Living organisms use oxygen for energy production and generates reactive oxygen species (ROS) in the process. ROS damage DNA, cells and cause various organ disorders. Therefore, the body synthesizes antioxidants and traps ROS by ingesting them externally to protect the body from oxidative damages. When the balance between antioxidants and ROS is disrupted, oxidative stress occurs, leading to various diseases such as atherosclerosis and Alzheimer's disease.

Bilirubin, a heme metabolite and bile pigment, is a potent low molecular weight antioxidant in the body. Unlike antioxidant nutrients, bilirubin does not fluctuate with intake, so blood levels are usually constant. In addition, bilirubin can interconvert from hydrophobic to hydrophilic due to its unique molecular structure that forms intramolecular hydrogen bonds, allowing it to freely cross cell membranes. This makes it an ideal low-molecular-weight antioxidant that is ubiquitous in the blood circulation system, tissues and cells.

Biopyrrin is the final oxidation product of the reaction of the suicidal antioxidant bilirubin with reactive oxygen species. Unlike other antioxidant nutrients such as vitamins C and E, biopyrine is not reduced to bilirubin in the redox environment in the body and is rapidly excreted in the urine. Therefore, an increase in the amount of bilirubin consumed due to oxidative stress manifests itself as an increase in the amount of urinary biopyrine. Because of these characteristics, biopyrine is expected to be a real-time oxidative stress marker of redox status in vivo. Urinary biopyrin levels have been reported to respond to surgical stress, ischemic heart disease, sepsis, mental disorders, and social stress.

This kit is a competitive ELISA (enzyme-linked immuno sorbent assay) kit using anti-bilirubin monoclonal antibody 24G7, which is specific for bilirubin and its oxidized metabolite, biopyrrin.

(1) The HRP (Horseradish peroxidase)-labeled antibody is reacted with biopyrrin in the sample.

(2) The reaction solution is transferred to wells on which the antigen has been immobilized. Unreacted labeled antibody will bind to the wells via the antigen.

(3) Antibodies that do not bind to the wells are removed by washing.

(4) A chromogenic substrate is added, the absorbance is measured, and the biopyrrin concentration is calculated from the calibration curve.

◯ Urine

Dilute the sample at least 4xwith sample diluent.

• Yamaguchi, T., Shioji, I., Sugimoto, A., Komoda, Y., & Nakajima, H. Epitope of 24G7 anti- bilirubin monoclonal antibody. Biochimica et Biophysica Acta (BBA)-General Subjects, 1289 (1), 110-114. 1996

• Yamaguchi T.and Sugimoto A. Anti-oxidative effects of bilirubin as a low molecular antioxidant. Journal of Analytical Bio-Science, Vol.32, No 4. 2009

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