Method Description

This US Food and Drug Administration-approved kit utilizes monoclonal antibodies in a solid-phase sandwich type enzyme immunoassay (EIA). Plastic microtiter wells are coated with a monoclonal antibody directed against the product of the sixth viral gene (VP6), which is the group-specific antigen for all known human rotaviruses. An aliquot of fecal suspension is added to the well and incubated simultaneously with an anti-rotavirus monoclonal antibody conjugated to horseradish peroxidase, resulting in the rotavirus antigen being sandwiched between the solid-phase and enzyme-linked antibodies. After 60 minutes incubation at ambient temperature, the specimen well is washed to remove unbound enzyme-labeled antibodies. Enzyme substrate (urea peroxidase) and chromogen tetra methylbenzidine are added to the wells and incubated for 10 minutes at ambient temperature. The enzyme bound in the wells converts the colorless substrate to a blue color. The intensity of the blue color is directly proportional to the concentration of rotavirus antigen in the specimen.(Package insert: Premier Rotaclone EIA for detection of Rotavirus Antigen in Human Fecal Samples. Meridian Bioscience; 01/2015)