Heterologous Immunization, a Way out of the Problem of Monoclonal Antibody Production against Carcinoma Antigen 125

Background: Monoclonal antibodies (mAbs) are essential tools for many molecular im-munology investigations, epitope mapping and molecular modelling, clinical laboratory di-agnostic tests and immunotherapy. Humoral immune response of immunized animals largely depends on the nature of antigen and the immunization technique. Polysaccharides and heavily-glycosylated proteins are very elusive targets incapable of mounting long-lasting, high affinity antibody responses. Carcinoma antigen 125 (CA 125), a well known tumor marker of ovarian cancer, is a mucin type antigen consisting of repetitive units of heavily glycosylated moieties which render production of mAbs very difficult. Objective: To evaluate the efficacy of heterologous antigen preparations as a way of mouse immuniza-tion in the production of anti-CA 125 mAb. Methods: Two different protocols of immuni-zation were used for priming of NMRI mice. In the first method, mice conventionally im-munized by three intraperitoneal injections of purified CA 125 and boosted by the antigen three days before fusion. In the second approach, mice were primed by three intraperitoneal injections of living CA 125 positive cells of OVCAR-3 cell line, and boosted by intrave-nous injection of the purified extracellular domain of CA 125. Production of mAb was per-formed by standard hybridoma technology and mAbs were characterized by different im-munoassays. Results: The first method failed to produce stable clones despite six time fu-sion. A total of ten stable clones, however, were produced in the second approach. Some of the clones were characterized and found to have excellent immunoreactivity when tested by ELISA assay, western blotting, intracellular and surface immunofluorescent staining of OVCAR-3 cell line and immunohistochemical staining of ovarian cancer tissues. Conclusion: Altogether the results of the present study clearly showed that heterologous antigen preparation is the method of choice for immunization when production of mono-clonal antibody against highly glycosylated poorly immunogenic antigens is concerned.