Mingxia Sun, Dan Cao, Man Zhao, Xulong Zhu, Yue Sun, Qian Wang, Hongliang Zhang, Yongbo Yang, Xiaoxiao Tian, Haiwei Wang, Xuehui Cai , Tongqing An 

Int J Biol Macromol. 2025 May 14:144248. doi: 10.1016/j.ijbiomac.2025.144248. Online ahead of print.

Abstract

The pandemic of porcine reproductive and respiratory syndrome (PRRS) continues a global threat to the swine industry and causes huge economic losses. Large-scale serological surveillance, particularly through enzyme-linked immunosorbent assay (ELISA), is an effective measure for PRRS control. Nanobodies have been used in diagnostic immunoassays due to their high affinity and specificity. In this study, two novel nanobodies against N protein of PRRSV were generated that exhibited ability to block the recognition of serum antibodies to N protein. A high-affinity nanobody was conjugated with horseradish peroxidase (HRP), then nanobody-based competitive ELISA (cELISA) was subsequently developed to detect antibodies against both genotypes 1 and 2 PRRSV. Notably, the developed cELISA exhibited enhanced sensitivity, specificity and reproducibility. The comparative analysis demonstrated a high agreement of 96.69 % between the cELISA and a commercial ELISA kit. Collectively, the developed nanobody-based cELISA can be used for epidemiological study of PRRSV infection and immune evaluations with advantages of simple operation and low costing.

Keywords: Competitive ELISA; High specificity; Nanobody; PRRSV.