Oxiselect hne adduct competitive elisa kit

The quantity of hydroxynonenal (HNE) modified proteins in mouse serum was measured using commercially available OxiSelect™ HNE Adduct Competitive ELISA Kit (Cell Biolabs, Inc., USA) according to the manufacturer protocol. Shortly HNE-BSA standard and mouse serum samples were added to HNE conjugate coated ELISA plates. After incubation, an anti-HNE polyclonal antibodies were added, followed with an HRP-conjugated secondary antibodies. Substrate Solution was added and the enzymatic reaction was stopped by Stop Solution. The absorbance of each well was measured immediately at λ = 450 nm.

Levels of HNE–protein adducts in liver lysates prepared in RIPA buffer were measured in livers of mice using the OxiSelect HNE Adduct Competitive ELISA kit (Cell Biolabs) as formerly described^{75 (link)}.

Plasma samples were produced by centrifuging EDTA-blood at 2500 RPM for 5 minutes at 4°C. Plasma levels of 3-nitrotyrosine (3-NT), 4-hydroxynonenal (4-HNE), and 8-hydroxydeoxyguanosine (8-OHdG) were assessed by enzyme-linked immunosorbent assays (ELISA; OxiSelect™ Nitrotyrosine ELISA kit, OxiSelect™ HNE Adduct Competitive ELISA kit and OxiSelect™ oxidative DNA Damage ELISA kit, Cell Biolabs, Inc. San Diego, CA, USA) following the manufacturer’s instructions and previously reported methodologies [28 (link),29 (link)]. Briefly, 50 μL of each sample were added to designated wells and incubated at room temperature for 10 minutes. These samples were then incubated with an additional 50 μL of diluted primary antibody at room temperature for one hour. Thereafter, samples were washed with a wash buffer and incubated with 100 μL of diluted secondary antibody-enzyme conjugate for an additional hour. Finally, the samples were washed several times with a wash buffer and incubated with 100 μL of the substrate solutions for 30 minutes in the dark. The enzyme reaction was stopped by adding 100 μL of a stop solution. A standard curve was always generated for each assay run. Absorbancies were read in a microplate reader (infinite M200, TECAN, Switzerland) at 450 nm using a reference filter of 655 nm.