In the first incubation step, ß2-microglobulin is bound to an immobilized antibody. Then a peroxidase-labeled anti-ß2-microglobulin-antibody is added, and a sandwich of capture antibody – ß2-microglobulin – peroxidase-conjugate is formed.
Tetramethylbenzidine (TMB) is used as a peroxidase substrate. Finally, an acidic stop solution is added to terminate the reaction. The color changes from blue to yellow. The intensity of the color is directly proportional to the concentration of ß2-microglobulin.
A dose-response curve of the absorbance unit (optical density, OD at 450 nm) vs. concentration is generated using the values obtained from the standards. ß2-microglobulin, present in the samples, is determined directly from this curve.