To investigate the role of Reg Iα in human inflammatory bowel disease (IBD), we made two phage-displayed single chain variable fragment (scFv) libraries from rabbits immunized with recombinant or native human Reg Iα. After one to three rounds of panning, we were able to isolate phage-displaying scFvs, which bound to human Reg Iα. Anti-Reg Iα scFvs from both libraries showed similar immunoreactivity to different processed forms of the protein. Despite several DNA fingerprint patterns among these clones, their deduced amino acid sequences are highly homologous with 100% identity in the complementarity-determining regions (CDRs) of the variable segment of heavy chain (VH) region and a small variation in the CDR1 of the variable segment of light chain (VL) region. We also expressed and purified soluble myc-tagged or glutathione S-transferase (GST) fusion scFv proteins from bacteria. Immunohistochemical studies using one of our anti-Reg Iα scFv antibodies showed prominent staining in the metaplastic Paneth cell population and light staining in the lamina propria. This scFv antibody is now being used for studies of the role of Reg Iα in human IBD.
- Phage display library
- Rabbit single chain Fv
- Reg Iα or pancreatic stone protein