阅读说明：本技术 谷胱甘肽还原酶检测试剂盒及应用 (Glutathione reductase detection kit and application ) 是由 万蒙 周惠良 于 2019-11-01 设计创作，主要内容包括：本发明公开了一种谷胱甘肽还原酶检测试剂盒,包括以下组分：第一试剂,包括磷酸盐缓冲液、EDTA-NA2、抗坏血酸氧化酶、高铁氰化钾、防腐剂和氧化型谷胱甘肽；第二试剂,包括三羟甲基胺基甲烷缓冲液、EGTA、防腐剂和还原型辅酶。第一试剂和第二试剂均为液态,可在2～8℃环境下保存12个月；高铁氰化钾能氧化血红蛋白,生成稳定的氰化高铁血红蛋白,减小检测结果的影响；第二试剂采用EGTA能提高检测结果与吸光度变化率的线性关系,便于采用二点定标。本发明还公开了一种采用上述谷胱甘肽还原酶检测试剂盒的应用,步骤为：分别向样本液中加入第一试剂和第二试剂,放入生化分析仪内孵育,读取吸光度并计算吸光度变化率△A1,再计算样本的谷胱甘肽还原酶活性。(The invention discloses a glutathione reductase detection kit which comprises reagent, including phosphate buffer solution, EDTA-NA2, ascorbic acid oxidase, potassium ferricyanide, preservative and oxidized glutathione, second reagent, including tris buffer solution, EGTA, preservative and reduced coenzyme, reagent and the second reagent are both in liquid state and can be stored for 12 months at 2-8 ℃, potassium ferricyanide can oxidize hemoglobin to generate stable ferrihemoglobin cyanide and reduce the influence of the detection result, EGTA adopted by the second reagent can improve the linear relation between the detection result and the absorbance change rate, and two-point calibration is convenient to adopt.)

1, glutathione reductase assay kit, characterized by comprising the following components:

reagent comprising phosphate buffer solution, EDTA-NA2, ascorbic acid oxidase, potassium ferricyanide, antiseptic and oxidized glutathione;

and the second reagent comprises tris buffer solution, EGTA, preservative and reduced coenzyme.

2. The glutathione reductase assay kit of claim 1, wherein the concentration of the phosphate buffer solution is 50-400 mmol/L, the concentration of EDTA-NA2 is 1-5 mmol/L, the concentration of the ascorbate oxidase is 0.5-5 KU/L, the concentration of potassium ferricyanide is 0.01-0.3 mmol/L, the concentration of the preservatives is 0.1-1 g/L, the concentration of the oxidized glutathione is 1.5-8 mmol/L, the concentration of the tris buffer solution is 50-300 mmol/L, the concentration of the EGTA is 1-8 mmol/L, and the concentration of the reduced coenzyme is 0.2-2.0 mmol/L.

3. The glutathione reductase assay kit of claim 1, wherein the preservative is sodium azide or a biological preservative.

4. The glutathione reductase assay kit of claim 1, wherein the pH value of the reagent is adjusted to 6.5-7.5 by hydrochloric acid, and the pH value of the second reagent is adjusted to 9.0-10.0 by sodium hydroxide.

5. The use of the glutathione reductase assay kit of any of claims 1-4, comprising the steps of:

taking fresh blood to prepare a sample solution;

adding the th reagent into the sample solution, and uniformly mixing to obtain a mixed solution;

placing the mixed solution in a biochemical analyzer, adjusting the temperature to be 30-40 ℃, and incubating for 300 seconds;

adding a second reagent, adjusting the temperature to be 30-40 ℃, and incubating for 60 seconds;

setting the main wavelength of the biochemical analyzer to be 340nm and the auxiliary wavelength to be 546nm, reading the absorbance, and calculating the average change rate △ A1 of the absorbance of the sample liquid;

obtaining average change rate △ A2 and activity B2 of preset calibrator absorbance, calculating activity B1 ═ △ A1 ÷ △ A2 × B2 × k of the sample, wherein k is an empirical parameter, and taking activity B1 of the sample as an index for evaluating glutathione reductase in the sample solution.

6. The use of the glutathione reductase assay kit of claim 5, wherein the sample fluid is a serum sample, a plasma sample or a red blood cell sample, wherein k is 1 when the sample fluid is a serum sample or a plasma sample, and k is 20 ÷ M when the sample fluid is a red blood cell sample, wherein M is the hemoglobin level of the sample fluid.

7. The use of the glutathione reductase assay kit of claim 6, wherein the preparation of the red blood cell sample comprises:

taking fresh blood with volume V, centrifuging to remove blood plasma to obtain red blood cells;

carrying out suspension cleaning on the red blood cells by adopting a 0.3-0.9% sodium chloride solution;

centrifuging and layering, removing supernatant, and adding distilled water to volume V;

standing for 10 minutes at the temperature of 2-8 ℃, centrifuging and layering, taking the upper-layer lysate liquid, and removing the tube bottom matrix of the lysate liquid to obtain a sample liquid;

and adding the sodium chloride solution into the sample solution to obtain the erythrocyte sample, wherein the volume ratio of the sample solution to the added sodium chloride solution is 5: 15-20.

8. The application of the glutathione reductase detection kit according to claim 7, wherein the temperature of the distilled water is 0-5 ℃.

9. The use of the glutathione reductase assay kit of claim 5, wherein the step of reading the absorbance is:

the absorbance was read 6 times in succession, each time at 20 second intervals.

10. The use of the glutathione reductase assay kit of claim 7, wherein the concentration of the sodium chloride solution is 0.9%.

Technical Field

The invention relates to the technical field of biochemical detection, in particular to glutathione reductase detection kits and application thereof.

Background

Glutathione Reductase (GR) is flavoenzymes, which can make thiol (-SH) containing enzyme in reduction state and activity state, maintain the integrity of erythrocyte, and prevent hemoglobin oxidation.

At present, methods for measuring glutathione reductase mainly comprise an Elisa method and an ultraviolet enzyme method, wherein the Elisa method needs manual operation, is long in time consumption, complicated in steps and high in cost, and the ultraviolet enzyme method is simple in steps and low in cost and is widely applied to by .

The existing kit for glutathione reductase by using an ultraviolet enzyme method is mainly an imported dry powder reagent, and needs to be dissolved by buffer solution and distilled water before use, and the dissolved reagent solution can be only stored for two days at the temperature of 2-8 ℃, so that waste can be caused when the reagent solution is inexhaustible.

Disclosure of Invention

aims to provide glutathione reductase detection kits which are not easy to cause waste, and the reagents are liquid.

glutathione reductase detection kit, comprising the following components:

reagent comprising phosphate buffer solution, EDTA-NA2, ascorbic acid oxidase, potassium ferricyanide, antiseptic and oxidized glutathione;

and the second reagent comprises tris buffer solution, EGTA, preservative and reduced coenzyme.

The kit has the advantages that the th reagent and the second reagent are both in liquid state, and can be stored for 12 months under the environment of 2-8 ℃ through thermal stability test and thermal acceleration investigation, potassium ferricyanide can oxidize ferrous ions (Fe2+) in hemoglobin into ferric ions (Fe3+) to generate stable cyanide methemoglobin, the influence on the detection result of the reagents is reduced, the second reagent adopts EGTA can improve the linear relation between the detection result and the absorbance change rate, and two-point calibration is convenient to adopt.

In addition, the glutathione reductase detection kit provided by the invention can also have the following additional technical characteristics:

, wherein the concentration of the phosphate buffer solution is 50-400 mmol/L, the concentration of EDTA-NA2 is 1-5 mmol/L, the concentration of the ascorbic acid oxidase is 0.5-5 KU/L, the concentration of the potassium ferricyanide is 0.01-0.3 mmol/L, the concentration of the preservative is 0.1-1 g/L, the concentration of the oxidized glutathione is 1.5-8 mmol/L, the concentration of the tris (hydroxymethyl) aminomethane buffer solution is 50-300 mmol/L, the concentration of the EGTA is 1-8 mmol/L, and the concentration of the reduced coenzyme is 0.2-2.0 mmol/L.

Further , the preservative is sodium azide or a biological preservative.

, the reagent adopts hydrochloric acid to adjust the pH value to 6.5-7.5, and the second reagent adopts sodium hydroxide to adjust the pH value to 9.0-10.0.

Another of the invention aims to provide applications of the glutathione reductase detection kit, which comprises the following steps:

taking fresh blood to prepare a sample solution;

adding the th reagent into the sample solution, and uniformly mixing to obtain a mixed solution;

placing the mixed solution in a biochemical analyzer, adjusting the temperature to be 30-40 ℃, and incubating for 300 seconds;

adding a second reagent, adjusting the temperature to be 30-40 ℃, and incubating for 60 seconds;

setting the main wavelength of the biochemical analyzer to be 340nm and the auxiliary wavelength to be 546nm, reading the absorbance, and calculating the average change rate △ A1 of the absorbance of the sample liquid;

obtaining average change rate △ A2 and activity B2 of preset calibrator absorbance, calculating activity B1 ═ △ A1 ÷ △ A2 × B2 × k of the sample, wherein k is an empirical parameter, and taking activity B1 of the sample as an index for evaluating glutathione reductase in the sample solution.

Further , the sample fluid is a serum sample, a plasma sample or a red blood cell sample, where k is 1 when the sample fluid is a serum sample or a plasma sample, and k is 20 ÷ M when the sample fluid is a red blood cell sample, where M is the hemoglobin level of the sample fluid.

Further , the red blood cell sample preparation step includes:

taking fresh blood with volume V, centrifuging to remove blood plasma to obtain red blood cells;

carrying out suspension cleaning on the red blood cells by adopting a 0.3-0.9% sodium chloride solution;

centrifuging and layering, removing supernatant, and adding distilled water to volume V;

standing for 10 minutes at the temperature of 2-8 ℃, centrifuging and layering, taking the upper-layer lysate liquid, and removing the tube bottom matrix of the lysate liquid to obtain a sample liquid;

and adding the sodium chloride solution into the sample solution to obtain the erythrocyte sample, wherein the volume ratio of the sample solution to the added sodium chloride solution is 5: 15-20.

, the temperature of the distilled water is 0-5 ℃.

Further , the step of reading the absorbance is:

the absorbance was read 6 times in succession, each time at 20 second intervals.

Further , the concentration of the sodium chloride solution is 0.9%.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a graph comparing the effect of washing red blood cells with sodium chloride of various concentrations according to the present invention;

FIG. 2 is a 320U/L reaction curve of the enzymatic activity of the reagent of comparative example 1 of the present invention;

FIG. 3 is a 320U/L reaction curve of the reagent enzyme activity of example 1 of the present invention;

FIG. 4 is a 320U/L reaction curve of the reagent enzyme activity of example 2 of the present invention;

FIG. 5 is a 320U/L reaction curve of the reagent enzyme activity of example 3 of the present invention;

FIG. 6 is a 320U/L reaction curve of the reagent enzyme activity of example 4 of the present invention.

Detailed Description

In order to make the objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. Several embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.