Production of Monoclonal Antibody Against Alkaline Phosphatase (Pages: 129-136)

MM. Eftekharian *, SM. Moazzeni * , AA. Pourfathollah ,


Introduction: Production of Monoclonal antibody against Alkaline phosphatase for application in immunohistochemical and immunocytochemical techniques such as alkaline phosphatase anti-alkaline phosphatase (APAAP) method. Material and Methods: In this investigation female Balb/c mice were immunized by several injections of alkaline phosphatase and the antibody titer in their sera were measured after each injection. The spleen lymphocytes of immunized mice and Sp2/0 myeloma cells were fused using 50% polyethylen glycol as fusing agent and hybridoma cells were selected by HAT medium. Identification and selection of anti-alkaline phosphatase producing clones were done by performing ELISA test on supernatants of all the resulting clones. Limiting dilution was performed twice on antiboby producing clones for their seperation and the resulted subclones were propagated. Since APAAP complex must be enzymatically active for using in immunohistochemical techniques the adhesion of Ab molecule to enzyme molecule must not affect the enzyme activity. For investigation of this effect, an ELISA technique was planned and the supernatants of selected hybridoma clones were studied by this method. For production of concentrated Ab the hybridoma cells were injected to peritoneal cavity of mice and the ascetic fluids were obtained. Finally the antibodies isotypes were determined. Results: After 6 fusion experiments 104 hybridoma clones were abtained and two clones (A1G9 and A1G8) which were antibody producing and had the highest absorbance in ELISA test were selected. Using the limiting dilution method finally two monoclonal subclones A1G8F7 and A1G9G3 were selected. ELISA experiments showed that antibodies which were produced by selected hybridoma clones did not react with active site of the enzyme and did not interfer with enzymatic activity. Electrophoresis of ascetic fluids of hybridoma injected mice showed a prominent band in g position. Isotype determination of monoclonal antibodies showed that both hybridoma clones produce antibody from IgG class with k light chain. Conclusion: Because monoclonal antibodies which are produced by the obtained hybridoma cell lines are from IgG class and do not affect the enzyme activity, it seem's that they are suitable for APAAP complex formation. Other steps of this study are now being performed until APAAP complex formation and it's application in immunohistochemistry.