Screening and epitope characterization of Nidogen-2-specific nanobodies.

NID2 is a key component of the BM and plays an important role in ECM organization and tumor-associated microenvironmental remodeling. In this study, full-length recombinant NID2 was used as an antigen to immunize camels, and a nanobody phage display library was constructed from peripheral blood lymphocytes. Using phage display-based screening, nanobodies recognizing distinct epitopes within the NID2 G1G2 and rod-G3 domains were isolated and characterized. Epitope specificity and competition were further analyzed by BLI, allowing the identification of nonoverlapping nanobody pairs. Based on these results, two high-affinity nanobodies were selected to establish a nanobody-based sandwich ELISA for NID2 detection. This assay enabled the detection of recombinant NID2 at concentrations down to 10 ng·mL⁻¹ in human serum. Although the analytical sensitivity is lower than that of some commercial antibody-based ELISA kits, the nanobody-based format offers advantages in terms of recombinant production, scalability, and assay design flexibility. Collectively, this study provides a panel of domain-selective nanobodies against NID2 and establishes a nanobody-based sandwich ELISA as a methodological platform for future investigations of serum NID2 in diagnostic and translational research contexts.