CN110387398B - Alanine aminotransferase reagent and preparation method and application thereof - Google Patents
Alanine aminotransferase reagent and preparation method and application thereof Download PDFInfo
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- CN110387398B CN110387398B CN201910651575.2A CN201910651575A CN110387398B CN 110387398 B CN110387398 B CN 110387398B CN 201910651575 A CN201910651575 A CN 201910651575A CN 110387398 B CN110387398 B CN 110387398B
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- 102100036475 Alanine aminotransferase 1 Human genes 0.000 title claims abstract description 183
- 108010082126 Alanine transaminase Proteins 0.000 title claims abstract description 183
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 77
- 238000002360 preparation method Methods 0.000 title abstract description 14
- 239000000126 substance Substances 0.000 claims abstract description 43
- 239000000758 substrate Substances 0.000 claims abstract description 24
- 239000005515 coenzyme Substances 0.000 claims abstract description 22
- 150000003839 salts Chemical class 0.000 claims abstract description 19
- 239000000872 buffer Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical group CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 26
- 235000002639 sodium chloride Nutrition 0.000 claims description 24
- 150000001720 carbohydrates Chemical class 0.000 claims description 20
- 235000004279 alanine Nutrition 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000003755 preservative agent Substances 0.000 claims description 14
- 230000002335 preservative effect Effects 0.000 claims description 13
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims description 13
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims description 13
- 229960001327 pyridoxal phosphate Drugs 0.000 claims description 13
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 12
- 229920000642 polymer Polymers 0.000 claims description 11
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- PPCXFTKZPBHXIW-UHFFFAOYSA-N ethyl ethanesulfonate Chemical compound CCOS(=O)(=O)CC PPCXFTKZPBHXIW-UHFFFAOYSA-N 0.000 claims description 8
- 229960005141 piperazine Drugs 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 229940098773 bovine serum albumin Drugs 0.000 claims description 7
- 230000001105 regulatory effect Effects 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 5
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 5
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 5
- 235000014633 carbohydrates Nutrition 0.000 claims description 5
- -1 2-hydroxy-3-morpholinoalanine Chemical compound 0.000 claims description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- 229920002472 Starch Polymers 0.000 claims description 4
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 4
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 4
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 239000001488 sodium phosphate Substances 0.000 claims description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 4
- 235000019698 starch Nutrition 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 3
- 229920002307 Dextran Polymers 0.000 claims description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 3
- 229930182566 Gentamicin Natural products 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 229960002518 gentamicin Drugs 0.000 claims description 3
- 229920002521 macromolecule Polymers 0.000 claims description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 3
- 235000011009 potassium phosphates Nutrition 0.000 claims description 3
- VVJYUAYZJAKGRQ-UHFFFAOYSA-N 1-[4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C(O)C1 VVJYUAYZJAKGRQ-UHFFFAOYSA-N 0.000 claims description 2
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 235000011008 sodium phosphates Nutrition 0.000 claims description 2
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims 1
- 125000000185 sucrose group Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 23
- 239000007853 buffer solution Substances 0.000 abstract description 11
- 230000001681 protective effect Effects 0.000 abstract description 6
- 239000000243 solution Substances 0.000 abstract description 6
- 239000006076 specific stabilizer Substances 0.000 abstract description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 19
- 229960003767 alanine Drugs 0.000 description 19
- 230000000052 comparative effect Effects 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 239000002994 raw material Substances 0.000 description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Substances OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 6
- 230000000087 stabilizing effect Effects 0.000 description 6
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 239000003381 stabilizer Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 3
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 229960003237 betaine Drugs 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 2
- 108090000340 Transaminases Proteins 0.000 description 2
- 150000004716 alpha keto acids Chemical class 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide 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](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000014898 transaminase activity proteins Human genes 0.000 description 2
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 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
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 1
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 150000002016 disaccharides Chemical group 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 229960002668 sodium chloride Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 230000002195 synergetic effect Effects 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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/52—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving transaminase
-
- 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.)
- G01N2333/91188—Transferases (2.) transferring nitrogenous groups (2.6)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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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 relates to an alanine aminotransferase reagent, a preparation method and application thereof, wherein the alanine aminotransferase reagent comprises alanine aminotransferase, an alanine aminotransferase substrate, alanine aminotransferase coenzyme, salt substances, a buffer medium and water. According to the reagent system, alanine aminotransferase is dissolved in a buffer solution system containing protective substances such as salt substances, and an alanine aminotransferase substrate and alanine aminotransferase coenzyme are used as specific stabilizers, so that the stability effect of ALT in a solution can be remarkably improved; and the preparation is simple, and the ALT calibration substance can be ensured to be reliable in calibration when the ALT calibration substance is applied to the calibration of alanine aminotransferase.
Description
Technical Field
The invention belongs to the field of quality control, and particularly relates to an alanine aminotransferase reagent and a preparation method and application thereof, in particular to an alanine aminotransferase reagent with good stability and a preparation method and application thereof.
Background
Alanine aminotransferase (alanine aminotransferase, ALT) is a transaminase that catalyzes the aminotransferase reaction between alanine and alpha-keto acid. ALT is mainly present in the liver and is a sensitive index reflecting liver injury. ALT assay is mainly used for experimental diagnosis of liver diseases and is one of the most commonly used items in daily tests in clinical laboratories. Methods for routine clinical laboratory detection of ALT typically use calibration material calibration with a calibration value traceable to an internationally approved reference method to achieve the accuracy of the ALT assay. However, ALT has poor stability in solution, ALT activity in the calibration substance is easy to be influenced by various factors such as time, storage temperature and the like to change obviously, especially the ALT method calibration reliability is directly influenced by a few units of decrease of enzyme activity per hour in high-temperature weather, so that the effect of the calibration substance is lost, and obvious errors occur in clinical sample measurement results. At present, most of common ALT stabilizers at home and abroad are sugar alcohols such as trehalose, but the addition of the stabilizers can cause a detection system to generate matrix effects with different degrees so as to influence a detection result.
CN101775380a discloses the stabilization of alanine aminotransferase ALT by betaine and a cereal alanine aminotransferase calibrator composition. An alanine aminotransferase calibrator composition comprising: human serum and betaine, the concentration of betaine is 1mol/L. The calibrator composition provided by the invention has a good stabilizing effect, particularly has a short-term stabilizing effect at high temperature, has no obvious collective effect in a common detection system, and is suitable for calibrating all detection systems for detecting the alanine aminotransferase ALT in a clinical laboratory.
CN106501514a discloses a kit for detecting alanine aminotransferase with good stability, which makes the result of detecting alanine aminotransferase more accurate and reliable. The kit of the invention comprises a reagent 1 and a reagent 2: wherein the components of reagent 1 comprise: tris-HCl buffer solution, L-alanine, reduced coenzyme (NADH), lactate Dehydrogenase (LDH), enzyme protectant, stabilizer and antiseptic; the components of reagent 2 include: tris-HCl buffer solution, alpha-ketoglutaric acid, stabilizer and preservative. The ALT detection kit provided by the invention is added with the enzyme protecting agent and the stabilizing agent, so that the stability of the alanine aminotransferase detection kit is improved, the ALT detection kit is suitable for various full-automatic biochemical analyzers, and the ALT detection kit is simple and safe to operate and is convenient for clinical application and popularization.
CN104195220a discloses a buffer solution for human serum alanine aminotransferase assay, the buffer solution is used in a kit by using Tris-citric acid as a buffer system, the kit comprises a first reagent and a second reagent, the first reagent comprises Tris, alanine, NADH, lactate dehydrogenase, sodium azide and citric acid, and the second reagent comprises Tris, alpha-ketoglutarate, sodium azide and citric acid. The buffer solution can provide benign conditions for enzymatic reaction of alanine aminotransferase in human serum, and the kit compatible with the buffer solution is superior to the buffer solution reported in the prior art in terms of accuracy and stability of measured data.
However, in the prior art, the strategy for better improving the stability of alanine aminotransferase is limited and the effect is not ideal, so that it is very significant to develop a new strategy for obviously improving the stability of alanine aminotransferase.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an alanine aminotransferase reagent and a preparation method and application thereof, in particular to an alanine aminotransferase reagent with good stability and a preparation method and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in one aspect, the invention provides an alanine aminotransferase reagent comprising an alanine aminotransferase, an alanine aminotransferase substrate, an alanine aminotransferase coenzyme, a salt, a buffer medium, and water.
According to the alanine aminotransferase reagent system, the alanine aminotransferase is dissolved in a buffer solution system containing protective substances such as salt substances, and an alanine aminotransferase substrate and alanine aminotransferase coenzyme are used as specific stabilizers, so that the stability effect of ALT in the solution can be remarkably improved. Wherein the alanine aminotransferase substrate acts like a shield by binding to the reaction site of the alanine aminotransferase without actually catalyzing the reaction, protecting the reaction site of ALT; wherein the alanine aminotransferase coenzyme functions similarly to the alanine aminotransferase substrate and also acts like a shield by binding sites on the alanine aminotransferase without actually catalyzing the reaction; wherein, the salt substance can simulate the ionic strength environment of the actual serum, so that the alanine aminotransferase is more stable; while the buffer medium plays an important role in maintaining the pH environment of the system. The reagent system can remarkably improve the stability of alanine aminotransferase, so that the activity of the alanine aminotransferase is basically stable within 7 days at 37 ℃, and the residual activity is up to 97%; the system can be directly used without re-dissolving, and overcomes the defects that the alanine aminotransferase freeze-dried product in the prior art needs re-dissolving when in use and the stability is still low after re-dissolving.
Preferably, the alanine aminotransferase is at a concentration of 80-200U/L, such as 80U/L, 90U/L, 100U/L, 110U/L, 120U/L, 130U/L, 140U/L, 150U/L, 160U/L, 170U/L, 180U/L, 190U/L, 200U/L, or the like.
The alanine aminotransferase can be derived from animal tissue or expressed recombinantly.
Preferably, the alanine aminotransferase substrate has a concentration of 100-300mmol/L, such as 100mmol/L, 120mmol/L, 150mmol/L, 180mmol/L, 200mmol/L, 220mmol/L, 240mmol/L, 250mmol/L, 280mmol/L, 300mmol/L, etc.
The concentration of the alanine aminotransferase substrate should be selected within the above range of 100 to 300mmol/L because a further high concentration cannot increase its stabilizing effect and causes a risk of reagent instability, and a further low concentration makes the stabilizing effect of alanine aminotransferase poor.
Preferably, the concentration of the alanine aminotransferase coenzyme is 10 to 100. Mu. Mol/L, for example, 10. Mu. Mol/L, 20. Mu. Mol/L, 30. Mu. Mol/L, 40. Mu. Mol/L, 50. Mu. Mol/L, 60. Mu. Mol/L, 70. Mu. Mol/L, 80. Mu. Mol/L, 90. Mu. Mol/L, 100. Mu. Mol/L, or the like.
The concentration of the alanine aminotransferase coenzyme should be selected within the above range of 10 to 100. Mu. Mol/L, since a further higher concentration accelerates the deterioration of the stability of the coenzyme itself and a further lower concentration does not exert the effect of stabilizing the alanine aminotransferase.
Preferably, the concentration of the salt substance is 75-300mmol/L, such as 75mmol/L, 80mmol/L, 100mmol/L, 120mmol/L, 150mmol/L, 180mmol/L, 200mmol/L, 250mmol/L, 280mmol/L or 300mmol/L, etc.
The concentration of the salt substance should be selected within the above 75-300mmol/L, because a further higher concentration would risk damaging the hydration layer of alanine aminotransferase in the reagent, and a further lower concentration would result in poor stabilizing effect.
Preferably, the alanine aminotransferase reagent further includes any one or a combination of at least two of a saccharide, a polymer, or a preservative, for example, a combination of a saccharide and a polymer, a combination of a polymer and a preservative, a combination of a saccharide and a preservative, and the like.
The addition of a carbohydrate to the alanine aminotransferase reagent can further improve the stability of the alanine aminotransferase, the carbohydrate has a polyhydroxy structure, and the carbohydrate combines with polar bonds on the alanine aminotransferase through intermolecular hydrogen bonding, and shields the action of water molecules on the alanine aminotransferase, thereby improving the stability.
The stability of the alanine aminotransferase is further improved by adding the high molecular substance into the alanine aminotransferase reagent, and the high molecular substance can form a package for the alanine aminotransferase so as to prevent the alanine aminotransferase from being adsorbed on the inner wall of the container; meanwhile, the high molecular substance can improve the consistency of the system and better simulate the serum environment, so that the stability of the alanine aminotransferase is further improved.
Preferably, the alanine aminotransferase substrate comprises alanine or alpha-ketoglutarate, preferably alanine.
Alanine amino transferase substrate needs to select alanine or alpha-ketoglutarate alternatively, presumably because alanine is neutral amino acid, has a certain antioxidation synergistic effect, and has better effect of maintaining alanine amino transferase stability than alpha-ketoglutarate.
As known to those skilled in the art: the alanine aminotransferase catalytic system is a dual substrate reaction, and can only perform actual catalytic reaction when both alanine and alpha-keto acid are required to be present.
Preferably, the alanine aminotransferase coenzyme is pyridoxal phosphate.
Pyridoxal phosphate is an essential substance for the catalytic activity of alanine aminotransferase, and is bound to alanine aminotransferase in a reagent, thereby specifically protecting the catalytic active center thereof.
Preferably, the salt substance includes any one of sodium chloride, potassium chloride, sodium phosphate or potassium phosphate, or a combination of at least two of them, for example, a combination of sodium chloride and potassium chloride, a combination of potassium chloride and sodium phosphate, a combination of sodium phosphate and potassium phosphate, or the like.
Preferably, the buffer medium comprises any one or a combination of at least two of 2-hydroxy-3-morpholinoalanine, 1, 4-piperazinediethanesulfonic acid, 4-hydroxyethylpiperazine ethanesulfonic acid or tris-hydroxymethyl aminomethane, for example, a combination of 2-hydroxy-3-morpholinoalanine and 1, 4-piperazinediethanesulfonic acid, a combination of 1, 4-piperazine diethylsulfonic acid and 4-hydroxyethylpiperazine ethanesulfonic acid, a combination of 4-hydroxyethylpiperazine ethanesulfonic acid and tris-hydroxymethyl aminomethane, and the like, preferably 1, 4-piperazine diethylsulfonic acid.
The 1, 4-piperazine diethyl sulfonic acid has the best effect of maintaining the stability of alanine aminotransferase in a plurality of buffer media, has two sulfonic groups in the molecular structure, and has stronger buffer capacity.
Preferably, the concentration of the buffer medium is 25-200mmol/L, e.g., 25mmol/L, 50mmol/L, 80mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, etc.
Preferably, the alanine aminotransferase reagent has a pH of 6.8 to 7.5, such as 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5, etc.
Preferably, the saccharide substance includes any one or a combination of at least two of sucrose, trehalose, lactose or dextran, for example, a combination of sucrose and trehalose, a combination of lactose and dextran, a combination of trehalose and lactose, or the like, preferably sucrose.
Sucrose has the best effect of maintaining the stability of alanine aminotransferase among a plurality of saccharide substances, and is probably because sucrose is disaccharide consisting of one molecule of glucose and one molecule of fructose, has stable chemical property and is in an amorphous structure, and has remarkable effect of preventing the secondary structure of enzyme from being changed, stretched and aggregated.
Preferably, the polymer substance includes any one or a combination of at least two of polyvinylpyrrolidone, bovine serum albumin, or starch, for example, a combination of starch and polyvinylpyrrolidone, a combination of polyvinylpyrrolidone and bovine serum albumin, a combination of bovine serum albumin and starch, or the like, preferably bovine serum albumin.
Preferably, the preservative comprises sodium azide and/or gentamicin.
Preferably, the concentration of the carbohydrate is 10-50g/L, such as 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, etc.
The concentration of the saccharide should be selected within the above range of 10-50g/L, since a further higher concentration would not increase the protective effect on the enzyme but rather decrease the protective effect, and a further lower concentration would not make the protective effect obvious.
Preferably, the concentration of the polymer is 10-50g/L, for example 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, etc.
The concentration of the polymer substance should be selected within the range of 10-50g/L, and the concentration is too high to improve the viscosity of the reagent and affect the accuracy of daily micro liquid taking, and too low to make the protection function poor.
Preferably, the preservative is present at a concentration of 0.1-0.5g/L, such as 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, or 0.5g/L, etc.
As a preferable technical scheme of the invention, the alanine aminotransferase reagent comprises 80-200U/L of alanine aminotransferase, 100-300mmol/L of alanine aminotransferase substrate, 10-100 mu mol/L of alanine aminotransferase coenzyme, 75-300mmol/L of salt substance, 10-50g/L of saccharide substance, 10-50g/L of macromolecule substance, 0.1-0.5g/L of preservative, 25-200mmol/L of buffer medium and water.
In another aspect, the present invention provides a method for preparing an alanine aminotransferase reagent as described above, comprising: and mixing alanine aminotransferase, an alanine aminotransferase substrate, alanine aminotransferase coenzyme, salt substances, a buffer medium and water according to the formula concentration, and regulating the pH value to obtain the alanine aminotransferase reagent.
Preferably, the preparation method comprises the following steps: mixing alanine aminotransferase, alanine aminotransferase substrate, alanine aminotransferase coenzyme, salt substances, saccharide substances, polymer substances, preservative, buffer medium and water according to the concentration of the formula, and regulating pH to obtain the alanine aminotransferase reagent.
In yet another aspect, the invention provides the use of an alanine aminotransferase reagent as described above for alanine aminotransferase calibration.
The alanine aminotransferase reagent has reasonable formula and simple preparation, and can ensure the reliability of ALT calibration substances during calibration when being applied to the calibration of alanine aminotransferase.
Compared with the prior art, the invention has the following beneficial effects:
according to the alanine aminotransferase reagent system, alanine aminotransferase is dissolved in a buffer solution system containing protective substances such as salt substances, and an alanine aminotransferase substrate and alanine aminotransferase coenzyme are used as specific stabilizers, so that the stability effect of ALT in the solution can be remarkably improved, the activity of ALT in 7 days at 37 ℃ is basically maintained stable, and the residual activity is up to 97%; the system can be directly used without re-dissolving, and overcomes the defects that the alanine aminotransferase freeze-dried product in the prior art needs re-dissolving when in use and the stability is still low after re-dissolving. The alanine aminotransferase reagent has reasonable formula and simple preparation, and can ensure the reliability of ALT calibration substances during calibration when being applied to the calibration of alanine aminotransferase.
Detailed Description
In order to further describe the technical means adopted by the present invention and the effects thereof, the following describes the technical scheme of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
Example 1
This example provides an alanine aminotransferase reagent comprising 100U/L of alanine aminotransferase from porcine heart, 200mmol/L alanine, 15. Mu. Mol/L pyridoxal phosphate, 150mmol/L sodium chloride, 50 mmol/L1, 4-piperazine diethyl sulfonic acid and water. The preparation method comprises the following steps: mixing the raw materials according to the concentration of the formula, and regulating the pH value to 6.8 to obtain the alanine aminotransferase reagent.
Example 2
This example provides an alanine aminotransferase reagent comprising 80U/L of alanine aminotransferase from porcine heart, 100mmol/L alanine, 10. Mu. Mol/L pyridoxal phosphate, 75mmol/L sodium chloride, 25 mmol/L1, 4-piperazine diethyl sulfonic acid and water. The preparation method comprises the following steps: mixing the raw materials according to the concentration of the formula, and regulating the pH value to 7.2 to obtain the alanine aminotransferase reagent.
Example 3
This example provides an alanine aminotransferase reagent comprising 200U/L of alanine aminotransferase from porcine heart, 300mmol/L of alanine, 100. Mu. Mol/L of pyridoxal phosphate, 300mmol/L of sodium chloride, 200mmol/L of 1, 4-piperazine diethyl sulfonic acid and water. The preparation method comprises the following steps: mixing the raw materials according to the concentration of the formula, and regulating the pH value to 7.5 to obtain the alanine aminotransferase reagent.
Example 4
This example provides an alanine aminotransferase reagent which differs from example 1 only in that "200 mmol/L of alanine" is replaced with "50 mmol/L of alanine", with the other conditions being identical.
Example 5
This example provides an alanine aminotransferase reagent which differs from example 1 only in that "200 mmol/L of alanine" is replaced with "350 mmol/L of alanine", with the other conditions being identical.
Example 6
This example provides an alanine aminotransferase reagent whose raw material for production differs from that of example 1 only in that "pyridoxal phosphate 15. Mu. Mol/L" is replaced with "pyridoxal phosphate 5. Mu. Mol/L", with the other conditions being identical.
Example 7
This example provides an alanine aminotransferase reagent whose raw material for production differs from that of example 1 only in that "pyridoxal phosphate 15. Mu. Mol/L" is replaced with "pyridoxal phosphate 120. Mu. Mol/L", with the other conditions being identical.
Example 8
This example provides an alanine aminotransferase reagent that differs from example 1 only in that "alanine" is replaced with "alpha-ketoglutarate", with the other conditions being identical.
Example 9
This example provides an alanine aminotransferase reagent which differs from example 1 only in that "1, 4-piperazine-diethyl-sulfonic acid" is replaced with "2-hydroxy-3-morpholinoalanine", with the other conditions being identical.
Example 10
This example provides an alanine aminotransferase reagent, which comprises 30g/L sucrose as a raw material for preparing the same as that of example 1, except that the raw material is identical.
Example 11
This example provides an alanine aminotransferase reagent, which comprises 50g/L of bovine serum albumin on the basis of example 10, and the other conditions are identical.
Example 12
The present example provides an alanine aminotransferase reagent, which further comprises gentamicin 0.2g/L on the basis of example 11, and the other conditions are identical.
Comparative example 1
This example provides an alanine aminotransferase reagent which is different from example 12 in that it does not contain alanine, pyridoxal phosphate and sodium chloride, and the other conditions are the same.
Comparative example 2
This example provides an alanine aminotransferase reagent which is different from example 12 in that it does not contain alanine or pyridoxal phosphate and has the same conditions as those of the other components.
Comparative example 3
This example provides an alanine aminotransferase reagent which is different from example 12 in that it does not contain pyridoxal phosphate and has the same conditions as those of the other components.
Comparative example 4
This example provides an alanine aminotransferase reagent which is different from example 12 in that the alanine is not contained in the raw material and the other conditions are the same.
Evaluation experiment:
the alanine aminotransferase reagents prepared in examples 1-12 and comparative examples 1-4 were subjected to an accelerated test at 37℃to determine the activity of alanine Aminotransferase (ALT) every day, and the remaining percentage of ALT activity in each reagent set was counted in Table 1.
TABLE 1
Group of | Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
Example 1 | 100% | 100% | 98% | 99% | 97% | 96% | 95% | 94% |
Example 2 | 100% | 100% | 99% | 97% | 98% | 97% | 95% | 94% |
Example 3 | 100% | 100% | 98% | 98% | 97% | 96% | 94% | 93% |
Example 4 | 100% | 100% | 98% | 97% | 95% | 95% | 92% | 90% |
Example 5 | 100% | 100% | 97% | 96% | 94% | 93% | 92% | 89% |
Example 6 | 100% | 99% | 98% | 95% | 94% | 91% | 88% | 86% |
Example 7 | 100% | 100% | 98% | 99% | 96% | 94% | 93% | 91% |
Example 8 | 100% | 98% | 97% | 97% | 95% | 93% | 92% | 91% |
Example 9 | 100% | 98% | 98% | 96% | 95% | 93% | 91% | 90% |
Example 10 | 100% | 100% | 99% | 99% | 98% | 97% | 95% | 95% |
Example 11 | 100% | 100% | 101% | 99% | 99% | 98% | 96% | 96% |
Example 12 | 100% | 100% | 100% | 101% | 101% | 99% | 98% | 97% |
Comparative example 1 | 100% | 89% | 55% | 40% | 20% | 8% | 6% | 6% |
Comparative example 2 | 100% | 91% | 84% | 78% | 75% | 71% | 68% | 66% |
Comparative example 3 | 100% | 97% | 95% | 93% | 90% | 87% | 85% | 82% |
Comparative example 4 | 100% | 98% | 98% | 95% | 94% | 91% | 88% | 87% |
From the data of examples 1-3 in Table 1, it can be seen that the alanine aminotransferase reagent of the present invention significantly improves ALT stability in solution.
The data in examples 4-5 show that the stability effect is slightly deteriorated when the concentration of alanine aminotransferase substrate is not in the range of 100-300 mmol/L.
The data in examples 6 to 7 show that the stability effect is slightly deteriorated when the concentration of alanine aminotransferase coenzyme is not in the range of 10 to 100. Mu. Mol/L.
The data in examples 8 and 9 demonstrate that the choice of substrate or buffer type is also a critical factor affecting reagent stability.
The data of examples 10-12 show that the addition of saccharides, macromolecules or preservatives to the reagent further promotes the stability of the alanine aminotransferase in the reagent, and that this promotion is additive.
As can be seen from the comparison of the data of comparative examples 1-4, the salt species, alanine aminotransferase substrate and alanine aminotransferase coenzyme are key factors in maintaining ALT stability in the reagents of the invention, and the lack thereof significantly reduces ALT stability.
The applicant states that the present invention is illustrated by the above examples as an alanine aminotransferase reagent and methods of making and using the same, but the present invention is not limited to, i.e., does not mean that the present invention must be practiced in dependence upon, the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. An alanine aminotransferase reagent, comprising an alanine aminotransferase, an alanine aminotransferase substrate, an alanine aminotransferase coenzyme, a salt, a saccharide, a polymer, a buffer medium and water;
the alanine aminotransferase coenzyme is pyridoxal phosphate;
the alanine aminotransferase substrate is alanine;
the salt substance comprises any one or a combination of at least two of sodium chloride, potassium chloride, sodium phosphate or potassium phosphate;
the buffer medium comprises any one or a combination of at least two of 2-hydroxy-3-morpholinoalanine, 1, 4-piperazine diethyl sulfonic acid, 4-hydroxyethyl piperazine ethane sulfonic acid or tris;
the saccharide substance comprises any one or a combination of at least two of sucrose, trehalose, lactose or dextran;
the high molecular substance comprises any one or a combination of at least two of polyvinylpyrrolidone, bovine serum albumin or starch;
the concentration of the alanine aminotransferase is 80-200U/L;
the concentration of the alanine aminotransferase substrate is 100-300mmol/L;
the concentration of the alanine aminotransferase coenzyme is 10-100 mu mol/L;
the concentration of the salt substance is 75-300mmol/L;
the concentration of the buffer medium is 25-200mmol/L;
the concentration of the saccharide is 10-50g/L;
the concentration of the macromolecular substance is 10-50g/L;
the pH value of the alanine aminotransferase reagent is 6.8-7.5.
2. The alanine aminotransferase reagent of claim 1, further comprising a preservative.
3. The alanine aminotransferase reagent of claim 2, wherein the preservative includes sodium azide and/or gentamicin.
4. The alanine aminotransferase reagent of claim 2, wherein the preservative is at a concentration of 0.1 to 0.5g/L.
5. The alanine aminotransferase reagent of claim 1, wherein the buffer medium is 1, 4-piperazine diethyl sulfonic acid.
6. The alanine aminotransferase reagent of claim 1, wherein the carbohydrate is sucrose.
7. The alanine aminotransferase reagent of claim 1, wherein the polymeric material is bovine serum albumin.
8. The alanine aminotransferase reagent of claim 1, wherein the alanine aminotransferase reagent includes 80 to 200U/L alanine aminotransferase, 100 to 300mmol/L alanine aminotransferase substrate, 10 to 100. Mu. Mol/L alanine aminotransferase coenzyme, 75 to 300mmol/L salt, 10 to 50g/L saccharide, 10 to 50g/L polymer, 0.1 to 0.5g/L preservative, 25 to 200mmol/L buffer medium and water.
9. The method for producing an alanine aminotransferase reagent according to any one of claims 1 to 8, wherein the method for producing is: and mixing alanine aminotransferase, an alanine aminotransferase substrate, alanine aminotransferase coenzyme, salt substances, saccharide substances, high polymer substances, buffer medium and water according to the formula concentration, and regulating pH to obtain the alanine aminotransferase reagent.
10. The method for producing an alanine aminotransferase reagent as claimed in claim 9, wherein the method for producing is: mixing alanine aminotransferase, alanine aminotransferase substrate, alanine aminotransferase coenzyme, salt substances, saccharide substances, polymer substances, preservative, buffer medium and water according to the concentration of the formula, and regulating pH to obtain the alanine aminotransferase reagent.
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