CN110669822A - Lipase kit and preparation method thereof - Google Patents
Lipase kit and preparation method thereof Download PDFInfo
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- CN110669822A CN110669822A CN201911080549.5A CN201911080549A CN110669822A CN 110669822 A CN110669822 A CN 110669822A CN 201911080549 A CN201911080549 A CN 201911080549A CN 110669822 A CN110669822 A CN 110669822A
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- reagent
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- tartaric acid
- raw materials
- lipase
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- 102000004882 Lipase Human genes 0.000 title claims abstract description 42
- 108090001060 Lipase Proteins 0.000 title claims abstract description 42
- 239000004367 Lipase Substances 0.000 title claims abstract description 41
- 235000019421 lipase Nutrition 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 72
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 47
- 239000002994 raw material Substances 0.000 claims abstract description 25
- 239000007853 buffer solution Substances 0.000 claims abstract description 22
- AWDRATDZQPNJFN-VAYUFCLWSA-N taurodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 AWDRATDZQPNJFN-VAYUFCLWSA-N 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 239000003398 denaturant Substances 0.000 claims abstract description 12
- 239000004094 surface-active agent Substances 0.000 claims abstract description 12
- OBKXEAXTFZPCHS-UHFFFAOYSA-N 4-phenylbutyric acid Chemical compound OC(=O)CCCC1=CC=CC=C1 OBKXEAXTFZPCHS-UHFFFAOYSA-N 0.000 claims abstract description 11
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 9
- 239000001110 calcium chloride Substances 0.000 claims abstract description 9
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 9
- 102000005311 colipase Human genes 0.000 claims abstract description 9
- 108020002632 colipase Proteins 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims abstract description 8
- 229960003964 deoxycholic acid Drugs 0.000 claims abstract description 8
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims abstract description 8
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 29
- 238000003756 stirring Methods 0.000 claims description 20
- 239000000872 buffer Substances 0.000 claims description 10
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical group OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 10
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 10
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical group [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 9
- 239000012535 impurity Substances 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 9
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 8
- 235000002906 tartaric acid Nutrition 0.000 claims description 8
- 239000011975 tartaric acid Substances 0.000 claims description 8
- 238000004806 packaging method and process Methods 0.000 claims description 6
- 239000008118 PEG 6000 Substances 0.000 claims description 5
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000011265 semifinished product Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 14
- 238000001514 detection method Methods 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 3
- FEWJPZIEWOKRBE-LWMBPPNESA-N levotartaric acid Chemical compound OC(=O)[C@@H](O)[C@H](O)C(O)=O FEWJPZIEWOKRBE-LWMBPPNESA-N 0.000 abstract 3
- 229940040461 lipase Drugs 0.000 description 30
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 8
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 8
- 239000000758 substrate Substances 0.000 description 6
- 206010033645 Pancreatitis Diseases 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 206010033647 Pancreatitis acute Diseases 0.000 description 4
- 201000003229 acute pancreatitis Diseases 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 235000019626 lipase activity Nutrition 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- -1 methyl resorufin Chemical compound 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 238000009739 binding Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- KQNPFQTWMSNSAP-UHFFFAOYSA-N isobutyric acid Chemical compound CC(C)C(O)=O KQNPFQTWMSNSAP-UHFFFAOYSA-N 0.000 description 2
- XZBACIYYJPQCJG-UHFFFAOYSA-N 2,3-didodecoxypropan-1-ol Chemical compound CCCCCCCCCCCCOCC(CO)OCCCCCCCCCCCC XZBACIYYJPQCJG-UHFFFAOYSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
- FQISKWAFAHGMGT-SGJOWKDISA-M Methylprednisolone sodium succinate Chemical compound [Na+].C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)COC(=O)CCC([O-])=O)CC[C@H]21 FQISKWAFAHGMGT-SGJOWKDISA-M 0.000 description 1
- 208000016222 Pancreatic disease Diseases 0.000 description 1
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000012482 calibration solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229960004584 methylprednisolone Drugs 0.000 description 1
- 210000004798 organs belonging to the digestive system Anatomy 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 description 1
- 229940045946 sodium taurodeoxycholate Drugs 0.000 description 1
- YXHRQQJFKOHLAP-FVCKGWAHSA-M sodium;2-[[(4r)-4-[(3r,5r,8r,9s,10s,12s,13r,14s,17r)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 YXHRQQJFKOHLAP-FVCKGWAHSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 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
-
- 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)
- G01N2333/92—Triglyceride splitting, e.g. by means of lipase
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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 a lipase kit and a preparation method thereof, wherein the lipase kit comprises a reagent R1, a reagent R2 and a reagent R3, and the reagent R1 comprises the following raw materials: 80-120 mmol/l tartaric acid buffer solution (pH 7.0); 3.0-5.0 mmol/l taurodeoxycholic acid; 0.05-0.08mmol/l phenylbutyric acid; 450-550 KU/l colipase; 10-15mmol/l calcium chloride; 2.0-3.0mmol/l deoxycholic acid; the reagent R2 comprises the following raw materials: 80-120 mmol/l tartaric acid buffer solution (pH 7.0); 0.5 to 1.0mmol/l surfactant; 0.2-0.8 mmol/protein denaturant; 2.0-3.0mmol/l diethyl ether; the reagent R3 comprises the following raw materials: 80-120 mmol/l tartaric acid buffer solution (pH 7.0); 0.15-0.35 mmol/l 6-methyl resorufin ester; 3.0-5.0 mmol/l taurodeoxycholic acid. The method has high continuous sensitivity and high detection precision, can continuously maintain the activity of the lipase after reacting for a period of time, and improves the reaction accuracy of the later stage.
Description
The technical field is as follows:
the invention relates to the technical field of medical detection reagents, in particular to a lipase kit and a preparation method thereof.
Background art:
lipase (Lip), also known as triacylglycerol hydrolase, is a generic name for enzymes that hydrolyze triacylglycerol of long-chain fatty acids. The main synthesis site of human lipase is in the pancreatic acinus, and this kind of lipase is also called pancreatic lipase, and is the main source of serum lipase. In addition, some digestive organs such as stomach, duodenum, esophagus, and white blood cells, adipose tissue, lung, vascular endothelium may also secrete small amounts of lipase and enter the blood. Serum lipase activity assays are useful for the diagnosis of pancreatic disease, particularly in acute pancreatitis. The level difference of human serum lipase of patients with acute pancreatitis, patients with non-acute pancreatitis and patients with normal pancreatitis is larger, and lipase has better sensitivity and specificity in diagnosis of acute pancreatitis compared with amylase.
The detection of the existing lipase usually uses a lipase kit, for example, the invention patent with the application number of CN201811635803.9 discloses a lipase detection kit and a production process, the lipase detection kit comprises a reagent R1 and a reagent R2 which are independent from each other, and the reagent R1 comprises the following components: the reagent R2 comprises the following components: the lipase detection kit is ready to use. The principle of lipase is: the 1, 2-o-dilauryl-rac-glycerol-3-glutaric acid (6 '-methyl resorufin) ester is hydrolyzed under the action of lipase to produce 1, 2-o-dilauryl-rac-glycerol and glutaric acid (6' -methyl resorufin) ester. Wherein glutaric acid (6' -methyl resorufin) ester is hydrolyzed by further action of lipase to produce glutaric acid and methyl resorufin, which appears red in solution. The rate of production of red methylprednisolone detected at a wavelength of 570nm, which is proportional to the lipase activity in the sample, is calculated to give the lipase activity in the sample.
However, in the above technical scheme, because the lipase activity is detected for a period of time, after the substrate is subjected to enzymolysis for a period of time, the enzymolysis reaction speed is slowed down due to the production of reactants, so the color deepening degree is slowed down, the reaction color change cannot be observed at the later stage, the absorbance change is not obvious, and the reference significance of the later measured data is small.
The invention content is as follows:
the invention aims to solve the technical problem of providing a method which has high continuous sensitivity and high detection precision, can continuously maintain the activity of lipase after reacting for a period of time and improves the reaction accuracy of the later stage.
In order to solve the technical problems, the invention adopts a lipase kit which comprises a reagent R1, a reagent R2 and a reagent R3, and is characterized in that the reagent R1 comprises the following raw materials:
80-120 mmol/l tartaric acid buffer solution (pH 7.0);
3.0-5.0 mmol/l taurodeoxycholic acid;
0.05-0.08mmol/l phenylbutyric acid;
450-550 KU/l colipase;
10-15mmol/l calcium chloride;
2.0-3.0mmol/l deoxycholic acid;
the reagent R2 comprises the following raw materials:
80-120 mmol/l tartaric acid buffer solution (pH 7.0);
0.5 to 1.0mmol/l surfactant;
0.2-0.8 mmol/protein denaturant;
2.0-3.0mmol/l diethyl ether;
the reagent R3 comprises the following raw materials:
80-120 mmol/l tartaric acid buffer solution (pH 7.0);
0.15-0.35 mmol/l 6-methyl resorufin ester;
3.0-5.0 mmol/l taurodeoxycholic acid.
Adopt above-mentioned technical scheme: the phenylbutyric acid can keep the activity of lipase cells, and after reacting for a period of time, the activity of the lipase can be continuously maintained, so that the reaction accuracy of the later stage is improved; in addition, the phenylbutyric acid can be removed to reduce protein, the protein can affect a substrate, the stability of the reagent can be enhanced after the phenylbutyric acid is removed, and the accuracy of a test result can be improved; glutaric acid can be dissolved in ether in a large amount, and the ether component is arranged, so that on one hand, a large amount of glutaric acid is absorbed, the reaction product is reduced, the reaction is promoted to be rapidly continued, the absorbance difference in unit time is obviously improved, on the other hand, a competitive inhibitor is arranged between the generated glutaric acid and the lipase, and the situation that the glutaric acid and the lipase compete for a binding site on the lipase is avoided by reducing the contact between the glutaric acid and the lipase, so that the binding reaction between the lipase and the substrate is realized; therefore, the activity of the lipase can be combined with the continuous sites of the substrate, the enzymolysis reaction is continued, and the reaction accuracy of the later stage is improved.
Preferably, the reagent R1 comprises the following raw materials:
100mmol/l tartaric acid buffer solution (pH 7.0);
3.4mmol/l taurodeoxycholic acid;
0.06mmol/l phenylbutyric acid;
500KU/l colipase;
12mmol/l calcium chloride;
2.6mmol/l deoxycholic acid;
the reagent R2 comprises the following raw materials:
100mmol/l tartaric acid buffer solution (pH 7.0);
0.8mmol/l surfactant;
0.6 mmol/protein denaturant;
2.5mmol/l diethyl ether;
the reagent R3 comprises the following raw materials:
100mmol/l tartaric acid buffer (pH 7.0);
0.27mmol/l 6-methyl resorufin ester;
3.4mmol/l taurodeoxycholic acid.
Preferably, the protein denaturant is guanidine hydrochloride.
Preferably, the surfactant is PEG2000, PEG6000 or PEG 8000.
Preferably, the volume ratio of the reagent R1 to the reagent R2 to the reagent R3 is 1:1: 1.
In order to solve the technical problems, the invention discloses a preparation method of a lipase kit, which comprises the following steps: it comprises the following steps: step S1, weighing each component of the reagent R1, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S2, weighing each component of the reagent R2, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, weighing each component of the reagent R3, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, checking the semi-finished product; step S4, cleaning a packaging appliance, drying, and filling reagents R1, R2 and R3 respectively; and step S5, packaging, inspecting finished products, and warehousing for storage.
Preferably, in step S2, the surfactant, the protein denaturant, and the ether are mixed and stirred, and then the tartaric acid buffer is added to the system.
Preferably, the surfactant, the protein denaturant and the ether are heated for 10min in the process of mixing and stirring, the heating temperature is 40-50 ℃, and the mixture is kept stand to room temperature after the heating is finished.
Preferably, the tartaric acid buffer is added to the system in multiple portions.
Compared with the prior art, the invention has the following advantages: calcium chloride is an enzyme reaction accelerator, can activate the activity of enzyme and provide the ionic environment required by the reaction, and can increase the salt ion effect in the reagent. PEG2000, PEG6000 or PEG 8000 belong to surfactants, have stronger emulsification, improve the melting property and stability of various raw materials, and the stability of the reagent in opening the bottle and the long-term storage stability are better. The sodium taurodeoxycholate plays a role of a stabilizer and an accelerator in the reaction, accelerates the lipase reaction and ensures the high-efficiency reaction. Colipases are catalytic enzymes of reactions required in the reaction. Guanidine hydrochloride is a protein denaturant, removes the influence of hybrid proteins on a substrate, enhances the stability of the reagent, and is beneficial to improving the accuracy of a test result. 1, 2-o-dilauryl-rac-glycerol-3-glutaric acid (6 '-methyl resorufin) ester is a substrate for 6' -methyl resorufin reaction, is mainly used for generating methyl resorufin, and is a chromogen in colorimetry.
The specific implementation mode is as follows:
the present invention will be further described with reference to the following specific examples.
In the first embodiment, the reagent components comprise the following components: the reagent R1 comprises the following raw materials:
100mmol/l tartaric acid buffer solution (pH 7.0);
3.4mmol/l taurodeoxycholic acid;
0.06mmol/l phenylbutyric acid;
500KU/l colipase;
12mmol/l calcium chloride;
2.6mmol/l deoxycholic acid;
the reagent R2 comprises the following raw materials:
100mmol/l tartaric acid buffer solution (pH 7.0);
0.8mmol/l PEG 2000；
0.6 mmol/guanidine hydrochloride;
2.5mmol/l diethyl ether;
the reagent R3 comprises the following raw materials:
100mmol/l tartaric acid buffer (pH 7.0);
0.27mmol/l 6-methyl resorufin ester;
3.4mmol/l taurodeoxycholic acid.
The preparation method comprises the following steps: step S1, weighing each component of the reagent R1, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S2, weighing each component of the reagent R2, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, weighing each component of the reagent R3, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, checking the semi-finished product; step S4, cleaning a packaging appliance, drying, and filling reagents R1, R2 and R3 respectively; and step S5, packaging, inspecting finished products, and warehousing for storage. In step S2, guanidine hydrochloride, PEG2000 and ether are mixed and stirred, and then tartaric acid buffer solution is added into the system. Firstly, heating guanidine hydrochloride, PEG2000 and diethyl ether for 10min in the process of mixing and stirring, wherein the heating temperature is 40-50 ℃, and standing to room temperature after heating. Adding tartaric acid buffer solution into the system for three times.
Example two, the reagent components included the following: the reagent R1 comprises the following raw materials:
120mmol/l tartaric acid buffer solution (pH 7.0);
3.0mmol/l taurodeoxycholic acid;
0.08mmol/l phenylbutyric acid;
450KU/l colipase;
10mmol/l calcium chloride;
3.0mmol/l deoxycholic acid;
the reagent R2 comprises the following raw materials:
100mmol/l tartaric acid buffer solution (pH 7.0);
0.8mmol/l PEG 6000；
0.2 mmol/guanidine hydrochloride;
3.0mmol/l diethyl ether;
the reagent R3 comprises the following raw materials:
100mmol/l tartaric acid buffer (pH 7.0);
0.27mmol/l 6-methyl resorufin ester;
3.4mmol/l taurodeoxycholic acid. The preparation method is the same as the first embodiment.
In the third embodiment, the reagent components comprise the following components: the reagent R1 comprises the following raw materials:
80mmol/l tartaric acid buffer solution (pH 7.0);
5.0mmol/l taurodeoxycholic acid;
0.05mmol/l phenylbutyric acid;
450KU/l colipase;
15mmol/l calcium chloride;
2.0mmol/l deoxycholic acid;
the reagent R2 comprises the following raw materials:
100mmol/l tartaric acid buffer solution (pH 7.0);
0.5mmol/l PEG 6000；
0.8 mmol/guanidine hydrochloride;
2.0mmol/l diethyl ether;
the reagent R3 comprises the following raw materials:
100mmol/l tartaric acid buffer (pH 7.0);
0.35mmol/l 6-methyl resorufin ester;
5.0mmol/l taurodeoxycholic acid. The preparation method is the same as the first embodiment.
Comparative example one differs from example three in that the reagent R1 comprises 0.05mmol/l benzoic acid; the rest is the same. The preparation method is the same as that of the embodiment.
Comparative example two, which differs from example two in that the reagent R1 comprises 0.05mmol/l isobutyric acid; the rest is the same. The preparation method is the same as the example.
Example four differs from example two in that the reagent R2 comprises 0.8 mmol/urea; the rest is the same. The preparation method is the same as the first embodiment.
Example five differs from example one in that the reagent R2 comprises 0.5mmol/l sodium dodecylbenzenesulfonate; the rest is the same. The preparation method is the same as the first embodiment.
Comparative example three differs from example one in that the reagent R2 comprises 2.0mmol/l methyl ether; the rest is the same. The preparation method is the same as the first embodiment.
Example six differs from example one in that, in step S2, guanidine hydrochloride, PEG2000 and diethyl ether are mixed and stirred with tartaric acid buffer. The rest is the same. The components and contents of the reagents are the same as in the first example.
The difference between the comparative example four and the example one is that, in the step S2, guanidine hydrochloride, PEG2000 and diethyl ether are heated for 10min while being mixed and stirred, the heating temperature is 80 ℃, and the mixture is left to stand to room temperature after the heating is finished. Adding tartaric acid buffer solution into the system for three times; the rest is the same. The components and contents of the reagents are the same as in the first example.
The determination method for detecting the stability of the lipase detection kit disclosed by the invention comprises the following steps:
a Hitachi 7060 full-automatic biochemical analyzer is selected, the main wavelength is set to be 570nm, the auxiliary wavelength is set to be 700nm, and a rate method is adopted. Adding 3 mul of sample, calibration solution or quality control product, then adding 100 mul of each of the reagent R1 and R2, mixing uniformly, incubating at 37 ℃ for 3 min-5 min, then adding 100 mul of the reagent R3, mixing uniformly, incubating at 37 ℃ for 100s, continuously detecting the absorbance change of each tube at the measuring wavelength of 1min, 2min, 4min, 6min and 8min, setting the basic value of absorbance as A, and calculating A + delta A of each tube, wherein delta A is the change value of the absorbance of each tube along with the time.
Claims (9)
1. A lipase kit comprises a reagent R1, a reagent R2 and a reagent R3, and is characterized in that the reagent R1 comprises the following raw materials:
80-120 mmol/l tartaric acid buffer solution (pH 7.0);
3.0-5.0 mmol/l taurodeoxycholic acid;
0.05-0.08mmol/l phenylbutyric acid;
450-550 KU/l colipase;
10-15mmol/l calcium chloride;
2.0-3.0mmol/l deoxycholic acid;
the reagent R2 comprises the following raw materials:
80-120 mmol/l tartaric acid buffer solution (pH 7.0);
0.5 to 1.0mmol/l surfactant;
0.2-0.8 mmol/protein denaturant;
2.0-3.0mmol/l diethyl ether;
the reagent R3 comprises the following raw materials:
80-120 mmol/l tartaric acid buffer solution (pH 7.0);
0.15-0.35 mmol/l 6-methyl resorufin ester;
3.0-5.0 mmol/l taurodeoxycholic acid.
2. The lipase kit as claimed in claim 1, wherein the reagent R1 comprises the following raw materials:
100mmol/l tartaric acid buffer solution (pH 7.0);
3.4mmol/l taurodeoxycholic acid;
0.06mmol/l phenylbutyric acid;
500KU/l colipase;
12mmol/l calcium chloride;
2.6mmol/l deoxycholic acid;
the reagent R2 comprises the following raw materials:
100mmol/l tartaric acid buffer solution (pH 7.0);
0.8mmol/l surfactant;
0.6 mmol/protein denaturant;
2.5mmol/l diethyl ether;
the reagent R3 comprises the following raw materials:
100mmol/l tartaric acid buffer (pH 7.0);
0.27mmol/l 6-methyl resorufin ester;
3.4mmol/l taurodeoxycholic acid.
3. The lipase kit as claimed in claim 1, wherein the surfactant is PEG2000, PEG6000 or PEG 8000.
4. The lipase kit as claimed in claim 1, wherein the volume ratio of the reagent R1, the reagent R2 and the reagent R3 is 1:1: 1.
5. The lipase kit as claimed in claim 1, wherein the protein denaturant is guanidine hydrochloride.
6. A method for producing a lipase kit according to any of claims 1 to 5, characterized in that it comprises the following steps: step S1, weighing each component of the reagent R1, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S2, weighing each component of the reagent R2, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, weighing each component of the reagent R3, adding the weighed components into a container, stirring, standing after uniformly stirring, filtering and removing impurities; step S3, checking the semi-finished product; step S4, cleaning a packaging appliance, drying, and filling reagents R1, R2 and R3 respectively; and step S5, packaging, inspecting finished products, and warehousing for storage.
7. The method of claim 6, wherein in step S2, the surfactant, the protein denaturant and the ether are mixed and stirred, and then the tartaric acid buffer is added into the system.
8. The method for preparing the lipase kit according to claim 7, wherein the surfactant, the protein denaturant and the ether are heated for 10min while being mixed and stirred, the heating temperature is 40-50 ℃, and the mixture is allowed to stand to room temperature after the heating is finished.
9. The method of claim 8, wherein the tartaric acid buffer is added to the system in multiple times.
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| CN104198474A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Detection kit for measuring content of lipase in serum by colorimetric method |
| CN104215632A (en) * | 2014-08-28 | 2014-12-17 | 宁波瑞源生物科技有限公司 | Stable lipase kit |
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| CN105755103A (en) * | 2014-12-16 | 2016-07-13 | 上海复星长征医学科学有限公司 | Reagent for measuring lipase activity by stable enzymic method |
| CN107782680A (en) * | 2016-08-26 | 2018-03-09 | 山东博科生物产业有限公司 | A kind of antiheparin fat enzyme detection kit of stabilization |
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| CN104198474A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Detection kit for measuring content of lipase in serum by colorimetric method |
| CN104215632A (en) * | 2014-08-28 | 2014-12-17 | 宁波瑞源生物科技有限公司 | Stable lipase kit |
| CN104328132A (en) * | 2014-09-04 | 2015-02-04 | 中山大学 | Lipase, and coding gene and application thereof |
| CN105755103A (en) * | 2014-12-16 | 2016-07-13 | 上海复星长征医学科学有限公司 | Reagent for measuring lipase activity by stable enzymic method |
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