A receptor-based immunoassay to detect Staphylococcus enterotoxin B in biological fluids

A rapid, simple, and inexpensive sandwich enzyme-linked receptor based immunodot assay was developed for the detection of staphylococcal enterotoxin B (SEB) in human fluids by using purified glycosphingolipid digalactosylceramide (diGalCer) receptor for SEB. Three micrograms of diGalCer was immobilized on a polyvinyledene difluoride membrane and the membrane was subsequently incubated with primary and secondary alkaline- phosphatase-labeled antibodies. A positive reaction was discerned as a blue spot. As little as 1 ng/ml of SEB could be detected in the assay. SEB did not bind to structurally related glycosphingolipids, such as glucosylceramide, galactosylceramide, and lactosylceramide in this assay. Of five monoclonal anti-SEB antibodies and commercial anti-SEB antiserum tested, the latter was the most sensitive in our assay. The specificity of SEB assay was assessed by comparison with structurally related toxins, for example, staphylococcal enterotoxin A, and toxic shock syndrome toxin 1 (TSST-1). TSST-1 was not detected in the assay. This was because these toxins were not recognized by the anti-SEB antibody and did not bind to diGalCer. In conclusion, we believe that this assay may be widely applicable because it is highly specific for SEB, it does not require special equipment, and the results can be obtained within few hours with the naked eye. Since the receptor for SEB has a long shelf life, it can be easily stored and used for a long time.