Figure 6
Enhanced phagocytosis induced by the monoclonal antibody produced by CpG-DNA 1826-stimulated mouse peritoneal cavity B cells. (A) Production of the bacteria-reactive monoclonal antibody. Ascites from mice induced by the 3F5H6 clone were isolated, and the resulting monoclonal antibody was purified by Protein A affinity column chromatography, subjected to SDS-PAGE, and stained with Coomassie brilliant blue R-250 solution. R, reducing. NR, non-reducing. (B) The isotype of the monoclonal antibody was determined by ELISA using S. aureus MW2-coated plates. (C) The bacteria-reactivity of the antibody was assessed by ELISA using plates coated with the indicated Gram-positive bacteria (n = 3/group). (D–G) FITC-labeled S. aureus MW2 cells (3 × 108 CFU/mL) were incubated with PBS, normal mouse IgG, or 3F5H6 mIg (10 μg/mL) for 1 h then added to RAW 264.7 cells (D,E) and peritoneal cells (F,G) in vitro. After 1 h, the cells were washed with PBS, fixed, and stained with Hoechst No. 33258 to visualize the nuclei (blue). (D,F) Confocal images revealed phagocytosis of S. aureus MW2. Scale bars, 10 μm. MW2, S. aureus MW2. (E,G) The phagocytic index was analyzed (n = 3/group). (H,I) Phagocytosis was enhanced by the bacteria-reactive monoclonal antibody. FITC-labeled S. aureus MW2 cells (3 × 108 CFU/mL) were incubated with normal mouse IgG or 3F5H6 mIgG (10 μg/mL) for 1 h and i.p. injected into BALB/c mice. After 1 h, peritoneal cells were harvested from the mice and stained with specific markers for macrophages (H) and dendritic cells (I). The phagocytic levels were analyzed by flow cytometry (n = 3/group). The results presented are representative of three experiments. *p < 0.05, **p < 0.005, ***p < 0.0005.
