A glutathione reductase detection kit and its preparation method and application

Abstract

The present invention belongs to the technical field of detection reagents, and specifically relates to a glutathione reductase detection kit and its preparation method and application. The present invention provides a glutathione reductase detection kit and its preparation method and application, and tris (2-carboxyethyl) phosphine hydrochloride can improve the stability of NADPH in the glutathione reductase detection reagent, and keep the NADPH activity to the greatest extent. Utilize tris (2-carboxyethyl) phosphine hydrochloride and NADPH to prepare glutathione reductase detection reagent, combine other specific buffers and other components of suitable concentration, can improve the stability of reagent and the accuracy of glutathione reductase detection result, and extend the shelf life of reagent. The results of the embodiment show that the obtained kit is higher than the industry standard and commercially available control kit in precision, linearity and stability, and has high clinical use value.

Description

Technical Field

[0001] The invention belongs to the technical field of detection reagents, and specifically relates to a glutathione reductase detection kit and a preparation method and application thereof. Background Art

[0002] Glutathione reductase (GR) is one of the most important enzymes in the human redox system and is the main flavoenzyme that maintains the reduced glutathione content in cells. At present, the method for determining glutathione reductase is mainly the glutathione substrate method. The detection principle is that glutathione reductase catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) with the participation of NADPH. NADPH has a specific absorption peak at a wavelength of 340nm, and its oxidation rate is proportional to the activity of GR in serum. The rate of decrease of NADPH absorbance at 340nm can be measured to calculate the GR activity. However, NADPH in existing detection reagents is easily inactivated, resulting in unstable measurement values ​​and affecting the test results. Summary of the invention

[0003] The purpose of the present invention is to make up for the deficiencies of the prior art, protect the activity and stability of NADPH, improve the stability of glutathione reductase detection reagents, and improve the accuracy of glutathione reductase detection results.

[0004] In order to achieve the above object, the present invention provides a glutathione reductase detection kit, which comprises an independently packaged reagent R1 and an independently packaged reagent R2;

[0005] The reagent R1 comprises: 20-100 mmol / L of TRIS buffer with a pH value of 8.0-8.5, 8-25 g / L of oxidized glutathione, 0.1-0.5 g / L of an interfering agent, 1-15 g / L of a surfactant, and 0.1-2 g / L of PROCLIN300; the interfering agent comprises disodium ethylenediaminetetraacetic acid and / or ethylene glycol bis(2-aminoethyl)tetraacetic acid;

[0006] The reagent R2 comprises: 80-200 mmol / L of TRIS buffer with a pH value of 9.0-9.5, 0.1-2 g / L of PROCLIN300, 1-15 g / L of a surfactant, 0.5-4 g / L of sodium chloride, 1-3 mmol / L of NADPH, and 0.5-3 g / L of tris(2-carboxyethyl)phosphine hydrochloride.

[0007] Preferably, the reagent R1 comprises: 30-70 mmol / L TRIS buffer with a pH value of 8.0-8.5, 10-20 g / L oxidized glutathione, 0.2-0.4 g / L interfering agent, 5-10 g / L surfactant, and 0.5-1 g / L PROCLIN300;

[0008] The reagent R2 comprises: 100-150 mmol / L of TRIS buffer with a pH value of 9.0-9.5, 0.5-1 g / L of PROCLIN300, 5-10 g / L of a surfactant, 1-2 g / L of sodium chloride, 1-2 mmol / L of NADPH, and 1-2 g / L of tris(2-carboxyethyl)phosphine hydrochloride.

[0009] Preferably, the reagent R1 comprises: 50-60 mmol / L TRIS buffer with a pH value of 8.1, 15 g / L oxidized glutathione, 0.3 g / L interfering agent, 8 g / L surfactant, and 0.7 g / L PROCLIN300;

[0010] The reagent R2 includes: 110-140 mmol / L of TRIS buffer with a pH value of 9.4, 0.7 g / L of PROCLIN300, 8 g / L of a surfactant, 1.5 g / L of sodium chloride, 1.5 mmol / L of NADPH, and 1.8 g / L of tris(2-carboxyethyl)phosphine hydrochloride.

[0011] Preferably, the volume ratio of the reagent R1 to the reagent R2 is 4:1.

[0012] Preferably, the surfactants in the reagent R1 and the reagent R2 respectively include tween 20.

[0013] The present invention also provides a method for preparing the glutathione reductase detection kit described in the above technical solution, comprising the following steps:

[0014] According to the component contents of reagent R1, TRIS buffer, oxidized glutathione, interfering agent, PROCLIN300 and surfactant are dissolved in pure water in sequence, and the pH is adjusted to 8.0-8.5 to obtain the reagent R1;

[0015] According to the component contents of reagent R2, TRIS buffer, PROCLIN300, surfactant, sodium chloride and tri(2-carboxyethyl)phosphine hydrochloride are dissolved in pure water in sequence, the pH is adjusted to 9.0-9.5, and then NADPH is added to adjust the pH to 9.0-9.5 to obtain the reagent R2.

[0016] The present invention also provides the use of the glutathione reductase detection kit described in the above technical solution in detecting glutathione reductase;

[0017] The use is for non-diagnostic and non-therapeutic purposes.

[0018] The present invention also provides a method for detecting glutathione reductase for non-diagnostic and non-therapeutic purposes, using the glutathione reductase detection kit described in the above technical solution and using a fully automatic biochemical analyzer for detection, with the main detection wavelength being 340nm and the secondary wavelength being 380nm.

[0019] Preferably, the volume ratio of reagent R1 to the sample to be tested in the glutathione reductase detection kit is 12:1.

[0020] Beneficial effects:

[0021] The invention provides a glutathione reductase detection kit, comprising an independently packaged reagent R1 and an independently packaged reagent R2; the reagent R1 comprises: 20-100 mmol / L of TRIS buffer with a pH value of 8.0-8.5, 8-25 g / L of oxidized glutathione, 0.1-0.5 g / L of an interfering agent, 1-15 g / L of a surfactant, and 0.1-2 g / L of PROCLIN300; the interfering agent comprises disodium ethylenediaminetetraacetic acid and / or ethylene glycol bis(2-aminoethyl)tetraacetic acid; the reagent R2 comprises: 80-200 mmol / L of TRIS buffer with a pH value of 9.0-9.5, 0.1-2 g / L of PROCLIN300, 1-15 g / L of a surfactant, 0.5-4 g / L of sodium chloride, 1-3 mmol / L of NADPH, and 0.5-3 g / L of tris(2-carboxyethyl)phosphine hydrochloride. NADPH is unstable in aqueous solution, and is extremely easy to be oxidized to cause inactivation, resulting in unstable measured values, affecting the test results. Tris(2-carboxyethyl)phosphine hydrochloride has electron-rich phosphorus and oxygen atoms, and can be combined with a variety of metals, such as rhodium, iridium, and par. At room temperature, it can be quickly complexed with rhodium and iridium in dioxane, can reduce disulfide bonds in various peptides and proteins, has a protective effect on the sulfhydryl groups in NADPH, can improve the stability of NADPH in glutathione reductase detection reagents, and maintain NADPH activity to the greatest extent, thereby not affecting the performance of the reagents, improving the stability of the reagents and the accuracy of the glutathione reductase detection results, extending the shelf life of the reagents, and the reagents are at least stable for 12 months under 2~8°C. The present invention uses tris(2-carboxyethyl)phosphine hydrochloride as a kit component, combined with other specific buffers and other components of suitable concentrations, and the obtained kit is higher than the industry standard and commercially available control kit in precision, linearity, and stability performance, and has a higher clinical use value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 This is a graph showing the correlation results of the glutathione reductase detection kit and the control kit in detecting common samples in Example 1;

[0024] Figure 2 This is a graph showing the correlation results of the glutathione reductase detection kit and the control kit in Example 2 for detecting common samples;

[0025] Figure 3 This is a graph showing the correlation between the glutathione reductase detection kit and the control kit in comparative example 1 for detecting common samples;

[0026] Figure 4 This is a graph showing the correlation results of the glutathione reductase detection kit and the control kit in comparative example 2 for detecting common samples. DETAILED DESCRIPTION

[0027] The present invention provides a glutathione reductase detection kit, which comprises an independently packaged reagent R1 and an independently packaged reagent R2;

[0028] The reagent R1 comprises: 20-100 mmol / L of TRIS buffer with a pH value of 8.0-8.5, 8-25 g / L of oxidized glutathione, 0.1-0.5 g / L of an interfering agent, 1-15 g / L of a surfactant, and 0.1-2 g / L of PROCLIN300; the interfering agent comprises disodium ethylenediaminetetraacetic acid and / or ethylene glycol bis(2-aminoethyl)tetraacetic acid;

[0029] The reagent R2 comprises: 80-200 mmol / L of TRIS buffer with a pH value of 9.0-9.5, 0.1-2 g / L of PROCLIN300, 1-15 g / L of a surfactant, 0.5-4 g / L of sodium chloride, 1-3 mmol / L of NADPH, and 0.5-3 g / L of tris(2-carboxyethyl)phosphine hydrochloride.

[0030] Unless otherwise specified, the present invention has no special requirements on the sources of the components in the kit, and commercially available products known to those skilled in the art may be used.

[0031] In the present invention, the tris(2-carboxyethyl)phosphine hydrochloride is a useful reducing agent for sulfur-containing and nitrogen-containing compounds. Tris(2-carboxyethyl)phosphine hydrochloride and its oxides are water-soluble and have a molecular formula of C9H 15O6P·HCl, manufactured by Shanghai Myril Biochemical Technology Co., Ltd., CAS No. 51805-45-9, product No. M18330, structural formula as shown in Formula I:

[0032] Formula Ⅰ.

[0033] In the present invention, as an embodiment, the reagent R1 includes 30-70 mmol / L of TRIS buffer with a pH value of 8.0-8.5; as another embodiment, the reagent R1 includes 50-60 mmol / L of TRIS buffer with a pH value of 8.0-8.5. As an embodiment, the pH value of the TRIS buffer is 8.1-8.4; as another embodiment, the pH value of the TRIS buffer is 8.1. The present invention adjusts the pH value and concentration of the reagent through the TRIS buffer, thereby improving the sensitivity of the detection.

[0034] In the present invention, as one embodiment, the reagent R1 includes 10-20 g / L of oxidized glutathione; as another embodiment, the reagent R1 includes 12-18 g / L of oxidized glutathione; as another embodiment, the reagent R1 includes 15 g / L of oxidized glutathione. The oxidized glutathione of the present invention is a reaction substrate.

[0035] In the present invention, as an embodiment, the reagent R1 includes 0.2-0.4 g / L of an interfering agent; as another embodiment, the reagent R1 includes 0.3 g / L of an interfering agent. As an embodiment, the reagent interfering agent is disodium ethylenediaminetetraacetate. The interfering agent of the present invention has the function of removing the interference of metal ions on the reagent.

[0036] In the present invention, as an embodiment, the reagent R1 includes 5-10 g / L of a surfactant; as another embodiment, the reagent R1 includes 8 g / L of a surfactant. As an embodiment, the surfactant is tween 20. The surfactant of the present invention has the effect of improving the accuracy of the reaction result.

[0037] In the present invention, as one embodiment, the reagent R1 includes PROCLIN300 0.5-1 g / L; as another embodiment, the reagent R1 includes PROCLIN300 0.7 g / L. PROCLIN300 in the present invention acts as a preservative, inhibiting the growth of bacteria and extending the shelf life of the kit.

[0038] In the present invention, as an embodiment, the reagent R2 includes 100-150 mmol / L of TRIS buffer with a pH value of 9.0-9.5; as another embodiment, the reagent R2 includes 110-140 mmol / L of TRIS buffer with a pH value of 9.0-9.5. As an embodiment, the pH value of the TRIS buffer is 9.2-9.4. The present invention limits the pH value and concentration of the TRIS buffer, thereby improving the sensitivity of the reagent.

[0039] In the present invention, as one embodiment, the reagent R2 includes PROCLIN300 0.5-1 g / L; as another embodiment, the reagent R2 includes PROCLIN300 0.7 g / L. PROCLIN300 in the present invention acts as a preservative, inhibits the growth of bacteria, and prolongs the shelf life of the kit.

[0040] In the present invention, as an embodiment, the reagent R2 includes 5-10 g / L of a surfactant; as another embodiment, the reagent R2 includes 8 g / L of a surfactant. As an embodiment, the surfactant is tween 20. The surfactant of the present invention has the effect of improving the accuracy of the reaction result.

[0041] In the present invention, as one embodiment, the reagent R2 includes 1-2 g / L of sodium chloride; as another embodiment, the reagent R2 includes 1.5 g / L of sodium chloride. The sodium chloride in the present invention has the function of providing ionic strength.

[0042] In the present invention, as one embodiment, the reagent R2 includes NADPH 1-2mmol / L; as another embodiment, the reagent R2 includes NADPH 1.5mmol / L. The NADPH described in the present invention has specific absorption at 340nm, and the glutathione reductase activity concentration is calculated by continuously monitoring the rate of decrease of absorbance at 340nm.

[0043] In the present invention, as an embodiment, the reagent R2 includes 1-2 g / L of tris(2-carboxyethyl)phosphine hydrochloride; as another embodiment, the reagent R2 includes 1.8 g / L of tris(2-carboxyethyl)phosphine hydrochloride. The tris(2-carboxyethyl)phosphine hydrochloride of the present invention has a better protective ability for the sulfhydryl group in NADPH and has better stability, thereby improving the stability of the reagent. The tris(2-carboxyethyl)phosphine hydrochloride described in the embodiment of the present invention was purchased from Shanghai Shanghai Mairui Biochemical Technology Co., Ltd., CAS No. 51805-45-9, item No. M18330.

[0044] In the present invention, as an embodiment, the volume ratio of the reagent R1 to the reagent R2 is 4:1.

[0045] The present invention also provides a method for preparing the glutathione reductase detection kit described in the above technical solution, comprising the following steps:

[0046] According to the component contents of reagent R1, TRIS buffer, oxidized glutathione, interfering agent, PROCLIN300 and surfactant are dissolved in pure water in sequence, and the pH is adjusted to 8.0-8.5 to obtain the reagent R1;

[0047] According to the component contents of reagent R2, TRIS buffer, PROCLIN300, surfactant, sodium chloride and tri(2-carboxyethyl)phosphine hydrochloride are dissolved in pure water in sequence, the pH is adjusted to 9.0-9.5, and then NADPH is added to adjust the pH to 9.0-9.5 to obtain the reagent R2.

[0048] The present invention can avoid the influence of a large amount of pH regulator on the activity of NADPH by adjusting the pH value first, then adding NADPH, and finally adjusting the pH value. In the present invention, as an embodiment, the pH regulator includes hydrochloric acid or NaOH.

[0049] The present invention adds tris(2-carboxyethyl)phosphine hydrochloride at an appropriate concentration to the reagent and combines it with a specific buffer and other components at an appropriate concentration, which can effectively protect the NADPH in the reagent without affecting the accuracy of the test result, solves the problem of poor stability of the glutathione reductase kit, and the reagent is stable for at least 12 months under 2-8°C. In addition, the kit provided by the present invention is higher than the industry standard and the commercially available control kit in terms of precision, linearity and stability, and has a high clinical use value.

[0050] In view of the advantages of the glutathione reductase detection kit provided by the present invention, the application of the glutathione reductase detection kit in detecting glutathione reductase also belongs to the protection scope of the present invention. In the present invention, the application is for non-diagnostic and non-therapeutic purposes.

[0051] The present invention also provides a method for detecting glutathione reductase for non-diagnostic and non-therapeutic purposes, using the glutathione reductase detection kit described in the above technical solution and using a fully automatic biochemical analyzer for detection, with the main detection wavelength being 340nm and the secondary wavelength being 380nm.

[0052] In the present invention, as an embodiment, the volume ratio of the reagent R1 and the sample to be tested in the glutathione reductase detection kit is 12:1.

[0053] In order to further illustrate the present invention, a glutathione reductase detection kit provided by the present invention and its preparation method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] A glutathione reductase detection kit, comprising independently packaged reagents R1 and R2, wherein the volume ratio of the reagents R1 to R2 is 4:1;

[0056] (1) Reagent R1 consists of the following components: 50 mmol / L TRIS buffer with a pH value of 8.1, 15 g / L oxidized glutathione, 0.3 g / L disodium ethylenediaminetetraacetic acid, 0.7 g / L PROCLIN300, and 8 g / L Tween20;

[0057] The specific preparation method is as follows: take 800 mL of purified water, add TRIS according to the above components, fully dissolve, then add oxidized glutathione, disodium ethylenediaminetetraacetic acid, PROCLIN300 and Tween20 in sequence, finally add purified water to make the volume to 1000 mL, and adjust the pH to 8.1 with hydrochloric acid and sodium hydroxide.

[0058] (2) Reagent R2 consists of the following components: 110 mmol / L TRIS buffer with a pH value of 9.4, 0.7 g / L PROCLIN 300, 8 g / L Tween 20, 1.5 g / L sodium chloride, 1.5 mmol / L NADPH and 1.8 g / L tris(2-carboxyethyl)phosphine hydrochloride; tris(2-carboxyethyl)phosphine hydrochloride was purchased from Shanghai Merrill Biochemical Technology Co., Ltd., CAS No. 51805-45-9, Catalog No. M18330.

[0059] The specific preparation method is as follows: take 200 mL of purified water, add TRIS according to the above components, fully dissolve, then add PROCLIN300, Tween20, sodium chloride and tri(2-carboxyethyl)phosphine hydrochloride in sequence, adjust the pH to 9.4 with hydrochloric acid and sodium hydroxide, then add NADPH, and finally add purified water to make the volume to 250 mL, and adjust the pH to 9.4 with hydrochloric acid and sodium hydroxide.

[0060] Example 2 and Comparative Examples 1 to 4

[0061] A glutathione reductase detection kit was prepared in the manner of Example 1, wherein the formula of Example 2 was the same as that of Example 1, except that the concentrations of the components in the formula were different; the formulas of Comparative Examples 1 to 3 were different from those of Example 1; the pH of reagents R1 and R2 in Comparative Example 4 was different from that in Example 1, and the specific parameters are listed in Table 1.

[0062] Table 1 Kit components of Examples 1 to 2 and Comparative Examples 1 to 4

[0063]

[0064] Application Example 1

[0065] Correlation Detection

[0066] Fifty fresh sera were collected from the hospital, numbered 1 to 50, and the glutathione reductase detection kits of Examples 1 to 2, Comparative Example 1 and Comparative Example 3, and a control kit (purchased from Beijing Jiuqiang Technology Co., Ltd., with the catalog number GSGR) were used to detect the content of glutathione reductase (GR) in the samples. The results are shown in Tables 2 and Figures 1 to 4 ;in,

[0067] The instrument used for the test was the fully automatic biochemical analyzer TBA-2000FR (manufacturer: Toshiba, Japan), and the detection parameters were set as follows: main wavelength 340 nm, sub-wavelength 380 nm, sample volume 20 μl, reagent R1 volume 240 μl, and reagent R2 volume 60 μl.

[0068] Table 2 Sample correlation test results (unit: U / L)

[0069]

[0070] According to Table 2 and Figures 1 to 4 It can be seen that the correlation between Example 1 and the control kit sample is 0.9912, the correlation between Example 2 and the control kit sample is 0.964, the correlation between Comparative Example 1 and the control kit sample is 0.9898, and the correlation between Comparative Example 3 and the control kit sample is 0.8904. From Example 1 and Comparative Example 1, it can be seen that the addition of tris(2-carboxyethyl)phosphine hydrochloride has no effect on the sample correlation, from Example 1 and Example 2, it can be seen that the correlation of each component at the preferred concentration is better, and from Example 1 and Comparative Example 3, it can be seen that Tween20 has a great influence on the reagent sample correlation.

[0071] Application Example 2

[0072] Precision testing

[0073] Serum samples of two patients were collected from the hospital, one of which had a high glutathione reductase content in serum (about 90U), and the other had a low glutathione reductase content in serum (about 30U). The samples of the two patients were recorded as high-value samples and low-value samples respectively; the glutathione reductase detection kits of Examples 1-2 and Comparative Examples 1-4, and the control kit (purchased from Beijing Jiuqiang Technology Co., Ltd., with the item number GSGR) were used to detect high-value samples and low-value samples, respectively, and the determination was repeated 10 times to obtain 10 measurement values, respectively, and the mean (X), standard deviation (S), coefficient of variation (CV), precision, and test results of the measurement results were calculated as shown in Tables 3 and 4. Among them,

[0074] The instrument used for the test was the fully automatic biochemical analyzer TBA-2000FR (manufacturer: Toshiba, Japan), and the detection parameters were set as follows: main wavelength 340 nm, sub-wavelength 380 nm, sample volume 20 μl, reagent R1 volume 240 μl, and reagent R2 volume 60 μl.

[0075] Table 3 Precision test results of high-value samples (unit: U / L)

[0076]

[0077] Table 4 Precision test results of low-value samples (unit: U / L)

[0078]

[0079] It can be seen from Table 3 and Table 4 that both the kit of the present invention and the control kit can meet the industry standard coefficient of variation <5%, and the kit prepared in Example 1 performs best. The addition of Tween 20 and pH have an impact on the precision of the reagent.

[0080] Application Example 3

[0081] Glutathione reductase (Source Leaf Biotechnology, Article No. S10151) was added to purified water to prepare a standard solution. The standard solution with a concentration of 400U / L was diluted with purified water to 6 test samples with different concentrations (concentrations: 400U / L, 200U / L, 100U / L, 50U / L, 25U / L, 12.5U / L). The glutathione reductase detection kits of Examples 1 to 2 and Comparative Example 1, and the control kit (purchased from Beijing Jiuqiang Technology Co., Ltd., Article No. GSGR) were used to detect standard solutions of different concentrations. The test samples of each concentration were measured 3 times, and the mean (yi) of the test results was calculated. The linear regression equation was obtained with the dilution concentration (xi) as the independent variable and the mean (yi) of the test results as the dependent variable. The correlation coefficient (r) of the linear regression was calculated. The results are shown in Table 5. Among them,

[0082] The instrument used for the test was the fully automatic biochemical analyzer TBA-2000FR (manufacturer: Toshiba, Japan), and the detection parameters were set as follows: main wavelength 340 nm, sub-wavelength 380 nm, sample volume 20 μl, reagent R1 volume 240 μl, and reagent R2 volume 60 μl.

[0083] Table 5 Linearity results of different groups (unit: U / L)

[0084]

[0085] It can be seen from Table 5 that both the kit provided by the present invention and the control kit can meet the linearity requirements specified in the industry standard, and the addition of tris(2-carboxyethyl)phosphine hydrochloride does not affect the test results.

[0086] Application Example 4

[0087] Acceleration stability

[0088] The glutathione reductase detection kits of Examples 1-2 and Comparative Examples 1-2, and the control kit (purchased from Beijing Jiuqiang Technology Co., Ltd., with the article number GSGR) were used as the detection objects, and the reagents in the detection objects were taken respectively, placed in the instrument with the lid opened and in a constant temperature incubator at 37°C for acceleration. The 4°C reagent was calibrated with Jiuqiang GR calibrator, article number GC-GR (target value 70U / L), and then taken out 1-2 times a week to test Jiuqiang GR quality control level 1 and level 2, article number GQ-GR (target values ​​50U / L and 100U / L, respectively) under the 4°C calibration channel. The measurement results are shown in Table 6; wherein, the instrument used for detection is the fully automatic biochemical analyzer TBA-2000FR (manufacturer: Toshiba, Japan), and the detection parameters are set as follows: main wavelength 340nm, sub-wavelength 380nm, sample volume 20μl, reagent R1 volume 240μl, reagent R2 volume 60μl.

[0089] Table 6 Linearity results of different groups (unit: U / L)

[0090]

[0091] As shown in Table 6, the stability of the kit of the present invention can be maintained for one month after opening the bottle, and the accelerated stability can be maintained for 14 days. The comparative examples without tris(2-carboxyethyl)phosphine hydrochloride and replaced with dithiothreitol have poor opening and accelerated stability, indicating that tris(2-carboxyethyl)phosphine hydrochloride can effectively improve the stability of the reagent. The results of this application example show that the kit of the present invention is superior to the control reagent and comparative examples 1 and 2 in terms of stability, and the kit prepared in Example 1 has better stability.

[0092] Application Example 5

[0093] Long-term stability

[0094] Since accelerated stability cannot completely replace long-term stability, the glutathione reductase detection kits of Examples 1-2 and Comparative Examples 1-2, and the control kit (purchased from Beijing Jiuqiang Technology Co., Ltd., with the catalog number GSGR) were used as the test objects, and the reagents were placed in a refrigerator at 2-8°C. The 4°C reagent was calibrated with Jiuqiang GR calibrator, catalog number GC-GR (target value 70U / L), and then taken out once every 3 months to test Jiuqiang GR quality control level 1 and level 2, catalog number GQ-GR (target values ​​50U / L and 100U / L, respectively) and blank absorbance (NADPH activity can be reflected by blank absorbance) under the 4°C calibration channel. The test results are shown in Tables 7 and 8; wherein,

[0095] The instrument used for the test was the fully automatic biochemical analyzer TBA-2000FR (manufacturer: Toshiba, Japan), and the detection parameters were set as follows: main wavelength 340 nm, sub-wavelength 380 nm, sample volume 20 μl, reagent R1 volume 240 μl, and reagent R2 volume 60 μl.

[0096] Table 7 Linearity results of different groups (unit: U / L)

[0097]

[0098] Table 8 Blank absorbance results of different groups

[0099]

[0100] It can be seen from Tables 7 and 8 that the long-term stability of the kit of the present invention can be maintained for 12 months, and NADPH can also maintain a high activity after 12 months. The long-term stability of the kit prepared in Example 1 is better.

[0101] Application Example 6

[0102] 300mL 110mmol / L TRIS buffer was prepared, pH was adjusted to 9.4, 1.5mmol / L NADPH was added, and it was randomly divided into three portions, each 100mL, one of which (blank group) was not added, 1.8g / L tris(2-carboxyethyl)phosphine hydrochloride was added to the second portion, and 1.8g / L dithiothreitol was added to the third portion. At the same time, it was placed in a 37℃ constant temperature incubator, and the blank absorbance of accelerated 7 days and zero time was tested at a wavelength of 340nm on a UV spectrophotometer to determine whether the two raw materials can effectively protect dithiothreitol (the activity of NADPH can be reflected by the blank absorbance). The results are shown in Table 9; the instrument used for the detection was the fully automatic biochemical analyzer TBA-2000FR (manufacturer: Toshiba, Japan), and the detection parameters were set as follows: main wavelength 340nm, sub-wavelength 380nm, sample volume 20μl, reagent R1 volume 240μl, reagent R2 volume 60μl.

[0103] Table 9 Blank absorbance results of different groups

[0104]

[0105] It can be seen from Table 9 that tris(2-carboxyethyl)phosphine hydrochloride can more effectively protect NADPH than dithiothreitol, so that it maintains a higher activity.

[0106] It can be seen from the above content that the kit provided by the present invention is superior to industry standards and commercially available kits in terms of precision, linearity and stability, and has high clinical use value.

[0107] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A glutathione reductase detection kit, characterized in that: The glutathione reductase detection kit comprises an independently packaged reagent R1 and an independently packaged reagent R2; The reagent R1 includes: 20-100 mmol / L of TRIS buffer with a pH value of 8.0-8.5, 8-25 g / L of oxidized glutathione, 0.1-0.5 g / L of an interfering agent, 1-15 g / L of a surfactant, and 0.1-2 g / L of PROCLIN300; the interfering agent is disodium ethylenediaminetetraacetate; The reagent R2 comprises: 80-200 mmol / L of TRIS buffer with a pH value of 9.0-9.5, 0.1-2 g / L of PROCLIN300, 1-15 g / L of a surfactant, 0.5-4 g / L of sodium chloride, 1-3 mmol / L of NADPH, and 0.5-3 g / L of tris(2-carboxyethyl)phosphine hydrochloride; The surfactants in the reagent R1 and the reagent R2 are tween20 respectively.

2. The glutathione reductase detection kit according to claim 1, characterized in that The reagent R1 includes: 30-70 mmol / L TRIS buffer with a pH value of 8.0-8.5, 10-20 g / L oxidized glutathione, 0.2-0.4 g / L interfering agent, 5-10 g / L surfactant, and 0.5-1 g / L PROCLIN300; The reagent R2 comprises: 100-150 mmol / L of TRIS buffer with a pH value of 9.0-9.5, 0.5-1 g / L of PROCLIN300, 5-10 g / L of a surfactant, 1-2 g / L of sodium chloride, 1-2 mmol / L of NADPH, and 1-2 g / L of tris(2-carboxyethyl)phosphine hydrochloride.

3. The glutathione reductase detection kit according to claim 1, characterized in that The reagent R1 includes: 50-60 mmol / L TRIS buffer with a pH value of 8.1, 15 g / L oxidized glutathione, 0.3 g / L interfering agent, 8 g / L surfactant, and 0.7 g / L PROCLIN300; The reagent R2 includes: 110-140 mmol / L of TRIS buffer with a pH value of 9.4, 0.7 g / L of PROCLIN300, 8 g / L of a surfactant, 1.5 g / L of sodium chloride, 1.5 mmol / L of NADPH, and 1.8 g / L of tris(2-carboxyethyl)phosphine hydrochloride.

4. The glutathione reductase detection kit according to any one of claims 1 to 3, characterized in that The volume ratio of the reagent R1 to the reagent R2 is 4:

1.

5. The method for preparing the glutathione reductase detection kit according to any one of claims 1 to 4, characterized in that: The steps include: According to the component contents of reagent R1, TRIS buffer, oxidized glutathione, interfering agent, PROCLIN300 and surfactant are dissolved in pure water in sequence, and the pH is adjusted to 8.0-8.5 to obtain the reagent R1; According to the component contents of reagent R2, TRIS buffer, PROCLIN300, surfactant, sodium chloride and tri(2-carboxyethyl)phosphine hydrochloride are dissolved in pure water in sequence, the pH is adjusted to 9.0-9.5, and then NADPH is added to adjust the pH to 9.0-9.5 to obtain the reagent R2.

6. Use of the glutathione reductase detection kit according to any one of claims 1 to 4 or the glutathione reductase detection kit prepared by the preparation method according to claim 5 in detecting glutathione reductase; The use is for non-diagnostic and non-therapeutic purposes.

7. A method for detecting glutathione reductase for non-diagnostic and non-therapeutic purposes, characterized in that: The glutathione reductase detection kit according to any one of claims 1 to 4 or the glutathione reductase detection kit prepared by the preparation method according to claim 5 is used for detection using a fully automatic biochemical analyzer, with the main detection wavelength being 340 nm and the secondary detection wavelength being 380 nm.

8. The method according to claim 7, characterized in that The volume ratio of the reagent R1 to the sample to be tested in the glutathione reductase detection kit is 12:1.

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