Clostridium Difficile Toxin A/B ELISA

The C. difficile Toxin A/B ELISA employs monoclonal antibodies in a sandwich-type method. These monoclonal antibodies against C. difficile toxins A and B are attached to the surface of each well in the microplate. A pipette is used to place a suspension of the stool sample to be examined as well as controls into the well of the microplate together with biotinylated anti-toxin A/B antibodies (Conjugate 1) for incubation at room temperature (20 to 25° C). After washing, streptavidin poly-peroxidase conjugate (Conjugate 2) is added and incubated again at room temperature (20 to 25° C). Any toxins specific to C. difficile present in the sample forms a sandwich complex consisting of immobilized antibodies, toxins, and conjugated antibodies. Unattached streptavidin poly-peroxidase conjugate is removed in a subsequent washing step. After addition of the substrate, the attached enzyme changes the previously colorless solution to blue in the wells that contain bound toxins. Upon addition of the stop solution, the color changes from blue to yellow. The absorbance is proportional to the concentration of toxins A and B present in the sample.