Related Experiment Videos
[Production of phage-displayed single chain variable fragments of monoclonal antibody MGb1]
1Institute of Digestive Disease, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, China.
Objective:
To lay a foundation for obtaining a tumor-targeting vehicle for in vivo study on diagnosis and treatment of gastric carcinoma by generating single chain variable fragments (ScFv) of monoclonal antibody MGb1 directed against the cancer.
Methods:
mRNA was isolated from MGb1-producing mouse hybridoma cell line, and the variable regions of heavy and light chain cDNAs were amplified separately and assembled into ScFv DNAs with a specially constructed linker DNA by PCR. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage, which display ScFv fragments as a fusion with gene 3 protein on the tips of the phage M13. After two rounds of panning with gastric carcinoma cell line KATO III highly expressing MGb1-binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by enzyme-linked immunosorbent assay (ELISA) from the enriched phages. The affinity of the positive phage clones was detected by competition ELISA.
Results:
The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. 17 phage clones displayed ScFv of MGb1 were selected from 40 enriched phage clones. 4 out of the 17 phage clones could strongly compete with the original hybridoma antibody MGb1 for binding to the antigen expressed on KATOIII cells.
Conclusion:
The phage-displayed ScFv fragments of monoclonal antibody MGb1 are successfully produced by phage antibody technology, which may be useful to widen the range of application of the antibody.