A rapid and quantitative method for the evaluation of V gene usage, specificities and the clonal size of B cell repertoires

The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of immune responses to self and nonself-antigens, the development and screening of synthetic antibodies and the accelerated study and screening of neutralizing antibodies to pathogenic threats.

► 5 days of single B cell cultures retrieve individual IgM specificities and genes.
► Paired mRNA/cDNA amplification of VH and VL with promiscuous primers.
► Limiting dilution analysis of B cell repertoire: frequencies and clonal sizes.