FIG 4.

Mouse serum in vitro antibody neutralizing activity against STa toxin. Intracellular cGMP levels in T-84 cells measured with an EIA cGMP ELISA kit (Assay Design) were used to show anti-STa antibody neutralization activity. If antibodies in serum (or fecal suspension) are neutralizing against STa toxin, these antibodies will prevent the toxin from stimulating intracellular cGMP in T-84 cells, which will result in a low cGMP level (lower than that of the control group [the “−” column]). The serum sample (30 μl; in a final dilution of 1:33.3) pooled from each immunization group or the control group (−) was incubated with STa toxin (2 ng, in 150 μl of cell culture medium) for 1 h at room temperature, and the serum-toxin mixture was added to T-84 cells (1 ml of final volume with cell culture medium). The intracellular cGMP level in the T-84 cells was measured after 1 h of incubation, with the mean cGMP level and standard deviation (from two replicates) of each group indicated as columns and lines. Labels on the x axis indicate the mouse groups immunized with different fusion antigens and the control group (“−”), from which serum samples were used for the antibody neutralization assay. STa toxin (without serum sample) was used as the control to show STa toxicity in stimulation of cGMP, and cell culture medium (without STa toxin and serum) was used as the control to show baseline cGMP level in T-84 cells.