[Improving the affinity of an anti-TNF-alpha scFv by random mutation].
OBJECTIVE
To improve the affinity of an anti-TNF-alpha scFv.


METHODS
A mutant phage antibody library derived from an anti-TNF-alpha scFv gene was generated by error-prone PCR. The mutated genes were then subjected to DNA shuffling. Mutants with improved affinity were selected by bio-panning. Affinity improvement of the selected mutants was verified by dot blot ELISA and thiocyanate elusion ELISA.


RESULTS
One mutant was obtained with relative affinity index (1.37 mol/L) higher than that of the parent scFv (0.48 mol/L).


CONCLUSIONS
Error-prone PCR plus DNA shuffling is effective in improving the affinity of antibodies.
Central Lab., Navy General Hospital, Beijing 100037, China.
Bao-an
BA
Wang
Xiao-sui
XS
Chen
Yu-xiao
YX
Wang
Jia
J
Qu
Li-jun
LJ
Zhou
Yan
Y
Wang
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000915:Antibody Affinity / Q000235:genetics*; D001435:Bacteriophages / Q000276:immunology; D004252:DNA Mutational Analysis; D032621:DNA Shuffling; D004797:Enzyme-Linked Immunosorbent Assay; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D016296:Mutagenesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D014409:Tumor Necrosis Factor-alpha