Department of Molecular Pathophysiology, School of Medicine, Shinshu University.
Institute for Biomedical Sciences, Shinshu University.
J Atheroscler Thromb. 2019 Nov 1;26(11):947-958. doi: 10.5551/jat.47183. Epub 2019 Apr 3.
A functional abnormality in high-density lipoprotein (HDL) particles rather than a quantitative abnormality in HDL cholesterol levels has been suggested to promote atherosclerosis. The modification of HDL may underlie functional changes to HDL such as gaining the ability to bind and activate the lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1). We aimed to develop a novel method for measuring modified HDL on the basis of its binding to LOX-1.
We designed a LOX-1 binding-based enzyme-linked immunosorbent assay (ELISA) with recombinant LOX-1 and anti-apoAI antibody. A lipid-free standard was devised by making a chimeric fusion protein containing anti-LOX-1 antibody and human apoAI fragment. We used this system to detect modified HDL, designated as LOX-1 ligand containing apoAI (LAA).
With our ELISA system, we detected HDL modified by copper oxidation, hypochlorous acid, 4-hydroxynonenal, and potassium cyanate, but not native HDL. Upon oxidation, HDL showed increased LOX-1 binding activity and decreased cholesterol efflux and paraoxonase-1 activities. In the ELISA, the chimeric fusion protein standard showed minimal variation in reference binding curves in contrast to copper-oxidized HDL preparations, suggesting better quality control of the chimeric fusion protein as the standard for measuring modified HDL activity. LAA was detectable in the plasma of healthy individuals and of mice fed a high-fat diet.
We have developed a novel ELISA by using recombinant LOX-1 and anti-apoAI antibody to measure the activity of modified HDL in plasma.
高密度脂蛋白（HDL）颗粒的功能异常而不是 HDL 胆固醇水平的定量异常被认为可促进动脉粥样硬化。HDL 的修饰可能是 HDL 功能变化的基础，例如获得与凝集素样氧化型低密度脂蛋白（LDL）受体-1（LOX-1）结合和激活的能力。我们旨在基于其与 LOX-1 的结合开发一种测量修饰型 HDL 的新方法。
我们设计了一种基于 LOX-1 结合的酶联免疫吸附测定（ELISA），使用重组 LOX-1 和抗载脂蛋白 AI（apoAI）抗体。通过制作包含抗 LOX-1 抗体和人 apoAI 片段的嵌合融合蛋白，设计了一种无脂质标准。我们使用该系统检测修饰型 HDL，命名为载脂蛋白 AI 包含 LOX-1 配体（LAA）。
使用我们的 ELISA 系统，我们检测到了铜氧化、次氯酸、4-羟基壬烯醛和氰酸钾修饰的 HDL，但未检测到天然 HDL。HDL 氧化后，LOX-1 结合活性增加，胆固醇流出和对氧磷酶-1 活性降低。在 ELISA 中，与铜氧化的 HDL 制剂相比，嵌合融合蛋白标准的参考结合曲线变化最小，表明嵌合融合蛋白作为测量修饰型 HDL 活性的标准具有更好的质量控制。在健康个体和高脂饮食喂养的小鼠的血浆中可检测到 LAA。
我们使用重组 LOX-1 和抗 apoAI 抗体开发了一种新型 ELISA，用于测量血浆中修饰型 HDL 的活性。
