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TECO® αGST is an ELISA for the quantitative determination of alpha Glutathione S-Transferase (αGST) in human serum, plasma or urine.

αGST  is a metabolic enzyme involved with cellular detoxification reactions. It is approximately 51kDa and it comprises ~5% of soluble cytosolic protein in hepatocytes. Upon cellular damage, αGST is rapidly released from cells. An increased concentration in blood is a sensitive indicator of acute hepatic injury. Studies have shown that increased serum or plasma αGST can be indicative of hepatotoxicity, allograft rejection, or viral hepatitis.

In the kidney,  αGST comprises ~2% of the cytosolic soluble protein content in the epithelial cells of the proximal tubule. Like with hepatocytes, it is rapidly released upon damage to the cells. Increased urine concentrations are indicative of acute kidney injury.

  • Unlike with other toxicity biomarkers, αGST has not been shown to increase with muscle damage, hemolysis or general inflammation (i.e. Rheumatoid arthritis).
  • αGST is rapidly released upon cellular damage and thus useful as an early indicator of acute liver (or kidney) damage in research settings.
  • Suitable to use with the M30 Apoptosense® ELISA (apoptosis detection), M65® ELISA (necrosis detection), and TECO® Hyaluronic Acid ELISA (chronic, fibrosis detection) as a panel of research assays useful for assessing liver damage.
  • Applications for cell culture and culture spheroids.
  • Sandwich ELISA with a 96-well plate in a convenient, ready-to-use format.
  • Easy to perform assay with minimal pipetting steps.
  • Antibody Coated Microassay Plate: 96 well (12×8 break-apart well strips coated with IgG directed against αGST)
  • Calibrator, 0.2mL (2mg/L): Purified αGST in stabilizing diluent containing ProClin 950 and Bronidox L as preservatives. 25x Concentrate.
  • Sample Diluent, 30mL: Protein containing solution with added stabilizers and ProClin 950 and Bronidox L as preservatives.
  • Wash Buffer, 45mL: Tris-buffered saline/Tween-20 (TBST) containing ProClin 950 as preservative. 25x Concentrate.
  • Positive Control, 1.5mL: Purified αGST in stabilizing diluent containing ProClin 950 and Bronidox L as preservatives.
  • Enzyme Conjugate, 11mL: Antibody solution containing anti-αGST IgG labelled with horseradish peroxidase and ProClin 950 and Bronidox L as preservatives.
  • Substrate, 11mL: Stabilized liquid TMB solution.
  • Stop Solution, 11mL: 0.5M Sulphuric Acid
  • Urine Stabilising Buffer (USB), 10mL: Protein containing solution with added stabilizers and ProClin 950 and Bronidox L as preservatives.

The α-GST assay is a standard sandwich ELISA. The 96-well microplate is coated with anti-αGST IgG. Sample added to the wells for an incubation binds to the microwell-coated antibody.  Addition of antibody-enzyme conjugate binds in a sandwich fashion to samples containing alpha-GST and then TMB substrate allows for colorimetric determination of αGST concentration. The color intensity is directly proprotional to the amount of αGST in a sample and the assay range is 2.5 – 80 μg/L.

 

 

TE1051_Rev04-2013_TECO_EKFAlphaGST-ELISA-InstructionsForUse

Typical calibration curve obtained using the Alpha GST EIA. 4-parameter plot of A450/630nm versus [alphaGST] μg/L. Assay range is 2.5 – 80 μg/L alphaGST.

Alpha GST ELISA Assay measurement test kitIn liver,  αGST is located in the hepatocytes whereas pi GST is found in the intrahepatic bile duct cells. This heterogeneous GST subclass distribution suggests that the isoenzymes have unique in vivo functions in different hepatic regions and that the detection of GST subclass levels in biological fluids could be of significant use in monitoring the integrity of specific hepatic regions. Currently, liver injury is studied by the measurement of liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). A disadvantage of these markers is that they are not distributed uniformly throughout the liver and that their periportal concentrations are greater than their centrilobular concentrations. In contrast, αGST has been found to be equally distributed in both the centrilobular and periportal regions of the liver. Since the centrilobular hepatocytes are very susceptible to damage in a variety of conditions including allograft rejection, viral hepatitis, and hepatotoxicity, αGST is a more sensitive indicator of hepatic injury. The Alpha GST EIA is a specific, precise immunoassay  and, being a quantitative test,  it is not affected by modulators of enzyme activity such as bile salts and bilirubin.

In kidney, αGST is found in the proximal tubule region. In healthy individuals, low levels of αGST are released into the urine as confirmed by immunoassay and Western blot analyses. Events that lead to proximal tubular damage often cause increased release of αGST into urine. Elevated urinary αGST levels have been shown to indicate proximal tubule damage due to nephrotoxicity, environmental toxicity, surgery, acute renal failure and transplantation.

αGST is often described as a “leakage” biomarker since it is rapidly released from damaged cells. Though intended for research use only in the US and Canada, the Alpha GST EIA can be a useful indicator of acute liver (or kidney) damage and is thus a useful research assay for assessing toxicity. The TECO Alpha GST EIA can also be used with human liver cell culture samples, providing an application for in vitro drug screening.