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.