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) to MG7 monoclonal antibody (McAb) 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 MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

Antiidiotypic antibody related to the 84 kD human sperm membrane protein

Wistar rats were inoculated with purified YWK-I antibody. The anti-idiotypie antibodies were isolated from rat sera by successive passage over affinity chromatography columns of YWK-I mAb and normal mouse Igs. Specificity of anti-Id antibody was established by ELISA. The 84 kD protein inhibited the binding of anti-Id to YWK-I mAb, but failed to repress antibody against normal mouse Ig binding to YWK-I mAb. In competitive inhibition assay, 84 kD protein had shown the ability to compete with anti-Id binding to YWK-I mAb in a dose-dependent manner. Crude sperm extract showed a lower competitive ability. No effeet was found with the irrelevant 36 kD sperm protein. The antisera from the Ba1b/e mice immunized with AId contained Ab3 that reacted with 84 kD sperm protein. The binding of anti-Id to YWK-I mAb was inhibited by Ab3 in a dose-dependent fashion and Ab3 was shown to be able to induce human sparm agglutination. These results indicate that anti-Id which may mimio an epitope of the 84 kD protein could be exploited as an antigen to raise antibodies against sperm protein.

PREPARATION OF ANTI-IDIOTYPIC ANTIBODIES SPECIFIC FOR ANTI-HEL AND ANALYSIS OF THEIR FUNCTIONAL MIMICRY

Objective. This study is to investigate the functional mimicry by using antiidiotypic antibodies of enzymes. Methods.Monoclonal antiidiotypic antibodies against antiHEL(hen eggwhite lysozyme, HEL) antibodies were obtained by fusion of Sp2/0 myeloma cells with spleen cells of syngeneic mice immunized with monoclonal antiHEL antibodies against HELs different antigenic epitopes. Then bacteriolysis of the antiidiotypic antibodies were observed. Results.Eight hybridomas strains secreting antiidiotypic antibodies were selected and characterized. It was shown that two of eight antiidiotypic antibodies secreted by two hybridomas(1A 10 C 9 and 2A 11 C 1B 3) could mimic HEL catalytic activity to lyse Micrococcus lysodeikticus and that the catalytic effect of mixed antiidiotypic antibodies of 1A 10 C 9 and 2A 11 C 1B 3 was stronger than that of one of them, but less than HEL. Conclusion. The results demonstrated that the antiidiotypic antibodies that could mimic enzyme activity existed in the idiotype network during antienzymatic immune response.