This is a method by which bilirubin reacts with diazotised sulphanilic acid to produce azobilirubin
Both total and direct bilirubin measure the formation of azobilirubin
, which is detected at 570/660 nm.
The conjugated bilirubin assay is an endpoint assay that couples conjugated bilirubin (direct bilirubin) with the diazonium salt of 3,5-dichloroaniline at low pH to form azobilirubin
It is based on the creation of azo dyes, the so-called azobilirubin
that acts as an acid-basic indicator.
Bilirubin estimation was done by Diazo method which is based on the principle that bilirubin reacts with diazotised sulphanilic acid in acidic medium to form pink coloured azobilirubin
with absorbance directly proportional to bilirubin concentration.
Direct" assays involve a chemical reaction with diazo dyes, followed by quantification of azobilirubin
produced over a specified time.
In the latter, the absorbance of Hb at low bilirubin concentrations overcompensates for decreased absorbance caused by the destruction of the azobilirubin
, whereas at high bilirubin concentrations Hb does not compensate for this decrease.
The color (pink) intensity of the azobilirubin
produced is proportional to the total bilirubin concentration.