There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions. The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples. AFTER MODIFICATION AND VALIDATION OF THE PROTOCOL FOR BOTH ANTIBODIES, SAMPLES OF TWO GROUPS WERE ANALYZED: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group.
Keywords: ACR, Acrolein; Antibodies; BSA, Bovine serum albumin; Cys, Cysteine; DEPC, Diethyl pyrocarbonate; ELISA; ELISA, Enzyme-linked immunosorbent assay; HCl, Hydrochloric acid; HNE; HNE, 4-Hydroxy-trans-2-nonenal; HPLC, High performance liquid chromatography; HRP, Horseradish peroxidase; His, Histidine; Human plasma; KLH, Keyhole limpet hemocyanin; LOD, limit of detection; LOQ, Limit of quantification; Lipid peroxidation; Lys, Lysine; MDA; MDA, Malondialdehyde; Obesity; Oxidative stress; PQL, Practical quantitation limit; PUFA, Polyunsaturated fatty acid; ROS, Reactive oxygen species; c-Ab, Commercial antibody; nc-Ab, Non-commercial antibody.