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Original

Enzyme-linked immunosorbent assay for 4-hydroxynonenal–histidine conjugates

, , &
Pages 809-820 | Received 01 Feb 2006, Published online: 07 Jul 2009
 

Abstract

Highly reactive aldehyde 4-hydroxynonenal (HNE) is the final product of lipid peroxidation, known as a second messenger of free radicals and a signaling molecule. It forms protein conjugates involved in pathology of various diseases. To determine cellular HNE–protein conjugates we developed indirect ELISA based on well-known, monoclonal antibody against HNE–histidine (HNE–His) adducts. The method was calibrated using HNE–albumin conjugates as standards (R2 = 0.999) and validated on human osteosarcoma cell cultures (HOS). The ELISA showed good sensitivity (8.1 pmol HNE–His/mg of protein), precision ( ± 8% intra-assay and ± 12% inter-assay) and spiking recovery ( ± 9%). The assay revealed 60-fold increase of cellular HNE–His adducts upon copper-induced lipid peroxidation of HOS. The ELISA matched HNE-immunocytochemistry of HNE-treated HOS cells and quantified the increase of cellular HNE–His conjugates in parallel to the decrease of free HNE in culture medium. The ELISA was developed as ELISA Stress for severe lipid peroxidation and ELISA Fine for studies on HNE physiology.

Abbreviations
BSA=

bovine serum albumin

DAB=

3,3′-diaminobenzidine tetrahydrochloride

ddH2O=

double-distilled water

DMEM=

Dulbecco's Modified Eagle's Medium

ELISA=

enzyme-linked immunosorbent assay

FCS=

fetal calf serum

His=

histidine

HNE=

4-hydroxynonenal

HPLC=

high performance liquid chromatography

HRP=

horseradish peroxidase

MDA=

malondialdehyde

PBS=

phosphate-buffered saline

PMSF=

phenylmethylsulphonylfluoride

ROS=

reactive oxygen species

TBARS=

thiobarbituric acid reactive substances

TMB=

3,3,5,5-tetramethylbenzidine

Abbreviations
BSA=

bovine serum albumin

DAB=

3,3′-diaminobenzidine tetrahydrochloride

ddH2O=

double-distilled water

DMEM=

Dulbecco's Modified Eagle's Medium

ELISA=

enzyme-linked immunosorbent assay

FCS=

fetal calf serum

His=

histidine

HNE=

4-hydroxynonenal

HPLC=

high performance liquid chromatography

HRP=

horseradish peroxidase

MDA=

malondialdehyde

PBS=

phosphate-buffered saline

PMSF=

phenylmethylsulphonylfluoride

ROS=

reactive oxygen species

TBARS=

thiobarbituric acid reactive substances

TMB=

3,3,5,5-tetramethylbenzidine

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