Clostridium Difficile GDH ELISA

The C. difficile GDH ELISA employs monoclonal antibodies in a sandwich-type method. These monoclonal antibodies against the glutamate dehydrogenase of C. difficile 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-GDH 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 GDH specific to C. difficile present in the sample forms a sandwich complex consisting of immobilized antibodies, GDH, and conjugated antibodies. Unattached streptavidin poly-peroxidase conjugate is removed in a subsequent washing step. After addition of the substrate, the colorless solution will turn blue in wells containing bound GDH of C. difficile. Upon addition of the stop solution, the color changes from blue to yellow. The absorbance is proportional to the concentration of GDH of C. difficile present in the sample.