Ab194225

The immunoreactivity to malondialdehyde (MDA) was measured by duplex ELISA [27 (link)]. Frontal lobe homogenates (50 ng/50 μl in 0.1 M carbonate-bicarbonate buffer, pH 9.6) were adsorbed onto the bottoms of 96-well MaxiSorp plates by overnight incubation at 4°C. Nonspecific binding was blocked with 1% bovine serum albumin (BSA) in TRIS buffered saline (TBS). After rinsing with TBS + 0.05% Tween 20, the samples were incubated overnight at 4°C with a cocktail of primary antibodies to MDA, including 0.0125 μg/ml of ab194225 and 0.02 μg/ml of ab27642 (Abcam, Cambridge, MA) to detect protein-acetaldehyde adducts [28 (link)]. Immunoreactivity was detected with horseradish peroxidase (HRP)-conjugated secondary antibody and Amplex UltraRed soluble fluorophore. Fluorescence was measured (Ex 530 nm/Em 590 nm) in a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). Results were normalized to ribosomal protein (RPLPO) detected in the same samples using biotinylated anti-RPLPO, alkaline phosphatase-conjugated streptavidin, and 4-methylumbelliferyl phosphate (4-MUP) as substrate. Fluorescence intensities (Ex 360/Em 450) were measured in a SpectraMax M5. The MDA/RPLPO ratios of immunoreactivity were used for statistical analysis. Negative control assays included incubations with primary, secondary or both antibodies omitted.