Development of monoclonal antibodies targeting human bocavirus capsid for pathobiological studies and antigen detection

Human bocavirus type 1 (HBoV1) has emerged as a clinically relevant respiratory pathogen frequently detected in pediatric populations with acute respiratory tract infections worldwide. Although previous studies have developed monoclonal antibodies (mAbs) using virus-like particles (VLPs) as immunogens, immunological tools for detecting diverse capsid antigens in physiologically relevant infection models remain limited. In this study, we employed an alternative immunization strategy using recombinant HBoV1 VP1 capsid protein produced by a wheat germ cell-free protein synthesis system to generate mouse mAbs. From 44 hybridoma clones, we selected three representative mAbs, designated #1, #38 and #44, and characterized them by ELISA, western blotting, and immunofluorescence microscopy. These antibodies detected HBoV1 capsid antigens in fixed sections of HBoV1-infected polarized primary human airway epithelial cells cultured under three-dimensional (3D) air-liquid interface (ALI) conditions. Analysis of truncation and deletion mutants revealed that the mAbs recognized two distinct antigenic regions within the conserved C-terminal domain shared by VP1, VP2, and VP3. Notably, all three mAbs reacted with recombinant VP1 proteins from all four HBoV genotypes, HBoV1 to HBoV4, while showing only weak cross-reactivity with the closely related human parvovirus B19. We further identified optimal antibody pairs for a sandwich ELISA capable of detecting capsid antigen released from heat-treated HBoV1 particles, with a detection limit of 2.5 × 10 7 genome copies/mL. Collectively, these mAbs represent useful research tools for investigating HBoV1 biology and may also support the development of antigen-detection assays for future diagnostic applications.