Application of chemically desialylated and degalactosylated human glycophorin for induction and characterization of anti-Tn monoclonal antibodies.
Human erythrocyte glycophorin was desialylated by mild acid hydrolysis and degalactosylated by Smith degradation. Two monoclonal antibodies (Tn5 and Tn56) obtained by immunization of mice with this 'artificial' Tn antigen were characterized and compared in some experiments with two antibodies (BRIC111 and LM225) obtained in other laboratories by immunization with Tn erythrocytes. The specific binding of the antibodies to glycophorins desialylated and degalactosylated on the nitrocellulose blot and to asialo-agalactoglycophorin-coated ELISA plates, and reactions with authentic Tn antigen served for identification of their anti-Tn specificity. The antibodies were further characterized in inhibition assay with various glycoproteins. The antibody Tn5 (similar to BRIC111) was shown to be specific for human erythrocyte Tn antigen, whereas Tn56 reacted strongly with different glycoproteins carrying O-linked GalNAc alpha- residues, and was strongly bound to the murine adenocarcinoma cell line Ta3-Ha. The antibodies Tn5, Tn56 and BRIC111 were similarly inhibited by ovine submaxillary mucin (OSM) and asialoOSM, but the antibody LM225 showed a distinct preference in reaction with OSM (sialosyl-Tn antigen). The results show that Tn antigen, obtained by chemical modifications of human glycophorin, enables the preparation and characterization of anti-Tn monoclonal antibodies, without using rare Tn erythrocytes.
Duk
M
M
Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Wrocław, Poland.
Steuden
I
I
Duś
D
D
Radzikowski
C
C
Lisowska
E
E
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology*; D015295:Antigens, Tumor-Associated, Carbohydrate; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004910:Erythrocyte Membrane / Q000276:immunology* / Q000473:pathology; D005434:Flow Cytometry; D005690:Galactose / Q000172:deficiency; D006021:Glycophorins / Q000737:chemistry / Q000276:immunology*; D006386:Hemagglutination Tests; D006801:Humans; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000172:deficiency; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D006021:Glycophorins; D012794:Sialic Acids; C040286:Tn antigen; D019158:N-Acetylneuraminic Acid; D005690:Galactose