GM1 ELISA was used to quantify the concentration of CT in OMV and cell-free supernatant samples as described previously (67 (link)). Equal volumes of supernatant and equal volumes of OMV samples were serially diluted. Different concentrations of purified CT with known concentrations were used as the standards. Ninety-six-well polystyrene microtiter plates were coated with GM1 ganglioside overnight, and 1% (wt/vol) fatty acid-free bovine serum albumin (BSA) was used to block the GM1-coated plates for 1 h at room temperature. Next, 12 μl of crude OMVs and 260 μl of supernatant were added to the wells in duplicate and incubated for 1 h at room temperature. Subsequently, a rabbit anti-CT polyclonal antibody (1:10,000) and then an HRP-linked goat ani-rabbit IgG antibody (1:2,000) were added to the wells and allowed to incubate for 1 h at room temperature each. For development of the CT-antibody complex, tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific) was used according to the manufacturer’s protocol. The color intensity in each well was measured at 485 nm in a plate reader. CT amounts in the samples were estimated by comparison to the standard curve.
Free full text: Click here
