Screening and preparation of nanobodies for SIGLEC-15 detection.

As an important member of the Siglec family, SIGLEC-15 plays an important role in osteoclast differentiation, bone remodeling, and tumor immune evasion. In the tumor microenvironment, SIGLEC-15 functions independently of the B7-H1/PD-1 pathway. In this study, the SIGLEC-15 fusion protein (SIGLEC-15-Fc) was successfully expressed and purified using a eukaryotic expression system. This protein was then used as an antigen to immunize camelids, inducing the production of specific nanobodies (VHHs) targeting SIGLEC-15. The resulting nanobodies exhibited a molecular weight of approximately 15 kDa. After screening, we identified two nanobody strains, Nb1C8 and Nb2D7, both of which bind SIGLEC-15 without competition. We confirmed the nanobodies' high affinity and stability through Octec platform and stability analyses. Flow cytometry analysis demonstrated that Nb1C8 and Nb2D7 specifically binds to SIGLEC-15 which naturally expressed on bladder cancer cells. This study marks the first development of nanobodies specifically targeting SIGLEC-15, providing a solid foundation for the development of SIGLEC-15-related diagnostic tools and antibody therapeutics.