Bilirubin quantification is a diagnostic technique designed to measure both conjugated (direct) and total bilirubin levels in serum. The calculation of unconjugated (indirect) bilirubin is derived from the difference between total and conjugated bilirubin. Measuring serum bilirubin is crucial for assessing liver damage and is particularly significant in diagnosing jaundice. Bilirubin detection approaches encompass the diazo reagent method, vanadate oxidation method, and bilirubin oxidase method, among others.

 

 

Diazo Reagent Method 

Detection Principle: Themodified J-G method, also known as the p-aminobenzene sulfonic acid technique, is utilized. Conjugated bilirubin reacts directly with the diazo reagent to produce a purple azo-bilirubin compound. Unconjugated bilirubin requires an accelerator (such as caffeine, sodium benzoate, and sodium acetate) to break the hydrogen bonds and enable its combination with bilirubin before forming the azo-bilirubin complex. The intensity of the resulting color is directly proportional to the bilirubin concentration. Colorimetry is performed at a wavelength of 600 nm, with the calibration curve used to determine the levels of total and conjugated bilirubin.

 

 

 

Bilirubin Oxidase Method 

Detection Principle: Bilirubin exhibits a peak absorption around 450 nm. The enzyme bilirubin oxidase (BOD) catalyzes the conversion of bilirubin into biliverdin, leading to a decrease in absorbance at 450 nm. The extent of this decrease correlates with the amount of bilirubin that has been oxidized. At pH 8.0, both unconjugated and conjugated bilirubin are oxidized, so the reduction in absorbance at 450 nm reflects the total bilirubin level. The addition of sodium dodecyl sulfate (SDS) and bile enhances this oxidation process. In a pH range of 3.7-4.5, BOD exclusively catalyzes the oxidation of conjugated bilirubin, while unconjugated bilirubin remains unaffected. Ditaurobilirubin (DTB) dissolved in human serum serves as the standard, and the change in absorbance at 450 nm is measured to calculate the content of conjugated bilirubin.

 

 

 

Vanadate Oxidation Method 

Detection Principle: Total bilirubin in serum is oxidized to biliverdin by vanadate in a buffer at pH 3.0. The difference in absorbance before and after vanadate treatment allows for the calculation of total bilirubin levels. The presence of surfactants and indirect reaction inhibitors facilitates the oxidation of direct bilirubin to biliverdin by the vanadate oxidant. The yellow-specific absorbance of bilirubin diminishes, enabling the determination of direct bilirubin concentration through absorbance changes pre- and post-vanadate oxidation.

 

 

 

Calibrators and Quality Controls

For these methods, calibration and quality control products are essential. Seebio Biotech offers both ready-made calibration and quality control products, as well as raw materials for customers who prefer to prepare their own solutions.

 

 

 

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