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Method for determining urokinase activity by chromophoric substrate method

A chromogenic substrate, urokinase technology, applied in color/spectral characteristic measurement, measuring device, material analysis through optical means, etc., can solve the problem that the sample cannot be fully reacted, cannot meet the production detection requirements, and affects the optimal enzyme Respond to problems such as pH, achieve objective test results, meet production testing needs, and have a wide range of urokinase detection

Pending Publication Date: 2022-03-15
BEIJING SAISHENG PHARMA
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Problems solved by technology

[0008] However, the above two methods using the standard curve require additional reagents to force the reaction to be terminated, and there are problems that some samples are not completely reacted, and the measurement results are not accurate enough; and the detection time is long, and the detection efficiency is relatively low for industrial production; At the same time, the r value of its standard curve is relatively small, and there is a problem that it is easy to make the result calculated by substituting the sample into the standard curve differ greatly from the actual value; the applicable detection range of urokinase is narrow, and it cannot meet the production detection of existing industrial multi-spec doses need
[0009] In addition, the substrate concentration of the former method is low, the reaction temperature is relatively low, and there is a problem that the enzyme activity is relatively weak, and the sample cannot be fully reacted in a short time; the latter method uses pure water to prepare a relatively high concentration of Substrate solution, there is a problem that the substrate solution is inconsistent with the sample solution system, and the optimum reaction pH of the enzyme is affected after mixing

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  • Method for determining urokinase activity by chromophoric substrate method

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Embodiment 1

[0042] Embodiment 1 The establishment of linear relationship regression equation

[0043] The present embodiment provides the method for the establishment of linear relationship regression equation, and concrete steps are as follows:

[0044] (1) Prepare gelatin-Tris buffer solution (pH8.8):

[0045] Tris 6.06g and sodium chloride 2.22g are mixed, add 700mL purified water to dissolve, obtain mixed liquid A;

[0046] Separately get 10g of gelatin and add an appropriate amount of purified water for heating and dissolving, cool the solution to room temperature and mix it with mixed solution A, shake it up to obtain mixed solution B;

[0047] Adjust the pH value of the mixture B to 8.8 with dilute hydrochloric acid, and then add purified water to dilute to 1000 mL.

[0048] (2) Preparation of chromogenic substrate solution:

[0049] Prepare the chromogenic substrate solution that concentration is 0.5mmol / L with gelatin-Tris buffer solution (pH8.8), add in the enzyme standard we...

Embodiment 2

[0058] This embodiment provides a method for determining the activity of urokinase by a substrate chromogenic method, specifically as follows:

[0059] Selecting the batch number produced by Beijing Saisheng Pharmaceutical Co., Ltd. is 20091223 urokinase preparation as the sample to be tested, and the marked amount is 1139 units;

[0060] Quantitatively dilute with gelatin-Tris buffer to make a solution with a concentration of 20-100U / mL, add it to the enzyme-labeled well plate with substrate, and set 3 duplicate holes;

[0061] Put the above enzyme-labeled well plate into a microplate reader at 37°C for 23 minutes;

[0062] The absorbance of the enzymatic hydrolysis product p-nitroaniline (PNA) in each well at the wavelength of 405nm is 0.4988;

[0063] Substitute this value into the standard curve obtained in Example 1, and the calculated urokinase activity in this preparation is 1080.83 units.

[0064] The results show that the activity value obtained by the standard curv...

Embodiment 3

[0065] Embodiment 3 average rate of recovery

[0066] According to the solution preparation method in Example 1, 200 μl of chromogenic substrate S-2444 solution was added to the reaction wells of the microtiter plate, and a standard curve was prepared with urokinase as a standard, and 3 duplicate holes were set for each activity unit point of the curve , select the batch number 20091223 preparation produced by Beijing Saisheng Pharmaceutical Co., Ltd. as the test product (enzyme activity label value is 1139 units), select 60 units, 40 units and 20 units as high, medium and low concentrations respectively, each sample 3 duplicate holes.

[0067] Immediately put the above-prepared microplate plate into a microplate reader preheated to 37°C for 20 minutes, and then measure the absorbance value A405 of the enzymatic hydrolysis product p-nitroaniline (PNA) in each well at a wavelength of 405nm to make the enzyme Active standard curve, measured to obtain the absorbance A405 value o...

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Abstract

The invention relates to the field of medicines and biological products, in particular to a method for determining urokinase activity by using a chromogenic substrate method. According to the method for determining the activity of urokinase by the chromogenic substrate method, L-pyroglutamylglycine-L-arginine-paranitroaniline hydrochloride is taken as a chromogenic substrate, a linear relation between a light absorption value of a product and the amount of urokinase is established, and the corresponding enzyme activity of the urokinase sample is calculated according to the linear relation. A linear relation regression equation is as follows: y is equal to 0.0074 x + 0.0775, and r is equal to 0.9999; wherein x is the activity of the sample urokinase; y is the light absorption value of the enzymatic hydrolysate paranitroaniline of the sample urokinase at the wavelength of 405 nm. The urokinase activity determination method provided by the invention has the advantages of few used reagents, relatively simple operation, higher accuracy, good inter-plate and in-plate precision, and high average recovery rate. The method is shorter in detection time and wider in urokinase applicable detection range, can meet the production detection requirements of existing industrial multi-specification doses, and has the advantages of higher efficiency and higher economy.

Description

technical field [0001] The invention relates to the fields of medicines and biological products, in particular to a method for measuring urokinase activity by using a chromogenic substrate method. Background technique [0002] Urokinase is an alkaline protein hydrolyzate obtained after separation and purification from fresh human urine from healthy people. There are two main methods for the determination of urokinase activity: the bubble rising method used in the 2020 edition of the second volume of urokinase species in the Chinese Pharmacopoeia, and the chromogenic substrate method used in the Japanese Pharmacopoeia. [0003] The bubble rising method refers to the characteristic that urokinase can activate plasminogen and convert it into plasmin, which has a strong proteolytic ability; while fibrinogen is transformed into It is a fibrin clot, and under the action of plasmin, the fibrin clot is hydrolyzed into a water molecule polypeptide and dissolved, and bubbles will ris...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N21/01
CPCG01N21/31G01N21/01
Inventor 王晓丽张涵任立新齐硕
Owner BEIJING SAISHENG PHARMA
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