The Synermed® Direct Bilirubin Reagent measures the absorbance of an oxidation product of bilirubin at 700 nm. The absorbance of the cholecyanin oxidation product is directly proportional to bilirubin concentration.
Conjugated bilirubin is oxidized in the presence of the electron transfer agent, KHC3, to a blue-green infrared light absorbing chromophore. Unconjugated bilirubin is not significantly detected due to the fact that it is not soluble under the conditions of the direct reaction mixture and remains bound to albumin.
Unlike traditional diazo methods, there is no chemical interference from hemoglobin in the reaction and hemoglobin has no absorbance at 700 nm. Lipemia has very little absorbance at the 700 nm wavelength used for the detection. Urinary indicans form no color in the reaction and do not interfere.