Murine monoclonal antibodies against galactosyltransferase from the ascites of ovarian cancer patients.
Glycoprotein:galactosyltransferase is a promising enzyme marker for ovarian carcinoma. Five stable murine hybridoma monoclones that produce homogeneous antibodies against galactosyltransferase from the ascites of ovarian cancer patients have been established. Three of the monoclonal antibodies produced were immunoglobulin G1 isotype, while two were immunoglobulin M. All the antibodies showed linear Scatchard binding plots and had very high affinity for galactosyltransferase with equilibrium dissociation constants (Kd) ranging between 0.16 X 10(-9) M and 0.97 X 10(-9) M. Two of the monoclonal antibodies recognized adjacent epitopes on the enzyme molecule, two antibodies recognized two other unique epitopes, while the epitope recognized by the fifth was uncertain. Following polyacrylamide gel electrophoresis of the purified enzyme, in the presence of sodium dodecyl sulfate, the separated proteins were transferred to nitrocellulose filters (transblotting) and galactosyltransferase was detected on the filters by immunoperoxidase staining after treatment with monoclonal antibodies. A band at Mr 47,000 was detected by all of the monoclonal antibodies. One of them can detect, in addition, two bands at Mr 52,000 and Mr 54,000. Purified galactosyltransferase catalyzed the transfer of galactose to four types of acceptors: (a) alkali-stable N-glycosidic glycoproteins; (b) alkali-labile mucin-type acceptors; (c) N-acetylglucosamine; and (d) glucose in the presence of alpha-lactalbumin. All these transfer activities of the enzyme were present in the immunoprecipitates of the monoclonal antibodies. Transblotting of the enzyme from nondenaturing slab gels produced diffused stain patterns. Assay of the enzyme using the four types of acceptors in gel slices showed overlapping activity profiles, which coincided with the stained area on the filters, suggesting that the reactions are catalyzed by the same protein.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D005260:Female; D005700:Galactosyltransferases / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010051:Ovarian Neoplasms / Q000201:enzymology*; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D005700:Galactosyltransferases; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase