Production and characterization of monoclonal antibodies against the extracellular domain of CA 125.
Carcinoma antigen 125 (CA 125) is overexpressed in ovarian cancer and antibodies against it are widely employed for diagnostic purposes. The rarity of CA 125 antigenic domains and its highly glycosylated structure, however, is a problem that may prevent immunized mice from developing a diversified population of anti-CA 125 antibodies. In this study a prime-boost strategy, which potentially could augment the humoral immune responses against rare and poorly immunogenic determinants, was used for immunization of mice and monoclonal antibodies (mAbs) were produced by hybridoma technology. Reactivity of mAbs was then assessed by ELISA, western blotting, immunoprecipitation, immunohistochemistry and immunofluorescence staining of OVCAR-3 cell line. Altogether, 10 clones were produced, 3 of which had IgG isotype and the rest were IgM. Two-third of clones recognized cognate antigen in fixed and living cells and had strong immunoreactivity in IHC staining. In Western blotting, our antibodies recognized CA 125 as high molecular weight antigen mostly migrated in the 3% stacking gel. Immunoprecipitation of OVCAR-3 cell lysate by mAbs resulted in a very similar migration pattern that reconfirmed their specificities. The mAbs produced in this study are invaluable tools in diagnosis and research fields for assessment of CA 125 expression in cancerous ovarian tissues.
Department of Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Sorour
S
Shojaeian
Abdolamir
A
Allameh
Amir Hassan
AH
Zarnani
Mahmood
M
Chamankhah
Roya
R
Ghods
Ali Ahmad
AA
Bayat
Mahmood
M
Jeddi-Tehrani
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D015153:Blotting, Western; D018394:CA-125 Antigen / Q000737:chemistry* / Q000276:immunology; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007114:Immunization / Q000379:methods*; D007132:Immunoglobulin Isotypes / Q000276:immunology; D047468:Immunoprecipitation; D051379:Mice; D008815:Mice, Inbred Strains; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000276:immunology
D000911:Antibodies, Monoclonal; D018394:CA-125 Antigen; D007132:Immunoglobulin Isotypes