Hexanoyl Lysine ELISA

Oxidative damage to lipids (lipid peroxidation) has been found to play an important role in various disease and aging processes. During early stages of lipid peroxidation, lipid hydroperoxides (LOOH) are formed. These can react additionally to form later stage end products such as malondialdehyde (MDA) and hydroxynonenal (HNE). LOOH is sometimes measured to quantify early stage or acute lipid peroxidation while MDA is commonly measured to quantify late stage or chronic lipid peroxidation. More recently, it has been reported that 13-hydroperoxyoctadecanoic acid (13-HPODE), a precursor to 13-hydroxyoctadecanoic acid (13-HODE) can react with proteins to form measurable adducts by covalently binding to specific amino acid residues. The Hexanoyl-Lysine (HEL) adduct is formed upon oxidative modification of omega-6 fatty acids such as linoleic acid, the predominant polyunsaturated fatty acid (PUFA) in the human diet, and arachidonic acid. HEL may be another useful biomarker for detecting and quantifying the earlier stages of lipid peroxidation.

Introduction

Oxidative damage to lipids (lipid peroxidation) has been found to play an important role in various disease and aging processes. During early stages of lipid peroxidation, lipid hydroperoxides (LOOH) are formed. These can react additionally to form later stage end products such as malondialdehyde (MDA) and hydroxynonenal (HNE). LOOH is sometimes measured to quantify early stage or acute lipid peroxidation while MDA is commonly measured to quantify late stage or chronic lipid peroxidation. More recently, it has been reported that 13-hydroperoxyoctadecanoic acid (13-HPODE), a precursor to 13-hydroxyoctadecanoic acid (13-HODE) can react with proteins to form measurable adducts by covalently binding to specific amino acid residues. The Hexanoyl-Lysine (HEL) adduct is formed upon oxidative modification of omega-6 fatty acids such as linoleic acid, the predominant polyunsaturated fatty acid (PUFA) in the human diet, and arachidonic acid. HEL may be another useful biomarker for detecting the earlier stages of lipid peroxidation.

Summary of Test

The NWLSS™ Hexanoyl-Lysine ELISA is a competitive enzyme-linked immuno-assay wherein a murine monoclonal antibody to HEL (Primary Antibody) and sample or standard are added to a microtiter plate which has been precoated with HEL. Sample or calibrator HEL competes with plate-bound HEL for binding with the antibody. Accordingly, higher concentrations of sample or calibrator leads to reduced binding of the anti-HEL antibody to the HEL coated plate. A subsequent wash step removes free HEL/antibody adduct leaving stationary plate bound HEL complexed to antibody for later detection. Anti-murine IgG antibody conjugated to horse radish peroxidase (HRP-Conjugate) is then added to the plate. HRP-conjugate binds to remaining murine anti-8-OHdG and unbound HRP-conjugate is removed in another wash step. Addition of 3,3',5,5' tetramethylbenzidine (TMB Substrate) results in blue color development proportional to the amount of anti-HEL antibody bound to the plate and inversely proportional to the concentration HEL in samples or calibrators originally applied to the plate. The reaction is terminated by addition of phosphoric acid (Stop Solution) producing yellow color with measurable absorbance at 450 nm.