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 a lpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. This lipopolysaccharide is re cognized in a genus-specific pattern by murine monoclonal antibodies (mAbs) , S25-23 and S25-2 (both IgG1 kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively. The vari able domains of these mAbs were reverse transcribed from mRNA which was iso lated 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 exam ples of an antibody-carbohydrate interaction involving anionic carboxyl gro ups on the ligand, we report that the affinities of these antibodies are hi gher than usually observed in carbohydrate-protein interactions (K-D of 10( -3) to 10(-5) M). SPR analyses of monovalent Fab and scFv binding to the na tural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitti ng of several measurements at different antibody concentrations) of sensorg ram data to a one-to-one interaction model. Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Sc atchard 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 carboxy l-reduced Kdo ligands, S25-23 did not. Identification of amino acids in the complementarity determining regions revealed the presence of a large numbe r of positively charged amino acids which were located towards the center o f the combining site, thus suggesting a different recognition mechanism tha n that observed for neutral ligands, The latter mainly involves aromatic am ino acids for hydrophobic stacking interactions and hydrogen bonds.