Anti-IFN-γ Antibody ELISA Protocol

Anti-IFN-γ antibody in sera was determined by indirect enzyme-linked immunosorbent assay (ELISA), as previously detailed [9] (link). Briefly, Maxisorb immunoplates (Nalge Nunc International, Penfield, NY, USA) were coated with 1 µg/mL of recombinant human IFN-γ in bicarbonate buffer (pH 9.6) and kept overnight at 4°C. Plates were blocked with phosphate-buffered saline (PBS) containing 1% non-fat milk powder at 37°C for 1 hour. Sera diluted 1∶1,000 were incubated for 1 hour at 37°C in duplicate. Plates were subsequently developed with anti-human IgG horseradish peroxidase conjugate (Caltag Laboratories, Invitrogen Life Technologies, Thermo Fisher Scientific, Paisley, UK) followed by o-phenylenediamine substrate (Sigma-Aldrich, St. Louis, MO, USA). The enzyme reaction was terminated with sulfuric acid (2N) and read for absorbance at 492 nm on a Spectra MR plate reader (Dynex Technologies, Chantilly, VA, USA).