Rat tnf α elisa kit

The serum TNF-α assessment was done using ELISA method. For the present study, rat TNF-α ELISA kit [manufactured by Diaclone, France (Cat. No. 872.010.001)] was used. The serum TNF-α level was measured according to the manufacturer’s instructions. First of all, the microwell plate was coated with the capture antibody. After that 100 µL of standard diluents and 100 µL serum were added to the appropriate wells. Then 50 µL of diluted detection antibody was added to each well and incubated at room temperature for 3 h and covered properly. The content was then discarded and washed 3 times with 300 µL of washing solution. The 100 µL of streptavidin-HPR solution was then added to the each well and again incubated at room temperature for 30 min and covered properly. The content was then again discarded and washed 3 times with 300 µL of washing solution. The 100 µL of TMB substrate solution was then added to each well and incubated in dark for 5–15 min at room temperature while covered. The 100 µL of stop reagent was then added to each well. Absorbance was read at 450 nm (using a reference wavelength of 620–630 nm) through Merck ELISA reader in pg/mL value.