Glutathione S-transferases as biomarkers of organ damage: applications of rodent and canine GST enzyme immunoassays

Abstract

The cytosolic glutathione S-transferase (GST) enzymes serve as ideal biomarkers of organ damage as they exhibit many of the required characteristics, i.e. specific localisation, high cytosolic concentration and relatively short half-life. The role of GSTs as early indicators of organ damage is applicable to both human and animal models. Because of the regio-specific localisation of the different isoforms of GST in liver and kidney, simultaneous monitoring of classes of GSTs in biological matrices permits the identification of specific areas of damage within a particular organ. Immunoassays have been developed which quantify canine alpha GST and roden microGST (Yb1). The immunoassays are solid phase EIAs, where GST in the sample or standard is captured by a specific anti-GST antibody coated onto the solid phase. After washing, a specific enzyme-labelled IgG conjugate is added which binds to the captured GST. After a further washing step, substrate is added and a colour developed. The absorbance is measured on an ELISA plate reader and is directly proportional to the amount of GST present in the sample. The assays are performed at room temperature and can be completed within 3 h. The immunoassays are specific for each GST and have a range of 0-100 micrograms/l. A range of assay parameters were investigated to validate the EIAs for GST detection. The assays are sensitive and reproducible. CV for inter- and intra-assay variation were below 9% for Yb1 assay and below 20% for the canine alpha GST EIA. Recovery of spiked GST over the standard curve range was 102 and 99%, respectively. No prozone effect was observed and samples exhibited linearity of dilution in both assays. Validation has shown that using these enzyme immunoassay, Yb1 and canine alpha GST can be measured accurately and precisely in biological matrices, tissue homogenates and cell lines and that changes in GST levels can be detected. The use of these assays have important applications in both in vitro and in vivo toxicity studies, where GST's serve as sensitive marker of hepatocellular and renal cell integrity.