A novel cell-free, non-fluorescent method to measure lox-1-binding activity corresponding to the functional activity of hdl

Akemi Kakino, Yoko Usami, Sayaka Horiuchi, Yoshiko Fujita, Kazuhiko Kotani, Chu Huang Chen, Tomonori Okamura, Tatsuya Sawamura

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Aims: 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. Methods: 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). Results: 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. Conclusion: We have developed a novel ELISA by using recombinant LOX-1 and anti-apoAI antibody to measure the activity of modified HDL in plasma.

Original languageEnglish
Pages (from-to)947-958
Number of pages12
JournalJournal of atherosclerosis and thrombosis
Volume26
Issue number11
DOIs
Publication statusPublished - 2019

Keywords

  • ELISA
  • HDL quality
  • LOX-1
  • Modified HDL

ASJC Scopus subject areas

  • Internal Medicine
  • Cardiology and Cardiovascular Medicine
  • Biochemistry, medical

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