Figure 7. The C. perfringens agrB locus regulates CPA production.

A) ELISA analyses. Culture supernatants obtained, at the indicated time point, from strain 13 (S13), CPJV501 (ΔagrB), CPJVp1 (ΔagrB/P1), CPJVp2 (ΔagrB/P2), CPJVp3 (ΔagrB/P3) or purified CPA was used to coat a 96-well microplate overnight at 4°C. The wells were incubated with a mouse monoclonal anti-CPA antibody followed by a HRP-conjugated anti-mouse antibody. The bound antibody was detected with a TMB substrate solution and the color reaction stopped with sulphuric acid (0.18 M). A₄₅₀ was determined using an ELISA reader. Error bars represent the standard error of the mean calculated using data from three independent experiments. B) Western blot showing the agr locus regulates production of CPA. Strain 13 (S13), CPJV501 (ΔagrB) or CPJVp3 (ΔagrB/P3) was inoculated in TGY and incubated at 37°C for 4 h. Bacteria were then pelleted by centrifugation, resuspended in lysis buffer and sonicated. Equal amount (25 µl) of bacterial lysates was run in a 12% SDS-PAGE, transferred to nitrocellulose membrane and western blotted with a monoclonal anti-CPA antibody. As a control, 25 µl of CPA-containing concentrated supernatant proteins was added to the gel. The expected molecular weight in kDa of CPA is shown at the left. Shown is a representative figure of three independent experiments.