Anti-dsDNA IgG ELISA Assay

Serum samples were obtained following whole blood collection and coagulation, and stored at −20°C. Anti-double stranded (ds)DNA IgG levels in diluted serum samples was detected following a previously reported ELISA assay (12 (link)). Briefly, the plate was coated with 0.1 mg/ml of calf thymus DNA (Sigma) in 1× saline-sodium citrate buffer and incubated at 4°C overnight, followed by washing with PBS containing 0.05% Tween-20 (PBS-T). Wells were then blocked with PBS containing 1% BSA for 1 h at room temperature. Samples were added and incubated for 1 h at room temperature. After additional washes in PBS-T, horseradish peroxidase-conjugated goat anti-mouse IgG-Fc secondary antibody (Bethyl Laboratories, Montgomery, TX) was added and incubated for 1 h at room temperature, followed by further washing. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Biolegend) was then added, and the reaction was stopped by adding 2N H₂SO₄ stop solution. The plate was read at 450 nm using SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). Total IgG levels were determined using a mouse IgG ELISA kit (Bethyl Laboratories) according to the manufacturer's instructions. Serum endotoxin (lipopolysaccharide, or LPS) level was measured using Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific) following the manufacturer's procedures.