title: Heterologous immunization, a way out of the problem of monoclonal antibody production against carcinoma antigen 125.
abstract: BACKGROUND
  Monoclonal antibodies (mAbs) are essential tools for many molecular immunology investigations, epitope mapping and molecular modelling, clinical laboratory diagnostic 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 immunization in the production of anti-CA 125 mAb.
  
  
  METHODS
  Two different protocols of immunization were used for priming of NMRI mice. In the first method, mice conventionally immunized 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 intravenous injection of the purified extracellular domain of CA 125. Production of mAb was performed by standard hybridoma technology and mAbs were characterized by different immunoassays.
  
  
  RESULTS
  The first method failed to produce stable clones despite six time fusion. 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.
  
  
  CONCLUSIONS
  Altogether the results of the present study clearly showed that heterologous antigen preparation is the method of choice for immunization when production of monoclonal antibody against highly glycosylated poorly immunogenic antigens is concerned.
authors.affiliation: Department of Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Sorour
authors.initials: S
authors.lastname: Shojaeian
authors.forename: Amir Hassan
authors.initials: AH
authors.lastname: Zarnani
authors.forename: Mahmood
authors.initials: M
authors.lastname: Jeddi-Tehrani
authors.forename: Forouzandeh
authors.initials: F
authors.lastname: Fereidooni
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Torkabadi
authors.forename: Mahammad Mehdi
authors.initials: MM
authors.lastname: Akhondi
authors.forename: Akram Sadat
authors.initials: AS
authors.lastname: Tabatabaei-Panah
authors.forename: Abdolamir
authors.initials: A
authors.lastname: Allameh
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D018394:CA-125 Antigen / Q000008:administration & dosage / Q000276:immunology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007114:Immunization / Q000379:methods*; D007274:Injections, Intraperitoneal; D007275:Injections, Intravenous; D051379:Mice; D008815:Mice, Inbred Strains; D009368:Neoplasm Transplantation / Q000379:methods; D013997:Time Factors; D014183:Transplantation, Heterologous
chemical_list: D000911:Antibodies, Monoclonal; D018394:CA-125 Antigen