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.