Production, Characterization, and Diagnostic Applications of Monoclonal Antibodies Against the N Protein of Avian Metapneumovirus Subgroup B

Recently the avian metapneumovirus subgroup B (aMPV-B) was declared an emerging pathogen in U.S. poultry, resulting in substantial economic losses. This highlights the urgent need for accurate disease diagnosis and development of specific diagnostic tests. To address this challenge, we developed monoclonal antibodies (mAbs) against the highly conserved nucleoprotein (N protein). After successful production and purification of recombinant N protein, four successive intraperitoneal doses were administered to SPF BALB/c mice at 2-wk intervals. Two weeks after the last immunization, the splenocytes were fused to NS-1 myeloma cells to generate hybridomas. Out of dozens of reactive clones, four mAbs were selected using an indirect immunofluorescent assay (iIFA) against aMPV-B-infected Vero E6 cells, all of which belonged to the IgG1 subclass. Further characterization was conducted using Western blot (linear epitopes) and immunohistochemistry (IHC; linear and conformational epitopes), reinforcing their suitability for diagnostic applications. This showed the effective binding ability of the developed mAbs to the viral N protein. Interestingly, two mAbs showed cross-reactivity with subgroup A, while none were reactive with subgroup C by iIFA in infected Vero E6 cells. In contrast, the polyclonal antibodies against the N protein of aMPV-B recognized the three subgroups (aMPV-A, B, and C), highlighting the conserved antigenic regions across them. These mAbs have been successfully used to develop the aMPV-B-specific iIFA for precise, sensitive, and time-efficient detection of the viral antigen in tissue cultures, instead of relying on visualization of the cytopathic effect. Additionally, they were successfully used to develop an aMPV-B-specific IHC, detecting viral antigens in the respiratory tracts of infected cases. In conclusion, the newly developed assay will enhance the specific diagnosis of aMPV-B.