1.   Sample pretreatment

1)    For serum (plasma) and milk sample:

Take 0.45 mL of sample and add 0.45 of Reagent 2 application solution, mix fully, then add 0.1 of Reagent 3 application solution and incubate at 37℃ for 15 min.

2)    For tissue an cells sample:

Take 0.9 mL of sample and add 0.1 of Reagent 3 application solution, mix fully and incubate at 37℃ for 15 min.

2.   The measurement of samples

1)    Control tube: Add 3 mL of double distilled water, 0.2 mL of sample, 0.2 mL of Reagent 5 into 5 mL EP tubes.

Sample tube: Add 0.2 mL of sample, 0.2 mL of Reagent 5, 3 mL of chromogenic agent into 5 mL EP tubes.

2)    Oscillate fully with a vortex mixer and incubate for 30 min at 37℃.

3)    Add 0.05 mL of Reagent 8, oscillate fully with a vortex mixer and incubate for 10 min at 60℃.

4)    Centrifuge the tubes at 2325 × g for 10 min and take the supernatant for measuring the OD value.

5)    Set the spectrophotometer to zero with double distilled water and measure the OD value of each tube at 460 nm with 1.0 cm optical path cuvette immediately.