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A monoclonal antibody against a carbohydrate epitope in lipopolysaccharide differentiates Chlamydophila psittaci from Chlamydophila pecorum, Chlamydophila pneumoniae, and Chlamydia trachomatis
- Můller-Loennies, Sven, Gronow, Sabine, Brade, Lore, MacKenzie, Roger, Kosma, Paul, Brade, Helmut
- Glycobiology 2006 v.16 no.3 pp. 184-196
- Chlamydia trachomatis, Chlamydophila pecorum, Chlamydophila pneumoniae, Chlamydophila psittaci, bacteria, biosynthesis, carbohydrate binding, cross reaction, crystal structure, diagnostic techniques, disaccharides, enzyme-linked immunosorbent assay, epitopes, immunization, ligands, lipopolysaccharides, mice, monoclonal antibodies, surface plasmon resonance, thin layer chromatography, transferases, veterinarians
- Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-[alpha]-D-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2[rightwards arrow]8)[Kdo(2[rightwards arrow]4)] Kdo(2[rightwards arrow]4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2[rightwards arrow]8)[Kdo(2[rightwards arrow]4)]Kdo(2[rightwards arrow]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[rightwards arrow]8)[Kdo(2[rightwards arrow]4)]Kdo(2[rightwards arrow]4)Kdo(2[rightwards arrow]4) with a K[subscript d] of 10 [micro]M, as determined by surface plasmon resonance (SPR) for the monovalent interaction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2[rightwards arrow]4)Kdo(2[rightwards arrow]4) Kdo but not with Kdo(2[rightwards arrow]8)Kdo(2[rightwards arrow]4)Kdo, Kdo disaccharides in 2[rightwards arrow]4- or 2[rightwards arrow]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).