Selection and identification of human monoantibody TRAb Fab fragment / 天津医药

ABSTRACT

Objective To select and express a human thyrotrophin receptor antibody (TRAb) Fab fragment from phage antibody library constructed with phage display technology. Methods With immobilized antigen, the reconstructed humanized TRAb Fab library was enriched by five rounds panning (adsorption-elution-amplification). The TSAb Fab and TBAb Fab fragment were selected by coated fusion proteins of hTSHRn and hTSHRc. The positive clones were identified and selected by Phage-ELISA. TRAb positive clones were identified by PCR and double restriction enzyme digestion. The soluble TRAb (TSAb, TBAb) Fab fragments were expressed. TRAb (TSAb, TBAb) Fab fragments were identified by Western blotting assay. The DNA fragment was sequenced from the positive clones. Results Following five rounds of biopanning, TRAb (TSAb,TBAb) Fab phage antibody was screened. The enrichment effect reached to 77 times and 94 times. The soluble TRAb (TSAb,TBAb) Fab antibodies were expressed from positive clones and identified by phage ELISA. Western blotting analysis showed that the phage displaying Fab had significant binding activity with antigens. These sequence analysis showed that all of the heavy chain Fd gene and light chain gene were derived from human immunoglobulin variable region. The light chain variable region of the monoclonal 48 was homologous to the immunoglobulin light chain Vλ homology of 94.4%, and the heavy chain variable region of the monoclonal 48 was homologous to the immunoglobulin heavy Fd chain VH4 homology of 88.9%. The light chain variable region of the monoclonal 56 was homologous to the immunoglobulin light chain Vλ homology of 95.6%, and the heavy chain variable region of the monoclonal 56 was homologous to the immunoglobulin heavy Fd chain VH3 homology of 84.6%. Conclusion We have successfully selected TRAb (TSAb, TBAb) Fab fragment from a human phage display immune antibody library.