A semi-synthetic human scFv phage display library by randomizing amino acid residues at CDR3H was constructed using pIGT3 phagemid vector. Recombinant phages were rescued by super-infecting the JS5 E. coli library stock with Ex-phage, the mutant M13KO7 helper phage containing amber mutations at gIII. The library was composed of 2×108 independent clones, and selected for the specific binders against malonyl-CoA decarboxylase (MCD) by panning. Five soluble scFv clones specific for MCD were finally identified and classified into two groups based on the difference in their binding pattern to MCD. Two clones (M4 and M8) showed good binding reactivity to MCD in ELISA but not in Western blot, whereas, the rest three clones (M23, M28 and M41) reacted to the antigen in Western blot but not in ELISA implying they bound to somewhat different epitopes on MCD. DNA sequencing analysis of M4, M8, M23 and M28 showed that VH of all clones were belonged to VH3 subgroup. On the other hand, M4 and M8 utilized VLκ subgroup I, and M23 and M28 used VLκ subgroup IV, suggesting that difference in binding pattern between M4/M8 and M23/M28 against MCD might come from the different VL gene utilization. In conclusion, human monoclonal scFv antibodies specific for MCD were successfully isolated and we demonstrated that distinct populations of recombinant antibodies specific to the target antigen could be isolated by Ex-phage system.
All Science Journal Classification (ASJC) codes
- Immunology and Allergy