FIG. 7.

GG rTcPRAC immunization generates specific IgG that binds mitogenic rTcPRAC. rTcPRAC-immune (TcP-imm) IgG or control CRP-immune (CRP-imm) IgG was bound to protein A/G resin and then incubated with rTcPRAC. (A) Equal volumes of nonbound protein after incubation of 30 μg rTcPRAC with TcP-imm IgG (rTcPRAC depleted) or CRP-imm IgG (TcPRACA control) were added to splenocyte cultures. After stimulation, live cells were assessed for B-cell activation in terms of B-cell proliferation and surface phenotype (CD69 and CD86) by flow cytometry. IL-10 and antibody (IgM and IgG) secretion was assessed by ELISA. Data represent the means for triplicate repeats (±SEM) for each measure. (B) rTcPRAC eluted from TcP-imm IgG was compared to nonspecifically bound protein eluted from control CRP-imm IgG for the ability to stimulate B cells. (Left) Cells were stimulated with eluted protein. After stimulation, cells were analyzed for B-cell proliferation and activation (CD69 and CD86) by flow cytometry. Data represent the means for triplicate repeats (±SD) for each measure. (Right) Cells were stimulated with eluted protein. After stimulation, the culture supernatant was analyzed for IL-10 secretion by ELISA. Eluted rTcPRAC induced dose-dependent IL-10 secretion compared to control eluted protein. Data represent the means for triplicate repeats (±SEM). *, P < 0.05; **, P < 0.001; ***, P < 0.0001 (Bonferroni posttests after two-way ANOVA or Student's t test). (C) Application of rTcPRAC to TcPRAC-specific and control IgG resulted in rTcPRAC in the flowthrough fraction from the control IgG column and in the elution fraction from the TcPRAC-specific IgG column. TcPRAC was visualized by use of anti-TcPRAC IgG from polyclonal sera from protein-immunized rabbits.