Glutathione reductase determination kit
By adding enzyme reaction accelerator, DTPA and complex enzyme stabilizer to the glutathione reductase assay kit, the problems of inaccurate testing and inhibition of enzyme activity during dilution of high concentration samples were solved, and more accurate and stable measurement results were achieved.
Patent Information
- Application Number
- CN202411787458.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-03-07
AI Technical Summary
The existing glutathione reductase assay kits are inaccurate when diluting high concentration samples, and the metal ions in the reagent inhibit the enzyme activity, resulting in low measurement values and unstable opening measurement values.
Add an enzyme reaction accelerator to reagent 1 to expand the linear range, add DTPA to reagent 2 to reduce the inhibitory effect, and add a complex enzyme stabilizer to maintain the stability of NADPH.
The maximum dilution ratio of the kit is improved, ensuring the accuracy of the determination after dilution of high-value samples, stabilizing the enzyme activity, and enhancing the opening stability of the reagent.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biological detection, and in particular relates to a glutathione reductase assay kit. Background Art
[0002] Glutathione reductase is one of the most important enzymes in the human redox system and is the main flavoenzyme that maintains the content of reduced glutathione (GSH) in cells. With the participation of NADPH, oxidized glutathione is converted into reduced glutathione, which plays an important role in preventing the oxidative decomposition of hemoglobin, maintaining the activity of thiol proteins, ensuring the reducibility of thiol proteins and the integrity of cells.
[0003] Glutathione reductase (GR) has extremely important physiological functions and is widely present in serum, red blood cells, mononuclear macrophages, heart, liver, kidney and other tissue cells. It can be used to assist in the diagnosis of liver diseases. Elevated glutathione reductase in serum is common in the early stages of acute hepatitis.
[0004] At present, common laboratory diagnostic methods include enzyme ultraviolet enzyme method and glutathione substrate method. This reagent can be used in biochemical analyzers to measure the content of glutathione reductase in serum. Clinically, the indicators of some patients will exceed the linear range of the reagent test, so they need to be diluted for testing. The maximum dilution multiple of the glutathione reductase assay kit is generally 5 times, which leads to inaccurate test results when the concentration in the patient's serum is too high and diluted more than 5 times. In addition, during the normal storage of the reagent, the metal ions in the reagent will inhibit the enzyme activity, resulting in a decrease in the blank absorbance of the reagent, resulting in a low measured value; the reagent is in contact with carbon dioxide in the air during the bottle opening test, which reduces the pH of the reagent, and the NADPH in reagent 2 will be unstable, making the opening measurement unstable. Summary of the invention
[0005] In view of the shortcomings of the prior art, the present invention provides a glutathione reductase assay kit. An enzyme reaction accelerator is added to reagent 1 to increase the linear range of the reagent, and DTPA is added to reagent 2 to complex with metal ions in the reagent, thereby reducing the inhibition of metal ions on enzyme activity and making the enzyme activity unaffected; a composite enzyme stabilizer is added to make NADPH stable even under pH changes.
[0006] On the one hand, the present invention provides a kit, which comprises reagent 1 and reagent 2, wherein the main components of reagent 1 are: tris(hydroxymethylaminomethane) buffer (pH7.8), disodium ethylenediaminetetraacetic acid (EDTA-2NA), oxidized glutathione (GSSG), an enzyme reaction accelerator, and a preservative proclin-300; the main components of reagent 2 are: tris(hydroxymethylaminomethane) buffer (pH9.6), reduced coenzyme II (NADPH), diethyltriaminepentaacetic acid (DTPA), a composite enzyme protectant, and a preservative proclin-300.
[0007] Furthermore, in the reagent 1, the concentration of tris(hydroxymethyl)aminomethane buffer (pH7.8) is 50-150mmol / L, the concentration of disodium ethylenediaminetetraacetic acid (EDTA-2NA) is 0.1-1mmol / L, the concentration of oxidized glutathione (GSSG) is 1-5mmol / L, the concentration of enzyme reaction accelerator is 0.1-10mmol / L, and the concentration of preservative proclin-300 is 0.1-0.5%; in the reagent 2, the concentration of tris(hydroxymethyl)aminomethane buffer (pH9.6) is 50-150mmol / L, the concentration of reduced coenzyme II (NADPH) is 0.1-1mmol / L, the concentration of diethyltriaminepentaacetic acid (DTPA) is 0.1-1mmol / L, and the concentrations of each component in the composite enzyme protectant are 1-10g / L, and the concentration of preservative proclin-300 is 0.1-0.5%.
[0008] Furthermore, in the reagent 1, the concentration of tris(hydroxymethyl)aminomethane buffer (pH7.8) is 100mmol / L, the concentration of disodium ethylenediaminetetraacetic acid (EDTA-2NA) is 0.5mmol / L, the concentration of oxidized glutathione (GSSG) is 2.2mmol / L, and the concentration of preservative proclin-300 is 0.4ml / L; the concentration of tris(hydroxymethyl)aminomethane buffer (pH9.6) in the reagent 2 is 100mmol / L, the concentration of reduced coenzyme II (NADPH) is 0.6mmol / L, the concentration of diethyltriaminepentaacetic acid (DTPA) is 0.5mmol / L, and the concentration of preservative is 0.4ml / L.
[0009] Furthermore, the enzyme reaction accelerator is any one of 5 mmol / L MnCl2, 5 mmol / L KCl and 0.1 mmol / L FAD.
[0010] Furthermore, the composite enzyme protectant comprises bovine serum albumin at a concentration of 2 g / L, sucrose at a concentration of 5 g / L and Triton X-100 at a concentration of 2 g / L.
[0011] The present invention has the following beneficial effects: 1. By adding an enzyme reaction accelerator to reagent 1, the enzyme activity can be synergistically promoted. By changing the reaction rate, the maximum dilution factor of the glutathione reductase assay kit can be increased, thereby making the test more accurate when the patient's serum concentration is too high and diluted.
[0012] 2. By adding a composite enzyme stabilizer to reagent 2, NADPH can remain stable even under changes in pH, making the reagent opening more stable.
[0013] 3. By adding DTPA to reagent 2, it can complex with the metal ions in the reagent, reduce the inhibition of metal ions on enzyme activity, and make the enzyme activity unaffected. DETAILED DESCRIPTION
[0014] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.
[0015] Example 1 The present invention provides a glutathione reductase assay kit, the main components of which are as follows: Three test groups and one control group were set up according to different enzyme reaction accelerators. The group settings are as follows: Grouping Enzyme reaction accelerator concentration Experimental Group 1 MnCl 5 mmol / L Experimental Group 2 KCl 5 mmol / L Experimental Group 3 FAD 0.1 mmol / L Control group none Detection method: Detection was performed on Beckman automatic biochemical analyzer AU680, wavelength was set to 340nm, reagent 1: sample: reagent 2 = 150μL: 3μL: 50μL, reagent 1 and sample were added and mixed, incubated at 37℃ for 2 minutes, R2 was added, mixed, incubated at 37℃ for 1 minute, absorbance changes were continuously monitored at the measurement wavelength for 2 minutes, and the absorbance change rate was calculated. Sample concentration = [(△A / min sample-△A / min blank) / (△A / min calibration-△A / min blank)] × calibrator concentration.
[0016] At the same time, high concentration sample dilution tests were performed on 3 test groups and 1 control group, and the results are as follows: The test data showed that when the control group was diluted 8 times, the relative deviation exceeded 10%, which did not meet the test requirements. When the samples were diluted 16 times in the three test groups with enzyme reaction accelerators, the relative deviations were all less than 10%, which was within the qualified range. Adding enzyme reaction accelerators MnCl, KCl or FAD can increase the maximum dilution factor of the glutathione reductase assay kit, effectively ensuring the accuracy of the high-value sample dilution after clinical determination. Example 2
[0017] The present invention provides a glutathione reductase assay kit, the main components of which are as follows: A control group was set up. The difference between the control group and the experimental group was that no complex enzyme protectant (BSA, sucrose and Triton X-100) was added to reagent 2. After calibration of the experimental group and the control group, the target values were measured on the 0th, 10th, 20th, 25th, 30th, 32nd, 34th, 36th, 38th, 40th, 42nd and 44th days after opening the bottle, and the deviation between the measured value and the target value and the deviation from the first day were observed. The measurement results are as follows: The experimental results show that the deviation between the measured value and the target value of the control group was greater than 10% when the bottle was opened for 36 days, which could not meet the detection requirements. The deviation between the measured value and the target value and the first day of the test group was less than 10% when the bottle was opened for 44 days, which met the detection requirements. The stability of the test group reagent opening was significantly better than that of the control group. It can be concluded that adding a composite enzyme protectant to reagent 2 can improve the stability of the reagent opening.
[0018] Example 3
[0019] The present invention provides a glutathione reductase assay kit, the main components of which are as follows: A control group was set up. The difference between the control group and the test group was that the ion chelator diethyltriaminepentaacetic acid (DTPA) was not added to reagent 2. The reagent was sealed and stored at 2-8°C after preparation. The absorbance and target value were measured at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 months respectively. 20 samples were measured at 0 and 20 months respectively to compare their deviations. The experimental results are as follows: Blank absorbance and target value determination results Sample test results The experimental results show that the blank absorbance of the control group decreased from 1.5976 to 1.1549 from 0 to 18 months, and the blank absorbance of the test group decreased from 1.5985 to 1.3322 from 0 to 18 months. The blank absorbance of the control group decreased rapidly, and the decrease in blank absorbance would lead to lower measured values. The deviation between the control group and the target value was significantly greater than that of the test group from 0 to 18 months. 20 samples were measured in the control group and the test group at 0 and 18 months respectively to compare their deviations. The measured values of the control group were lower than those of the test group, with the deviations ranging from 0.35% to 2.57% in the 0th month and 5.08% to 14.48% in the 18th month. Therefore, adding DTPA to reagent 2 can slow down the decrease in blank absorbance and ensure that the test samples are more accurate within the storage validity period.
[0020] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A glutathione reductase assay kit, comprising reagent 1 and reagent 2, wherein the main components of reagent 1 are: tris(hydroxymethylaminomethane) buffer (pH 7.8), disodium ethylenediaminetetraacetic acid (EDTA-2NA), oxidized glutathione (GSSG), enzyme reaction accelerator, and preservative proclin-300; the main components of reagent 2 are: tris(hydroxymethylaminomethane) buffer (pH 9.6), reduced coenzyme II (NADPH), diethyltriaminepentaacetic acid (DTPA), a composite enzyme protectant, and preservative proclin-300.
2. A glutathione reductase assay kit according to claim 1, characterized in that: The concentration of Tris(hydroxymethyl)aminomethane buffer (pH7.8) in the reagent 1 is: 50-150mmol / L, the concentration of disodium ethylenediaminetetraacetic acid (EDTA-2NA) is: 0.1-1mmol / L, the concentration of oxidized glutathione (GSSG) is: 1-5mmol / L, the concentration of enzyme reaction accelerator is: 0.1-10mmol / L, and the concentration of preservative proclin-300 is: 0.1-0.5%; the concentration of Tris(hydroxymethyl)aminomethane buffer (pH9.6) in the reagent 2 is: 50-150mmol / L, the concentration of reduced coenzyme II (NADPH) is: 0.1-1mmol / L, the concentration of diethyltriaminepentaacetic acid (DTPA) is: 0.1-1mmol / L, the concentrations of each component in the composite enzyme protectant are: 1-10g / L, and the concentration of preservative proclin-300 is: 0.1-0.5%.
3. A glutathione reductase assay kit according to claim 2, characterized in that: The concentration of Tris(hydroxymethyl)aminomethane buffer (pH7.8) in reagent 1 is: 100mmol / L, the concentration of disodium ethylenediaminetetraacetic acid (EDTA-2NA) is: 0.5mmol / L, the concentration of oxidized glutathione (GSSG) is: 2.2mmol / L, and the concentration of preservative proclin-300 is: 0.4ml / L; the concentration of Tris(hydroxymethyl)aminomethane buffer (pH9.6) in reagent 2 is: 100mmol / L, the concentration of reduced coenzyme II (NADPH) is: 0.6mmol / L, the concentration of diethyltriaminepentaacetic acid (DTPA) is: 0.5mmol / L, and the concentration of preservative is 0.4ml / L.
4. A glutathione reductase assay kit according to claim 2, characterized in that: The enzyme reaction accelerator is any one of 5 mmol / L MnCl2, 5 mmol / L KCl and 0.1 mmol / L FAD.
5. A glutathione reductase assay kit according to claim 2, characterized in that: The composite enzyme protectant comprises a bovine serum albumin concentration of 2 g / L, a sucrose concentration of 5 g / L and a Triton X-100 concentration of 2 g / L.