Emc102a 96

Serum levels of IL-6, TNF-α (EMC004.96 and EMC102a.96, Neo Bioscience Technology, Shenzhen, China) were measured by Mouse ELISA Kits according to the manufacturer’s protocols. Reagents, samples, and standards were prepared as instructed. After adding 50 μl standards and serum samples per well, the 96-well plate was incubated for 2 hours at 37°C. Then the liquid was removed and washed. And then 100 μl of Biotin antibody was added to each well before incubating for 1 h at 37°C, and followed by washing the plate 5 times with a washing buffer. 100 μl of HRP-avidin was added to each well, incubated for 1 hour at 37°C, and washed 5 times. After adding 90 μl TMB substrate to each well, the plate was protected from light and incubated for 20 minutes at 37°C. Color formation was stopped by 50 μl stop solution, and the optical density (OD) value was read at wavelength of 450 nm on a plate microplate reader within 5 minutes. Corresponding concentrations were converted from OD values according to the standard curve. Each serum sample was tested twice and the average value was taken for analysis.

The level of TNF-α, IL-6, and IL-10 was determined by commercial kit (NEOBIOSCIENCE, EMC102a.96, EMC004.96, and EMC005.96) according to the manufacturer's protocol.