Mouse isotyping panel 1 kit

Antibody isotype concentrations were detected using a 1:10,000 dilution of serum with a Mouse isotyping panel 1 kit (Meso Scale Diagnostics). Values for mouse IgG2a were not reported due to the C57BL/6J background containing the IgG2c allotype. Additionally, values for IgG3 and IgE were below detectable levels. Lipid-specific antibody levels were detected by coating polystyrene 96-well plates (Biolegend) with antigen containing 1 µg/mL of human LDL, MDA-modified human LDL (Academy Bio-Medical Co), APOB100 (Abcam), or MDA-modified APOB100 in coating buffer (100 µL/well; 35 mmol/L sodium carbonate, 68 mmol/L sodium bicarbonate) and incubated overnight at 4°C. Serum antibodies were analyzed at a dilution of 1:1000 for IgM and 1:200 for IgG1 diluted in 5% BSA and 1x PBS. Detection antibodies (goat anti-mouse IgM-HRP or goat anti-mouse IgG1-HRP, Southern Biotech) were added at 1:10,000 dilution, and signal was detected with TMB peroxidase substrate (Vector Labs). oxLDL serum levels were measured using a 1:20 dilution of serum with the mouse oxidized low density lipoprotein ELISA kit (Mybiosource) per manufacture’s recommendation. Plates were read using a SpectraMax M2 plate reader (Molecular Devices) at 450 nm.

Fecal and plasma murine immunoglobulin levels were determined using the Mouse Isotyping Panel 1 Kit (Meso Scale Discovery, MD, USA). Fecal pellets were vortexed in 1 mL of PBS for 10 min, centrifuged at 12000 × g for 10 min twice, and supernatant collected for Ig level determination. Plasma was diluted at 1:10,000 and fecal supernatant was diluted at 1:100 for analysis. Fecal Ig levels were normalized per mg of total fecal protein content, as determined by Bradford Protein Assay (Bio-Rad, CA, USA). Plates were read using a MESO QuickPlex SQ 120, and analyzed using the MSD workbench software.