1.  Properly dilute the sample if the color is too dark, and multiply by dilution factor when calculating the result.

2.  It is recommended to use disposable plastic tubes to avoid contamination.

3.  Prepare fresh enzyme working solution for needed amount before use. The enzyme working solution cannot be store for a long time.
4.  The adhesion of enzyme stock solution is strong. It should be slowly absorbed when absorbing with pipette.
5.  The incubation time must be 10 min accurately after adding enzyme working solution. Therefore, it is better to make  batch operation if there are many samples to be detected. The number of operation in a batch should be less than 20. 

(1)    Blank tube: Add 0.02 mL of Double-distilled water into a 5 mL centrifuge tube.

 Standard tube: Add 0.02 mL of 10 mmol/L urea standard working solution into a 5 mL centrifuge tube.

 Sample tube: Add 0.02 mL of Sample into a 5 mL centrifuge tube.

 Control tube: Add 0.02 mL of Sample into a 5 mL centrifuge tube.

(2)    Add 0.25 mL of enzyme working solution to blank tube, standard tube and sample tube, add 0.25 mL of reagent 3 to control tube, mix fully with vortex mixer, then react at 37℃ for 10 min accurately.

(3)    Add 1 mL of reagent 4 and 1 mL of reagent 5, mix fully, react at 37℃ for 10 min accurately.

(4)    Set to zero with double distilled water and measure the OD values of each tube with 1 cm optical path cuvette at 580 nm.