Characterization of high affinity monoclonal antibodies specific for chlamydial lipopolysaccharide.
Pathogens belonging to the genus Chlamydia contain lipopolysaccharide with a 3-deoxy-D- manno- oct-2-ulosonic acid (Kdo) trisaccharide of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. This lipopolysaccharide is recognized in a genus-specific pattern by murine monoclonal antibodies (mAbs), S25-23 and S25-2 (both IgG1kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively. The variable domains of these mAbs were reverse transcribed from mRNA which was isolated from hybridomas and cloned as single-chain variable fragments (scFvs) in E.coli TG1. The kinetics of binding of whole antibodies, Fab fragments and scFvs to natural and synthetically modified ligands were determined by surface plasmon resonance (SPR) using synthetic neoglycoconjugates. As examples of an antibody-carbohydrate interaction involving anionic carboxyl groups on the ligand, we report that the affinities of these antibodies are higher than usually observed in carbo-hydrate-protein interactions (K(D)of 10(-3)to 10(-5)M). SPR analy-ses of monovalent Fab and scFv binding to the natural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitting of several measurements at different antibody concentrations) of sensorgram data to a one-to-one interaction model. Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Scatchard analysis of the data gave kinetic and affinity constants that were in good agreement with those obtained by global fitting. The SPR data also showed that while S25-2 bound well to several Kdo disaccharides and carboxyl-reduced Kdo ligands, S25-23 did not. Identification of amino acids in the complementarity determining regions revealed the presence of a large number of positively charged amino acids which were located towards the center of the combining site, thus suggesting a different recognition mechanism than that observed for neutral ligands. The latter mainly involves aromatic amino acids for hydrophobic stacking inter-actions and hydrogen bonds.
Müller-Loennies
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Division of Biochemical and Medical Microbiology, Borstel Research Center, Parkallee 22, D-23845 Borstel, Germany,
MacKenzie
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Patenaude
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Evans
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Kosma
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Brade
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Brade
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Narang
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D002240:Carbohydrate Sequence; D002689:Chlamydia / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D008070:Lipopolysaccharides / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D011994:Recombinant Proteins