Elisa plate reader

Maxisorp microtiter plates (Dutscher, France) were coated with 0.3 μg LMW630 recombinant protein in carbonate buffer (Na₂CO₃/NaHCO₃) for 2 h at room temperature (RT). Free sites were blocked by a 2-hour incubation at RT with 1X-PBS 1% BSA. Plates were washed three times with 1X-PBS 0.05% Tween 20 (PBS-T) before being co-incubated with serum, supernatants, or mAbs at different concentrations (from 10⁻⁶ μg/mL to 10 μg/mL) for 1 h at RT. After five washes, HRP-conjugated goat anti-mouse IgG Heavy and Light Chain antibody (Bethyl, TX, USA; dilution 1:20,000) was added for 1 h at RT, followed by incubation with OPD substrate for 10 min (Sigma-Aldrich, MO, USA). Absorbance was measured at 495 vs 620 nm on an ELISA plate reader (Berthold, France).

Maxisorp microtiter plates (Dutscher, #055260) were coated with a total of 0.3 μg per well of LMW recombinant proteins in carbonate-bicarbonate buffer (pH 9.6) for 2 h at room temperature (RT). Free sites were blocked by a 2-h incubation at RT with PBS 1% BSA. Plates were washed three times with PBS 0.05% Tween 20 (PBS-T) before being coincubated with serum, supernatants, or monoclonal antibodies at different concentrations (from 10^–6 μg/mL to 10 μg/mL) for 1 h at RT. After five washes, goat anti-mouse IgG-Fc fragment HRP conjugated antibody (Bethyl, dilution 1:20,000, #A90-131P) was added for 1 h at RT followed by incubation with OPD (o-phenylenediamine dihydrochloride) revelation substrate for 10 min (Sigma-Aldrich, #P8287). Absorbances were analyzed at 492 vs 620 nm on an ELISA plate reader (Berthold).

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.