Purification and Characterization of the CA 125 Tumor-Associated Antigen from Human Ascites

Abstract

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 12 5/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 immunora-diometric 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.

Specificity and Affinity of 26 Monoclonal Antibodies against the CA 125 Antigen: First Report from the ISOBM TD-1 Workshop

“…Papain and endoproteinase lys-c produced peptides tibodies are expressed on the same peptides. We have been unable to confirm the existence of peptides with molecular weight below 200 kDa, binding any of the monoclonal anti bodies as shown by others [18]. Analysis of the Report from the ISOBM TD-I Workshop low molecular weight peptides formed after reduction of SHIN-3 cell supernatant would be a much needed test for the existence of CA 125 epitopes which are not destroyed by re duction [19].…”

“…Elec trophoresis with SDS under nonreducing con ditions reveals a subtraction at 200-250 kDa [10,[13][14][15][16][17], In one study, gel filtration in 0.1% SDS detected most of the immunoreactive CA 125 as being excluded from a BioGel A 1.5 column. However, minor fractions were eluted corresponding to 750, 210, 55 and 15 kDa [ 18]. A molecule of 49 kDa secreted from the ovarian cancer cell line SHIN-3 bound OC 125 [19].…”

Synthesis and Secretion of the Ovarian Cancer Antigen CA 125 by the Human Cancer Cell Line NIH:OVCAR-3

“…In contrast, Hanisch et al [7] described CA 125 antigenicity in a mucus glycoprotein component from human milk with a high molecular weight. Other investigators have also reported very high molecular weights for CA 125 [3,8], whereas some have confirmed the 200-kD size range for the antigen and also detected even smaller species in the 55-to 162-kD range [9,10]. Nagata et al [11], on the other hand, suggested that CA 125 is a membrane-bound glycoprotein complex with a glycophosphatidylinositol anchor.…”

Conflicting Views on the Molecular Structure of the Cancer Antigen CA125/MUC16

“…It could not be determined whether these moieties represent oligomers of a smaller subunit, different glycoforms, or macromolecular complexes of distinct glycoproteins [44]. SDS-PAGE and gel filtration characterization showed that the purified antigen CA125 from human ascites exists as a high molecular weight 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 [45].…”

“…an additional 10431 amino acids have been found. The tandem repeat domain consists of 40-60 repeats, each 156 amino acids long with a disulphide bridge loop of 19 amino acids [42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]59,[59][60][61][62][63]. Although the overall structure is well conserved, only few repeats have identical amino acid sequences.…”