Abstract
Aim:
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).
Conclusion:
Error-prone PCR plus DNA shuffling is effective in improving the affinity of antibodies.
MeSH terms
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Amino Acid Sequence
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Animals
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Antibodies, Monoclonal / analysis
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Antibodies, Monoclonal / chemistry
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Antibodies, Monoclonal / genetics*
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Antibodies, Monoclonal / immunology*
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Antibody Affinity / genetics*
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Bacteriophages / immunology
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DNA Mutational Analysis
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DNA Shuffling
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Enzyme-Linked Immunosorbent Assay
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Immunoglobulin Fragments / genetics
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Immunoglobulin Fragments / immunology
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Immunoglobulin Variable Region / analysis
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Immunoglobulin Variable Region / chemistry
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Immunoglobulin Variable Region / genetics*
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Immunoglobulin Variable Region / immunology*
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Mutagenesis / genetics*
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Polymerase Chain Reaction
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Tumor Necrosis Factor-alpha / immunology*
Substances
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Antibodies, Monoclonal
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Immunoglobulin Fragments
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Immunoglobulin Variable Region
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Tumor Necrosis Factor-alpha