Isolation and characterization of ovarian cancer antigen CA 125 using a new monoclonal antibody (VK-8): identification as a mucin-type molecule

Abstract

A new murine monoclonal antibody (MAb VK-8), detecting the CA 125 ovarian cancer antigen, was used to purify this antigen from OVCAR-3 ovarian cancer cells by affinity chromatography. The biochemical properties of the purified antigen are characteristic of a mucin-type glycoprotein: (1) the molecule is highly glycosylated (77% w/w), mainly with galactose, N-acetylglucosamine, and N-acetylgalactosamine, (2) the protein moiety is rich in serine, threonine and proline, (3) many of the serine and threonine residues are glycosylated, (4) the glycan chains are almost entirely O-linked, with core 2 [Galb1 = 3(GlcNAcb1 = 6)GalNAc] structures predominating and (5) these chains carry fucosylated Type 2 (Le y and Le x , and H type 2) blood group structures. The antigen exhibited a very high m.w. (G10 3 kDa) in aqueous buffer as well as in urea, but was degraded by proteolytic enzymes to smaller fragments that no longer reacted with the antibody. Although this result, and other immunochemical data, indicate that OC125, the original MAb to CA125, and VK-8 antibodies detect epitopes on the protein portion of the molecule, the involvement of carbohydrate cannot be ruled out. Further insight into the structure and function of the CA125 antigen will come from cloning the gene coding for the peptide backbone, and from more detailed carbohydrate structural analysis. Int.

Molecular Cloning of the CA125 Ovarian Cancer Antigen

“…Anti-CA125 antibody mAb 1 OC125 was a generous gift from Dr. R. Bast, Jr. mAb VK-8, developed in our laboratory by immunization of mice with the human ovarian cancer cell line OVCAR-3, also identifies CA125 but reacts with a different epitope(s) than OC125 (15). Tumor cell lines were from the Sloan-Kettering Institute cell bank.…”

“…Purification of CA125 Antigen-CA125 was purified from the culture supernatant of NIH:OVCAR-3 cells in a simple two step procedure (15). Briefly, the cells were cultured as a monolayer in a synthetic medium (ITS, Life Technologies, Inc.) in RPMI medium containing 1% fetal bovine serum, and the culture medium was harvested every 7 days.…”

“…The VK-8 column was washed at 4°C with phosphate-buffered saline, then with 1 M NaCl in phosphatebuffered saline, and finally eluted with 3 M MgCl 2 . Fractions (6.0 ml) were collected and assayed for CA125 antigen by enzyme-linked immunosorbent assay with mAb VK-8 as described (15). Fractions from the MgCl 2 eluate containing CA125 reactivity were pooled and used in subsequent studies.…”

“…Thus, no consensus emerged from these studies concerning the biochemical nature of this antigen. Recently, however, our studies have strongly indicated that CA125 is a typical mucin molecule with a high carbohydrate content and a preponderance of serineand threonine-linked (O-linked) glycan chains (15,16). Possibly because of the mucinous nature of CA125 its peptide moiety has been very difficult to clone.…”

Progress and Challenges in Screening for Early Detection of Ovarian Cancer

“…Current immunoassays for the quantitation of serum CA125 levels are based on a heterologous assay (CA125 II) using both monoclonal antibodies (M11, OC125) in place of the original homologous assay with monoclonal antibody OC125 alone. Molecular analysis of the CA125 antigen has identified a mucin-type glycoprotein that is highly glycosylated with the protein moiety rich in serine, threonine, and proline (23). A serum CA125 of 35 U/ml, initially measured using the homologous assay and representing 1% of healthy female blood donors, is usually accepted as the upper limit of normal (24).…”