Identification, purification, and radioimmunoassay of NB/70K, a human ovarian tumor-associated antigen.

NB/70K, a tumor-associated antigen of human ovarian epithelial tumor Fraction OCA, has been purified and identified as a glycoprotein which is stable in 0.6 M perchloric acid, binds to concanavalin A, and migrates electrophoretically with alpha-like mobility in barbital-buffered agarose at pH 8.6. NB/70K does not appear to contain normal serum, normal ovary, normal lung, or carcinoembryonic antigen-like cross-reacting antigenic determinants as measured by radioimmunoassay. NB/70K has been purified from ovarian antigen Fraction OCA by chromatography on gamma-globulin coupled to Sepharose 4B and by elution from acrylamide gels. NB/70K migrates as a single band with an apparent molecular weight of 70,000 in sodium dodecyl sulfate:acrylamide gel electrophoresis. A rabbit antibody raised against NB/70K was able to precipitate a polypeptide with a molecular weight of 70,000 as visualized by autoradiography of sodium dodecyl sulfate:acrylamide gels. A radioimmunoassay has been developed for measuring NB/70K activity, using Staphylococcus aureus protein A as a precipitating agent.

Clinical correlations of ovarian cancer antigen NB/70K: a preliminary report.

NB/70K is a glycoprotein extracted from human ovarian cancer tissue. It was measurable postoperatively in the plasma of 89% of 127 women with epithelial cancer of the ovary; 60% of these women had levels in excess of 11 kU/mL compared with 5% of control subjects. The level of NB/70K correlated with FIGO stage and amount of residual tumor, but not with pathology subtype or tumor differentiation. Elevated NB/70K plasma levels also were found in patients with benign gynecologic neoplasms and a variety of systemic carcinomas, and modest elevation was observed in association with hepatic and renal decompensation. The highest levels, found preoperatively in women with ovarian cancer, decreased after tumor resection. These preliminary data indicate that the NB/70K assay has high sensitivity in epithelial ovarian cancer, and plasma levels appear to correlate with tumor volume.

The development of a double-antibody radioimmunoassay for detecting ovarian tumor-associated antigen fraction OCA in plasma.

Ovarian tumor-associated antigen isolated from human tumor tissue was shown to have a different mobility from that of carcinoembryonic antigen (CEA) in both acrylamide gel electrophoresis and immunoelectrophoresis in agarose. The ovarian tumor antigen is composed of six species with different electrophoretic mobility in acrylamide gel electrophoresis. Three of these species were detected in Sephadex G-100 ovarian fraction OCA (from the void volume peak) and the other three species of lower apparent molecular weight were detected in fraction OCD (from the second peak). Fractions OCA and OCD did not share common antigenic determinations as determined by immunodiffusion. CEA was shown to share antigenic determinants with both OCA and OCD. A double-antibody radioimmunoassay capable of detecting nanogram quantities of plasma OCA was developed. In a preliminary study of ovarian cancer patients, OCA appeared to be a more sensitive marker for ovarian cancer than CEA. There was vitually no correlation (r2 - 0.1) between OCA and CEA levels in these patients, as determined by radioimmunoassay.

Quantitation of antigens in normal and malignant ovarian tissue.

Evidence is presented that at least one of the antigens of human ovarian cancer tissue which appeared to be tumor-associated in immunodiffusion and immunoelectrophoresis experiments actually represents a quantitative rather than a qualitative difference between normal and malignant tissue. A glucoprotein band (Rf equals 0.01) believed to contain at least one tumor-associated antigen was isolated by disc-gel electrophoresis with 5.6 per cent SDS-acrylamide and was used to immunize rabbits. Immunodiffusion and immunoelectrophoresis experiments with the resulting antiserum indicated that the glycoprotein band contained two antigens, one which was present in normal extracts at a concentration approximately one tenth of that in tumor extracts and another which was detectable only in tumor tissue. The tenfold difference between normal and tumor tissue was confirmed by studies of the appearance and disappearance of the glycoprotein band when acrylamide gel electrophoresis was performed on varying amounts of normal and tumor extracts.

Purification of human ovarian tumor-associated antigen and demonstration of circulating tumor antigen in patients with advanced ovarian malignancy.

Human ovarian tumor-associated antigen (TAA) has been purified from ovarian tumor tissue by affinity chromatography on concanavallin A-Sepharose and three different gamma globulin-Sepharose columns. The resulting ovarian TAA appears to be contaminated by one normal antigen or family of antigens. Rabbit antiserum prepared against this purified ovarian TAA (antiserum 404) was coupled to CNBr-activated Sepharose 4B. This coupled Sepharose was added to fractionated serum from ovarian cancer patients with Stage III and IV malignancy. Bound protein was eluted with 0.2M glycine buffer and tested against antiserum 404. The bound protein contained TAA identical to the TAA isolated from ovarian tumor tissue.