Then the liquid was poured out of the wells and wells were washed with PBS-Tween 20
buffer solution three times.Wells were tapped face down on a layer of absorbent to remove the residual wash buffer. In the next stage,100 ml of tetramethylbenzidine (TMB) as enzyme substrate was added into each well and incubation was allowed for 10 min at room temperature in the dark, and then 100 m1 of the stop solution was added to the microplate wells, which changes the color from blue to yellow. The optical density (OD) was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., USA). All samples were run in duplicates. For low concentration range assay, 200 ml samples and standards were directly added into the assay well and incubated for 2 h. Then the liquid was poured out of the wells and wells were washed with buffer solution, and similar procedurewas followed as described above in order to measure the
OD at 450 nm microplate reader