Autoimmune ELISA kits measure autoimmune antibodies in the serum. It is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay utilizes a specific antigen for immobilization on the microtiter wells and anti-human IgG antibody conjugated to horseradish peroxidase (HRP) for detection. The test sample is allowed to react simultaneously with the two components, resulting in autoimmune antibodies being sandwiched between the solid phase and enzyme-linked antibodies. After incubation, the wells are washed to remove unbound-labeled antibodies. A HRP substrate, TMB, is added to result in the development of a blue color. The color development is then stopped with the addition of Stop Solution changing the color to yellow. The concentration of autoimmune antibodies is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.