Biochemical analysis of human ovarian cancer-associated antigens defined by murine monoclonal antibodies.
Two monoclonal antibodies (MOv1 and MOv2) raised against a membrane preparation of a human surgical specimen from a mucinous ovarian cystoadenocarcinoma were used to biochemically define their target antigens. The heating of peritumoral mucus-soluble extracts and the sialidase treatment of crude membrane preparations did not affect the binding capacity of MOv1 and MOv2, which, on the contrary, was significantly reduced by periodate oxidation of the same materials. Pronase digestion completely solubilized MOv1-defined antigens, whereas MOv2-defined antigens were only partially solubilized. This, however, did not affect antibody binding with digested products. These data suggest that carbohydrate residues of recognized molecules constitute the antigenic determinants and that sialic acid residues are not involved. Gel filtration on Sepharose 4B of the peritumoral mucus, solubilized either by 200 mM NaCl or Pronase, revealed that most of the antigenic activity eluted in the void-volume fractions with a high carbohydrate content and in the included fractions before the elution volume of the ferritin standard protein. When CsCl gradient equilibrium ultracentrifugation of the solubilized mucus was used, MOv1-recognized antigens sedimented with a density of 1.45 g/ml, while the MOv2-defined epitope was carried by molecules with a density of 1.52 g/ml as well as by molecules with a lower density. Using thin-layer chromatography of organic solvent extracts obtained from mucus and crude membrane preparations, only MOv2-positive molecules could be resolved as a single band of glycolipid. Altogether, these data suggest that the antigens detected by MOv1 are mainly mucins whereas the determinant recognized by MOv2 is carried by both mucins and a glycolipid. To analyze the diagnostic potential of MOv1- and MOv2-recognized molecules, we tested their presence, as soluble products, in supernatants of tumor cell lines and in peritoneal effusions from cancer patients. To this aim, we developed an immunoradiometric assay using the same monoclonal antibody in insolubilized and soluble form. Whereas MOv1-immunoradiometric assay was always negative, by MOv2-immunoradiometric assay it was possible to detect the relevant antigen in 8 of the 10 effusions from patients with well-differentiated ovarian tumors and in 5 of the 11 effusions from patients with poorly differentiated ovarian tumors, whereas the 10 control effusions from patients with various diseases were negative.
Miotti
S
S
Aguanno
S
S
Canevari
S
S
Diotti
A
A
Orlandi
R
R
Sonnino
S
S
Colnaghi
M I
MI
D002288:Adenocarcinoma, Mucinous / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000032:analysis*; D002460:Cell Line; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D051379:Mice; D009093:Mucus / Q000032:analysis; D010051:Ovarian Neoplasms / Q000276:immunology*; D012995:Solubility; D014461:Ultracentrifugation
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm