Enhancing the sensitivity of nanobodies through covalent and non-covalent polymerization.

The precise identification of disease using antibodies is crucial for guiding therapeutic interventions. Among these binding proteins, the nanobody is the smallest known antigen-binding fragment. This study aimed to enhance antigen detection sensitivity by integrating covalent dimerization and non-covalent heptamerization strategies to increase the density of nanobodies for improved antigen capture. First, we synthesized a SpyTag-Catcher-fused nanobody and a heptamerization protein using a prokaryotic expression system. Western blot was used to preliminarily verify the nanobody's antigen-binding capability. Surface Plasmon Resonance (SPR) analysis revealed that the enhanced immunoactivity of the nanobody polymerization resulted from their increased affinity. Finally, we employed these nanobodies to improve the sensitivity of sandwich immunoassays, specifically immuno-magnetic beads and ELISA, for detecting antigens in solution. Our findings demonstrate that the 14-mer nanobody, formed by covalent dimers and non-covalently self-assembled heptamers, enhanced antigen capture capability by more than tenfold.