A Novel Monoclonal Antibody Targeting the A29 Protein of Monkeypox Virus and Its Application in Immunoassay

Background: The monkeypox virus (MPXV) has attracted considerable global attention due to its potential to cause widespread outbreaks, necessitating the development of rapid and accurate diagnostic methods of significant clinical importance. A29, a key envelope protein of MPXV, represents a promising diagnostic target. Methods: A novel monoclonal antibody, D10, was isolated from the human Tomlinson I+J phage display library by biopanning against the recombinant A29 protein. The D10 Fab fragment was expressed and purified, and its binding affinity was characterized by biolayer interferometry. Molecular docking was performed to predict potential interacting residues. Specificity and detection performance were evaluated by direct and competitive enzyme-linked immunosorbent assay (ELISA). Results: D10 possesses a unique complementarity-determining region sequence and exhibits strong binding affinity toward the A29 protein. Structural modeling analysis suggested potential interacting residues of A29, including Gln67, Arg74, Asn75, Arg81, and Asn84, which may primarily interact with Ser10, Thr5, Gly49, Gly47, and Glu97 in the heavy chain of D10. The binding affinity, determined by biolayer interferometry, showed a dissociation equilibrium constant of 6.44 nM, indicating strong binding capability. Furthermore, competitive ELISA demonstrated that D10 binds selectively to the A29 protein, with a half-maximal inhibitory concentration of 1.88 μg/mL and a limit of detection of 0.12 μg/mL. Conclusions: Overall, this monoclonal antibody provides a valuable tool for the immunological detection of MPXV and holds potential for future clinical diagnostic applications.