mAb BW835 (IgG1) has been generated to breast cancer cell lines by alternating injections of MCF-7 or SW-613 cells and has been demonstrated to be of value in the serodiagnosis of mammary carcinoma. BW835 defines a carbohydrate epitope on integrated or secreted MUC1 glycoforms from carcinoma cells and human milk. To identify BW835-reactive glycopeptides on MUC1, proteolytic fragments of the mucin obtained by digestion with the Gly-C-specific endopeptidase IV from papaya corresponding to low

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... cular mass fragments (< 10 kilodaltons) of the tandem repeat domain were screened. A glycosylated fragment (glycopeptide 17) containing the mAb HMFG-2-defined epitope was highly reactive to BW835 antibody, while nonglycosylated tandem repeat peptide TAP25 or its in vitro-glycosylated N-acetylgalactosamine (GalNAc) derivatives were unreactive. Glycopeptide 17 bound to peanut agglutinin and to a Thomsen-Friedenreich antigen (TF alpha)-specific mAb (A78-G/A7). Binding of BW835 to glycopeptide 17 or to MUC1 was competitively inhibited by peanut agglutinin and by the synthetic glycopeptides TF alpha Ser or TF alpha Thr but not by their beta-anomers. Evidence for site specificity of binding by BW835 to glycopeptide 17 was revealed by demonstrating nonreactivity of the antibody to other TF alpha-expressing glycoproteins with peptide moieties lacking MUC1-specific motifs at putative glycosylation sites. The epitope of BW835 was localized to threonine within the VTSA-peptide motif by site-specific enzymatic beta-galactosylation of the synthetic tandem repeat peptide TAP25-GalNAc1 TAPPAHGVT(-O-alpha GalNAc)SAPDTRPAPGSTAPPA. This is the first report on a TF alpha-specific mAb that shows a strict peptide sequence dependency of binding.