Development of monoclonal antibody-based methods for detecting mpox virus

The World Health Organization declared mpox a public health emergency of international concern in both 2022 and 2024, highlighting the critical need for rapid and reliable diagnostic solutions. To address this challenge, we developed monoclonal antibodies against four mpox virus (MPXV) antigens (A29L, A35R, H3L, and E8L) using hybridoma technology. Epitope binning analyses, performed using competitive enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry, identified non-overlapping antibody pairs (e.g., 18F7–27C3 for A29L and 4E7–6C11 for A35R), which served as the foundation for sandwich ELISA assays exhibiting nanogram-level sensitivity. These antibody pairs demonstrated high specificity, effectively distinguishing MPXV antigens from homologous proteins of cowpox virus, vaccinia virus, and variola virus, while maintaining reactivity toward cultured MPXV. Collectively, this work establishes a robust immunodiagnostic platform with strong translational potential for point-of-care applications during mpox outbreaks.