High sensitivity streptavidin hrp conjugate

C. difficile 630Δerm and 630ΔermΔPaloc were grown in 6-well plates containing 2 mL TY medium for either 24 h or 48 h with 0.2 mg/mL of anti–LMW mAbs when specified. Absorbance was measured at 600 nm, and the cultures were harvested and centrifuged at 4,000 rpm for 5 min. Toxins were assessed in the supernatants using ELISA. Maxisorp microtiter plates (Dutscher, France) were coated with 5 μg/mL anti-TcdB capture antibody (BBI Solutions, Madison, WI) or anti-TcdA capture antibody (Novus Biological, CO, USA). Purified toxins A and B (Sigma-Aldrich) were used as the standards. The supernatants were added for 1h30 at RT. After washing, the anti-toxin B biotinylated antibody (BBI solutions, Madison, WI, USA) followed by high-sensitivity streptavidin-HRP conjugate (ThermoFisher, Waltham, MA), or anti-toxin A HRP-conjugated antibody (LSBio, WA, USA) signal was detected with TMB substrate (ThermoFisher, Waltham, MA) at 450 nm using an ELISA plate reader (Berthold, France). Toxin concentrations were normalized to the OD_600 nm values for each well.