The DBC rT3 ELISA is a competitive enzyme immunoassay, where the antigen (rT3 present in calibrators, controls and patient samples) competes with a biotin-labelled antigen (rT3-Biotin conjugate) for a limited quantity of antibody which is coated on the microplate wells. After one hour incubation followed by the first washing, unbound materials are removed and a Streptavidin-HRP conjugate is added and incubated for 30 minutes. Following a second washing, the TMB substrate is added. The enzymatic reaction is terminated by addition of the stopping solution, upon which the colour intensity is measured with a microplate reader. The colour intensity is inversely proportional to the concentration of rT3 in the sample. The set of kit calibrators that are run simultaneously
with the samples is used to plot a calibration curve and determine the concentration of rT3 in samples and controls.