Method for detecting content of urate oxidase in serum
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A technique for urate oxidase and serum, which is applied in the field of detection of urate oxidase in serum, can solve the problems of inaccurate and rapid detection of serum urate oxidase content, and achieve cost saving, high accuracy and precision, and a simple and fast method Effect
Inactive Publication Date: 2020-10-09
修正生物医药(杭州)研究院有限公司
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[0006] In summary, there are many technical problems in the prior art, and it is impossible to accurately and rapidly detect the urate oxidase content in serum
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Embodiment 1
[0044] 1. Preparation of samples to be tested
[0045] Uric acid oxidase (protein content 1.5 mg / mL, enzyme specific activity 16.3 U / mg) was diluted 100 times with commercially available blank rabbit serum so that the final protein concentration was 0.015 mg / mL. The sample to be tested for urate oxidase was obtained.
[0046] 2. Solution preparation:
[0047] Preparation of Tris-HCl solution: Weigh 6.05g Tris, dissolve in 500mL distilled water, and adjust the pH to 8.04.
[0048] Preparation of solution A: Weigh 0.0067g 4-AAP (4-aminoantipyrine), 0.0017g potassium ferrocyanide, dissolve in 100mL Tris-HCl solution.
[0049] Preparation of solution B: Weigh 0.212g DHBS (sodium 3,5-dichloro-2-hydroxybenzenesulfonate), 0.5g Triton X-100, dissolve in 100mL Tris-HCl solution.
[0050] Preparation of uric acid solution: Weigh 0.0168g uric acid and dissolve it in 100mL Tris-HCl solution.
[0051] Preparation of Tris-HCl-B solution: take 50mL Tris-HCl solution, adjust the pH to 6.0...
Embodiment 2
[0076] Embodiment 2 adopts the method of the present invention and the control test of existing uric acid method to detect the protein content of the sample to be tested
[0077] 1. Sample to be tested
[0078] The samples to be tested are serum from cynomolgus monkeys used in the pharmacokinetic experiments of pegylated urate oxidase, which are blood samples collected before administration, blood samples taken 30 minutes after drug administration, and blood samples collected 4 days after drug administration. The specific activity of pegylated urate oxidase used for administration is 3.8 U / mg.
[0079] 2. Solution preparation:
[0080] Preparation of TEA buffer solution: Weigh 0.29g EDTA and 7.45g triethanolamine, dissolve them in 900mL distilled water, and adjust the pH to 8.9. Finally, make up to 1L with distilled water.
[0081] Preparation of uric acid solution (T): Weigh 0.017g of uric acid and dissolve it in 500mL of TEA buffer.
[0082] Preparation of stop solution ...
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Abstract
The invention provides a method for detecting the content of urate oxidase in serum, which is characterized by comprising the following steps: 1) preparation of a standard substance: taking urate oxidase with known enzymatic specific activity, and diluting with a Tris-HCl solution; 2) standard curve making: adding a working solution into a test tube, adding a standard substance and blank serum, adding a stop solution to stop the reaction after constant-temperature water bath reaction, and detecting a light absorption value; 3) sample detection: adding a working solution into the test tube, then adding a to-be-detected sample, adding a stop solution to stop the reaction after the constant-temperature water bath reaction, and detecting a light absorption value; 4) data determination and calculation: calculating the enzyme activity of the standard substance at each point according to the enzyme specific activity of the standard substance and the protein content of the standard substance at each point of the standard curve; fitting a standard curve for the enzyme activity of the standard substance at each point according to the light absorption value of each point of the standard curve; substituting the light absorption value of the to-be-detected sample into the standard curve, and obtaining the protein content of the to-be-detected sample through a calculation formula. The methodis accurate, simple and rapid, and the cost is greatly saved compared with an existing complex method.
Description
technical field [0001] The invention relates to the field of drug analysis, in particular to a method for detecting urate oxidase in serum. Background technique [0002] Several products of urate oxidase have been launched or are in the stage of non-clinical research and clinical research for the treatment of hyperuricemia, gout and other diseases. For non-clinical research and clinical research, the content of urate oxidase in serum samples of experimental animals or subjects is a key indicator for evaluating the metabolism of urate oxidase in vivo. [0003] At present, the detection methods of urate oxidase mainly include immunoassay and activity method. Among them, the activity method can be divided into two methods for detecting the decrease of uric acid and the increase of allantoin according to the different detection substances. [0004] The immunization method requires the corresponding anti-uricase antibody. If there is no commercial antibody from the correspondin...
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