Isolation of two distinct populations of recombinant antibody molecules specific for rat malonyl-CoA decarboxylase from a semi-synthetic human scFv display library using Ex-phage system.
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 x 10(8) 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 VLkappa subgroup I, and M23 and M28 used VLkappa 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.
Baek
Hyun-jung
HJ
IG Therapy Co., Room 112, Nong 3rd Building, Kangwon National University, Chunchon 200-701, South Korea.
Hur
Byung-woong
BW
Cho
Jin-won
JW
Lee
Hee-kyung
HK
Kim
Nam-il
NI
Oh
Mi-young
MY
Cha
Sang-hoon
SH
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002262:Carboxy-Lyases / Q000276:immunology*; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D051381:Rats; D016415:Sequence Alignment; D012995:Solubility
D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase