Purification and characterization of the CA 125 tumor-associated antigen from human ascites.
CA 125 is an antigenic determinant associated with epithelial ovarian carcinomas, which is recognized by a monoclonal antibody, OC 125. The biochemical structure, the immunological characteristics and the physiological function of CA 125 are unknown, principally because the molecule expressing it has not been purified to homogeneity. In the present study, we developed a single, one-step method for purifying CA 125 by column affinity chromatography, using the OC 125 antibody as immobilized ligand. The column proved to be highly specific for the purification of CA 125 from human ascites (HA). The antigen that eluted from the column has a specific activity of 6,240 +/- 120 U of CA 125/mg protein, the specific activity in the initial HA samples being 100 +/- 12 U/mg protein. The purified, immunoreactive CA 125 (IR-CA 125) was shown to be proteinaceous in nature. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration characterization showed that the purified antigen exists as a high molecular weight (MW) complex, of up to 1.5 million daltons, which could be dissociated under strong denaturing conditions, giving rise to moieties with an apparent MW of 205 and 55 kD. IR-CA 125 was also associated with a lower MW protein, with an apparent MW of 10-15 kD. The 205-kD MW protein was immunoreactive CA 125, as measured by immunoradiometric assay after being electroeluted from the polyacrylamide gel. Furthermore, when the affinity-purified antigen was subjected to SDS-PAGE, followed by immunoblotting, the lane which was reactive with the iodinated OC 125 antibody gave rise to a band with a molecular mass of 205 kD. Our results suggest that, on an analytical scale, the affinity column is useful for the purification of CA 125. The purified antigen is being used to investigate the possible role of CA 125 in the growth, development and physiological characteristics of human ovarian carcinomas in in vitro studies.
de los Frailes
M T
MT
Department of Obstetrics and Gynecology, Frauenklinik Universität Erlangen-Nürnberg, (FRG).
Stark
S
S
Jaeger
W
W
Hoerauf
A
A
Wildt
L
L
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000302:isolation & purification*; D001201:Ascites / Q000276:immunology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D010051:Ovarian Neoplasms / Q000276:immunology*; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D000939:Epitopes