توليد آنتي بادي مونوكلونال عليه پراكسيداز

The Production of Monoclonal Antibody against Peroxidase

Introduction: Monoclonal antibody has many applications against peroxidase among which peroxidase-anti peroxidase (PAP) complex formation is of great importance. This complex is used in many immunohistochemical and immunocytochemical staining techniques. The aim of this study was the production of a mouse antiperoxidase of hybridoma cell line to be used in peroxidase - antiperoxidase immunostaining. Materials & Methods: The Balb/c mice were first immunized by repeated injections of horseradish peroxidase (HRP). After the confirmation of their immunization by ELISA, the spleen lymphocytes and SP210 myeloma cells were hybridized by using PEG. The hybridoma cells were then selected through the selective HAT medium. From seven rounds of cell fusion, 224 hybridoma clones were obtained, from which six clones produced monoclonal antibody against HRP. Once again from these six clones, two clones were selected and developed. Limiting dilution was performed twice and monospecific clones were developed. Results: Using the Isostrip kit revealed that both monoclonal anti peroxidase antibodies (P1 F11 D2, P2F6F3) were from IgG1 isotype class. In addition, ELISA test showed that the monoclonal antibodies donʹt affect the active site of the enzyme. Therefore it seems that the produced antibodies are suitable for formation of PAP immune complex. Conclusion: At present, this study is being carried out farther to produce PAP complex and investigate its practical usage in immunohistochemical and immunocyto chemical techniques.