In Vitro Affinity Maturation Of Human BFGF Fab Antibody

Objective: construct mutational human phage antibody library of bFGF by rite-directed mutagenesis and CDR walking and then select the anti-bFGF phage antibody from the library to obtain anti-bFGF antibody of higher affinity.Methods: Eight site in CDR3 of heavy chain with the oligonucleotides synthesized by a reported condon-based mutagenesis procedure, the Fd chain was inserted into the phagemid which have inserted light chain , The recombinant phagemid then was electro-transformed into XL1-Blue cells to construct the mutation library, The recombinant phage was rescued by coculturing with helper phage VCSM13. After three rounds of panning, the higher specific phage antibodies against bFGF were selected.Results: Build a mutational library contained 2.5×10~6 clones Under the super-infection of helper phage VCSM13, After three rounds of "adsorption-elution-amplification", phage antibodies against bFGF have been enriched effectively. 50 phage antibody clones were rescued and 2 Fab phage antibody clones with high affinity and specificity to bFGF were obtained by phage-ELISA.,when measure the affinity constant, one clones' affinity was improved.Conclusion: construct mutational human phage antibody library by rite-directed mutagenesis and CDR walking, and select heigh affinity Fab antibody from them, this provid a feasible way for affinity maturation of Fab antibody.