On the validity of using lipopolysaccharide-driven limiting dilution systems for clonable B-cells to analyse functional antibody repertoires.

Analyses of B-cell repertoires by mitogen-driven limiting dilution systems (LDA) followed by specific antibody detection on immobilized antigens (ELISA), while constituting the only method available to determine absolute frequencies of any given specificity, provide no indications as to the functional ability of clonal precursors scored as positive to actually respond to antigen in vivo. We have now addressed this question by quantitating dextran alpha 1----6-specific clonal precursors among small, resting, and large activated B cells in the spleens of mice, before and after priming with optimal doses of antigen. The results demonstrate that virtually all B cells scored as antigen-specific in LDA/ELISA systems do respond to antigen and undergo blast transformation after priming in vivo.