Pierce 96 well polystyrene plate

Microtiter plates (Thermo Scientific Pierce 96-well polystyrene plates) were coated with 100 μl of heat-killed bacteria (∼10⁷ CFU/well) in coating buffer (0.05 M carbonate buffer pH 9.6) for 18 h at 4°C. Following incubation in blocking buffer (5% bovine skim milk in TBS) for 1 h at 37°C, the plates were further incubated with 1:100 of the indicated hybridoma supernatant concentrate or with 1:2000 mouse anti-Brucella O-PS (M84) mAb [35 (link)] for 1 h at RT in blocking buffer. Detection of antibodies with secondary antibody, reaction development and absorbance measurement were carried out as described in glycoprotein indirect-enzyme-linked immunosorbent assay.

Glyco-iELISA was performed as described previously [23 (link)], with minor modifications. Briefly, microtiter plates (Thermo Scientific Pierce 96-well polystyrene plates) were coated with 100 μl of AcrA, O157-AcrA or O145-AcrA (125 ng/well) in coating buffer (0.05 M carbonate buffer pH 9.6) for 18 h at 4°C. The plates were blocked in blocking buffer (5% bovine skim milk in TBS) for 1 h at 37°C and subsequently incubated with the indicated hybridoma supernatant or mice sera dilution for 1 h at RT in blocking buffer. Following four washing steps in TBS Tween-20 0.05%, plates were further incubated for 1 h at RT with HRP goat anti-mouse IgG secondary antibody (Sigma-Aldrich) at a 1:6000 dilution in blocking buffer. Finally, plates were washed four times in TBS 0.05% Tween-20 and after incubation with the substrate (0.3% H₂O₂, 0.1% 3,3',5,5'- tetramethylbenzidine [TMB] in 0.1 M citric acid pH 5) for 5 to 20 min at RT, the reaction was stopped with 0.2 M H₂SO₄. The absorbance at 450 nm was measured with a FilterMax F5 Multi-Mode microplate reader (Molecular Devices).