Rotavirus ELISA

The Rotavirus ELISA employs monoclonal antibodies in a sandwich-type method. A monoclonal antibody to the product of the 6th viral gene (VP6) is coated to the surface of each well of the microplate. This is a group specific antigen that is found in all rotaviruses that cause disease in humans. 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 monoclonal anti-rotavirus 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). With the presence of rotaviruses in the stool sample, a sandwich complex forms which consists of immobilized antibodies, the rotavirus antigens, and the antibodies conjugated with the biotin-streptavidin-peroxidase complex. 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 rotaviruses present in the sample.