Principle of Assay
Universal TNF alpha ELISA Kit (A335459) is based on Double Antigen Sandwich ELISA technology for the quantitative measurement of universal TNF alpha in serum, plasma, cell culture supernatant, cell or tissue lysate, and other liquid samples. The antigen has been pre-coated onto a 96-well microplate, and antibodies present in the sample bind to the immobilized antigen. A Biotinylated antigen is then added to detect the captured antibodies. The samples and standards are added to the wells and antibodies in each sample form a sandwich with the immobilized and biotinylated antigens. After washing with wash buffer to remove unbound components, HRP-Streptavidin Conjugate is added and incubated. After another wash, TMB substrate solution is used to visualize the HRP enzymatic reaction, producing a blue-colored product that changes to yellow after adding acidic stop solution. The intensity of the yellow color is proportional to the amount of antibodies captured on the plate. The concentration of antibodies can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
