| With the development of tumor immunologic technique,immuneologic function which was due to bifunctional antibody(also called bifunctional antibody) has become the major research aspect of anti-tumor.Bispecific antibody(Anti-bladder cancer/anti-CD3) can identify both CD3 molecule in subset of T lymphocyte and antigen on cell membrane of bladder transitional cell cancer(abbreviation:bladder cancer).It can stimulate T lymphocyte and increase its cytotoxicity.At the same time,it can direct the stimulated T lymphocyte to bladder cancer cells and produce cytotoxicity.In our study,production,and lethal effect in vitro,and synergistic action with IL-2 was done. Samples of bladder cancer patients in out department was collected and washed.Vessels and other things were removed and cut into pieces. Homogenate suspension was made by tripsis.50 ml liquid of the upper was purified by DEAE-Sephadex-50 gel.The density of the purified antigen was 0.35 mg/L. Mice were immuned by the antigen with intraperitoneal injection once a week for 5 times. Splenocyte and myeloma cell of mice were infused together under 50% PEG and hybridoma cells formed.Hybridoma cells were cultured with HAT and HT nutrient medium.Hybridoma cells which were positive to bladder cancer antigen and negative to normal bladder membrane were separated by indirect ELISA.Then hybridoma cells were cloned by limiting dilution assay and inoculated to mice.When abdomen of mice became larger, abdominal dropsy was gathered,in which there was F(ab)1 of monoclonal antibody of anti-human bladder cancer.Antibody subtype was identified by gel double diffusion method and its specificity was examined by indirect ELISA. Abdominal dropsy with monoclonal antibody of anti-human bladder cancer was purified by gel column. Pepsin was added at 37 ℃for 16 hours.Then PH was made to 8.0 by adding NAHCO3 and abdominal dropsy was dialyzed by PBS at 4 ℃for 24 hours.The fragment was gathered after laminar analysis and concentrated.After isolation and depuration,we got the purified F(ab`)2 fragment of monoclonal antibody of anti-human bladder cancer antibody.It could be concentrated by polyethylene glycol.With the same method,we could get F(ab`)2 fragment of anti-CD3 antibody. Dithiothreitol was added and F(ab`) fragment of monoclonal antibody of anti-human bladder cancer was purified by Sephadex G-25 gel column. With the same method,we could get F(ab`) fragment of anti-CD3 antibody. DTNB was added to F(ab`) fragment of anti-CD3 antibody at 37 ℃for 30 minutes.The F(ab`)-NB fragment was gathered by chromatographic fractionation.Then it was mixed with F(ab`) fragment of monoclonal antibody of anti-human bladder cancer at 37 ℃for 4 hours.Finally F(ab`)2 fragment of bispecific antibody(anti-bladder cancer/anti-CD3) was got by chromatographic fractionation. Monocyte was separated from normal people and patients with bladder cancer and sent to culture medium with IL-2.Then LAK cells was got by culture and induction.Target cells was BIU-87 cells of bladder cancer,K562 cells and Raji cells. LAK cells were added to liquid of target cells with different ratio.After centrifuge and Giemsa stain,the combination rate was calculated under high power lens. The combination rate(%)=LAK cells which were binded to BIU-87 cells/200 LAK cells×100%. Bispecific antibody was added to LAK cells at 37 ℃for enougn time and then target cells were added.After enough function, parenzyme was used. Scintillation fluid was used and cpm value was got by specifical machine.The blank was RPMI 1640 and target cells. The activity of cytotoxicity(%)=(1-cpm of experiment group/cpm of blank group) ×100%. The molecular weight by SDS-PAGE was 140×103-150×103 of monoclonal...
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