Phosphatase substrate p nitrophanylphosphate disodium

To measure Ig isotypes and subclasses in plasma, the Antibody Isotyping 7-Plex Mouse ProcartaPlex™ Panel was performed with a MAGPIX luminex reader (ThermoFisher). For total IgG and IgM quantification, single ELISA kits were used (ThermoFisher). Anti-MDA specific IgGs were measured by coating MaxiSorp plates (NuncTM, city, Roskilde, Denmark) with purified, mouse-derived delipidated apolipoprotein MDA for 1 h at 37° C. After being washed, all wells were blocked for 1 h with 2% BSA in a PBS at 37° C. Mouse samples were also added to a non-coated well in order to assess individual non-specific binding. After washing, 50 μl/well of the alkaline phosphatase-conjugated anti-mouse IgG was added (Sigma-Aldrich), it was diluted at 1:1000 in a PBS/BSA 2% solution, and this was added and incubated for 1 h at 37° C. After washing, phosphatase substrate p-nitrophanylphosphate disodium (Sigma-Aldrich) dissolved in a diethanolamine buffer (pH 9.8) was added and incubated for 30 min at 37° C. Optical density (OD) was determined at 405 nm (Filtermax 3, Molecular DevicesTM, San Jose, CA) and each sample was tested in duplicate. Corresponding non-specific binding was subtracted from mean OD for each sample.