CN113528609A - Glutathione reductase detection kit and preparation method and application thereof - Google Patents
Glutathione reductase detection kit and preparation method and application thereof Download PDFInfo
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- 108010063907 Glutathione Reductase Proteins 0.000 title claims abstract description 42
- 102100036442 Glutathione reductase, mitochondrial Human genes 0.000 title claims abstract description 42
- 238000001514 detection method Methods 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 105
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 46
- 239000000022 bacteriostatic agent Substances 0.000 claims abstract description 32
- 108010053070 Glutathione Disulfide Proteins 0.000 claims abstract description 28
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 claims abstract description 28
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- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 claims abstract description 24
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- 239000007853 buffer solution Substances 0.000 claims abstract description 23
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- LCTORFDMHNKUSG-XTTLPDOESA-N vancomycin monohydrochloride Chemical group Cl.O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 LCTORFDMHNKUSG-XTTLPDOESA-N 0.000 claims description 13
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- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 5
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- 108010088751 Albumins Proteins 0.000 claims description 3
- SFNALCNOMXIBKG-UHFFFAOYSA-N ethylene glycol monododecyl ether Chemical compound CCCCCCCCCCCCOCCO SFNALCNOMXIBKG-UHFFFAOYSA-N 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 238000003149 assay kit Methods 0.000 claims description 2
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
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- 238000003556 assay Methods 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 229920000136 polysorbate Polymers 0.000 description 20
- 102000004190 Enzymes Human genes 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 16
- 229930183010 Amphotericin Natural products 0.000 description 14
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 description 14
- 229940009444 amphotericin Drugs 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 10
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- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
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- 239000002211 L-ascorbic acid Substances 0.000 description 5
- 235000000069 L-ascorbic acid Nutrition 0.000 description 5
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
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- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 3
- 230000020477 pH reduction Effects 0.000 description 3
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- 239000002994 raw material Substances 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
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- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
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- 235000019154 vitamin C Nutrition 0.000 description 2
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- 241000894006 Bacteria Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 108010042687 Pyruvate Oxidase Proteins 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 231100000354 acute hepatitis Toxicity 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
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- 239000005515 coenzyme Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 239000000852 hydrogen donor Substances 0.000 description 1
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- 230000000670 limiting effect Effects 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000006864 oxidative decomposition reaction Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
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- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a glutathione reductase detection kit, a preparation method and application thereof, wherein the kit comprises a reagent R1 and a reagent R2, and the reagent R1 comprises: potassium phosphate buffer solution, EDTA, GSSG, ascorbic acid oxidase, bilirubin oxidase, potassium ferricyanide, surfactant, first bacteriostatic agent and second bacteriostatic agent; the reagent R2 includes: sodium bicarbonate buffer solution, NADPH, a first bacteriostatic agent, a surfactant and a stabilizing agent. Compared with the prior art, the glutathione reductase detection kit has the advantages of good airborne stability, high sensitivity and strong antibacterial ability, and can meet the clinical application of detecting glutathione reductase.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnostic reagents, in particular to a glutathione reductase detection kit and a preparation method and application thereof.
Background
Glutathione Reductase (GR) is one of important enzymes of human redox system, reduced coenzyme II (NADPH) is used as a hydrogen donor to reduce oxidized glutathione (GSSG) to reduced Glutathione (GSH) under the catalytic action of GR, and GSH can prevent the oxidative decomposition of hemoglobin to maintain the integrity of cells. GR can reflect liver injury early, acute hepatitis responds earlier than transaminase, and GR can reflect jaundice type, and activity of GR is normal level.
At present, methods for detecting glutathione reductase comprise an ELISA method and an ultraviolet enzyme method, wherein the ELISA method is complex in operation and time-consuming in detection, a main reagent of the ultraviolet enzyme method is a dry powder reagent, redissolution is needed before use, the reagent needs to be used in a very short time after redissolution, and otherwise waste is caused. The detection reagent in the ultraviolet enzyme method can also adopt a liquid double reagent, but the current domestic liquid double reagents have the defects of poor airborne stability, low sensitivity, poor antibacterial capability and the like.
Chinese patent CN110734952A discloses a glutathione reductase detection kit and application thereof, the components are as follows: r1 phosphate buffer solution, EDTA, ascorbic acid oxidase, potassium ferricyanide, preservative and oxidized glutathione; r2: TRIS buffer solution, EGTA and preservative reduced coenzyme. The invention discloses the application of the kit, but the kit has poor stability and weak anti-interference capability.
Chinese patent CN111321198A discloses a glutathione reductase assay kit and a preparation method and application thereof, and the components are as follows: r1: phosphate buffer, ascorbic acid oxidase, pyruvate oxidase, FAD, potassium ferrocyanide, oxidized glutathione, MgCl2Preservatives and surfactants; r2: glycine buffer solution, NADPH, EGTA, dithiothreitol, preservative and protective agent. The invention discloses the actual preparation and application of the kit, but the kit has poor airborne stability, low sensitivity and weak antibacterial ability.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the glutathione reductase detection kit and the preparation method and the application thereof.
The invention adopts the following technical scheme: a glutathione reductase detection kit comprises a reagent R1 and a reagent R2, wherein:
the reagent R1 includes: potassium phosphate buffer solution, EDTA, GSSG, ascorbic acid oxidase, bilirubin oxidase, potassium ferricyanide, surfactant, first bacteriostatic agent and second bacteriostatic agent;
the reagent R2 includes: sodium bicarbonate buffer solution, NADPH, a first bacteriostatic agent, a surfactant and a stabilizing agent.
Compared with the prior art, the invention has the following advantages:
(1) in the prior art, the phenomenon of obvious pH reduction can occur after an R2 reagent prepared from a glycine buffer solution is opened, and the pH reduction speed can be slowed down by adopting a sodium bicarbonate buffer solution, so that the stable pH environment of an enzyme is ensured;
(2) the interference of bilirubin and vitamin C can be effectively removed by bilirubin oxidase and ascorbic acid oxidase;
(3) the potassium ferricyanide can oxidize iron ions in the hemoglobin, and the interference of the hemoglobin is effectively reduced.
Further, in the reagent R1, the concentration of potassium phosphate buffer solution is 50-250mmol/L, the concentration of EDTA is 0.5-2mmol/L, the concentration of GSSG is 0.5-2mmol/L, the concentration of ascorbic acid oxidase is 1-5KU/L, the concentration of bilirubin oxidase is 0.5-2KU/L, and the concentration of potassium ferricyanide is 0.05-0.1 g/L.
Preferably, in the reagent R1, the concentration of potassium phosphate buffer solution is 50mmol/L, the concentration of EDTA is 1mmol/L, the concentration of GSSG is 1.5mmol/L, the concentration of ascorbic acid oxidase is 3KU/L, the concentration of bilirubin oxidase is 1KU/L, and the concentration of potassium ferricyanide is 0.05 g/L.
Further, in the reagent R2, the concentration of sodium bicarbonate buffer solution was 20 to 100mmol/L, and the concentration of NADPH was 0.5 to 2.5 g/L.
Preferably, in the reagent R2, the concentration of sodium bicarbonate buffer solution is 25mmol/L and the concentration of NADPH is 1.5 g/L.
Further, the pH of the reagent R1 is 7.0 to 7.5, and the pH of the reagent R2 is 9.5 to 10.5. The reagent R1 and the reagent R2 are kept in a slightly alkaline environment, so that the detection of the detection reagent is more accurate and stable.
Further, the volume ratio of the reagent R1 to the reagent R2 was 5: 1.
Further, the surfactant is selected from one or more of P123, F108, Brij35, Triton405, Triton100, Tween 20 and Tween 80. P123 in the surfactant is matched with Tween 80 or F108 is matched with Tween 80, so that the airborne stability of the reagent is effectively improved.
Preferably, the surfactant is P123 at a concentration of 0.05g/L and Tween 80 at a concentration of 5 ml/L.
Preferably, the surfactant is F108 at a concentration of 0.01g/L and Tween 80 at a concentration of 5 ml/L.
Further, the first bacteriostatic agent is water-soluble amphotericin B, and the second bacteriostatic agent is vancomycin hydrochloride. The first bacteriostatic agent adopts water-soluble amphotericin broad-spectrum antifungal, the second bacteriostatic agent adopts vancomycin hydrochloride broad-spectrum antibacterial, and the first bacteriostatic agent and the second bacteriostatic agent do not interfere with other components in the reagent; in addition, the water-soluble amphotericin is composed of amphotericin deoxycholate sodium in a high proportion, and the amphotericin deoxycholate sodium can enhance the stability and antibacterial capacity of reductase, thereby improving the airborne stability of the reagent
Further, the stabilizer is a mixture of bovine serum albumin, sorbitol and riboflavin, and the mass ratio of albumin, sorbitol and riboflavin in the stabilizer is 0.1-2: 2-5: 0.5-1.
Preferably, the mass ratio of the bovine serum albumin, the sorbitol and the riboflavin is 1: 2: 0.5. the stabilizer which is composed of the bovine serum albumin, the sorbitol and the riboflavin with the optimal proportion (the mass ratio of 1: 2: 0.5) has the effect of stabilizing the NADPH, and can further improve the stability of the reagent.
The invention also provides a preparation method of the glutathione reductase detection kit, which comprises the following steps:
s1, preparation reagent R1: taking a set amount of water, adding potassium phosphate buffer solution, EDTA, GSSG, potassium ferricyanide, surfactant, first bacteriostatic agent and second bacteriostatic agent, adjusting the pH value to 7.0-7.5, adding a set amount of water to a set volume, adding ascorbic acid oxidase and bilirubin oxidase, and uniformly stirring at a low speed to obtain a reagent R1;
s2, preparation reagent R2: taking a set amount of water, adding a sodium bicarbonate buffer solution, a first bacteriostatic agent, a surfactant and a stabilizer, adjusting the pH value to 9.5-10.5, adding a set amount of water to a set volume, adding NADPH, and uniformly stirring at a low speed to obtain the reagent R2.
When the preparation method of the invention is used for preparing the reagent, the method of firstly adding raw materials except the enzyme, adjusting the pH value to a constant volume and then adding the enzyme is strictly adopted, so that the loss of the enzyme in the preparation process can be effectively reduced, and the stability and the sensitivity of the reagent are improved.
The invention also provides application of the glutathione reductase detection kit in non-disease determination of the glutathione reductase.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
In the reagent R1 of the embodiment of the invention, the concentration of potassium phosphate buffer solution is 50-250mmol/L, the concentration of EDTA is 0.5-2mmol/L, the concentration of GSSG is 0.5-2mmol/L, the concentration of ascorbic acid oxidase is 1-5KU/L, the concentration of bilirubin oxidase is 0.5-2KU/L, and the concentration of potassium ferricyanide is 0.05-0.1 g/L; in the reagent R2 of the example, the concentration of sodium bicarbonate buffer solution was 20 to 100mmol/L, and the concentration of NADPH was 0.5 to 2.5 g/L. The mass ratio of albumin, sorbitol and riboflavin in the stabilizer is 0.1-2: 2-5: 0.5-1.
In the raw materials in the embodiment of the invention, bilirubin oxidase and ascorbate oxidase can effectively remove interference of bilirubin and vitamin C; the potassium ferricyanide can oxidize iron ions in the hemoglobin, thereby effectively reducing the interference of the hemoglobin; p123 in the surfactant is matched with Tween 80 or F108 is matched with Tween 80, so that the airborne stability of the reagent is effectively improved; the first bacteriostatic agent adopts water-soluble amphotericin broad-spectrum antifungal, the second bacteriostatic agent adopts vancomycin hydrochloride broad-spectrum antibacterial, and the first bacteriostatic agent and the second bacteriostatic agent do not interfere with other components in the reagent; in addition, the water-soluble amphotericin is composed of amphotericin deoxycholate sodium in a high proportion, and the amphotericin deoxycholate sodium can enhance the stability and antibacterial capability of reductase, so that the airborne stability of the reagent is improved; the sodium bicarbonate buffer solution can slow down the PH dropping speed, and ensures the stable PH environment of the enzyme.
The kit of the embodiment of the invention is prepared by the following method:
s1, preparation reagent R1: taking a set amount of water, adding potassium phosphate buffer solution, EDTA, GSSG, potassium ferricyanide, surfactant, first bacteriostatic agent and second bacteriostatic agent, adjusting the pH value to 7.0-7.5, adding a set amount of water to a set volume, adding ascorbic acid oxidase and bilirubin oxidase, and uniformly stirring at a low speed to obtain a reagent R1;
s2, preparation reagent R2: taking a set amount of water, adding a sodium bicarbonate buffer solution, a first bacteriostatic agent, a surfactant and a stabilizer, adjusting the pH value to 9.5-10.5, adding a set amount of water to a set volume, adding NADPH, and uniformly stirring at a low speed to obtain the reagent R2.
When the reagent is prepared, the method of adding the raw materials except the enzyme first and then adding the enzyme after the pH is adjusted to a constant volume is strictly adopted, so that the loss of the enzyme in the preparation process can be effectively reduced, and the stability and the sensitivity of the reagent are improved;
example 1
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: 100mmol/L, EDTA1mmol/L, GSSG 1mmol/L potassium phosphate buffer solution, 3KU/L ascorbate oxidase, 1KU/L bilirubin oxidase, 0.1g/L, Triton 1000.1.1% potassium ferricyanide, 200.5% Tween, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 50mmol/L, NADPH 2g/L of sodium bicarbonate buffer solution, 1000.1% of water-soluble amphotericin B, Triton 1000.1, 200.5% of Tween, 1g/L of bovine serum albumin, 2g/L of sorbitol and 0.5g/L of riboflavin.
Example 2
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: 50mmol/L potassium phosphate buffer solution, 1.5mmol/L, GSSG 1.5.5 mmol/L EDTA1.5mmol/L ascorbic acid oxidase 3KU/L, 1KU/L bilirubinoxidase, 0.1g/L potassium ferricyanide, Brij351000.1%, Tween 200.5%, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 80mmol/L, NADPH 2g/L of sodium bicarbonate buffer solution, 1000.1% of water-soluble amphotericin B, Triton 1000.1, 200.5% of Tween, 1.5g/L of bovine serum albumin, 2.5g/L of sorbitol and 0.5g/L of riboflavin.
Example 3
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: 50mmol/L potassium phosphate buffer solution, 1.5mmol/L, GSSG 1.5.5 mmol/L EDTA1.5mmol/L, GSSG, 3KU/L ascorbic acid oxidase, 1KU/L bilirubinoxidase, 0.1g/L, Triton 4050.1% potassium ferricyanide, 200.5% Tween, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 80mmol/L, NADPH 2g/L of sodium bicarbonate buffer solution, B, Triton 4050.1% of water-soluble amphotericin, 200.5% of Tween, 1g/L of bovine serum albumin, 2g/L of sorbitol and 0.5g/L of riboflavin.
Example 4
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: 50mmol/L, EDTA 1.5.5 mmol/L, GSSG 1.5.5 mmol/L potassium phosphate buffer, 3KU/L ascorbate oxidase, 1KU/L bilirubin oxidase, 0.1g/L, Tween 800.5% potassium ferricyanide, 200.5% Tween, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 80mmol/L, NADPH 2g/L of sodium bicarbonate buffer solution, B, Tween 800.5% of water-soluble amphotericin, 200.5% of Tween, 1g/L of bovine serum albumin, 2g/L of sorbitol and 0.5g/L of riboflavin.
Example 5
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: 50mmol/L potassium phosphate buffer solution, 1.5mmol/L, GSSG 1.5.5 mmol/L EDTA1.5mmol/L, GSSG, 3KU/L ascorbic acid oxidase, 1KU/L bilirubin oxidase, 0.1g/L, P1230.05% potassium ferricyanide, Tween 800.5%, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 80mmol/L, NADPH 2g/L of sodium bicarbonate buffer solution, B, P1230.05% of water-soluble amphotericin, 800.5% of Tween, 1g/L of bovine serum albumin, 2g/L of sorbitol and 0.5g/L of riboflavin.
Example 6
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: 50mmol/L potassium phosphate buffer solution, 1.5mmol/L, GSSG 1.5.5 mmol/L EDTA1.5mmol/L, GSSG, 3KU/L ascorbic acid oxidase, 1KU/L bilirubin oxidase, 0.1g/L, P1230.05% potassium ferricyanide, Tween 800.5%, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 80mmol/L, NADPH 2g/L of sodium bicarbonate buffer solution, B, F1080.01% of water-soluble amphotericin, 800.5% of Tween, 1g/L of bovine serum albumin, 2g/L of sorbitol and 0.5g/L of riboflavin.
Example 7
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: potassium phosphate buffer 50mmol/L, EDTA 1.5.5 mmol/L, GSSG 1.5.5 mmol/L, ascorbic acid oxidase 3KU/L, P1230.05%, Tween 800.5%, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 80mmol/L, NADPH 2g/L of sodium bicarbonate buffer solution, B, F1080.01% of water-soluble amphotericin, 800.5% of Tween, 1g/L of bovine serum albumin, 2g/L of sorbitol and 0.5g/L of riboflavin.
Example 8
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: 50mmol/L potassium phosphate buffer solution, 1.5mmol/L, GSSG 1.5.5 mmol/L EDTA1.5mmol/L, GSSG, 3KU/L ascorbic acid oxidase, 1KU/L bilirubin oxidase, 0.1g/L potassium ferricyanide, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 80mmol/L, NADPH 2g/L sodium bicarbonate buffer solution, water-soluble amphotericin B, 1g/L bovine serum albumin, 2g/L sorbitol and 0.5g/L riboflavin.
Example 9
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: 50mmol/L, EDTA 1.5.5 mmol/L, GSSG 1.5.5 mmol/L potassium phosphate buffer, 3KU/L ascorbate oxidase, 1KU/L bilirubin oxidase, 0.1g/L, P1230.05% potassium ferricyanide, 800.5% Tween, water-soluble amphotericin B, and vancomycin hydrochloride; the reagent R2 comprises 80mmol/L, NADPH 2g/L sodium bicarbonate buffer, B, F1080.01% of water-soluble amphotericin and 800.5% of Tween.
Example 10
The glutathione reductase detection kit comprises reagents R1 and R2, wherein the reagent R1 comprises: potassium phosphate buffer 50mmol/L, EDTA 1.5.5 mmol/L, GSSG 1.5.5 mmol/L, ascorbic acid oxidase 3KU/L, bilirubin oxidase 1KU/L, potassium ferricyanide 0.1g/L, P1230.05%, Tween 800.5%; the reagent R2 comprises sodium bicarbonate buffer solution 80mmol/L, NADPH 2g/L, F1080.01%, Tween 800.5%, bovine serum albumin 1g/L, sorbitol 2g/L and riboflavin 0.5 g/L.
Comparative example 1
The kit in patent CN 110734952A.
Comparative example 2
The kit in patent CN 111321198A.
Test example 1
The airborne stability test comprises the following test methods:
the reagents R1 and R2 of examples 1-10 and comparative examples 1-2 were placed on a 7180 full-automatic biochemical analyzer, and the detection was performed on days 0, 3, 7, 14, 21, 28, 35, 45, and 56, and the values of the quality control at two levels of 53.5U/L and 121.5U/L were monitored for three times, and the data were recorded and analyzed statistically for trend changes, the results are shown in tables 1 and 2.
TABLE 1
TABLE 2
As can be seen from the data in tables 1 and 2, the onboard stability of the kits according to examples 5 to 7 of the present invention is better, and the onboard stability of the kit according to example 6 is the best, which indicates that the onboard stability of the kit can be significantly improved by adding the combination of P123 and tween 80 to R1 and the combination of F108 and tween 80 to R2.
In addition, the mean values of examples 1-3 and comparative examples 1-2 all had a significant downward trend, indicating that their stability was decreasing. Brij35 of example 2 had inhibitory effect on enzyme activity.
In addition, the first bacteriostatic agent and the second bacteriostatic agent were not added to the reagents R1 and R2 of example 10, and the mean value was found to be significantly increased when the reagent samples were cultured on the airplane for 56 days, and fungi and bacteria were found to propagate.
In comparison, the reagent of comparative examples 1 and 2 has a larger deviation between 28 days and 56 days compared with the examples, because the reagent of the present invention adopts a method of firstly adjusting pH and then adding the enzyme in the preparation process, so that the stability of the enzyme is ensured; in addition, the pH value of the R2 reagent containing glycine buffer in the comparative example 2 is obviously reduced after the machine is opened, while the pH value of the R2 reagent in the example is reduced by sodium bicarbonate buffer, so that the pH reduction speed is reduced, the stable pH environment of the enzyme is ensured, and the formula of the reagent is better.
Test example 2
Accelerated stability test at 37 ℃ for 10 days, the test method is as follows:
the reagents R1 and R2 of examples 1 to 10 and comparative examples 1 to 2 were placed in an incubator at 37 ℃ and the test was carried out on days 0, 3, 5, 7 and 10, and the target value was monitored to be 53.5U/L, the quality control level 1, three times per repetition, data was recorded, and the trend change was statistically analyzed, and the results are shown in Table 3.
TABLE 3
As can be seen from the data in Table 3, the thermal stability of examples 5-7 is better, and the thermal stability of example 6 is the best, which indicates that the thermal stability of the kit can be remarkably improved by adding the combination of P123 and Tween 80 in R1 and the combination of F108 and Tween 80 in R2. In addition, the mean values of examples 8-9 and comparative examples 1-2 all had a significant decline, indicating that their stability was decreasing. Therefore, the kit can be stored for about 15 months at the temperature of 2-8 ℃ in a dark environment.
Test example 3
The antibacterial test comprises the following test methods: each of the reagents R1 and R2 of examples 1 to 10 and comparative examples 1 to 2 was placed on a 7180 full-automatic biochemical analyzer, 20. mu.l was sampled on each of days 0, 28, 45 and 56, and after plating, the samples were incubated at 28 ℃ for 3 days to observe the growth of colonies, less than 5 were represented by +, (5-15) were represented by + +, 15-30 were represented by + + + and more than 30 were represented by + + +, with the results shown in Table 4.
TABLE 4
As can be seen from the data in Table 4, the reagents in examples 6-7 have strong antibacterial ability, which indicates that the kit of the invention can achieve good antibacterial effect, and the quality of the reagent on board can be guaranteed. The reagents of comparative examples 1-2 did not have antimicrobial capabilities.
The foregoing has described preferred embodiments of the present invention and is not to be construed as limiting the claims. The invention is not limited to the above examples, the specific process of which is susceptible of variation, and all variations which come within the scope of the independent claims are within the scope of the invention.
Claims (11)
1. A glutathione reductase detection kit comprises a reagent R1 and a reagent R2, and is characterized in that:
the reagent R1 comprises: potassium phosphate buffer solution, EDTA, GSSG, ascorbic acid oxidase, bilirubin oxidase, potassium ferricyanide, surfactant, first bacteriostatic agent and second bacteriostatic agent;
the reagent R2 comprises: sodium bicarbonate buffer solution, NADPH, a first bacteriostatic agent, a surfactant and a stabilizing agent.
2. The glutathione reductase detection kit of claim 1, wherein: in the reagent R1, the concentration of potassium phosphate buffer solution is 50-250mmol/L, the concentration of EDTA is 0.5-2mmol/L, the concentration of GSSG is 0.5-2mmol/L, the concentration of ascorbic acid oxidase is 1-5KU/L, the concentration of bilirubin oxidase is 0.5-2KU/L, and the concentration of potassium ferricyanide is 0.05-0.1 g/L.
3. The glutathione reductase detection kit of claim 1, wherein: in the reagent R2, the concentration of sodium bicarbonate buffer solution is 20-100mmol/L, and the concentration of NADPH is 0.5-2.5 g/L.
4. The glutathione reductase detection kit of claim 1, wherein: the PH value of the reagent R1 is 7.0-7.5, and the PH value of the reagent R2 is 9.5-10.5.
5. The glutathione reductase detection kit of claim 1, wherein: the volume ratio of the reagent R1 to the reagent R2 is 5: 1.
6. The glutathione reductase detection kit of claim 1, wherein: the surfactant is selected from one or more of P123, F108, Brij35, Triton405, Triton100, Tween 20 and Tween 80.
7. The glutathione reductase detection kit of claim 1, wherein: the first bacteriostatic agent is water-soluble amphotericin B.
8. The glutathione reductase detection kit of claim 1, wherein: the second bacteriostatic agent is vancomycin hydrochloride.
9. The glutathione reductase detection kit of claim 1, wherein: the stabilizer is a mixture of bovine serum albumin, sorbitol and riboflavin, and the mass ratio of albumin, sorbitol and riboflavin in the stabilizer is 1-1.5: 2-5: 0.5-1.
10. A preparation method of a glutathione reductase detection kit is characterized by comprising the following steps: the method comprises the following steps:
s1, preparation reagent R1: taking a set amount of water, adding potassium phosphate buffer solution, EDTA, GSSG, potassium ferricyanide, surfactant, first bacteriostatic agent and second bacteriostatic agent, adjusting the pH value to 7.0-7.5, adding a set amount of water to a set volume, adding ascorbic acid oxidase and bilirubin oxidase, and uniformly stirring at a low speed to obtain a reagent R1;
s2, preparation reagent R2: taking a set amount of water, adding a sodium bicarbonate buffer solution, a first bacteriostatic agent, a surfactant and a stabilizer, adjusting the pH value to 9.5-10.5, adding a set amount of water to a set volume, adding NADPH, and uniformly stirring at a low speed to obtain the reagent R2.
11. Use of the glutathione reductase assay kit of any one of claims 1 to 9 in a non-disease assay for glutathione reductase.
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