Enhancement of the antigen-binding capacity of incomplete IgG antibodies to Brucella melitensis through Fc region interactions with staphylococcal protein A

Abstract

Incomplete IgG anti- Brucella antibodies in human sera were detected using both a conventional ELISA and a modified method. In the ELISA procedure serum IgG was allowed to bind solid-phase B. melitensis antigen and, after washing, biotinylated staphylococcal protein A (BioSPA) was used as an Fc-specific tracer followed by streptavidin-HRP conjugate. In the modified method, serum IgG was co-incubated with a defined quantity of BioSPA in the presence of solid-phase antigen. BioSPA bound Fc regions of serum IgG irrespective of antigen specificity whilst antibodies which were specific for Brucella bound the solid-phase antigen through their Fab regions and were detected subsequently by streptavidin-HRP. IgG anti- Brucella antibodies were detectable with a 5–25-fold increase in sensitivity when they were thus ‘activated’ in situ with BioSPA. In contrast with the IgG antibodies of untreated human sera, BioSPA-activated IgG showed strong antigen binding capacity and resisted the dissociation effect of the chaotropic agent, guadinene hydrochloride. In similar experiments, BioSPA did not enhance the affinity of IgG anti- Salmonella antibodies of human sera towards S. typhi antigen. The activating effect of BioSPA on the incomplete IgG anti- Brucella antibodies from patients with brucellosis possibly involves a re-orientation of Fab sites.