Fig. 2. Ag-specific B-cell selection in bulk, V-gene recovery from single B cells, expression and quantitation of r-IgG.

a A schematic diagram shows antigen-specific B-cell selection and isolation from mice immunized with a target antigen. IgG⁺ B cells (cell surface) were enriched by depletion of non-B cells and IgM⁺ and IgD⁺ (cell surface). Biotinylated antigen was added in solution and target-specific IgG⁺ B cells were isolated by magnetic separation. Viable Ag-specific single B cells were sorted in each well of a 96-well plate. Single B-cell expression cloning was performed to generate rIgG heavy and kappa chains. b Recovery of paired V_H and Vκ was determined by eGel analysis of amplified DNA. Bar graph shows percent of clones, which were amplified to yield V_K and V_H genes from single B cells. Representative eGel analysis of amplified V_H and VK genes are shown at the bottom for total B cells, IgG⁺ B-cells (after IgM⁺/IgD⁺ cells were depleted) and IgM⁺/IgD⁺ B cells. c Schematic representation of expression of individual rIgGs recovered from single antigen-selected B cells. Paired rIgG heavy and kappa chains were co-expressed in Expi293F cells for transient expression of individual antibodies. Antibody concentrations in conditioned media from transfected Expi293F cultures was measured by Octet Red96 (ForteBio). Representative quantitation of 94 IgG samples in Expi293F conditioned media is shown in the graph. A representative heatmap demonstrates the range of individual rIgG expression from Expi293F cells in each well of a 96-well culture plate, 6 days post-transfection.