Enzyme-linked immunosorbent assay for 4-hydroxynonenal-histidine conjugates.
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 (R(2) = 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.
Borovic
Suzana
S
Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia. borovic@irb.hr
Rabuzin
Filip
F
Waeg
Georg
G
Zarkovic
Neven
N
D000447:Aldehydes / Q000494:pharmacology*; D002138:Calibration; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005609:Free Radicals; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007150:Immunohistochemistry; D015227:Lipid Peroxidation; D018384:Oxidative Stress; D011485:Protein Binding; D017382:Reactive Oxygen Species; D012680:Sensitivity and Specificity
D000447:Aldehydes; D003432:Cross-Linking Reagents; D005609:Free Radicals; D017382:Reactive Oxygen Species; D006639:Histidine; C027576:4-hydroxy-2-nonenal