title: Antigen-binding specificity of anti-αGal reagents determined by solid-phase glycolipid-binding assays. A complete lack of αGal glycolipid reactivity in α1,3GalT-KO pig small intestine.
abstract: BACKGROUND
  αGal-specific lectins, monoclonal and polyclonal antibodies (Abs) are widely used in xenotransplantation research. Immunological assays such as immunohistochemistry, flow cytometry, Western blot and thin layer chromatography are often the only applicable characterization procedures when limited amount of tissue is available and biochemical characterization is impossible. Hence, detailed knowledge of the Ab/lectin carbohydrate-binding specificity is essential.
  
  
  METHODS
  The binding specificity of human blood group AB serum, three different affinity-purified human polyclonal anti-Gal Ab batches, and two anti-Gal mAb clones (TH5 and 15.101) as well as Griffonia simplicifolia isolectin B4 and Marasmius oreades agglutinin were examined for reactivity with glycolipid fractions isolated from human and pig (wild-type and α1,3GalT-KO) tissues using thin layer chromatogram and microtiter well binding assays.
  
  
  RESULTS
  All anti-Gal-specific reagents reacted with the pentaglycosylceramide Galα1,3nLc4, and several 6-12 sugar compounds in wild-type pig kidneys. However, their staining intensity with different αGal antigens varied considerably. Some, but not all, anti-Gal reagents cross-reacted with a pure iGb3 glycolipid reference compound. No reactivity with glycolipids isolated from α1,3GalT-KO pig small intestine or human tissues was found, confirming the specificity of the anti-Gal reagents in those tissues for α1,3Gal-epitopes produced by the α1,3GalT (GGTA1).
  
  
  CONCLUSIONS
  Different anti-Gal reagents vary in their carbohydrate epitope specificity. Mono-/polyclonal Abs and lectins have different carbohydrate epitope fine specificity toward pig glycolipids as well as purified Galα1,3nLc4, and iGb3. Despite the difference in αGal specificity, all reagents were completely non-reactive with glycolipids isolated from α1,3GalT-KO pig small intestine.
authors.affiliation: Department of Surgery, Sahlgrenska Academy at Gothenburg University, Sahlgrenska University Hospital, Gothenburg, Sweden. mette.diswall@gu.se
authors.forename: Mette
authors.initials: M
authors.lastname: Diswall
authors.forename: Anki
authors.initials: A
authors.lastname: Gustafsson
authors.forename: Jan
authors.initials: J
authors.lastname: Holgersson
authors.forename: Mauro S
authors.initials: MS
authors.lastname: Sandrin
authors.forename: Michael E
authors.initials: ME
authors.lastname: Breimer
mesh_terms: D000017:ABO Blood-Group System / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology*; D005690:Galactose / Q000737:chemistry / Q000276:immunology*; D005700:Galactosyltransferases / Q000235:genetics* / Q000276:immunology; D055785:Gene Knockdown Techniques; D006017:Glycolipids / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007421:Intestine, Small / Q000276:immunology*; D011485:Protein Binding; D013552:Swine
chemical_list: D000017:ABO Blood-Group System; D000911:Antibodies, Monoclonal; D000941:Antigens; D006017:Glycolipids; D005700:Galactosyltransferases; D005690:Galactose