A novel method for determining paratope, idiotype, and IgV and C gene frequencies within populations of B lymphocytes has been established. Short term clones of mitogen- or antigen-activated B cells are established as pure colonies (single founder lymphocytes) of antibody-secreting cells (ASC) on filter paper discs. Colonies are immobilized within the paper and clonality is maintained by spatial isolation. Colonies may be detected and characterized by microscopy, immunoblotting, and in situ Northern hybridization. Depending upon the density of splenocytes plated, the ratio of ASC colonies to Ig+ C57BL/6 splenocytes ranges from 1:6 to 1:23. This method will be used to characterize the kappa+ and lambda+ antibody repertoires for the frequency of paratopes specific for: random amino acid copolymers and structurally defined proteins; auto-, allo-, and xenogeneic protein homologs including Class I MHC molecules, antibody, and proteins associated with pathological autoimmunity, pathogenic, commensual, and irrelevant bacteria viruses, and helminths. Determination of the frequencies for specific paratopes in mice bearing the Igh-Va or Igh-Vb locus will measure the importance of VH allelism on the paratopic repertoire. Comparison of K+ and lambda +1 paratope frequencies for the same antigens will provide a direct measure of the diversity V kappa provides the antibody repertoire. Combination of specific (i.e., antigen or idiotype) immunoblotting followed by in situ hybridization of the same filter paper disc will permit paratopic and idiotypic specificities to be mapped to expressed VH families. Finally, this survey of paratope frequencies will be used to define the antibody repertoire as random or biased. Paratopic bias would imply that natural selection has dominated the evolution of the Ig-V genes. In contrast, failure to detect bias would suggest that non-selective modes of evolution, so-called molecular evolution, have dominated the history of these loci.
Showing the most recent 10 out of 53 publications
