The enzymatic immunoassay (ELISA) is based on the competition between unlabelled LEAP-2 and Biotin labelled LEAP-2 for limited specific rabbit anti-LEAP-antiserum sites.
The complex rabbit antiserum – LEAP-2 (free LEAP-2 or Labelled) binds to the mouse monoclonal anti-rabbit antibody coated in the well.
After the washing step, the complex binds to the mouse monoclonal anti-rabbit antibody is revealed by Streptavidine_AChE (Tracer).
Finally, Ellman’s reagent (enzymatic substrate for AChE® and chromogen) is added to the wells.
The AChE® tracer acts on the Ellman’s Reagent to form a yellow compound that strongly absorbs at 414 nm.
The intensity of the color, determined by spectrophotometry, is proportional to the amount of tracer bound to the well and is inversely proportional to the amount of free LEAP-2 present in the well during the immunological incubation.