GLUTATHIONE S-TRANSFERASES AS BIOMARKERS OF ORGAN DAMAGE - APPLICATIONS OF RODENT AND CANINE GST ENZYME IMMUNOASSAYS

The cytosolic glutathione S-transferase (GST) enzymes serve as ideal b iomarkers of organ damage as they exhibit many of the required charact eristics, i.e. specific localisation, high cytosolic concentration and relatively short half-life. The role of GSTs as early indicators of o rgan damage is applicable to both human and animal models. Because of the regio-specific localisation of the different isoforms of GST in li ver and kidney, simultaneous monitoring of classes of GSTs in biologic al matrices permits the identification of specific areas of damage wit hin a particular organ. Immunoassays have been developed which quantif y canine alpha GST and rodent mu GST (Yb1). The immunoassays are solid phase EIAs, where GST in the sample or standard is captured by a spec ific anti-GST antibody coated onto the solid phase. After washing, a s pecific enzyme-labelled IgG conjugate is added which binds to the capt ured GST. After a further washing step, substrate is added and a colou r developed. The absorbance is measured on an ELISA plate reader and i s directly proportional Co the amount of GST present in the sample. Th e 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 mu g/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 ove r the standard curve range was 102 and 99%, respectively. No prozone e ffect was observed and samples exhibited linearity of dilution in both assays. Validation has shown that using these enzyme immunoassay, Yb1 and canine aGST can be measured accurately and precisely in biologica l matrices, tissue homogenates and cell lines and that changes in GST levels can be detected. The use of these assays have important applica tions in both in vitro and in vivo toxicity studies, where GSTs serve as sensitive marker of hepatocellular and renal cell integrity. (C) 19 98 Elsevier Science Ireland Ltd. All rights reserved.