Tetramethylbenzidine tmb substrate solution

The equilibrium dissociation constant (K_d) values of ErbB2 aptamer were determined using an enzyme-linked oligonucleotide assay (ELONA). Briefly, 96-well ELISA plates (Corning^®, Sigma-Aldrich) were coated with ErbB2 (Cat. No. 1129-ER, R&D system) at 4 ºC for 16 h. The treated wells were blocked with Pierce^™ Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h, followed by three washes with PBS. The biotin-labeled aptamers were added and incubated for 1 h at room temperature. After extensive washing with washing buffer (10 mM PBS, 0.05% Tween-20, pH 7.4), Pierce^™ High Sensitivity Streptavidin HRP (Thermo Fisher Scientific) was added to each well to bind biotin-conjugated aptamer. After incubation for 1 h at room temperature, the plates were washed again as described above. Tetramethyl benzidine (TMB) substrate solution (Thermo Fisher Scientific) was added to each well and incubated for 30 min at room temperature. Then, the reaction was quenched by addition of 2 M H₂SO₄. The protein-bound aptamer-streptavidin complexes were quantified by determining the absorbance at 450 nm using GloMax^® Discover System (Promega, Madison, WI, USA). The saturation curve was plotted and K_d was analyzed with Sigma Plot 12.5 (https://systatsoftware.com/products/sigmaplot/) software.

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.