Abstract:
:The present experiments address functional antibody diversity and clonal distribution in murine available repertoires. IgM-containing supernatants were prepared by unbiased, polyclonal stimulation of resting splenic B cells from C57BL/6 mice, to ensure similar numbers of responding clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively assayed by limiting dilution analysis (LDA) on immunoblots of sodium dodecylsulfate polyacrylamide gel electrophoresis of homologous liver extracts, allowing to determine specific clonal frequencies towards the many hundred blotted antigens. The clonal frequency of reactivity of B cells with the extract was shown to be a bi-modal distribution of specific frequencies between 1/220 and 1/100,000. Cross-correlation analysis of reactivity to different bands in individual supernatants revealed low levels of cross-reactivity, suggesting that the blotted extract provides a very diverse set of antigens. Investigation of the affinity/concentration thresholds for detection of antigen-antibody interactions 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 correlated with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphocytes, 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.
journal_name
Eur J Immunoljournal_title
European journal of immunologyauthors
Nobrega A,Grandien A,Haury M,Hecker L,Malanchère E,Coutinho Adoi
10.1002/(SICI)1521-4141(199804)28:04<1204::AID-IMMsubject
Has Abstractpub_date
1998-04-01 00:00:00pages
1204-15issue
4eissn
0014-2980issn
1521-4141pii
10.1002/(SICI)1521-4141(199804)28:04<1204::AID-IMMjournal_volume
28pub_type
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