DEVELOPMENT AND CHARACTERIZATION OF AN ANTIBODY-DIRECTED TO AN ALPHA-N-ACETYL-D-GALACTOSAMINE GLYCOSYLATED MUC2 PEPTIDE

In an attempt to raise anti-Tn antibodies, an alpha-N-acetyl-D-galacto samine glycosylated peptide based on the tandem repeat of the intestin al mucin MUC2 was used as an immunogen. The MUC2 peptide (PTTTPISTTTMV TPTPTPTC) was glycosylated in vitro using concentrated alpha-N-acetylg alactosaminyltransferases activity from porcine submaxillary glands wh ich resulted in the incorporation of 8-9 mol of Ga/NAc. Rabbits and mi ce developed specific anti-MUC2-GalNAc glycopeptide antibodies and no detectable anti-Tn antibodies. Anti-glycopeptide antibodies did not sh ow reactivity with the unglycosylated MUC2 peptide or with other GalNA c glycosylated peptides. A mouse monoclonal antibody (PMH1) representa tive of the observed immune response was generated and its immunohisto logical reactivity analysed in normal tissues. PMH1 reacted similarly to other anti-MUC2 peptide antibodies. However, in some cells the stai ning was not restricted to the supranuclear area but extended to the e ntire cytoplasm. In addition, PMH1 reacted with purified colonic mucin by Western blot analysis suggesting that PMH1 reacted with some glyco forms of MUC2. The present work presents a useful approach for develop ment of anti-mucin antibodies directed to different glycoforms of indi vidual mucins.