Effect of VL and VH consensus sequence-specific primers on the binding and expression of a mini-molecule antibody directed towards human gastric cancer.
OBJECTIVE
To construct ScFv and Fab from murine anti-gastric cancer monoclonal antibody (mAb)3H11.


METHODS
At first, 3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1. The bacterial expressed 3H11 Ab fragments showed no antigen binding activity. Then, phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was perfomed. Again the identification of positive clone was failed. Finally the N-terminal sequences of V regions were resumed to 3H11 original sequences by site-directed mutagenesis via PCR.


RESULTS
Binding activity to gastric cancer cells was detected only from N-terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected. Correction of either VL or VH N-terminal sequences could partially resume the antigen binding activity.


CONCLUSIONS
Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.
Li
J
J
Navy General Hospital, Beijing 100037.
Wang
Y
Y
Wang
Z
Z
Dong
Z
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000912:Antibodies, Neoplasm / Q000235:genetics; D001665:Binding Sites; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D013274:Stomach Neoplasms / Q000276:immunology* / Q000473:pathology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region