Accurate serological surveillance is crucial for porcine reproductive and respiratory syndrome (PRRS) control. Current ELISAs primarily target the nucleocapsid (N) protein, while the diagnostic utility of membrane protein antibodies remains poorly understood. Herein, an M protein-based blocking ELISA (M-B-ELISA) was developed using whole PRRSV antigen and an M-specific mAb. Using a cut‑off of percent inhibition (PI) ≥ 40%, the M-B-ELISA specifically recognized PRRSV-2 antibodies without cross-reacting with PRRSV-1 or other swine pathogens, showing analytical sensitivity comparable to a commercial kit. In 331 sera from actively immunized or infected pigs, the M-B-ELISA demonstrated high agreement (>95%) with a commercial kit and an in-house N-B-ELISA. However, among the 304 serum samples collected from suckling piglets, the agreement rate between the M-B-ELISA and the N-based detection method decreased to 74.01%. This decrease was mainly attributed to the presence of 87 samples that were M antibody-positive but N antibody-negative; IFA further confirmed that 84 of these samples were PRRSV-positive. Temporal analysis revealed that active M-specific seroconversion was delayed by ~1 week compared to N-antibodies post-immunization. However, M-antibodies exhibited a higher mean PI and prolonged detectability during the maternal antibody persistence phase (1~6 weeks of age). Consequently, the PRRSV-2 M-B-ELISA provides superior accuracy over traditional N-based assays for detecting maternal antibodies in piglets under two months old, offering a robust tool for PRRSV serosurveillance and eradication programs.

Keywords: Blocking ELISA; M protein antibodies; Maternal antibodies; PRRSV.