Wang Shujie, Chen Tianfeng, Yang Yong-Bo, Wang Hongfeng, Sun Mingxia, Gao Kexin, Wei Ziyi, Bai Yuanzhe, Tang Yan-Dong, Cai Xuehui, An Tongqing
State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, Heilongjiang, PR China; Heilongjiang Provincial Key Laboratory of Veterinary Immunology, Harbin, PR China.
State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, Heilongjiang, PR China.
Int J Biol Macromol. 2025 Mar;296:139670. doi: 10.1016/j.ijbiomac.2025.139670. Epub 2025 Jan 8.
Porcine reproductive and respiratory syndrome virus (PRRSV) demonstrates a significantly high prevalence among swine populations. Monoclonal antibodies (mAbs) with high affinity for conserved epitopes of PRRSV can facilitate the development of a broad-spectrum detection method for this virus. This study identified two PRRSV-specific mAbs, designated 2B1 and 2C6, which recognized two conformation-dependent epitopes through indirect immunofluorescence assay (IFA) and Western blot analysis. Further investigation via immunoprecipitation and eukaryotic expression studies confirmed that both mAbs specifically target the Nucleocapsid (N) protein of PRRSV. Importantly, these two epitopes exhibit high conservation across PRRSV isolates, including DV, CH-1a, HuN4, NADC30-like strains, NADC34-like strains and VR2332. The mAb 2C6 was effectively blocked by sera from pigs positive for PRRSV-1 and PRRSV-2. Consequently, a blocking enzyme-linked immunosorbent assay (b-ELISA) based on 2C6 was developed to detect anti-PRRSV antibodies, achieving enhanced sensitivity and specificity. The results obtained using this method demonstrated a higher concordance rate compared to those derived from commercial kit. Additionally, a total of 451 animals from various provinces in China were sampled, revealing an overall IgG antibody seropositivity against PRRSV of 77.38 % (349/451), with nursery pigs at 33.48 % (151), growing pigs at 15.96 % (72), fattening pigs at 39.69 % (179) and sows at 10.86 % (49). Collectively, the established b-ELISA represents an optimal method for large-scale serological investigations into PRRSV antibodies within farming operations.
猪繁殖与呼吸综合征病毒（PRRSV）在猪群中具有显著的高流行率。对PRRSV保守表位具有高亲和力的单克隆抗体（mAb）有助于开发针对该病毒的广谱检测方法。本研究鉴定出两种PRRSV特异性单克隆抗体，命名为2B1和2C6，通过间接免疫荧光试验（IFA）和蛋白质印迹分析识别出两个构象依赖性表位。通过免疫沉淀和真核表达研究进一步调查证实，这两种单克隆抗体均特异性靶向PRRSV的核衣壳（N）蛋白。重要的是，这两个表位在PRRSV分离株中具有高度保守性，包括DV、CH-1a、HuN4、NADC30样毒株、NADC34样毒株和VR2332。mAb 2C6被PRRSV-1和PRRSV-2阳性猪的血清有效阻断。因此，开发了一种基于2C6的阻断酶联免疫吸附测定（b-ELISA）来检测抗PRRSV抗体，提高了灵敏度和特异性。使用该方法获得的结果与商业试剂盒的结果相比显示出更高的符合率。此外，对来自中国不同省份的451只动物进行了采样，结果显示PRRSV的总体IgG抗体血清阳性率为77.38%（349/451），其中保育猪为33.48%（151只），生长猪为15.96%（72只），育肥猪为39.69%（179只），母猪为10.86%（49只）。总体而言，所建立的b-ELISA是养殖场内大规模血清学检测PRRSV抗体的最佳方法。
