估检测的诊断性能，和未接种疫苗的动物，ABrC-ELISA 显示出 95.88 % (95 % CI: 92.56–98.01 %) 和 98.77 % (95 % CI: 96.43–99.74 %) 的相对诊断敏感性和 97.97 % (95 %) 的诊断特异性CI: 94.19–99.58 %) 和 90.54 % (95 % CI: 84.64–94.73 %) 分别针对本土 PPR 竞争性 ELISA 试剂盒和 IDvet Screen® PPR 竞争性试剂盒。研究表明，ABrC-ELISA 快速、灵敏、特异，可作为检测小反刍动物血清中 PPRV 抗体的更好替代方法，用于 PPR 的血清监测/血清监测，不仅在根除和根除后阶段在疾病控制的流行国家以及 PPR 非流行国家。77 % (95 % CI: 96.43–99.74 %) 和 97.97 % (95 % CI: 94.19–99.58 %) 和 90.54 % (95 % CI: 84.64–94.73 %) 与本土 PPR 竞争性 ELISA 试剂盒和 IDvet Screen 的诊断特异性® PPR 比赛套件，分别。研究表明，ABrC-ELISA 快速、灵敏、特异，可作为检测小反刍动物血清中 PPRV 抗体的更好替代方法，用于 PPR 的血清监测/血清监测，不仅在根除和根除后阶段在疾病控制的流行国家以及 PPR 非流行国家。77 % (95 % CI: 96.43–99.74 %) 和 97.97 % (95 % CI: 94.19–99.58 %) 和 90.54 % (95 % CI: 84.64–94.73 %) 与本土 PPR 竞争性 ELISA 试剂盒和 IDvet Screen 的诊断特异性® PPR 比赛套件，分别。研究表明，ABrC-ELISA 快速、灵敏、特异，可作为检测小反刍动物血清中 PPRV 抗体的更好替代方法，用于 PPR 的血清监测/血清监测，不仅在根除和根除后阶段在疾病控制的流行国家以及 PPR 非流行国家。