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 phys iological function of CA 125 are unknown, principally because the mole cule expressing it has not been purified to homogeneity. In the presen t study, we developed a single, one-step method for purifying CA 125 b y column affinity chromatography, using the OC 125 antibody as immobil ized ligand. The column proved to be highly specific for the purificat ion of CA 125 from human ascites (HA). The antigen that eluted from th e column has a specific activity of 6,240 +/- 120 U of CA 125/mg prote in, 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 purifi ed 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 ap parent MW of 10-15 kD. The 205-kd MW protein was immunoreactive CA 125 , as measured by immunoradiometric assay after being electroeluted fro m the polyacrylamide gel. Furthermore, when the affinity-purified anti gen was subjected to SDS-PAGE, followed by immunoblotting, the lane wh ich was reactive with the iodinated OC 125 antibody gave rise to a ban d with a molecular mass of 205 kD. Our results suggest that, on an ana lytical scale, the affinity column is useful for the purification of C A 125. The purified antigen is being used to investigate the possible role of CA 125 in the growth, development and physiological characteri stics of human ovarian carcinomas in in vitro studies.