CN112611727A - Glutathione transferase detection kit, preparation method and application - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a glutathione transferase detection kit, a preparation method and application thereof, wherein the glutathione transferase detection kit comprises the following components: the first reagent comprises buffer solution, 2, 4-dinitrochlorobenzene, ethanol or methanol, ascorbic acid oxidase, surfactant and preservative; the second reagent comprises buffer solution, reduced glutathione, stabilizer, surfactant and preservative. The ascorbic acid oxidase can oxidize reductive ascorbic acid, so that the influence of the ascorbic acid on a detection result is reduced, and the ethanol or the methanol can help the 2, 4-dinitrochlorobenzene to be better dissolved. The stability of the reduced glutathione can be improved by adding the stabilizer into the second reagent, and the accuracy of the detection result is ensured.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a glutathione transferase detection kit, a preparation method and application thereof.
Background
Glutathione transferase, i.e. Glutathione S-transferases (GST), is a group of isoenzymes related to the liver detoxification function, and the main function of the GST is to catalyze the coupling of electrophilic groups of certain endogenous or external harmful substances and thiol groups of reduced Glutathione, increase the hydrophobicity of the GST so that the GST can easily pass through cell membranes to be eliminated from the body, thereby achieving the purpose of detoxification. Thus, GST plays an important biological role in protecting cellular DNA and some proteins from damage by electrophilic compounds. In addition, GST also has anti-mutagenic, anti-tumor effects for reducing tumorigenesis, and can increase the metabolic capacity of cancer cells to chemotherapeutic drugs, resulting in drug resistance.
GST is widely distributed, with the highest content in the liver, accounting for about 5% of liver soluble proteins, and is rapidly released into the blood when hepatocytes are injured. Therefore, changes in the content and activity of this enzyme in peripheral blood reflect to some extent liver function and damage. GST is reported to be elevated significantly in acute hepatitis, and appears earlier than ALT (alanine aminotransferase) and AST (aspartate aminotransferase). Therefore, the GST assay has certain advantages over the ALT assay in early liver injury.
At present, the method for measuring glutathione transferase mainly comprises an Elisa method and a Radioimmunoassay (RIA), wherein the Elisa method needs manual operation, takes longer time, has complicated steps and higher cost, and the RIA method needs higher laboratory conditions besides the hidden dangers of radioactive pollution and human body injury.
At present, no glutathione transferase detection kit product exists at home and abroad.
Disclosure of Invention
The invention aims to provide a simple and quick glutathione transferase detection kit.
A glutathione transferase detection kit comprises the following components:
a first reagent comprising a buffer, 2, 4-dinitrochlorobenzene (CDNB), ethanol or methanol, ascorbate oxidase, a surfactant, and a preservative;
the second reagent comprises buffer solution, reduced Glutathione (GSH), stabilizer, surfactant and preservative.
The invention has the beneficial effects that: the ascorbic acid oxidase can oxidize reduced ascorbic acid, reduce the influence of ascorbic acid on the detection result, and the ethanol or methanol can help the 2, 4-dinitrochlorobenzene (CDNB) to be better dissolved. The stability of reduced Glutathione (GSH) can be improved by adding the stabilizer into the second reagent, and the accuracy of the detection result is ensured.
In addition, the glutathione transferase detection kit provided by the invention can also have the following additional technical characteristics:
further, the first reagent buffer and the second reagent buffer may be a phosphate buffer, a MOPS, a MOPSO buffer, or a Tris buffer.
Further, the surfactant is one or the combination of Triton X-100 and Tween 20.
Further, the stabilizer is one or a combination of TCEP, EGTA, EDTA-2Na and vitamin C magnesium stearate.
Further, the preservative is sodium azide or a biological preservative.
Further, the concentration of buffer solution in the first reagent and the concentration of buffer solution in the second reagent are both 50-200 mmol/L, the concentration of 2, 4-dinitrochlorobenzene is 1-5 mmol/L, the concentration of ascorbic acid oxidase is 0.5-5 KU/L, the content of ethanol or methanol is 8-20%, the content of surfactant in the first reagent and the second reagent is 0.2-0.8%, the concentration of reduced glutathione is 5-50 mmol/L, the concentration of stabilizer is 5-20 mmol/L, and the concentration of preservative in the first reagent and the second reagent is 0.1-1 g/L.
Further, the pH value of the buffer solution in the first reagent is adjusted to be 6.0-7.5, and the pH value of the buffer solution in the second reagent is adjusted to be 3.0-7.5.
The invention also aims to provide a preparation method of the glutathione transferase detection kit, which comprises the following steps:
weighing 2, 4-dinitrochlorobenzene (CDNB), slowly adding ethanol or methanol, and ultrasonically dissolving to obtain a CDNB dissolved solution;
adding ascorbic acid oxidase, a surfactant and a preservative into a buffer solution for full dissolution;
and adding the CDNB dissolved solution into the buffer solution under the stirring state, and uniformly mixing to obtain the first reagent.
Dissolving a stabilizer in a buffer solution to obtain a stabilizer buffer solution;
and adding reduced glutathione, surfactant and preservative, and dissolving completely to obtain the second reagent.
The invention also aims to provide an application of the glutathione transferase detection kit, which comprises the following steps:
placing the first reagent and the second reagent in a biochemical analyzer, and adjusting the temperature to 37 deg.C
(1) Mixing and reacting a sample with the first reagent to obtain a reaction solution 1, and incubating for 300 seconds;
(2) mixing the reaction solution 1 obtained in the step (1) with the second reagent for reaction to obtain a reaction solution 2, and incubating for 60 seconds;
(3) and (3) reading the change rate value of the absorbance of the reaction liquid 2 obtained in the step (2) at 19-25 points at the main wavelength of 340nm and the sub-wavelength of 405nm, and calculating by using a standard liquid sample calibrated by the same parameters to obtain the content of the glutathione transferase.
The apparatus used in the above measurement method is a biochemical analyzer conventionally used in the art, and includes a Hitachi 7180 full-automatic biochemical analyzer or a Beckmann series full-automatic biochemical analyzer.
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 reaction line in the detection of a sample in example 1 of the present invention;
FIG. 2 is a reaction line in the case of detecting a specimen in example 2 of the present invention;
FIG. 3 is a reaction line in the case of detecting a sample in example 3 of the present invention;
FIG. 4 is a reaction line in the case of detecting a sample in example 4 of the present invention;
FIG. 5 is a reaction line in the case of detecting a specimen in example 5 of the present invention;
FIG. 6 is a reaction line in the case of detecting a specimen in example 6 of the present invention;
FIG. 7 is a reaction line in the case of detecting a specimen in comparative example 2 of the present invention;
FIG. 8 is a reaction line in the case of detecting a specimen in comparative example 3 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.
Example 1
A glutathione transferase detection kit comprises the following components:
a first reagent, which comprises phosphate buffer solution, 2, 4-dinitrochlorobenzene (CDNB), ethanol, ascorbic acid oxidase, Triton X-100 and sodium azide;
and the second reagent comprises phosphate buffer solution, TCEP, reduced Glutathione (GSH), Triton X-100 and sodium azide.
The invention has the advantages that:
the ascorbic acid oxidase can oxidize reduced ascorbic acid, reduce the influence of ascorbic acid on detection results, and the ethanol can help 2, 4-dinitrochlorobenzene (CDNB) to be better dissolved. The stability of reduced Glutathione (GSH) can be improved by adding the stabilizer TCEP in the second reagent, and the accuracy of a detection result is ensured.
Specifically, the concentration of the phosphate buffer solution is 50mmol/L, the concentration of 2, 4-dinitrochlorobenzene (CDNB) is 1mmol/L, the concentration of ascorbic acid oxidase is 0.5KU/L, the concentration of ethanol is 8%, the concentration of a stabilizer TCEP is 5mmol/L, the concentration of reduced glutathione is 5mmol/L, the concentration of a surfactant Triton X-100 is 0.2%, and the concentration of a preservative sodium azide is 0.5 g/L.
The first reagent buffer solution was adjusted to a pH of 6.0, and the second reagent buffer solution was adjusted to a pH of 3.0. Adjusting the pH ensures that the difference between the enzymatic and non-enzymatic reactions is maximized and that the starting material is relatively stable at this pH.
The application of the glutathione transferase detection kit comprises the following steps:
placing the first reagent and the second reagent in a biochemical analyzer, and adjusting the temperature to 37 deg.C
(1) Mixing and reacting a sample with the first reagent to obtain a reaction solution 1, and incubating for 300 seconds;
(2) mixing the reaction solution 1 obtained in the step (1) with the second reagent for reaction to obtain a reaction solution 2, and incubating for 60 seconds;
(3) and (3) reading the change rate value of the absorbance of the reaction liquid 2 obtained in the step (2) at 19-25 points at the main wavelength of 340nm and the sub-wavelength of 405nm, and calculating by using a standard liquid sample calibrated by the same parameters to obtain the content of the glutathione transferase.
The application of the glutathione transferase detection kit comprises the following specific test conditions:
the method comprises the following steps: a rate method;
primary/secondary wavelength: 340nm/405 nm;
temperature: 37 ℃;
the correction type is as follows: linearity;
the calibration method comprises the following steps: calibrating at two points;
the reaction direction is as follows: upwards.
And (4) calculating a result:
sample GR Activity (U/L) ═ sample Δ A/min/calibrator Δ A/min × calibrator GR Activity (U/L)
TABLE 1 glutathione transferase assay reagent protocols
Sample requirements:
serum or plasma without haemolysis.
Sample stability: the specimen can be stored stably for 3 days at the temperature of 2-8 ℃ and for 2 weeks at the temperature of-20 ℃.
In this example, the biochemical analyzer was a Hitachi 7180 full-automatic biochemical analyzer, the calibrator was a mixture of phosphate buffer, bovine serum and sucrose, the value of GST activity (U/L) of the calibrator was known, and the calibrator Δ A/min was obtained when the calibrator was used for calibration.
Example 2
This embodiment is substantially the same as embodiment 1 except that:
this example includes the following components:
a first reagent, which comprises MOPSO buffer solution, 2, 4-dinitrochlorobenzene (CDNB), ethanol, ascorbic acid oxidase, Tween 20 and sodium azide;
and the second reagent comprises MOPSO buffer solution, TCEP, reduced Glutathione (GSH), Tween 20 and sodium azide.
Specifically, the concentration of the MOPSO buffer solution is 50mmol/L, the concentration of 2, 4-dinitrochlorobenzene (CDNB) is 1mmol/L, the concentration of ascorbic acid oxidase is 0.5KU/L, the concentration of ethanol is 8%, the concentration of a stabilizer TCEP is 5mmol/L, the concentration of reduced glutathione is 5mmol/L, the concentration of a surfactant Tween 20 is 0.5%, and the concentration of a preservative sodium azide is 0.5 g/L.
The first reagent buffer solution was adjusted to a pH of 6.5, and the second reagent buffer solution was adjusted to a pH of 6.5. Adjusting the pH ensures that the difference between the enzymatic and non-enzymatic reactions is maximized and that the starting material is relatively stable at this pH.
Example 3
This embodiment is substantially the same as embodiment 1 except that:
this example includes the following components:
a first reagent which comprises phosphate buffer solution, 2, 4-dinitrochlorobenzene (CDNB), ethanol, ascorbic acid oxidase, Tween 20 and sodium azide;
the second reagent comprises Tris buffer solution, EGTA, reduced Glutathione (GSH), Tween 20 and Proclin-300.
Specifically, the concentration of the phosphate buffer solution is 100mmol/L, the concentration of the Tris buffer solution is 80mmol/L, the concentration of 2, 4-dinitrochlorobenzene (CDNB) is 5mmol/L, the concentration of ascorbic acid oxidase is 0.8KU/L, the concentration of ethanol is 12%, the concentration of a stabilizer EGTA is 8mmol/L, the concentration of reduced glutathione is 12mmol/L, the concentration of surfactant Tween 20 is 0.4%, the concentration of a preservative sodium azide is 0.5g/L, and the concentration of the preservative Proclin-300 is 0.05%.
The first reagent buffer solution was adjusted to a pH of 6.5, and the second reagent buffer solution was adjusted to a pH of 4.0.
Example 4
This embodiment is substantially the same as embodiment 1 except that:
this example includes the following components:
the first reagent comprises MOPS buffer solution, 2, 4-dinitrochlorobenzene (CDNB), ethanol, ascorbic acid oxidase, Tween 20 and sodium azide;
the second reagent comprises Tris buffer solution, EDTA-2Na, reduced Glutathione (GSH), Triton X-100 and Proclin-300.
Specifically, the concentration of the MOPS buffer solution is 100mmol/L, the concentration of the Tris buffer solution is 100mmol/L, the concentration of 2, 4-dinitrochlorobenzene (CDNB) is 3mmol/L, the concentration of ascorbic acid oxidase is 2KU/L, the concentration of ethanol is 18%, the concentration of EDTA-2Na serving as a stabilizer is 10mmol/L, the concentration of reduced glutathione is 10mmol/L, the concentration of Tween 20 serving as a surfactant is 0.4%, the concentration of Triton X-100 serving as a surfactant is 0.3%, the concentration of sodium azide serving as a preservative is 0.6g/L, and the concentration of Proclin-300 serving as a preservative is 0.08%.
The first reagent buffer solution was adjusted to a pH of 6.7, and the second reagent buffer solution was adjusted to a pH of 3.5.
Example 5
This embodiment is substantially the same as embodiment 1 except that:
this example includes the following components:
the first reagent comprises MOPS buffer solution, 2, 4-dinitrochlorobenzene (CDNB), ethanol, ascorbic acid oxidase, Tween 20 and sodium azide;
the second reagent comprises phosphate buffer solution, EDTA-2Na, vitamin C magnesium stearate, reduced Glutathione (GSH), Triton X-100, and Proclin-300.
Specifically, the concentration of the MOPS buffer solution is 100mmol/L, the concentration of the phosphate buffer solution is 100mmol/L, the concentration of 2, 4-dinitrochlorobenzene (CDNB) is 3mmol/L, the concentration of ascorbic acid oxidase is 3KU/L, the concentration of ethanol is 10%, the concentration of EDTA-2Na serving as a stabilizer is 10mmol/L, the concentration of magnesium stearate serving as vitamin C is 5mmol/L, the concentration of reduced glutathione is 10mmol/L, the concentration of Tween 20 serving as a surfactant is 0.3%, the concentration of Triton X-100 serving as a surfactant is 0.3%, the concentration of sodium azide serving as a preservative is 0.6g/L, and the concentration of Proclin-300 serving as a preservative is 0.08%.
The first reagent buffer solution was adjusted to a pH of 6.5, and the second reagent buffer solution was adjusted to a pH of 6.5.
Example 6
This embodiment is substantially the same as embodiment 1 except that:
this example includes the following components:
a first reagent which comprises phosphate buffer solution, 2, 4-dinitrochlorobenzene (CDNB), ethanol, ascorbic acid oxidase, Tween 20 and sodium azide;
the second reagent comprises MOPS buffer solution, TCEP, EDTA-2Na, vitamin C magnesium stearate, reduced Glutathione (GSH), Triton X-100, and Proclin-300.
Specifically, the concentration of the MOPS buffer solution is 80mmol/L, the concentration of the phosphate buffer solution is 120mmol/L, the concentration of 2, 4-dinitrochlorobenzene (CDNB) is 4mmol/L, the concentration of ascorbic acid oxidase is 3KU/L, the concentration of ethanol is 10%, the concentration of a stabilizer TCEP is 4mmol/L, the concentration of EDTA-2Na is 10mmol/L, the concentration of vitamin C magnesium stearate is 5mmol/L, the concentration of reduced glutathione is 10mmol/L, the concentration of a surfactant Tween 20 is 0.3%, the concentration of a surfactant Triton X-100 is 0.3%, the concentration of a preservative sodium azide is 0.6g/L, and the concentration of a preservative Proclin-300 is 0.08%.
The first reagent buffer solution was adjusted to a pH of 6.5, and the second reagent buffer solution was adjusted to a pH of 6.5.
Comparative example 1
This embodiment is substantially the same as embodiment 1 except that:
to the first reagent, 2, 4-dinitrochlorobenzene (CDNB) was dissolved without adding ethanol.
Comparative example 2
This embodiment is substantially the same as embodiment 1 except that:
in the second reagent, no stabilizer was added.
Comparative example 3
This embodiment is substantially the same as embodiment 1 except that:
the first and second reagents were formulated without following the procedure of example 1.
The results of measurements on samples of known activity using examples 1 to 6 and comparative examples 1 to 3, respectively, and the deviation from the known value are shown in Table 2.
TABLE 2
As can be seen from Table 1, the examples 1 to 6 all achieved good results, and the maximum deviation did not exceed 15%. In contrast, in the first reagent of comparative example 1, 2, 4-dinitrochlorobenzene (CDNB) was not dissolved in ethanol, and the result of the sample could not be detected; in the second reagent of comparative example 2, no stabilizer was added, and the detection result of the sample had a large deviation, which was more pronounced at low activity values; comparative example 3 although the first and second reagent components were not changed, the preparation was not carried out according to the procedure of example 1, so that the major component of 2, 4-dinitrochlorobenzene (CDNB) was not completely dissolved and the detection results were also significantly deviated.
The attached figures 1-6 show that the reaction curve of the detection sample of the normal and effective reagent in the examples 1-6 is a smooth straight line connected with each point in the service life, the reaction rate is high, and the slope of the reaction line is large.
FIG. 7 is a graph showing the reaction rate of the sample in comparative example 2, which is a smooth straight line connecting the points, but the reaction rate is decreased, the slope of the reaction line is decreased, and the value of the sample is slightly decreased.
FIG. 8 is a graph showing the reaction of the sample in comparative example 3, in which the reaction rate is further decreased, the slope of the reaction line is further decreased, and the deviation of the detection value of the sample is larger due to improper formulation of the first reagent and the second reagent.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. The glutathione transferase detection kit is characterized by comprising the following components:
the first reagent comprises buffer solution, 2, 4-dinitrochlorobenzene, ethanol or methanol, ascorbic acid oxidase, surfactant and preservative;
the second reagent comprises buffer solution, reduced glutathione, stabilizer, surfactant and preservative.
2. The glutathione transferase assay kit of claim 1, wherein the buffer in the first reagent is any one of phosphate buffer, MOPS, MOPSO buffer, and Tris buffer, and the buffer in the second reagent is any one of phosphate buffer, MOPS, MOPSO buffer, and Tris buffer.
3. The glutathione transferase assay kit of claim 1, wherein the surfactant is one or a combination of Triton X-100, Tween 20.
4. The glutathione transferase assay kit of claim 1, wherein the stabilizer is one or a combination of TCEP, EGTA, EDTA-2Na and magnesium stearate.
5. The glutathione transferase assay kit of claim 1, wherein the preservative is sodium azide or a biological preservative.
6. The glutathione transferase assay kit according to any one of claims 1 to 5, wherein the concentration of the buffer solution in the first reagent and the second reagent is 50 to 200mmol/L, the concentration of the 2, 4-dinitrochlorobenzene is 1 to 5mmol/L, the concentration of the ascorbate oxidase is 0.5 to 5KU/L, the content of ethanol or methanol is 8 to 20%, the content of the surfactant in the first reagent and the second reagent is 0.2 to 0.8%, the concentration of the reduced glutathione is 5 to 50mmol/L, the concentration of the stabilizer is 5 to 20mmol/L, and the concentration of the preservative in the first reagent and the second reagent is 0.1 to 1 g/L.
7. The glutathione transferase assay kit of claim 1 or 2, wherein the pH value of the buffer solution in the first reagent is adjusted to 6.0 to 7.5, and the pH value of the buffer solution in the second reagent is adjusted to 3.0 to 7.5.
8. The method for preparing the glutathione transferase assay kit of any one of claims 1 to 7, comprising the steps of:
weighing 2, 4-dinitrochlorobenzene, slowly adding ethanol or methanol, and ultrasonically dissolving to obtain a CDNB dissolving solution;
adding ascorbic acid oxidase, a surfactant and a preservative into a buffer solution for full dissolution;
adding the CDNB dissolved solution into the buffer solution under the stirring state, and uniformly mixing to obtain a first reagent;
dissolving a stabilizer in a buffer solution to obtain a stabilizer buffer solution;
and adding reduced glutathione, surfactant and preservative, and dissolving completely to obtain the second reagent.
9. The use of the glutathione transferase assay kit of any one of claims 1 to 7, comprising the steps of:
placing the first reagent and the second reagent in a biochemical analyzer, and adjusting the temperature to be 37 ℃;
(1) mixing the sample with the first reagent for reaction to obtain a reaction solution 1, and incubating for 300 seconds;
(2) mixing the reaction solution 1 obtained in the step (1) with the second reagent for reaction to obtain a reaction solution 2, and incubating for 60 seconds;
(3) and (3) reading the change rate value of the absorbance of the reaction liquid 2 obtained in the step (2) at 19-25 points at the main wavelength of 340nm and the sub-wavelength of 405nm, and calculating by using a standard liquid sample calibrated by the same parameters to obtain the content of the glutathione transferase.
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