*3.8. Cytokine Measurements by ELISA*

Capture antibodies (50 ΐL of 1 mg/mL in 0.05 M sodium carbonate, pH 9.6) were added to coat each well of Immuno 96-microwell plates (9018, Corning Costar, Ithaca, NY, USA) and incubated overnight at 4 °C. After washing wells twice with 0.15 M sodium phosphate buffer, phosphate buffered saline (PBS), pH 7.2, containing 0.05% Tween 20 (PBS-T), blocking buffer (PBS containing 0.05% Tween 20 and 0.5% bovine serum albumin (BSA)) was added and incubated for 30 min at room temperature. After removing blocking buffer, diluted sera (5 times with blocking buffer) were added and incubated for 2 h at room temperature or at 37 °C. After washing wells with PBS-T three times, 50 ΐL of 0.5 ΐg/mL biotin-coupled antibodies (detection antibodies) against each cytokine were added to each well and incubated at room temperature for 45 min. After washing with PBS-T three times, streptavidin-horseradish peroxidase (Invitrogen, Camarillo, CA, USA) was added and incubated at room temperature for 45 min, according to the manufacturer's protocol. After washing wells with PBS-T three times, the substrate solution containing freshly prepared 0.7 mg/mL of *o*-phenylenediamine dihydrochloride (WAKO Pure Chemical Industries Ltd., Osaka, Japan) in citric acid buffer, pH 5.0, containing 0.006% hydrogen peroxide was added and incubated for 10 min. Reactions were stopped by adding 50 ΐL of 10% sulfuric acid to each well. Cytokine concentration was estimated by measuring the optical density (OD) at 490 nm using  a micro plate reader (Model 680, Bio-Rad, Hercules, CA, USA) and a standard curve. Each cytokine level is expressed as the mean (pg/mL) ± SD. 
