[Production of phage-displayed anti-idiotypic antibody single chain variable fragments to MG7 monoclonal antibody directed against gastric carcinoma].
OBJECTIVE
To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti-Id ScFv) MG7 to monoclonal antibody directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.


METHODS
Balb/c mice were immunized i.p. with purified MG7 monoclonal antibody conjugated with keyhole limpet hemocyanin. mRNA was isolated from the spleens of immunized mice. Heavy and light chain genes (VH and VL) of antibody were amplified separately and assembled into ScFv genes with a specially constructed linker DNA by RT-PCR. The ScFv genes 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 displayed ScFv fragments as a fusion with gene 3 protein on the tips of M13 phage. After four rounds of panning with monoclonal antibody MG7, the MG7-positive clones were selected with the enzyme-linked immunosorbent assay (ELISA) from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were primarily identified by competition ELISA.


RESULTS
The VH, VL and ScFv DNAs were about 340, 320 and 750 bp respectively. Twenty-four MG7-positive clones were selected from 40 enriched phage clones. Five of the 24 clones displayed beta or gamma type anti-Id ScFv.


CONCLUSIONS
The anti-Id ScFv frangments to MG7 monoclonal antibody can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma using anti-Id ScFv.
He
F
F
Institute of Digestive Disease, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Nie
Y
Y
Han
Z
Z
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D013274:Stomach Neoplasms / Q000276:immunology*; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019444:Vaccines, DNA; C055966:immunoglobulin Fv