The tests were done in duplicate. Briefly, a microtiter plate (Costar, Cambridge, MA, USA) was coated with 100 μL/well of 5 μg/mL monoclonal mouse anti-human EPZ015666 research buy granulysin (clone RB1) (MBL International, Nagoya, Japan) in 0.05 M carbonate-bicarbonate buffer (pH 9.5) overnight at 4°C. The plates were washed with PBS containing 0.05% Tween 20 and blocked with buffered protein solution with ProClin-150 at room temperature for 1 hr. After being washed, the undiluted plasma was added and incubated for 2 hr at room temperature. The bound antigens were detected

with 0.1 μg/mL of monoclonal mouse anti-human granulysin biotin (RC8) (MBL International) and avidin-horseradish peroxidase (Av-HRP) conjugate (BD Biosciences Pharmingen) diluted to 1:1000. After incubation for 1 hr, the reactions were developed by coloring with TMB substrate (BD Biosciences Pharmingen) for 20 min in the dark. The reaction was stopped by 2N H2SO4 solution (BD Biosciences Pharmingen). Optical densities were measured at 450 nm wavelength by an ELISA reader (ELx808 IU ultra microplate reader, Bio-Tek instruments, Winooski, VT, USA). Granulysin

concentrations were selleck calculated from a standard curve using granulysin containing culture supernatant obtaining from Cos7 cell transfected with gene encoding 15K granulysin. The lower detection limit for granulysin was 0.047 ng/mL. Interferon-γ concentrations in plasma and stimulated PBMC supernatant were determined by ELISA according to the manufacturer’s instruction (BD Biosciences Pharmingen). The tests were done in duplicate. Briefly, a microplate (Costar) was coated with 100 μL/well of anti-human IFN-γ (diluted to 1:250 in 0.1 M sodium carbonate) and incubated overnight at 4°C. The plates were washed three times with PBS containing 0.05% Tween 20, blocked with 200 μL/well of buffered protein solution

with ProClin-150 and incubated at room temperature for 1 hr. After being washed, 100 μL of undiluted sample was added and incubated for 2 hr at room temperature. The bound antigen were detected with biotinylated anti-human IFN-γ monoclonal antibody and streptavidin-horseradish peroxidase conjugate (diluted to 1:250 with 10% FBS in PBS) and incubated for 1 hr at room temperature. Then, 100 μL of TMB substrate solution was added and incubated for 30 min at room temperature in the Dichloromethane dehalogenase dark. The reaction was stopped by 2N H2SO4 solution. Samples were analyzed at 450/550 nm wavelength with a microplate ELISA reader (ELx808 IU ultra microplate reader) and IFN-γ concentrations were calculated from a standard curve using recombinant human IFN-γ. The lower detection limit was 4.7 pg/mL. Statistical analyses were performed by SPSS software version 17.0. IFN-γ and granulysin concentrations in different independent subject groups were compared by Mann-Whitney U test. A P value < 0.05 was considered statistically significant.