title: Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci.
abstract: Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.4- and 2.4/2.4-linked, and a branched 2.4[2.8]2.4-linked Kdo tetrasaccharide occur in Chlamydiaceae. While the 2.8/2.4-linked trisaccharide contains a family-specific epitope, the branched Kdo oligosaccharide occurs only in Chlamydophila psittaci and antibodies against it will be useful in human and veterinarian diagnostics. To overcome the generation of cross-reactive antibodies that bind with high affinity to a dominant epitope formed by 2.4/2.4-linked Kdo, we immunized mice with a synthetic 2.4[2.8]-linked branched Kdo trisaccharide and used phage display of scFv to isolate recombinant antibody fragments (NH2240-31 and SAG506-01) that recognize the branched Kdo oligosaccharide with a K(D) of less than 10 nM. Importantly, although these antibodies used germline genes coding for an inherited Kdo recognition site, they were able clearly to distinguish between 2.4[2.8]2.4- and 2.4/2.4-linked Kdo. Sequence determination, binding data, and X-ray structural analysis revealed the basis for the improved discrimination between similar Kdo ligands and indicated that the alteration of a stacking interaction from a phenylalanine residue in the center of the combining site to a tyrosine residue facing away from the center favors recognition of branched 2.4[2.8]2.4-linked Kdo residues. Immunofluorescence tests of infected cell monolayers using this antibody show specific staining of C. psittaci elementary bodies that allow it to be distinguished from other pathogenic chlamydiae.
authors.affiliation: Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Gerstenbruch
authors.forename: Cory L
authors.initials: CL
authors.lastname: Brooks
authors.forename: Paul
authors.initials: P
authors.lastname: Kosma
authors.forename: Lore
authors.initials: L
authors.lastname: Brade
authors.forename: C Roger
authors.initials: CR
authors.lastname: Mackenzie
authors.forename: Stephen V
authors.initials: SV
authors.lastname: Evans
authors.forename: Helmut
authors.initials: H
authors.lastname: Brade
authors.forename: Sven
authors.initials: S
authors.lastname: Müller-Loennies
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology; D000888:Antibodies, Anti-Idiotypic / Q000235:genetics / Q000276:immunology*; D002241:Carbohydrates / Q000235:genetics / Q000276:immunology*; D002693:Chlamydiaceae / Q000235:genetics / Q000276:immunology; D002691:Chlamydophila psittaci / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D008070:Lipopolysaccharides / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D009844:Oligosaccharides / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D014312:Trisaccharides / Q000235:genetics / Q000276:immunology; D014965:X-Rays
chemical_list: D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D002241:Carbohydrates; D000939:Epitopes; D008070:Lipopolysaccharides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014312:Trisaccharides