Functional evaluation of biotinylated Fab fragments after photochemical re-bridging with monobromomaleimide

Re-bridging interchain disulfide bonds in antibody Fab fragments is essential for site-selective functionalization while preserving structural integrity. Here, we report a light-triggered [2 + 2] photocycloaddition strategy employing a biotinylated monobromomaleimide to covalently re-link reduced Fab fragments under mild conditions. While this photochemical reaction has been previously demonstrated in model systems, its impact on protein functionality remained untested. In this study, we applied the reaction to both polyclonal and monoclonal Fab fragments and verified successful re-bridging by non-reducing SDS-PAGE. Importantly, the conjugated Fabs retained their biotin-binding capability after UV irradiation, as confirmed by streptavidin-specific Western blotting. Antigen-binding activity was further validated by ELISA and SPR measurements. To our knowledge, this study is the first to demonstrate that photochemical disulfide re-bridging using monobromomaleimide derivatives can yield structurally and functionally competent Fab conjugates. This establishes a versatile platform for diagnostic and affinity-based applications.