Development of enzyme-linked immunosorbent assay for 8-iso-prostaglandin F2α, a biomarker of oxidative stress in vivo, and its application to the quantification in aged rats
Introduction
A series of prostaglandin F2-like compounds termed F2-isoprostanes are known to be produced in vivo nonenzymatically from arachidonic acid, an essential nutrient, in animals and humans predominantly by a free radical-induced lipid peroxidation [1], [2], [3]. A large body of evidence has suggested that the measurement of F2-isoprostanes is a reliable and useful approach to assess lipid peroxidation and oxidative stress in vivo [4]. Of these, the levels of 8-iso-prostaglandin F2α (8-iso-PGF2α) have been studied more extensively as a biomarker of oxidative marker in vivo until now. The majority of arachidonic acid is esterified to the sn-2 position of phospholipids in cell membranes of various tissues or blood cells. Earlier studies have shown that F2-isoprostanes including 8-iso-PGF2α are initially generated in situ from arachidonic acid esterified in phospholipids under the oxidative stress and subsequently released as a free form by the action of phospholipases [2]. Thus, 8-iso-PGF2α can be detectable as both free and esterified forms. The free form of 8-iso-PGF2α can be quantified to evaluate total generation of it in vivo that reflects the enhanced systemic lipid peroxidation. By contrast, the measurement of 8-iso-PGF2α esterified in phospholipids in tissues would provide the methods for assessing the oxidative injury in specific cells, tissues, or organs. For example, the administration of CCl4 to animals has been shown to cause the marked elevation of circulating levels of F2-isoprostanes [1], [2]. Alternatively, higher levels of F2-isoprostanes have been recognized in plasma of smokers due to free radicals in cigarette smoke, whereas the intake of antioxidants such as vitamin C decreased the plasma levels of F2-isoprostanes as biomarkers of lipid peroxidation [5]. Hence, the development of a sensitive and specific assay for 8-iso-PGF2α should be highly useful as a noninvasive approach by analyzing the levels in plasma and urine to assess the involvement of oxidative stress and lipid peroxidation in human or animal disease from the point of prognostic diagnosis and the evaluation of antioxidants in pharmaceuticals and nutraceuticals.
Until now, most studies have employed gas chromatography–mass spectrometry (GC–MS) to quantify the levels of 8-iso-PGF2α as a more sensitive and specific way [3], [6]. However, the mass spectrometric analysis requires high-cost apparatus and an analytical expert to operate the quantitative analysis. Additionally, the GC–MS analysis requires extensive sample preparations and derivatization of analytes. Moreover, the use of a deuterium-labeled internal standard required for the quantification with mass spectrometric quantification becomes more expensive for the analysis of multiple samples. As an alternative to GC–MS, liquid chromatography–mass spectrometry (LC–MS) is becoming useful for the analysis and quantification of analytes in clinical laboratories. However, many methods still require extensive sample purification prior to quantitative analysis. More recently, Saenger et al. has reported an improved assay for the measurement of urinary 8-iso-PGF2α by LC–MS without sample pretreatment [7]. Recent advances have been reviewed regarding measurement of oxidative stress parameters using LC–MS [8]. Nevertheless, this mass spectrometric approach is also limited by smaller numbers of analytes assayed from biological sources due to the sequential operation of expensive instrumentation as compared with immunological methods. Therefore, we planed to develop a convenient and sensitive immobilized enzyme-linked immunosorbent assay (ELISA) specific for 8-iso-PGF2α. This immunological method is applicable for many samples at the same time without high-cost facilities and an analytical expert. The present study was undertaken to prepare monoclonal antibodies specific for 8-iso-PGF2α. Then, using the specific monoclonal antibody, we attempted to develop the immobilized ELISA for 8-iso-PGF2α. Finally, we applied our method to the quantification of 8-iso-PGF2α free in plasma and esterified in plasma lipids as well as the form esterified in tissues lipids from liver and kidney in young and old rats.
Section snippets
Materials
8-iso-PGF2α, other related isoprostanes, and PGF2α were obtained from Cayman Chemical (Ann Arbor, MI, USA). Biotin-conjugated rabbit anti-mouse IgG (H + L) antibody was the product of Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Essentially fatty acid-free bovine serum albumin (BSA), bovine γ-globulin, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl, N-hydroxysuccinimide, ExtrAvidin-peroxidase conjugate, Dulbecco's modified Eagle's medium with 25 mM HEPES, penicillin G, and
Development of immobilized ELISA for 8-iso-PGF2α using a specific monoclonal antibody
A sensitive standard curve of ELISA for 8-iso-PGF2α was generated using a monoclonal antibody secreted by a hybridoma clone called 3B10-10G8-5H10. This monoclonal antibody was classified as IgG1 having isotypes of γ1 heavy chain and κ light chain. The typical standard curve enabled us to quantify the amount from 0.23 pg to 98.4 pg in each 96-well dish, corresponding to those of free 8-iso-PGF2α required to displace 10–90% of the maximal binding of the immobilized antigen to the used monoclonal
Conclusion
We developed an immobilized ELISA for 8-iso-PGF2α, an isomer of F2-isoprostanes that are generated by the free radical-induced peroxidation of arachidonic acid. The quantification of 8-iso-PGF2α has been regarded as a biomarker of oxidative stress in vivo. Our ELISA method was demonstrated to be highly sensitive and specific for 8-iso-PGF2α, allowing the determination of a number of samples at the same time. As well as sensitivity and specificity, the utility of our ELISA for the quantification
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research (C) 14560099 from the Japan Society for the Promotion of Science and grants from Shimane University. In addition, the present research was also supported by the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN).
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