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Screening and characterization of high affinity human anti-Met recombinant antibody scFv
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Abstract:
To prepare human high affinity antibody fragment scFv that specifically binds to cell receptor Met by phage display technique. Methods: The variable regions were amplified from VH and VL of anti-Met Fab genes screened from the natural immune Fab antibody phage display library. The scFv genes were amplified by overlap PCR with different VH and VL library genes assembled by pairs of different CDR mutation primers. scFv DNA was purified and digested with SfiI and was then inserted into pComb3XSS. Positive phage-displayed antibodies with high affinity were selected on live cell lines. Results: After 5 cycles of cell screening and 2 cycles of antigen screening, 60 positive clones were subjected to ELISA. The selected high affinity scFv gene was cloned into pBAD/gIII for expression and was then studied by SDS-PAGE and Western blotting; a band was showed at about 30 000 as expected. To analyze the immunological characters of scFv for Met binding, flow cytometry and immunoprecipitation assays were carried out with S114, MKN45 and NIH3T3 cell lines. The results of flow cytometry and immunoprecipitation demonstrated scFv could bind to natural Met specifically on the surface of S114 and MKN45 cells (Kd=4.763×10 -8 mol/L), and the affinity was about 100 times higher than before mutation (Kd= 5.24×10 -6 mol/L). The results of ELISA showed that there was no change in the binding site of antigen after maturation of the affinity. Conclusion: It is indicated that anti-Met scFv antibody fragment can recognize Met extracellular domain in natural conformation with relatively high affinity, and it may be a potential candidate for clinical diagnosis and therapy.
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