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Ehrlich's diazo reagent reacts with the direct bilirubin in the serum to form a pink to reddish-purple colored compound (Azobilirubin) read at one minute.
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. Conjugated bilirubin in the serum or plasma sample is directly proportional to the formation of azobilirubin, which is measured bichromatically at 540/600 nm.
It is based on the creation of azo dyes, the so-called azobilirubin that acts as an acid-basic indicator.
This is a method by which bilirubin reacts with diazotised sulphanilic acid to produce azobilirubin (Violet colour).
"Direct" assays involve a chemical reaction with diazo dyes, followed by quantification of azobilirubin produced over a specified time.
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.
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.
Total bilirubin was analyzed by measuring azobilirubin after the addition of accelerators and diazotized sulfanilic acid (17).