The method involves dissolving the CTH bottom spot in chloroform:methanol (2:1).
We prepared a working solution by mixing the dissolved CTH in methanol to a final concentration of 25 μg/mL. A coating of 100μL was pipetted onto the wells of the microtiter plate and allowed to evaporate in a fume-hood with constant air flow. Once the evaporation was complete, the uncoated sites in the well were blocked with blocking buffer (0.01M phosphate buffer saline, 4% BSA and 0.05% Tween-20) and incubated at 4oC overnight.
The next day, the wells were washed with PBS containing 0.05% Tween-20 (PBS-T), and 100μL of samples containing the Stx2 were added. Following a 1 hr incubation, the plates were washed with PBS-T to remove unbound Stx2 and mouse anti-Stx2 primary antibody was added to a final concentration of 0.1 μg/well. The primary antibody binds to the Stx2, which in turn is bound to the CTH receptor. After a 1 hour incubation, the wells were washed again and secondary antibody was added. The secondary antibody was goat anti-mouse antibody, which has a horse-radish peroxidase (HRP) conjugate. After a 1 hr incubation and washes, the substrate TMB (3,3_,5,5_-tetramethylbenzidine) was added. The HRP from the secondary antibody breaks down TMB giving a blue color. This reaction is stopped by adding 2M H2SO4. After addition of the stop solution, a yellow color develops and the intensity of this is quantified by a plate reader. The intensity of the color corresponds to the amount of Stx2 present in the sample. Standards were run with each reaction that allows the exact quantification of the Stx2. Our R-ELISA (Receptor ELISA) can successfully detect and quantify Stx2, and routinely a linear curve is obtained between 20 and 350 ng/mL Stx2.
This method can also be used to detect Stx1 by using different primary antibodies. Unlike many more expensive commercially available kits for detecting Stx, our method can distinguish Stx2 from Stx1. It also allows multiple samples to be processed at the same time, and all necessary components for performing this assay are commercially available.