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

1 Citation (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 Jan 1

Fingerprint

HDL Lipoproteins
Immunosorbents
Assays
Enzyme-Linked Immunosorbent Assay
Fusion reactions
Enzymes
Antibodies
Copper
Anti-Idiotypic Antibodies
Class E Scavenger Receptors
Ligands
Aryldialkylphosphatase
Plasmas
Hypochlorous Acid
Oxidation
Proteins
High Fat Diet
Nutrition
Quality Control
HDL Cholesterol

Keywords

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

ASJC Scopus subject areas

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

Cite this

A novel cell-free, non-fluorescent method to measure lox-1-binding activity corresponding to the functional activity of hdl. / Kakino, Akemi; Usami, Yoko; Horiuchi, Sayaka; Fujita, Yoshiko; Kotani, Kazuhiko; Chen, Chu Huang; Okamura, Tomonori; Sawamura, Tatsuya.

In: Journal of atherosclerosis and thrombosis, Vol. 26, No. 11, 01.01.2019, p. 947-958.

Research output: Contribution to journalArticle

Kakino, Akemi ; Usami, Yoko ; Horiuchi, Sayaka ; Fujita, Yoshiko ; Kotani, Kazuhiko ; Chen, Chu Huang ; Okamura, Tomonori ; Sawamura, Tatsuya. / A novel cell-free, non-fluorescent method to measure lox-1-binding activity corresponding to the functional activity of hdl. In: Journal of atherosclerosis and thrombosis. 2019 ; Vol. 26, No. 11. pp. 947-958.
@article{312579e13901476eb6e58dd0294e377f,
title = "A novel cell-free, non-fluorescent method to measure lox-1-binding activity corresponding to the functional activity of hdl",
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.",
keywords = "ELISA, HDL quality, LOX-1, Modified HDL",
author = "Akemi Kakino and Yoko Usami and Sayaka Horiuchi and Yoshiko Fujita and Kazuhiko Kotani and Chen, {Chu Huang} and Tomonori Okamura and Tatsuya Sawamura",
year = "2019",
month = "1",
day = "1",
doi = "10.5551/jat.47183",
language = "English",
volume = "26",
pages = "947--958",
journal = "Journal of Atherosclerosis and Thrombosis",
issn = "1340-3478",
publisher = "Japan Atherosclerosis Society",
number = "11",

}

TY - JOUR

T1 - A novel cell-free, non-fluorescent method to measure lox-1-binding activity corresponding to the functional activity of hdl

AU - Kakino, Akemi

AU - Usami, Yoko

AU - Horiuchi, Sayaka

AU - Fujita, Yoshiko

AU - Kotani, Kazuhiko

AU - Chen, Chu Huang

AU - Okamura, Tomonori

AU - Sawamura, Tatsuya

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - ELISA

KW - HDL quality

KW - LOX-1

KW - Modified HDL

UR - http://www.scopus.com/inward/record.url?scp=85074516339&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85074516339&partnerID=8YFLogxK

U2 - 10.5551/jat.47183

DO - 10.5551/jat.47183

M3 - Article

C2 - 30944265

AN - SCOPUS:85074516339

VL - 26

SP - 947

EP - 958

JO - Journal of Atherosclerosis and Thrombosis

JF - Journal of Atherosclerosis and Thrombosis

SN - 1340-3478

IS - 11

ER -