Mapping of antibody responses to the protective antigen of Bacillus anthracis by flow cytometric analysis.

BACKGROUND

Knowledge of the target and functional capability of the antibody response against an antigen provides more specific and relevant information about protective immunity than measuring the total amount of antibody produced against an antigen.

METHODS

Using flow cytometry, a competitive assay has been created for measuring the antibody response against important epitopes of an antigen. Monoclonal antibodies (mAbs) against the protective antigen (PA) component of Bacillus anthracis are available that neutralize the activity of lethal toxin (LeTx). Flow cytometric analysis revealed that these mAbs bind PA conjugated to polystyrene latex microspheres.

RESULTS

Unlabeled mAbs against PA competed with fluorescent mAbs that bind the homologous epitope but not with fluorescent mAbs that bind heterologous epitopes. Four-parameter logistic models were developed for measuring the antibody response against two epitopes of PA. Sera from anthrax-vaccinated rabbits inhibited the binding of fluorescent mAbs to either epitope; the degree of inhibition correlated with the dilution of sera.

CONCLUSIONS

The response in the rabbit sera to either epitope on PA depended on the dose of vaccine administered to the rabbits. No inhibition was seen with sera from control animals. With no species-specific components, this assay could be adapted readily for comparing responses between species.