Jelic Marija, Jokesch Philipp, Oskolkova Olga, Faustmann Gernot, Winklhofer-Roob Brigitte M, Ullrich Bernd, Krauss Jürgen, Übelhart Rudolf, Gesslbauer Bernd, Bochkov Valery
Department of Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, University of Graz, 8010 Graz, Austria.
Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria.
Antioxidants (Basel). 2025 Jul 24;14(8):903. doi: 10.3390/antiox14080903.
Oxidized phospholipids (OxPLs) are increasingly recognized as biologically active lipids involved in various pathologies. Both exposure to pathogenic factors and the efficacy of protective mechanisms are critical to disease development. In this study, we characterized an immunoassay that quantified the total capacity of the plasma to degrade or mask OxPLs, thereby preventing their interaction with cells and soluble proteins. OxLDL-coated plates were first incubated with human blood plasma or a control vehicle, followed by an ELISA using a monoclonal antibody specific to oxidized phosphatidylethanolamine. Pretreatment with the diluted blood plasma markedly inhibited mAb binding. The masking assay was optimized by evaluating the buffer composition, the compatibility with various anticoagulants, potential interfering compounds, the kinetic parameters, pre-analytical stability, statistical robustness, and intra- and inter-individual variability. We propose that this masking assay provides a simple immunological approach to assessing protective mechanisms against lipid peroxidation products. Establishing this robust and reproducible method is essential for conducting clinical association studies that explore masking activity as a potential biomarker of the predisposition to a broad range of lipid-peroxidation-related diseases.
氧化磷脂（OxPLs）越来越被认为是参与各种病理过程的生物活性脂质。暴露于致病因素和保护机制的功效对疾病发展都至关重要。在本研究中，我们描述了一种免疫测定法，该方法可量化血浆降解或掩盖OxPLs的总能力，从而防止它们与细胞和可溶性蛋白质相互作用。首先将氧化低密度脂蛋白（OxLDL）包被的平板与人血浆或对照载体孵育，然后使用针对氧化磷脂酰乙醇胺的单克隆抗体进行酶联免疫吸附测定（ELISA）。用稀释的血浆预处理可显著抑制单克隆抗体的结合。通过评估缓冲液组成、与各种抗凝剂的兼容性、潜在干扰化合物、动力学参数、分析前稳定性、统计稳健性以及个体内和个体间变异性，对掩盖测定法进行了优化。我们认为，这种掩盖测定法提供了一种简单的免疫学方法来评估针对脂质过氧化产物的保护机制。建立这种稳健且可重复的方法对于开展临床关联研究至关重要，这些研究将掩盖活性作为广泛的脂质过氧化相关疾病易感性的潜在生物标志物进行探索。
