Description
In the first step Insulin AAb from the diluted sample bind to human recombinant insulin coated on the microtiter plate. After an incubation of 60 minutes at 37 °C unbound components are removed by washing. In a next step bound antibodies reacts with the added anti-human-IgG horseradish peroxidase (HRP) complex. Excessive conjugate is removed after 15 minutes at 37 °C by another washing step. HRP converts the colorless substrate TMB added into a blue product. The enzyme reaction is stopped by adding an acid solution after 15 minutes at 37 °C. The absorbance of the resulting yellow product is measured at 450 / 620 nm within 30 minutes. The obtained OD is direct proportional to the amount of bound antibodies