Preparation of antibody against horseradish peroxidase using hybridoma technology

Monoclonal antibody against horseradish peroxidase (HRP) has many applications which peroxidase anti-peroxidase. Peroxidase-antiperoxidase (PAP) complex formation is its most known and important usage. This complex is used in many immunohistochemical and immunocytochemical staining techniques.

The aim of this study was the preparation of anti-HRP monoclonal antibody through hybridoma technology.

The BALB/c mice were immunized by repeated injections of HRP. After the confirmation of their immunization by ELISA test, the spleen lymphocytes and SP2/0 myeloma cells were hybridized using PEG as fusing agent. The hybridoma cells were then selected by culturing in HAT medium. Identification and selection of anti-HRP producing clones were done by ELISA test on culture supernatants of the obtained clones. To acquire the monoclones, limiting dilution was performed twice and the effect of finally obtained antibodies on enzyme activity was investigated by a specific ELISA test. In vivo tumor induction method was used for production of concentrated antibody. At last class and subclass of the obtained antibodies were determined by Isostrip Kit.

After seven rounds of cell fusions, 224 clones were obtained, from which, six ones were anti-HRP producers. Two clones (P1F11 and P2F6) with higher antibody secretion were selected and subcloned. Both derived hybridoma monoclones (P1F11D2 and P2F6F3) were producing antibodies from IgG1 subclass with kappa (Kappa) light chains which didn't affect the enzyme activity. The electrophoresis of ascetic fluid of tumor induced mice showed an obvious band in gamma (gamma) position.

The obtained monoclonal antibodies are from IgG class and don't affect the enzyme activity, therefore it seems that they are suitable for PAP complex production.