4 u00b0 c overnight after incubation plates were washed four times with pbst buffer to block non specific binding block buffer were added to each well and the plates were incubated at room temperature for 1 h after incubation plates were washed five times with elisa wash buffer appropriately diluted samples or standard solution were then added to the 96 microwell plate wells and the plates were incubated at room temperature for 2 u02dc 3 h after incubation plates were washed with buffer six times then secondary antibody for detection was added to each well plates were incubated for another 1 h after incubation plates were washed seven times with elisa wash buffer streptavidin hrp horseradish peroxidase was added to each well and the reaction was allowed to react for 30 mins after reaction the plates were washed with elisa buffer eight times and then hrp substrate solution was added to the 96 microwell plate wells plates were incubated at room temperature for 20 u02dc 30 mins to develop color the plates were measured the absorbance at 405 nm by elisa reader 1 ml trizol reagent invitrogen was added to the right lung tissue and homogenized on ice with electric homogenizer the homogenate.