Detection of PPMV-1 antibodies in pigeons using a blocking ELISA based on an NP-specific monoclonal antibody

Pigeon paramyxovirus type 1 (PPMV-1) is a prevalent infectious agent causing Newcastle disease in pigeons, yet serological methods for detecting PPMV-1-specific antibodies remain limited. In this study, we developed a blocking enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (mAb) targeting the nucleocapsid (NP) protein of PPMV-1. The B-cell epitopes of the NP protein were computationally predicted, and the core antigenic domain was expressed in Escherichia coli and purified to generate specific mAbs. Following hybridoma technology, a high-affinity mAb (6B8) was produced and used to establish the blocking ELISA. Assay conditions were systematically optimized using checkerboard titration, including antigen concentration, serum dilution, incubation time, blocking buffer, and blocking duration. Analysis of known pigeon serum samples established a cut-off value of 26.87% inhibition (PI%), with an analytical sensitivity of 1:400, representing the highest dilution at which all tested positive sera remained detectable. The assay showed no cross-reactivity with other common avian pathogens, except for limited cross-reaction with Newcastle disease virus La Sota strain. Intra- and inter-batch coefficients of variation were all below 10%. Clinical validation using 86 pigeon serum samples demonstrated a concordance rate of 97.67% (84/86) compared with the hemagglutination inhibition (HI) test. In conclusion, this monoclonal antibody-based blocking ELISA provides a sensitive, specific, and reliable tool for large-scale serological surveillance of PPMV-1 in pigeons.