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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 |