One-hundred millilitres of the standard solutions or the defatted
samples were added in wells and incubated for 30 min at room
temperature (20e25 C) in the dark. The liquidwas then sucked out
of the wells, and all the wells were filled with 250 mL of washing
buffer before sucking out the liquid again. The washing procedure
was repeated twice. Next, 100 mL of the diluted enzyme conjugate
was added in the wells and incubated for 15 min at room temperature
in the dark. This washing procedure was repeated two
more times. Then, 100 mL of chromogenwas added to each well and
incubated for 15 min at room temperature in the dark. Lastly,100 mL
of the stop solution was added to each well, and the absorbance
was measured at 450 nm by a microplate reader (Infinite 200, Tecan
Austria GmbH, Groedig, Austria)