Construction and screening of nanobody targeting macrophage membrane receptor Vsig4

Objective: To construct V-set and immunoglobulin domain containing 4 (Vsig4) nanobodies (Nbs) as specific macrophage probes so as to use them as molecular probes of macrophagocytes. Methods: A nanobody phage library was generated by using peripheral blood lymphocytes isolated from an alpaca immunized with recombinant Vsig4 protein. After three rounds of selection against recombinant Vsig4. The Nbs were subjected to sequencing and genome alignment to obtain VHH sequence. Nbs were isolated and tested for Vsig4 specificity in an ELISA using recombinant Vsig4. The affinity capacity of Nbs was verified by the cell line stably expressing Vsig4. Results: A nanobody phage library with an estimated 7.27×10⁷ clones with 70% insertion was successfully constructed. Totally 136 Vsig4-positive clones were sequenced and aligned according to different CDR3 sequences. In summary, 15 Vsig4 nanobodies were obtained and grouped into 3 different CDR3 epitopes. The affinity of representing nanobody and Vsig4 was analyzed via ELISA; Nb119 showed the highest affinity against both recombinant and native Vsig4. Conclusion: We successfully constructed and screened Vsig4 specific nanobody number 119 with high affinity and specificity. It can help with macrophage detection and in vivo monitoring.