Description
Principle of the Assay::Bilirubin Assay Kit measures the total and direct bilirubin within serum, plasma, urine, cell lysates, or tissue lysate samples. The assay is based on the Jendrassik-Grof method (Ref. 1) in which diazotized sulfanilic acid reacts with bilirubin to form azobilirubin, the latter of which can be detected at an OD of 540 nm (Figure 1). Since unconjugated bilirubin and bilirubin bound to albumin react very slowly, an accelerant is added to the reaction to allow for measurement of total bilirubin. In the absence of the accelerant, only the direct or conjugated bilirubin is measured.
Background/Introduction: Bilirubin is an open chain molecule containing four pyrrole-like rings that forms during the breakdown of heme. Bilirubin is excreted in urine and bile and can also be found in low levels in plasma. Three principle forms of bilirubin are found in plasma: conjugated (to glucuronic acid; also called direct bilirubin which makes bilirubin water soluble), unconjugated, or bound to serum albumin. Eventually, bilirubin is degraded in the liver to be removed from the body. While high levels of bilirubin in serum have been correlated with jaundice, hepatitis, Gilbert's syndrome, and drug toxicity, low levels of bilirubin have been correlated with cardiovascular disease, diabetes mellitus, and metabolic syndrome. Bilirubin Assay Kit is a simple colorimetric assay that measures the amount of total and direct (conjugated) bilirubin present in plasma, serum, urine, cell lysates, or tissue lysates in a 96-well microtiter plate format. The kit has a detection sensitivity limit of 0.5 mg/dL bilirubin. Each kit provides sufficient reagents to perform up to 200 assays*, including blanks, bilirubin standards and unknown samples. Sample bilirubin concentrations are determined by comparison with a known bilirubin standard