• Scientific questions: This study addresses the urgent need for rapid and highly specific diagnostic tools for detecting mpox virus (MPXV) during global outbreaks, with a focus on overcoming the antigenic cross-reactivity among orthopoxviruses that undermines the specificity of existing antigen-based detection methods. • Evidence before this study: Current MPXV diagnostic approaches rely primarily on PCR-based genomic detection, whereas antigen-based assays exhibit poor specificity due to the high sequence homology among orthopoxvirus antigens. Existing monoclonal antibody (mAb) platforms fail to reliably distinguish MPXV from closely related viruses such as cowpox, vaccinia, and variola, thereby limiting their clinical applicability. • New findings: This study successfully generated MPXV-specific monoclonal antibodies against four viral antigens (A29L, A35R, H3L, and E8L), identified non-overlapping antibody pairs (e.g., 18F7–27C3 for A29L and 4E7–6C11 for A35R), and established sandwich ELISA assays exhibiting nanogram-level sensitivity. These assays demonstrated high specificity for MPXV, effectively distinguishing it from other orthopoxviruses, while retaining reactivity in viral culture supernatants. • Significance of the study: The development of these mAb-based immunoassays establishes a robust, rapid, and accessible diagnostic platform for MPXV detection. This advancement possesses strong translational potential for point-of-care testing in resource-limited settings, thereby enhancing global outbreak response and surveillance capacity. 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.
Du et al. (Thu,) studied this question.