2.3. ELISA detection.
Detection of human PD1, PD-L1, IL-2, IL-6, IL-10, TGF-β and INF-γ concentrations in serum- plasma by ELISA kit(MLBio). The blood specimen was placed at room temperature for 2 hours or 4℃ overnight, and then 1000×g centrifuged for 20 mins to collect the supernatant. A sample, standard sample and HRP labeled antibody were added to the micropores of pre-coated antibody, incubated and washed thoroughly. The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a enzyme labeling(Bio-Rad). The color depth is positively correlated with the antigen in the sample.