Measurement of Cotinine Levels
The ELISA (Bioassay Technology, China) application standards were
prepared according to the manufacturer’s instructions. Briefly, 50 μl of
the standards were added to the standard wells, 40 μl of samples were
added to the sample wells, and 10 μl of anti-cotinine antibody was added
to the sample wells. Afterward, streptavidin-HRP is added to both
standard and sample wells, and the plate was incubated at 37 °C for 60
minutes. The plate was washed 5 times and 50 µl of substrate A and B
were added to each well and incubated in the dark for 10 min. The
reaction was stopped by adding 50 µl stop solution to each well and the
absorbance was determined by spectrophotometer at 450 nm.