Kilty C, Doyle S, Hassett B, Manning F
Biotrin, Mount Merrion, Dublin, Ireland.
Chem Biol Interact. 1998 Apr 24;111-112:123-35. doi: 10.1016/s0009-2797(97)00156-7.
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.
胞质谷胱甘肽S-转移酶（GST）是理想的器官损伤生物标志物，因为它们具有许多所需的特性，即特定的定位、高胞质浓度和相对较短的半衰期。GST作为器官损伤早期指标的作用适用于人类和动物模型。由于GST不同同工型在肝脏和肾脏中的区域特异性定位，同时监测生物基质中的GST类别可以确定特定器官内的特定损伤区域。已经开发了免疫测定法来定量犬α-GST和大鼠微GST（Yb1）。这些免疫测定法是固相酶免疫测定法，其中样品或标准品中的GST被包被在固相上的特异性抗GST抗体捕获。洗涤后，加入特异性酶标记的IgG缀合物，它与捕获的GST结合。再经过一次洗涤步骤后，加入底物并显色。在酶标仪上测量吸光度，其与样品中GST的含量成正比。测定在室温下进行，3小时内即可完成。这些免疫测定法对每种GST具有特异性，检测范围为0-100微克/升。研究了一系列测定参数以验证用于GST检测的酶免疫测定法。这些测定法灵敏且可重复。Yb1测定法的批间和批内变异系数分别低于9%，犬α-GST酶免疫测定法低于20%。在标准曲线范围内加标的GST回收率分别为102%和99%。未观察到前带效应，且两种测定法中样品均表现出稀释线性。验证表明，使用这些酶免疫测定法，可以在生物基质、组织匀浆和细胞系中准确、精确地测量Yb1和犬α-GST，并且可以检测到GST水平的变化。这些测定法在体外和体内毒性研究中都有重要应用，其中GST是肝细胞和肾细胞完整性的敏感标志物。
