Application

This kit can be used to measure the uric acid content in animal urine, serum, plasma samples.

Detection significance

Uric acid, a purine metabolite, is degraded into allantoin by uric acid enzymes in most mammals. Due to the absence of uric acid oxidase gene, uric acid is the final product of purine metabolism in humans, so the level of uric acid in human blood is higher than that in most mammals. Uric acid is a physiologically important plasma antioxidant that effectively protects biological targets from the oxidation of hydroxyl radicals, hypochloric acid and peroxynitrite.

Detection principle

Uric acid can be used as an antioxidant to remove peroxide, hydroxyl and oxygen free radicals, chelate and transfer metal ions, protect vascular endothelial cells from damage. Uric Acid in protein-free filtrate reduce phosphotungstic acid to form tungsten blue, allantoin and carbon dioxide, the depth of blue color is proportional to the concentration of uric Acid.

Experiment instruments

Micropipettor, Vortex mixer, Centrifuge, Spectrophotometer (690 nm).

Sample pretreatment

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment.

(1) Serum (plasma): Collect the serum or plasma samples by conventional methods. Detect the sample directly.

(2) Urine: Take the urine (the UA has a low solubility and is easy to form crystallization precipitation). Therefore, it should be heated to 50℃ and diluted 10 times with double-distilled water immediately. The final result should be multiplied by 10.

Operation steps

1. Dilution of standard

Dilute the 1 g/L UA standard stock solution to the concentrations in the below table with double-distilled water, the total volume is 1000 μL.

2. Operation processes

(1)   Standard tube: add 0.2 mL of diluted UA standard into a 5 mL EP tube (make duplicates for each concentration).

Blank tube: add 0.2 mL of double-distilled water into a 5 mL EP tube.

Sample tube: add 0.2 mL of sample into a 5 mL EP tube.

(2)  Add 2 mL of Reagent 2 to each tube and mix fully with the vortex mixer.

(3)  Stand the tubes for 10 min. Centrifuge at 1708 g for 5 min (The supernatant should be clarified, and if turbid, transfer the supernatant into the new EP tube and centrifuge again).

(4)  Take 1.6 mL of the supernatant, then add 0.5 mL of Reagent 3 and 0.5 mL of Reagent 4 orderly. Mix fully and stand the tubes at room temperature for 10 min.

(5)  Set the spectrophotometer to zero with double-distilled water and measure the OD value of each tube at 690 nm with 1 cm diameter cuvette. Calculate △A690=ASample -ABlank. (Note: The color stability of uric acid is poor, so it is suggested to finish the absorbance detection within 20 min, and the quantity of samples is better not over 20 for each batch.)

Note: It can be refer to the following operating table.

Technical parameter

1. The sensitivity of the kit is 0.58 mg/L.

2. The intra-assay CV is 1.8% and the inter-assay CV is 2.6%.

3. The recovery of the kit is105%.

4. The detection range of the kit is 0.58-100 mg/L.

Notes

1. This kit is for research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 3 months.

4. Do not use components from different batches of kit.

5. The supernatant after centrifugation must be clarified.

6. The color stability of uric acid is poor, so it is recommended to complete colorimetric analysis within 20 min after color development.