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Glycobiology Advance Access published online on November 10, 2005
Glycobiology, doi:10.1093/glycob/cwj055
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org
Received August 9, 2005
Revised November 2, 2005
Accepted November 3, 2005

Article

A Monoclonal Antibody against a Carbohydrate Epitope in Lipopolysaccharide Differentiates Chlamydophila psittaci from Chlamydophila pecorum, Chlamydophila pneumoniae and Chlamydia trachomatis

Sven Müller-Loennies 1, Sabine Gronow 1, Lore Brade 2, Roger MacKenzie 3, Paul Kosma 4, and Helmut Brade 2*

1 Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany; Institute for Biological Sciences; Both authors contributed equally to this study
2 Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany; Institute for Biological Sciences
3 Research Center Borstel, National Research Council Canada, K1A 0R6 Ottawa, ON, Canada; Department of Chemistry
4 Research Center Borstel, University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria

* To whom correspondence should be addressed.
Helmut Brade, E-mail: hbrade{at}fz-borstel.de


  Abstract

Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-{alpha}-D-manno-oct-2-ulosonic 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 C. pecorum, C. pneumoniae and C. trachomatis in double labelling experiments of infected cell monolayers and by 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 bovine serum albumin. The mAb bound preferentially Kdo(2->8)[Kdo(2->4)]Kdo(2->4)Kdo with a KD of 10 µM as determined by surface plasmon resonance 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 with Kdo monosaccharide. MAb S69-4 was able to detect LPS on thin layer chromatography plates in amounts of less than 10 ng by immuno-staining. 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, H. P., N. O. Seto, C. R. MacKenzie, L. Brade, P. Kosma, H. Brade, and S. V. Evans. 2003. Germline antibody recognition of distinct carbohydrate epitopes. Nat.Struct.Biol. 10:1019-1025].


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