FUNCTIONAL DIVERSITY AND CLONAL FREQUENCIES OF REACTIVITY IN THE AVAILABLE ANTIBODY REPERTOIRE

The present experiments address functional antibody diversity and clon al distribution in murine available repertoires. IgM-containing supern atants were prepared by unbiased, polyclonal stimulation of resting sp lenic. B cells from C57BL/6 mice, to ensure similar numbers of respond ing clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively ass ayed by limiting dilution analysis (LDA) on immunoblots of sodium dode cylsulfate polyacrylamide gel electrophoresis of homologous liver extr acts, allowing to determine specific clonal frequencies towards the ma ny hundred blotted antigens. The clonal frequency of reactivity of B c ells with the extract was shown to be a bi-modal distribution of speci fic frequencies between 1/220 and 1/100 000. Cross-correlation analysi s of reactivity to different bands in individual supernatants revealed low levels of cross-reactivity, suggesting that the blotted extract p rovides a very diverse set of antigens. Investigation of the affinity/ concentration thresholds for detection of antigen-antibody interaction s of our assay supports the notion that global repertoire analyses on immunoblots were highly discriminative and non-degenerate. Furthermore , reactivity patterns obtained with complex antibody mixtures correlat ed with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphoc ytes, indicating a minimal repertoire size that is orders of magnitude higher than previous theoretical proposals suggested, and extensively heterogeneous in the size of clonal specificities.