Quantifying Oxidized Apolipoprotein A1 by ELISA

Nunc MaxSorp ELISA plates (#446612, Thermo Fisher Scientific, Waltham, MA) were coated with the indicated purified native or oxidized protein (0.2 μg ml⁻¹) in Carbonate and Bicarbonate buffer, pH 9.6. Purified human apoA1, rh-apoA1 and mutant apoA1’s were modified by either the MPO/H₂O₂/Cl⁻ system, the MPO/H₂O₂/NO₂⁻ system or by multiple other oxidation systems as indicated at 10:1 molar ratio of oxidants/apoA1, unless otherwise specified, details of oxidation conditions are described in reference 25 (link). Primary antibody r8B5.2 (50 ng ml⁻¹) (Fig. 1d and Fig. 2a,e) was added into the 5% BSA blocked ELISA wells and incubated for 1 hr at room temperature. The plates were then washed with PBS, 0.05% Tween 20 (PBST) 4 times. The signal was detected by Peroxidase AffiniPure Goat Anti-Mouse IgG, F(ab’)2 Fragment Specific (1:10,000 dilution, Code # 115-036-006, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA)