ELISA was used to detect the expression levels of IL-6 (EK0410, Boster Biological Technology, Wuhan, China), TNF-α (EK0525, Boster Biological Technology, Wuhan, China), SOD (SES134Hu, Cloud-clone Corp, Houston, TX, USA) and MDA (CEA597Ge, Cloud-clone Corp, Houston, TX, USA) in the plasma, according to the protocols of the kits.
Plasma samples were collected and centrifuged at the speed of 1000 rpm for 20 min. Then 100 μl supernatant was added to the provided 96-well plate for 2h in 37 °C while standard curve was made in the same plate. A hundred milliliters diluted primary antibody was added to each well and the plate was incubated in 37 °C for 1h. Afterwards, each well was washed with wash buffer or 0.01 M PBS for 3 times. Then 100 μl diluted secondary antibody was then added and incubated in 37 °C for 30 min. The plate was then washed for 5 times in the same way. At last, reaction substrate and stop buffer was in turn added and the OD450 values were measured with the multiskan microplate reader.
Plasma samples were collected and centrifuged at the speed of 1000 rpm for 20 min. Then 100 μl supernatant was added to the provided 96-well plate for 2h in 37 °C while standard curve was made in the same plate. A hundred milliliters diluted primary antibody was added to each well and the plate was incubated in 37 °C for 1h. Afterwards, each well was washed with wash buffer or 0.01 M PBS for 3 times. Then 100 μl diluted secondary antibody was then added and incubated in 37 °C for 30 min. The plate was then washed for 5 times in the same way. At last, reaction substrate and stop buffer was in turn added and the OD450 values were measured with the multiskan microplate reader.