Molecular basis for the lack of mimicry of Brucella polysaccharide antigens by Ab2gamma antibodies.

The crystal structure of the complex of an anti-Id Fab with an Fab specific for a Brucella polysaccharide antigen has previously been reported (Evans et al., 1994, J. Mol. Biol. 241, 691-705). To complement this study, the binding characteristics and immunological properties of this Ab2 and two others raised with a second anti-Brucella antibody were investigated, including quantitative kinetic measurements by surface plasmon resonance. The affinities of the Fabs from the Ab2s for the Ab1s were three orders of magnitude greater than those estimated for the antigen, but the Ab2s failed to induce antigen-binding Ab3s, that is, they were of the Ab2gamma type. The avidities of the Ab1s for antigen were however within one order of magnitude of their avidities for Ab2. Tests of 16 other anti-Brucella polysaccharide antibodies showed that the two idiotopes were not present in them, and in confirmation of the lack of a dominant idiotope, N-terminal sequencing of their H and L chains showed a wide variety of V genes were employed in the immune response to the Brucella polysaccharides. The failure of the Ab2 to induce antigen-reactive Ab3 thus appears to be due to neither intrinsic affinity nor idiotope frequency, but arises instead from structural reasons, for example, the incomplete penetration of the Ab2 into the binding-site cleft of the Ab1. The surface topography of polysaccharide antigens and their binding-sites thus appears to be especially difficult for Ab2s to mimic and will restrict their routine use as surrogates for T-cell independent polysaccharide antigens.

Characterisation of residues in antibody binding sites by chemical modification of surface-adsorbed protein combined with enzyme immunoassay.

Specific functional group modification of an antibody adsorbed to microtitre plates has been used to probe the binding site residues that determine antigen specificity. Chemical modification of adsorbed protein in tandem with enzyme immunoassay (termed CMAP-EIA) consumes only modest amounts of antibody, while allowing a variety of reagents to be rapidly screened in situ. Modification of tyrosine and arginine residues with 1-fluoro-2,4-dinitrobenzene, and p-hydroxyphenylglyoxal resulted in reduced binding of polysaccharide antigen from Yersinia enterocolitica O-polysaccharide to its homologous monoclonal antibody, YsT9-1. Modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under various conditions indicated that carboxylate groups may also be involved. Parallel experiments with diethylpyrocarbonate and acetic anhydride were used to rule out the involvement of histidine and lysine residues respectively. In all cases, binding of an anti-idiotypic antibody, AJ5, could only be reduced at concentrations of modifying reagent substantially higher than those required to reduce polysaccharide antigen binding to YsT9-1. The results are discussed with regard to the structure of the combining site of YsT9-1 as determined by X ray crystallography and by modelling, and the role of particular residues in complex formation with antigen and in the idiotope.

High-performance liquid affinity chromatography: rapid immunoanalysis of transferrin in serum.

We describe a new method for quantitatively measuring substances of clinical interest by high-performance liquid affinity chromatography (HPLAC). As a model system we selected analysis for transferrin in human serum with immobilized antibodies in a high-performance liquid chromatographic system. SelectiSpher-10 Activated Tresyl columns (5 or 10 x 0.5 cm) were used for in situ coupling of polyclonal antibodies to transferrin. The amount of transferrin eluted was determined by integrating the eluted peak at 280 nm. The whole analytical procedure--including injection of sample, washing, elution, and analysis of data--takes only 7 min. We characterized the HPLAC system for analysis of transferrin in several ways: intra-assay CV approximately 3%; inter-assay CV 2-9%; linear response up to 1 mg/mL column volume; detection limit approximately 3 micrograms; analytical recovery 98% +/- 2%; purity of eluted sample greater than 95% (SDS-PAGE). The HPLAC method was compared with "rocket" immunoelectrophoresis, a commonly used method of analysis for transferrin, and there was excellent correlation between the two methods (r = 0.96, n = 60). Benefits of this HPLAC technique include high precision, rapid analysis, and simplified sample handling.