CN110846378A - 1, 5-sorbitan detection kit and preparation method thereof - Google Patents
1, 5-sorbitan detection kit and preparation method thereof Download PDFInfo
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- CN110846378A CN110846378A CN201911057350.0A CN201911057350A CN110846378A CN 110846378 A CN110846378 A CN 110846378A CN 201911057350 A CN201911057350 A CN 201911057350A CN 110846378 A CN110846378 A CN 110846378A
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- MPCAJMNYNOGXPB-SLPGGIOYSA-N 1,5-anhydro-D-glucitol Chemical compound OC[C@H]1OC[C@H](O)[C@@H](O)[C@@H]1O MPCAJMNYNOGXPB-SLPGGIOYSA-N 0.000 title claims abstract description 22
- MPCAJMNYNOGXPB-UHFFFAOYSA-N 1,5-Anhydro-mannit Natural products OCC1OCC(O)C(O)C1O MPCAJMNYNOGXPB-UHFFFAOYSA-N 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims description 56
- 238000001514 detection method Methods 0.000 title abstract description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 49
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 20
- 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 claims abstract description 20
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 108010024636 Glutathione Proteins 0.000 claims abstract description 10
- 239000007853 buffer solution Substances 0.000 claims abstract description 10
- 229960003180 glutathione Drugs 0.000 claims abstract description 10
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 10
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims abstract description 9
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108010021582 Glucokinase Proteins 0.000 claims abstract description 9
- 102000030595 Glucokinase Human genes 0.000 claims abstract description 9
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 9
- 108020005115 Pyruvate Kinase Proteins 0.000 claims abstract description 9
- 102000013009 Pyruvate Kinase Human genes 0.000 claims abstract description 9
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 9
- 229930029653 phosphoenolpyruvate Natural products 0.000 claims abstract description 9
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108010001816 pyranose oxidase Proteins 0.000 claims abstract description 9
- 239000004094 surface-active agent Substances 0.000 claims abstract description 8
- 239000003755 preservative agent Substances 0.000 claims abstract description 6
- 230000002335 preservative effect Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 41
- 238000003756 stirring Methods 0.000 claims description 33
- 239000000463 material Substances 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 29
- 239000002244 precipitate Substances 0.000 claims description 19
- 238000001914 filtration Methods 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 239000008213 purified water Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000013504 Triton X-100 Substances 0.000 claims description 7
- 229920004890 Triton X-100 Polymers 0.000 claims description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 6
- 239000007995 HEPES buffer Substances 0.000 claims description 6
- 239000007987 MES buffer Substances 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 6
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 6
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 6
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 claims description 6
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 6
- 229940033663 thimerosal Drugs 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- GIAVHGFPMPSIFI-UHFFFAOYSA-N 3-hydroxy-2,4,6-triiodobenzoic acid Chemical compound OC(=O)C1=C(I)C=C(I)C(O)=C1I GIAVHGFPMPSIFI-UHFFFAOYSA-N 0.000 claims description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 4
- 230000002421 anti-septic effect Effects 0.000 claims description 2
- -1 octyl phenyl Chemical group 0.000 claims description 2
- 229940051841 polyoxyethylene ether Drugs 0.000 claims description 2
- 229920000056 polyoxyethylene ether Polymers 0.000 claims description 2
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 2
- JKVUQLWTIZFTMF-UHFFFAOYSA-M potassium;2-oxopropanoate Chemical compound [K+].CC(=O)C([O-])=O JKVUQLWTIZFTMF-UHFFFAOYSA-M 0.000 claims description 2
- 238000003149 assay kit Methods 0.000 claims 5
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000001105 regulatory effect Effects 0.000 claims 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 abstract description 7
- 239000011591 potassium Substances 0.000 abstract description 7
- 229910052700 potassium Inorganic materials 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000000034 method Methods 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000003556 assay Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
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- 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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
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- 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
-
- 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
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
- G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Abstract
The invention relates to a 1, 5-sorbitan detection kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight: 20-100mmol/L of first buffer solution, 1-20KU/L of glucokinase, 0.5-5KU/L of pyruvate kinase, 0.2-1mmol/L of adenosine triphosphate, 1-3mmol/L of potassium phosphoenolpyruvate, 0.5-2mmol/L of 4-aminoantipyrine, 1-5ml/L of surfactant, 0.5-10 g/L of glutathione, 0.8-2.0ml/L of preservative, and 5-20mmol/L of magnesium chloride. The components and concentrations of the reagent R2 include: 20-100mmol/L of second buffer solution, 20-125KU/L of pyranose oxidase, 15-60KU/L of peroxidase, 1-10mmol of chromogen, 1-5ml/L of surfactant and 0.8-2.0ml/L of preservative. The invention has the advantages that: high sensitivity, good stability and wide linear range.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a 1, 5-sorbitan kit and a preparation method thereof.
Background
1,5-AG (1, 5-anhydro-sorbitol) shows obvious negative correlation with the existing diabetes index, and the degree of reduction of 1,5-AG in blood is obviously correlated with the severity of diabetes. 1,5-AG reflects the average blood glucose level of several days to 1 week, and thus the effect of 1, 5-sorbitan detection is increasingly emphasized.
The existing reagent and the existing detection method for detecting the 1, 5-anhydro-sorbitol have the defects of poor sensitivity and repeatability and low accuracy, and limit the application of the reagent and the detection method. Therefore, a new technical solution should be provided to solve the above problems.
Disclosure of Invention
The purpose of the invention is: provides a 1, 5-dehydrated sorbitol detection kit which can obviously improve the repeatability and the sensitivity and a preparation method thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
the 1, 5-sorbitan detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration:
the reagent R2 comprises the following components in percentage by weight:
the further technical scheme is as follows:
the first buffer solution in the reagent R1 and the second buffer solution in the reagent R2 are respectively one or a mixture of more than two of potassium phosphate buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.
The preservatives in the reagents R1 and R2 are respectively one or a mixture of more than two of sodium azide, Proclin950, Proclin300 and thimerosal.
The surfactant in the reagents R1 and R2 is one or a mixture of more than two of Tween 20, Triton X-100, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.
The chromogen in the reagent R2 is one of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and 3-hydroxy-2, 4, 6-triiodobenzoic acid.
A method for preparing a 1, 5-dehydrated sorbitol detection kit comprises the following steps,
(1) preparation of reagent R1
Adding part of purified water into a liquid preparation tank, adding a stirrer to a magnetic stirrer, adjusting the rotating speed of the stirrer to 200 plus 300rpm, keeping stirring in a uniform speed state, adding a first buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, then adding glucokinase, pyruvate kinase, adenosine triphosphate, phosphoenolpyruvate potassium pyruvate and 4-aminoantipyrine, sequentially adding glutathione, magnesium chloride, a surfactant and an antiseptic according to the feeding mode after the materials are completely dissolved, continuing stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank after the materials are completely dissolved, adjusting the pH value to be between 6.50 and 8.00, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
(2) preparation of reagent R2
Adding part of purified water into a liquid preparation tank, adding a second buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adding pyranose oxidase and peroxidase, sequentially adding chromogen, surfactant and preservative according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adjusting the pH value and fixing the volume to the final volume.
And filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution in a finished product tank for marking.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
1. according to the 1, 5-sorbitan detection kit, magnesium chloride, glutathione and triton X-100 are simultaneously added into an R1 reagent, so that the activity and interference removal performance of enzyme can be effectively improved, the analysis sensitivity and stability of the reagent are improved, and the accuracy of a detection result is ensured.
2. The invention has the advantages of simple operation, rapid detection, high sensitivity, good stability and wider linear range, and is suitable for being popularized and used in various large, medium and small hospitals.
Drawings
FIG. 1 calibration graph of a calibrator.
Wherein: the X-axis represents calibrator concentration and the Y-axis represents absorbance.
Detailed Description
The invention is further described below with reference to specific examples:
example 1:
a1, 5-dehydrated sorbitol detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises: tris buffer solution with the concentration of 20mmol/L, glucokinase with the concentration of 1KU/L, pyruvate kinase with the concentration of 0.5KU/L, adenosine triphosphate with the concentration of 0.2mmol/L, potassium phosphoenolpyruvate with the concentration of 1mmol/L, 4-aminoantipyrine with the concentration of 0.5mmol/L, triton X-100 with the concentration of 1ml/L, glutathione with the concentration of 0.5g/L, Proclin-300 with the concentration of 0.8ml/L and magnesium chloride with the concentration of 5 mmol/L.
The reagent R2 comprises: tris buffer solution with the concentration of 20mmol/L, pyranose oxidase with the concentration of 20KU/L, peroxidase with the concentration of 15KU/L, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt with the concentration of 1mmol/L, triton X-100 with the concentration of 1ml/L and Proclin-300 with the concentration of 0.8 ml/L.
The preparation method of the detection kit for the 1, 5-anhydro-sorbitol comprises the following steps,
(1) preparation of reagent R1
Adding part of purified water into a liquid preparation tank, adding a Tris buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate exists at the bottom of the liquid preparation tank, adding glucokinase, pyruvate kinase, adenosine triphosphate, potassium phosphoenolpyruvate and 4-aminoantipyrine, sequentially adding glutathione, magnesium chloride, Triton X-100 and Proclin-300 according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved and no precipitate exists at the bottom of the liquid preparation tank, adjusting the pH value to be 6.50, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
(2) preparation of reagent R2
Adding part of purified water into a liquid preparation tank, adding a magnetic stirrer, adjusting the rotating speed of the stirrer to 200rpm, keeping stirring in a constant speed state, adding a Tris buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, then adding pyranose oxidase and peroxidase, sequentially adding N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, Triton X-100 and Proclin-300 according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank after the feeding is finished, adjusting the pH value to be 7.4, and fixing the volume to the final volume.
And filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution in a finished product tank for marking.
Example 2
A1, 5-dehydrated sorbitol detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises: MES buffer solution with the concentration of 60mmol/L, glucokinase with the concentration of 10KU/L, pyruvate kinase with the concentration of 2.5KU/L, adenosine triphosphate with the concentration of 0.6mmol/L, potassium phosphoenolpyruvate with the concentration of 2mmol/L, 4-aminoantipyrine with the concentration of 1.2mmol/L, Tween 20 with the concentration of 3ml/L, glutathione with the concentration of 5g/L, Proclin-950 with the concentration of 1.4ml/L and magnesium chloride with the concentration of 12 mmol/L.
The reagent R2 comprises: MES buffer solution with the concentration of 60mmol/L, pyranose oxidase with the concentration of 83KU/L, peroxidase with the concentration of 40KU/L, 3-hydroxy-2, 4, 6-triiodobenzoic acid with the concentration of 6mmol/L, Tween 20 with the concentration of 3ml/L and Proclin-950 with the concentration of 1.4 ml/L.
The preparation method of the detection kit for the 1, 5-anhydro-sorbitol comprises the following steps,
(1) preparation of reagent R1
Adding part of purified water into a liquid preparation tank, adding MES buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adding glucokinase, pyruvate kinase, adenosine triphosphate, potassium phosphoenolpyruvate and 4-aminoantipyrine, sequentially adding glutathione, magnesium chloride, Tween 20 and Proclin-950 according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank after the feeding is finished, adjusting the pH value to be 7.20, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
(2) preparation of reagent R2
Adding part of purified water into a liquid preparation tank, adding MES buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adding pyranose oxidase and peroxidase, sequentially adding 3-hydroxy-2, 4, 6-triiodobenzoic acid, tween-20 and Proclin-950 according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved, ensuring that no precipitate is formed at the bottom of the liquid preparation tank, adjusting the pH value to be 8.8, and fixing the volume to the final volume.
And filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution in a finished product tank for marking.
Example 3
A1, 5-dehydrated sorbitol detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises: HEPES buffer solution with the concentration of 100mmol/L, glucokinase with the concentration of 20KU/L, pyruvate kinase with the concentration of 5KU/L, adenosine triphosphate with the concentration of 1mmol/L, potassium phosphoenolpyruvate with the concentration of 3mmol/L, 4-aminoantipyrine with the concentration of 2mmol/L, polyvinylpyrrolidone with the concentration of 5ml/L, glutathione with the concentration of 10g/L, thimerosal with the concentration of 2ml/L and magnesium chloride with the concentration of 20 mmol/L.
The reagent R2 comprises: HEPES buffer solution with the concentration of 100mmol/L, pyranose oxidase with the concentration of 125KU/L, peroxidase with the concentration of 60KU/L, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt with the concentration of 10mmol/L, polyvinylpyrrolidone with the concentration of 5ml/L and thimerosal with the concentration of 2 ml/L.
The preparation method of the detection kit for the 1, 5-anhydro-sorbitol comprises the following steps,
(1) preparation of reagent R1
Adding part of purified water into a liquid preparation tank, adding HEPES buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate exists at the bottom of the liquid preparation tank, adding glucokinase, pyruvate kinase, adenosine triphosphate, potassium phosphoenolpyruvate and 4-aminoantipyrine, sequentially adding glutathione, magnesium chloride, polyvinylpyrrolidone and thimerosal according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved and no precipitate exists at the bottom of the liquid preparation tank after the feeding is finished, adjusting the pH value to be 8, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
(2) preparation of reagent R2
Adding part of purified water into a liquid preparation tank, adding HEPES buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate exists at the bottom of the liquid preparation tank, adding pyranose oxidase and peroxidase, sequentially adding N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, polyvinylpyrrolidone and thimerosal according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved, adjusting the pH value to 10, and fixing the volume to the final volume.
And filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution in a finished product tank for marking.
Several important indexes of the 1, 5-sorbitan detection kit of the present invention are described below by specific experiments in combination with tables and graphs, specifically as follows:
1.1, scaling
Calibration was performed using the calibrator, and the results are shown in table 1, and the calibration curve is shown in fig. 1.
TABLE 1 calibration results for calibrators
1.2 precision CV detection:
selecting a low-value positive sample to be detected, and diluting the low-value positive sample with physiological saline to generate a sample close to the low-limit concentration level of the linear range of the method, wherein the concentration levels are generally 4, and are respectively as follows: 1.36, 2.03, 3.05 and 4.58 umol/L. The assay was repeated 10 times for each concentration level sample. And selecting the lowest concentration level with the CV value being equal to or less than the acceptable threshold value from the data as the lower limit of the detection range by taking the CV value being less than or equal to 8 percent as the acceptable threshold value.
CV > 8% when the sample concentration is below 2.03 umol/L. CV was < 8% when the sample concentration was higher than 3.05 umol/L. Therefore, the linear range is limited to 3.05 umol/L.
High-value samples are selected and diluted with physiological saline to generate samples close to the high-limit concentration level of the linear range of the method, generally 4 concentration levels are respectively: 279.00, 310.00, 341.00 and 372.00 umol/L. The assay was repeated 10 times for each concentration level sample. And selecting the highest concentration level with the CV value being equal to or less than the acceptable threshold value from the data as the upper limit of the detection range by taking the CV value being less than or equal to 8 percent as the acceptable threshold value.
When the sample concentration is higher than 310.00umol/L, the CV is more than 8 percent. CV is < 8% when the sample concentration is below 310.00 umol/L. Therefore, the linear range is highly limited to 310.00 umol/L.
And (4) conclusion: the linear range of the invention can be made to be 3.05-310.00 umol/L.
1.3 repetitive CV detection:
| 1 | 4.87 | 79.98 | 143.00 | 301.37 |
| 2 | 4.73 | 73.82 | 145.42 | 301.20 |
| 3 | 4.84 | 82.07 | 155.43 | 282.06 |
| 4 | 4.65 | 74.53 | 150.65 | 304.22 |
| 5 | 4.76 | 74.74 | 151.49 | 296.44 |
| 6 | 4.90 | 77.20 | 154.56 | 306.56 |
| 7 | 4.26 | 78.58 | 151.33 | 293.74 |
| 8 | 4.86 | 74.35 | 145.19 | 289.19 |
| 9 | 4.56 | 81.38 | 150.21 | 306.69 |
| 10 | 4.64 | 77.97 | 155.22 | 282.93 |
| mean value | 4.71 | 77.46 | 150.25 | 296.44 |
| SD | 0.194 | 3.045 | 4.403 | 9.214 |
| CV％ | 4.11％ | 3.93％ | 2.93％ | 3.11％ |
And (4) conclusion: the repeatability CV of the detected clinical serum sample is small and within 5 percent.
1.4, sensitivity detection:
clinical serum samples of known concentration were diluted to < 3.05umol/L with physiological saline and the test was repeated 10 times to calculate SD and CV. The Coefficient of Variation (CV) should be no more than 10%.
| 1 | 2.66 |
| 2 | 2.44 |
| 3 | 2.72 |
| 4 | 2.65 |
| 5 | 2.31 |
| 6 | 2.43 |
| 7 | 2.17 |
| 8 | 2.41 |
| 9 | 2.16 |
| 10 | 2.56 |
| Mean value | 2.45 |
| SD | 0.198 |
| CV％ | 8.08％ |
And (4) conclusion: when the concentration of the serum sample is less than 3.05umol/L, the repeatability is not more than 10 percent.
1.5, detecting thermal stability:
the kit is put into an electric heating constant temperature incubator, the temperature is accelerated for 10 days at 37 ℃, the detection is carried out on the days 0, 2,4,6, 8 and 10 respectively, and the thermal stability change of the kit is observed.
And (4) conclusion: when the detection is carried out on days 0, 2,4,6, 8 and 10, the relative deviation is within +/-5 percent, which indicates that the kit has good thermal stability and can be stably stored for at least 10 days at 37 ℃.
The foregoing is a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications or substitutions can be made without departing from the principle of the present invention, and these modifications or substitutions should also be considered as the protection scope of the present invention.
Claims (6)
1. The utility model provides a 1, 5-sorbitan assay kit which characterized in that: the reagent comprises a reagent R1 and a reagent R2, wherein each component and concentration of the reagent R1 comprises:
the reagent R2 comprises the following components in percentage by weight:
2. the 1, 5-sorbitan assay kit according to claim 1, wherein: the first buffer solution in the reagent R1 and the second buffer solution in the reagent R2 are respectively one or a mixture of more than two of potassium phosphate buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.
3. The 1, 5-sorbitan assay kit according to claim 1, wherein: the preservatives in the reagents R1 and R2 are respectively one or a mixture of more than two of sodium azide, Proclin950, Proclin300 and thimerosal.
4. The 1, 5-sorbitan assay kit according to claim 1 or 2, wherein: the surfactant in the reagents R1 and R2 is one or a mixture of more than two of Tween 20, Triton X-100, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.
5. The 1, 5-sorbitan assay kit according to claim 4, wherein: the chromogen in the reagent R2 is one of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and 3-hydroxy-2, 4, 6-triiodobenzoic acid (HTIB).
6. The method for producing a 1, 5-sorbitan test kit according to any one of claims 1 to 5, characterized in that: comprises the following steps of (a) carrying out,
(1) preparation of reagent R1
Adding part of purified water into a liquid preparation tank, adding a stirrer to a magnetic stirrer, regulating the rotating speed of the stirrer to 200-300rpm, keeping stirring in a constant speed state, adding a first buffer solution while stirring according to the concentration requirement of claim 1, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, then adding glucokinase, pyruvate kinase, adenosine triphosphate, phosphoenolpyruvate potassium pyruvate and 4-aminoantipyrine, sequentially adding glutathione, magnesium chloride, a surfactant and an antiseptic according to the feeding mode after the materials are completely dissolved, continuing stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adjusting the pH value to be 6.50-8.00, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
(2) preparation of reagent R2
Adding part of purified water into a liquid preparation tank, adding a second buffer solution while stirring according to the concentration requirement of claim 1, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adding pyranose oxidase and peroxidase, sequentially adding the chromogen, the surfactant and the preservative according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adjusting the pH value and fixing the volume to the final volume.
And filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution in a finished product tank for marking.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134201A (en) * | 2021-11-16 | 2022-03-04 | 苏州普瑞斯生物科技有限公司 | Production process of 1, 5-dehydration-D-sorbitol detection reagent by adopting enzyme method |
| CN115786450A (en) * | 2022-12-27 | 2023-03-14 | 广东优尼德生物科技有限公司 | 1,5-dehydration-D-sorbitol determination kit and preparation method thereof |
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| CN106047988A (en) * | 2016-04-28 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | A kit for measuring 1,5-anhydroglucitol and a preparing method thereof |
| CN109212238A (en) * | 2018-09-26 | 2019-01-15 | 山东博科生物产业有限公司 | A kind of glycosylated albumin assay kit |
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| US20140255960A1 (en) * | 2013-03-11 | 2014-09-11 | Polymer Technology Systems, Inc. | Systems and methods for test strips with extended dynamic ranges |
| CN104483487A (en) * | 2014-12-22 | 2015-04-01 | 宁波美康生物科技股份有限公司 | Kit for detecting 1, 5-anhydro-D-ghlcitol in human blood |
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| CN114134201A (en) * | 2021-11-16 | 2022-03-04 | 苏州普瑞斯生物科技有限公司 | Production process of 1, 5-dehydration-D-sorbitol detection reagent by adopting enzyme method |
| CN115786450A (en) * | 2022-12-27 | 2023-03-14 | 广东优尼德生物科技有限公司 | 1,5-dehydration-D-sorbitol determination kit and preparation method thereof |
| CN115786450B (en) * | 2022-12-27 | 2023-11-17 | 广东优尼德生物科技有限公司 | 1, 5-dehydration-D-sorbitol determination kit and preparation method thereof |
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