Hrp conjugated donkey anti mouse igg

Plasma was collected from fresh heparinized blood of each participant and stored at À80 C until analysis. Microtitre plates were coated with 100 mL of IFN-g (10 mg/mL; R&D Systems, Minneapolis, MN, USA) per well, incubated at 4 C overnight, and then washed three times with 200 mL of PBST (0.1% Tween 20/PBS, Sigma, St Louis, MO, USA). Recombinant mouse anti-human IFN-g antibodies (R&D Systems) were used as standards with serial dilution. Plasma was diluted 10 À5 times, and 100 mL of the diluent buffer (blank), samples, standards, positive and negative controls were added in duplicate to each well and incubated at room temperature for 3 hours. After washing three times with PBST, the standards were incubated with 100 mL of HRP-conjugated donkey anti-mouse IgG (Thermo Scientific, Rockford, IL, USA), while the patient plasma and control wells were incubated with 100 mL of HRP-conjugated sheep anti-human IgG (Thermo Scientific) for 1.5 hours. After three washes, 100 mL of TMB substrate (Thermo Scientific) was added to each well and incubated for 30 minutes, and 100 mL of stopping solution (Thermo Scientific) was added before determining the optical density at 450e550 nm. The normal range for the anti-IFN-g Abs concentration was defined by the 99th percentile for healthy volunteers. Concentrations higher than the normal range were classified as positive for anti-IFN-g Abs.