Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-α-D-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2→8)[Kdo(2→4)]Kdo(2→4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2→8)[Kdo(2→4)]Kdo(2→4) Kdo-BSA, we obtained the mouse monoclonal antibody (mAb) S69-4 which was able to differentiate C. psittaci from Chlamydophila pecorum, C. pneumoniae, and C. trachomatis in double labeling experiments of infected cell monolayers and by enzyme-linked immunosorbent assay (ELISA). The epitope specificity of mAb S69-4 was determined by binding and inhibition assays using bacteria, LPS, and natural or synthetic Kdo oligosaccharides as free ligands or conjugated to BSA. The mAb bound preferentially Kdo(2→8)[Kdo(2→4)]Kdo(2→4)Kdo(2→4) with a K(d) of 10 microM, as determined by surface plasmon resonance (SPR) for the monovalent interaction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2→4)Kdo(2→4)Kdo but not with Kdo(2→8)Kdo(2→4)Kdo, Kdo disaccharides in 2→4- or 2→8-linkage, or Kdo monosaccharide. MAb S69-4 was able to detect LPS on thin-layer chromatography (TLC) plates in amounts of <10 ng by immunostaining. Due to the high sensitivity achieved in this assay, the antibody also detected in vitro products of cloned Kdo transferases of Chlamydia. The antibody can therefore be used in medical and veterinarian diagnostics, general microbiology, analytical biochemistry, and studies of chlamydial LPS biosynthesis. Further contribution to the general understanding of carbohydrate-binding antibodies was obtained by a comparison of the primary structure of mAb S69-4 to that of mAb S45-18 of which the crystal structure in complex with its ligand has been elucidated recently (Nguyen et al., 2003, Nat. Struct. Biol., 10, 1019-1025).
Kdo, diagnostic, neoglycoconjugate, immunofluorescence
NCBI PubMed ID: 16282606Publication DOI: 10.1093/glycob/cwj055Journal NLM ID: 9104124Publisher: IRL Press at Oxford University Press
Correspondence: hebra@fz-borstel.de
Institutions: Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany, Institute for Biological Sci-ences, National Research Council Canada, Ottawa, Ontario, Canada K1A 0R6, Department of Chemistry, University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austri
Methods: serological methods, genetic methods
Found