CN113957120B - Cholinesterase determination kit - Google Patents
Cholinesterase determination kit Download PDFInfo
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- CN113957120B CN113957120B CN202111183125.9A CN202111183125A CN113957120B CN 113957120 B CN113957120 B CN 113957120B CN 202111183125 A CN202111183125 A CN 202111183125A CN 113957120 B CN113957120 B CN 113957120B
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- China
- Prior art keywords
- reagent
- cholinesterase
- butyrylthiocholine
- determination kit
- lhepes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 108090000322 Cholinesterases Proteins 0.000 title claims abstract description 17
- 229940048961 cholinesterase Drugs 0.000 title claims abstract description 17
- 102000003914 Cholinesterases Human genes 0.000 title claims 2
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 29
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 24
- AWBGQVBMGBZGLS-UHFFFAOYSA-N butyrylthiocholine Chemical compound CCCC(=O)SCC[N+](C)(C)C AWBGQVBMGBZGLS-UHFFFAOYSA-N 0.000 claims abstract description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 8
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 7
- 239000011780 sodium chloride Substances 0.000 claims abstract description 6
- 238000003149 assay kit Methods 0.000 claims description 2
- 102100032404 Cholinesterase Human genes 0.000 abstract description 23
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000000034 method Methods 0.000 abstract description 6
- 238000000354 decomposition reaction Methods 0.000 abstract description 5
- 101710083761 Cholinesterase Proteins 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 13
- 238000005259 measurement Methods 0.000 description 6
- 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 description 5
- 108010053652 Butyrylcholinesterase Proteins 0.000 description 5
- 239000007995 HEPES buffer Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000011088 calibration curve Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- GANZODCWZFAEGN-UHFFFAOYSA-N 5-mercapto-2-nitro-benzoic acid Chemical compound OC(=O)C1=CC(S)=CC=C1[N+]([O-])=O GANZODCWZFAEGN-UHFFFAOYSA-N 0.000 description 2
- 102100033639 Acetylcholinesterase Human genes 0.000 description 2
- 108010022752 Acetylcholinesterase Proteins 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 229940022698 acetylcholinesterase Drugs 0.000 description 2
- 230000001713 cholinergic effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 208000028399 Critical Illness Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 206010019755 Hepatitis chronic active Diseases 0.000 description 1
- 208000007964 Organophosphate Poisoning Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000002932 cholinergic neuron Anatomy 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000001640 nerve ending Anatomy 0.000 description 1
- 210000004738 parenchymal cell Anatomy 0.000 description 1
- 230000001242 postsynaptic effect Effects 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
- C12Q1/46—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase involving cholinesterase
-
- 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
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
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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 discloses a cholinesterase determination kit, which belongs to the technical field of in-vitro diagnostic reagents, wherein the reagent R1 comprises the following components in percentage by weight: 11.9g/LHEPES, 8g/L sodium chloride, 0.14g/L anhydrous calcium chloride, 0.12g/L5,5 dithio-2-2 nitrobenzoic acid; the reagent R2 comprises the following components in percentage by weight: 11.9g/LHEPES g/L butyrylthiocholine, 2.13g/L magnesium chloride, 500g/L dimethyl sulfoxide, 1.05g/L citric acid. The invention solves the problem of inaccurate clinical detection caused by the decomposition of butyrylthiocholine in the storage process of the butyrylcholine esterase determination kit, can better meet clinical requirements, and has more convenient and accurate use and longer effective period.
Description
Technical Field
The invention relates to the field of in vitro diagnostic reagents, in particular to a cholinesterase determination kit.
Background
Cholinesterase (cholinesterase, CHE) is an enzyme that catalyzes the hydrolysis reaction of acylcholines or cholinesters, and is composed mainly of true cholinesterase and pseudocholinesterase. True cholinesterase, also known as acetylcholinesterase (acetylcholinesterase), is found mainly in the synaptic cleft of cholinergic nerve endings, especially in the folds of postsynaptic membranes of motor nerve end plates, and also in cholinergic neurons and erythrocytes: pseudocholinesterase (PCHE) is present in serum or plasma and can act on other cholinergic compounds in addition to acetylcholine.
In clinic, measurement of pseudocholinesterase activity is often used as an important means to aid in diagnosing organophosphate poisoning and assessing hepatic parenchymal cell damage. Of the severe hepatitis patients, about four fifths of patients have CHE reduced to 60% of normal, and critically ill patients may fall to within 10% of normal, even complete deficiency. In addition, chronic active hepatitis and cirrhosis can all lead to decreased cholinesterase activity.
The cholinesterase determination kit generally adopts butyrylthiocholine to generate butyric acid and thiocholine under the catalysis of cholinesterase, and the thiocholine reacts with 5, 5-dithio-2-nitrobenzoic acid to generate 5-mercapto-2-nitrobenzoic acid and 2-nitrobenzoic acid-5-thiocholine; the activity of serum butyrylcholinesterase can be calculated by detecting the change in absorbance of 5-mercapto-2-nitrobenzoic acid at 405 nm; however, butyrylthiocholine is unstable and mainly decomposed by visible light, and cannot be completely protected from light in the clinical application process, so that the reagent is relatively fast in failure in the application process, and the clinical application is required to correct accuracy continuously through calibration, and even the reagent is invalid.
Disclosure of Invention
The invention aims to solve the technical problem of inaccurate clinical detection caused by decomposition of butyrylthiocholine in the storage process of the cholinesterase determination kit, and can better meet clinical requirements, and the cholinesterase determination kit is more convenient and accurate to use and longer in effective period.
In order to solve the technical problems, the invention adopts the following technical scheme:
a cholinesterase assay kit, consisting of a reagent R1 and a reagent R2 independent of each other, the components of the reagent R1 being: HEPES, sodium chloride, anhydrous calcium chloride, 5 dithio-2-2 nitrobenzoic acid;
the reagent R2 comprises the following components: HEPES, butyrylthiocholine, magnesium chloride, dimethyl sulfoxide, citric acid.
The technical scheme of the invention is further improved as follows:
The reagent R1 comprises the following components in percentage by weight: 11.8-12.0 g/LHEPES, 7.9-8.1 g/L sodium chloride, 0.13-0.15 g/L anhydrous calcium chloride, 0.11-0.13g/L5,5 dithio-2-2 nitrobenzoic acid;
the reagent R2 comprises the following components in percentage by weight: 11.8-12.0 g/LHEPES g/L butyrylthiocholine, 5.6-5.8 g/L magnesium chloride, 2.03-2.23 g/L dimethyl sulfoxide, 499.9-500.1 g/L citric acid, 1.04-1.06 g/L citric acid.
The technical scheme of the invention is further improved as follows:
the reagent R1 comprises the following components in percentage by weight: 11.9g/LHEPES, 8.0g/L sodium chloride, 0.14g/L anhydrous calcium chloride, 0.12g/L5,5 dithio-2-2 nitrobenzoic acid;
The reagent R2 comprises the following components in percentage by weight: 11.9g/LHEPES g/L butyrylthiocholine, 2.13g/L magnesium chloride, 500g/L dimethyl sulfoxide, 1.05g/L citric acid.
By adopting the technical scheme, the invention has the following technical progress:
1. According to the invention, 0.14g/L of anhydrous calcium chloride is added to the reagent 1, 500g/L of dimethyl sulfoxide is added to the reagent 2, so that the problem of instability of butyrylthiocholine as a reaction substrate in a butyrylcholine esterase detection kit is solved, the problems of inaccurate reagent detection result and long-term instability caused by decomposition of butyrylthiocholine by visible light are successfully solved, and the validity period of the cholinesterase detection kit is effectively prolonged to 18 months.
2. The invention solves the problem of inaccurate clinical detection caused by decomposition of butyrylthiocholine in the storage process of the butyrylcholine esterase determination kit, and better meets the clinical requirements; compared with the similar products in the current market, the method is more convenient and accurate to use and longer in effective period.
Drawings
FIG. 1 is a reaction curve of an embodiment of the present invention;
FIG. 2 is a calibration curve of an embodiment of the present invention;
FIG. 3 is a reaction curve of a comparative example of the present invention;
FIG. 4 is a calibration curve of a comparative example of the present invention.
Detailed Description
The invention is described in further detail below with reference to the attached drawings and examples:
Examples
The components and amounts of reagent R1:
| Name of the name | Dosage of | Unit (B) | Tolerance of |
| HEPES | 11.9 | g/L | ±0.1g |
| Sodium chloride | 8.0 | g/L | ±0.1g |
| Anhydrous calcium chloride | 0.14 | g/L | ±0.01g |
| 5,5 Dithio-2-2 nitrobenzoic acid (DTNB) | 0.12 | g/L | ±0.01g |
The components and amounts of reagent R2:
| Name of the name | Dosage of | Unit (B) | Tolerance of |
| HEPES | 11.9 | g/L | ±0.1g |
| Butyrylthiocholine | 5.7 | g/L | ±0.1g |
| Magnesium chloride | 2.13 | g/L | ±0.1g |
| Dimethyl sulfoxide | 500 | g/L | ±0.1g |
| Citric acid | 1.05 | g/L | ±0.01g |
Comparative example
The components and amounts of reagent R1:
The components and amounts of reagent R2:
| Name of the name | Dosage of | Unit (B) | Tolerance of |
| HEPES | 11.9 | g/l | ±0.1g |
| Butyrylthiocholine | 5.7 | g/L | ±0.1g |
| Magnesium chloride | 2.13 | g/l | ±0.1g |
| Citric acid | 1.05 | g/L | ±0.01g |
Comparison of test results for examples and comparative examples:
1. Quality control samples of three known values were measured simultaneously using the comparative and example reagents near the end of the effective period, and the measurement results are shown in table 1:
Table 1:
| Target value | 1 | 2 | 3 | Average value of | Relative deviation of | |
| Comparative example | 5705 | 5238 | 5276 | 5298 | 5271 | -7.61％ |
| 5285 | 4956 | 4943 | 4938 | 4946 | -6.42％ | |
| Examples | 5705 | 5699 | 5699 | 5698 | 5699 | -0.11％ |
| 5285 | 5278 | 5278 | 5280 | 5279 | -0.12％ |
As is apparent from the data in Table 1, the relative deviation between the results of the comparative examples and the target value of the quality control product is close to 7%, while the relative deviation between the results of the examples and the target value of the quality control product is less than 0.2%, and the results of the examples are superior to those of the comparative examples.
2. Embodiment verification: 30 clinical specimens were selected and tested simultaneously with the comparative and examples, and the results are shown in Table 2:
table 2:
As can be seen from Table 2, the absolute deviation between the measurement results of the comparative examples and the measurement results of the examples is between-2647 and-62, and the relative deviation is between-20.86% and-2.66%, and the measurement results of the examples are superior to the measurement results of the comparative examples.
The reaction curves of the examples are shown in FIG. 1.
The calibration curve of the example is shown in fig. 2.
The response curve of the comparative example is shown in FIG. 3.
The calibration curve of the comparative example is shown in fig. 4.
In summary, according to the invention, 0.14g/L of anhydrous calcium chloride is added to the reagent 1, 500g/L of dimethyl sulfoxide is added to the reagent 2, so that the problem of instability of butyrylthiocholine as a reaction substrate in a butyrylthiocholine esterase determination kit is solved, the problems of inaccurate reagent detection results and long-term instability caused by decomposition of butyrylthiocholine by visible light are successfully solved, and the validity period of the cholinesterase determination kit is effectively prolonged to 18 months.
Claims (1)
1. A cholinesterase assay kit consisting of a reagent R1 and a reagent R2 independent of each other, characterized in that:
the reagent R1 comprises the following components in percentage by weight: 11.9g/LHEPES, 8.0g/L sodium chloride, 0.14g/L anhydrous calcium chloride, 0.12g/L5,5 dithio-2-2 nitrobenzoic acid;
The reagent R2 comprises the following components in percentage by weight: 11.9g/LHEPES g/L butyrylthiocholine, 2.13g/L magnesium chloride, 500g/L dimethyl sulfoxide, 1.05g/L citric acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111183125.9A CN113957120B (en) | 2021-10-11 | 2021-10-11 | Cholinesterase determination kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111183125.9A CN113957120B (en) | 2021-10-11 | 2021-10-11 | Cholinesterase determination kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113957120A CN113957120A (en) | 2022-01-21 |
| CN113957120B true CN113957120B (en) | 2024-04-26 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111183125.9A Active CN113957120B (en) | 2021-10-11 | 2021-10-11 | Cholinesterase determination kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113957120B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04197199A (en) * | 1990-11-29 | 1992-07-16 | Iatron Lab Inc | Reagent for measuring choline esterase activity |
| US5859064A (en) * | 1996-03-13 | 1999-01-12 | The United States Of America As Represented By The Secretary Of The Navy | Chemical warfare agent decontamination solution |
| CN1978660A (en) * | 2005-12-08 | 2007-06-13 | 霍夫曼-拉罗奇有限公司 | Stabilized cholinesterase substrate solution |
| CN106680222A (en) * | 2016-12-23 | 2017-05-17 | 郑州科欣生物技术有限公司 | Reagent for determining cholinesterase and kit |
-
2021
- 2021-10-11 CN CN202111183125.9A patent/CN113957120B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04197199A (en) * | 1990-11-29 | 1992-07-16 | Iatron Lab Inc | Reagent for measuring choline esterase activity |
| US5859064A (en) * | 1996-03-13 | 1999-01-12 | The United States Of America As Represented By The Secretary Of The Navy | Chemical warfare agent decontamination solution |
| CN1978660A (en) * | 2005-12-08 | 2007-06-13 | 霍夫曼-拉罗奇有限公司 | Stabilized cholinesterase substrate solution |
| CN106680222A (en) * | 2016-12-23 | 2017-05-17 | 郑州科欣生物技术有限公司 | Reagent for determining cholinesterase and kit |
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| Publication number | Publication date |
|---|---|
| CN113957120A (en) | 2022-01-21 |
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