title: A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides.
abstract: BACKGROUND
  Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear.
  
  
  METHODS
  A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies.
  
  
  RESULTS
  Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats.
  
  
  CONCLUSIONS
  We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption.
  
  
  CONCLUSIONS
  The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.
authors.affiliation: Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, N22, W11 Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Matsushita
authors.affiliation: Sumitomo Bakelite Co., Ltd., Tokyo, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Takada
authors.affiliation: Sumitomo Bakelite Co., Ltd., Tokyo, Japan.
authors.forename: Kota
authors.initials: K
authors.lastname: Igarashi
authors.affiliation: Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12, Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Naruchi
authors.affiliation: Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12, Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Risho
authors.initials: R
authors.lastname: Miyoshi
authors.affiliation: Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, N22, W11 Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Fayna
authors.initials: F
authors.lastname: Garcia-Martin
authors.affiliation: Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, N22, W11 Kita-ku, Sapporo 001-0021, Japan; Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12, Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Maho
authors.initials: M
authors.lastname: Amano
authors.affiliation: Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, N22, W11 Kita-ku, Sapporo 001-0021, Japan; Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12, Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hinou
authors.affiliation: Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, N22, W11 Kita-ku, Sapporo 001-0021, Japan; Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12, Kita-ku, Sapporo 001-0021, Japan. Electronic address: shin@sci.hokudai.ac.jp.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000097:blood; D018604:Epitope Mapping; D006801:Humans; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology*; D019151:Peptide Library; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D018396:Mucin-1; D010446:Peptide Fragments; D019151:Peptide Library
keywords: Autoantibody; Epitope mapping; MUC1 glycopeptide; Microarray; Non-specific protein adsorption