Phage display technology has been demonstrated to be a powerful tool in identifying peptide motifs critical for cell adhesion [1]. We aim to use this technology to identify novel synovial determinants using the human synovium/SCID mouse transplantation model.
First, to validate the capacity of the library to identify specific ligands, we tested in vitro its ability to recognize the integrin αvβ3 which displays a specific RGD peptide binding motif [2]. Three rounds of selection were performed on a BSA blocked, αvβ3 (0.5 μg/well) coated microtiter plate. Bound phages were recovered by acid elution and amplified in E coli. As expected, a striking enrichment in the third round of selection was obtained for RGD containing clones. In addition, novel flanking sequences were identified in six of the clones.
Having validated the library, to select in vivo synovial determinants, three rounds of enrichment were performed using 6-week-old SCID mice transplanted with human synovium [3]. Four weeks post-transplantation, 109 TU/ml phages were injected intravenously into the tail vein. The phages were allowed to recirculate for 5 min and animal sacrificed following perfusion through the heart to remove unbound phages. In each round a significant enrichment for synovial tissue was obtained. We are currently engaged in the characterization of these novel determinants.