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Development and characterization of an antibody directed to an α-N-acetyl-D-galactosamine glycosylated MUC2 peptide

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Abstract

In an attempt to raise anti-Tn antibodies, an α-N-acetyl-D-galactosamine glycosylated peptide based on the tandem repeat of the intestinal mucin MUC2 was used as an immunogen. The MUC2 peptide (PTTTPISTTTMVTPTPTPTC) was glycosylated in vitro using concentrated α-N-acetylgalactosaminyltransferases activity from porcine submaxillary glands which resulted in the incorporation of 8–9 mol of Ga/NAc. Rabbits and mice developed specific anti-MUC2-GalNAc glycopeptide antibodies and no detectable anti-Tn antibodies. Anti-glycopeptide antibodies did not show reactivity with the unglycosylated MUC2 peptide or with other GalNAc glycosylated peptides. A mouse monoclonal antibody (PMH1) representative of the observed immune response was generated and its immunohistological reactivity analysed in normal tissues. PMH1 reacted similarly to other anti-MUC2 peptide antibodies. However, in some cells the staining was not restricted to the supranuclear area but extended to the entire cytoplasm. In addition, PMH1 reacted with purified colonic mucin by Western blot analysis suggesting that PMH1 reacted with some glycoforms of MUC2. The present work presents a useful approach for development of anti-mucin antibodies directed to different glycoforms of individual mucins.

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Authors and Affiliations

  1. Institute of Molecular Pathology and, Immunology of the University of Porto–IPATIMUP, Rua, 4200-, Porto, Portugal

    Celso A. Reis (Dr R. Frias s/n), Tina Sørensen & Leonor David

  2. School of Dentistry, Nørre Alle 20, DK 2200-, Copenhagen, Denmark

    Ulla Mandel, Ekaterina Mirgorodskaya & Peter Roepstorff

  3. Organic Chemistry, Umea University, S-90187, Umea, Sweden

    Jan Kihlberg

  4. Laboratory for Infectious Diseases, Hvidovre Hospital, 2650, Hvidovre, Denmark

    John-Erik Stig Hansen

  5. Faculty of Health Sciences, School of Dentistry, Nørre Alle 20, DK 2200-, Copenhagen, Denmark

    Henrik Clausen

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  1. Celso A. Reis
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  2. Tina Sørensen
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Reis, C.A., Sørensen, T., Mandel, U. et al. Development and characterization of an antibody directed to an α-N-acetyl-D-galactosamine glycosylated MUC2 peptide. Glycoconj J 15, 51–62 (1998). https://doi.org/10.1023/A:1006939432665

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