Quantification of MPO-DNA Complexes via ELISA

An in-house enzyme-linked immunosorbent assay (ELISA) was used to quantify MPO-DNA complexes [16 (link)]. Briefly, after overnight coating with anti-MPO antibody (2 µg/mL; 0400-0002, Bio-Rad, Hercules, CA, USA) at 4 °C, a 96-well plate was blocked with 2.5% bovine serum albumin in phosphate-buffered solution for 2 h at room temperature. The plate was subsequently washed before incubating for 90 min at room temperature with 20% plasma isolated from the NASH in blocking buffer. The plate was washed five times and then incubated for 90 min at room temperature with an anti-DNA antibody (1:10; Cell Death Detection ELISA, 11544675001, Sigma, St. Louis, MO, USA). After five washes, the plate was developed with an ABTS substrate (Sigma).