Development and characterization of monoclonal antibodies against a mucin-type glycoprotein.
The preparation of greater than 30 different hybridomas, all secreting IgM class antibodies against epiglycanin, a glycoprotein at the surface of the mouse mammary carcinoma cell line TA3-Ha, is described. The specificities of 10 of the antibodies, with affinity constants in the range of 10(8)-10(10) l/mol were compared in an enzyme competitive binding assay. The affinity of epiglycanin was strongly reduced for all antibodies tested by incubation with periodate (10 mM, 4 degrees C) and was reduced for most of the antibodies by endo-alpha-N-acetyl- D-galactosaminidase. This suggested that carbohydrate, and specifically the Gal beta (1----3)GalNAc disaccharide, formed an integral part of the epitopes of most of the antibodies. The isolated disaccharide, however, exhibited 250,000 times less inhibitory activity in the competitive binding assay than epiglycanin. The binding capacity of epiglycanin was also reduced by incubation with trypsin or pronase, suggesting a high molecular weight dependency for binding. Incubation with sialidase increased its affinity for the antibodies. The binding of the antibodies to epiglycanin was strongly inhibited by peanut agglutinin, and to a lesser extent by lectins from Triticum vulgaris, Ricinus communis, Pisum sativum and Phaseolus vulgaris. None of the antibodies bound to any of eight different gangliosides immobilized on HPTLC plates. Mono- (Fab) and divalent [F(ab')2] fragments of the antibodies possessed very low affinity for epiglycanin. The results demonstrated that the specificities of the antibodies are related, but distinguishable, and they suggest that this epiglycanin-IgM model may be useful for studies on the general principles of the interaction between IgM antibodies and mucin-type glycoproteins.
Haavik
S
S
Department of Pharmacognosy, Institute of Pharmacy, Oslo, Norway.
Codington
J F
JF
Davison
P F
PF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000918:Antibody Specificity; D001667:Binding, Competitive; D002240:Carbohydrate Sequence; D003429:Cross Reactions; D006825:Hybridomas / Q000276:immunology; D007075:Immunoglobulin M; D008325:Mammary Neoplasms, Experimental / Q000737:chemistry / Q000276:immunology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C016424:epiglycanin protein, mouse