CN107449746A - A kind of preparation method of fatty enzyme detection kit - Google Patents
A kind of preparation method of fatty enzyme detection kit Download PDFInfo
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- CN107449746A CN107449746A CN201710588808.XA CN201710588808A CN107449746A CN 107449746 A CN107449746 A CN 107449746A CN 201710588808 A CN201710588808 A CN 201710588808A CN 107449746 A CN107449746 A CN 107449746A
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- detection kit
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- enzyme detection
- fatty enzyme
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- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 23
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 239000002904 solvent Substances 0.000 claims abstract description 18
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 16
- 230000001681 protective effect Effects 0.000 claims abstract description 15
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 claims abstract description 13
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 229950006238 nadide Drugs 0.000 claims abstract description 8
- 239000000758 substrate Substances 0.000 claims abstract description 8
- GAFOTGQRSUWUEK-UHFFFAOYSA-N 2,3-di(dodecanoyloxy)propyl dodecanoate propane-1,2,3-triol Chemical compound O=C(OCC(OC(=O)CCCCCCCCCCC)COC(=O)CCCCCCCCCCC)CCCCCCCCCCC.OCC(O)CO GAFOTGQRSUWUEK-UHFFFAOYSA-N 0.000 claims abstract description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 6
- 238000003756 stirring Methods 0.000 claims abstract description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 13
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 10
- 229920000053 polysorbate 80 Polymers 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims description 7
- 229930006000 Sucrose Natural products 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
- 239000005720 sucrose Substances 0.000 claims description 7
- 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 6
- HHNJQTAVFGQHBP-UHFFFAOYSA-L C(C)(=O)O.C(C)(=O)[O-].[Mg+2].C(C)(=O)[O-] Chemical compound C(C)(=O)O.C(C)(=O)[O-].[Mg+2].C(C)(=O)[O-] HHNJQTAVFGQHBP-UHFFFAOYSA-L 0.000 claims description 6
- 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 6
- 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 4
- 230000003139 buffering effect Effects 0.000 claims description 3
- NDRRVVXLMFHSSV-UHFFFAOYSA-L B(O)(O)O.C(C)(=O)[O-].[Mg+2].C(C)(=O)[O-] Chemical compound B(O)(O)O.C(C)(=O)[O-].[Mg+2].C(C)(=O)[O-] NDRRVVXLMFHSSV-UHFFFAOYSA-L 0.000 claims description 2
- CKPFGBDNJJVFJB-UHFFFAOYSA-L C(C)(=O)[O-].[Mg+2].C(CC(O)(C(=O)O)CC(=O)O)(=O)O.C(C)(=O)[O-] Chemical compound C(C)(=O)[O-].[Mg+2].C(CC(O)(C(=O)O)CC(=O)O)(=O)O.C(C)(=O)[O-] CKPFGBDNJJVFJB-UHFFFAOYSA-L 0.000 claims description 2
- FCPXGQYSEJFCGW-UHFFFAOYSA-L C(C)(=O)[O-].[Mg+2].NCC(=O)O.C(C)(=O)[O-] Chemical compound C(C)(=O)[O-].[Mg+2].NCC(=O)O.C(C)(=O)[O-] FCPXGQYSEJFCGW-UHFFFAOYSA-L 0.000 claims description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 2
- 229930091371 Fructose Natural products 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- 229930195725 Mannitol Natural products 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- POULHZVOKOAJMA-UHFFFAOYSA-M dodecanoate Chemical compound CCCCCCCCCCCC([O-])=O POULHZVOKOAJMA-UHFFFAOYSA-M 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 229940070765 laurate Drugs 0.000 claims description 2
- 229960003511 macrogol Drugs 0.000 claims description 2
- 229940069446 magnesium acetate Drugs 0.000 claims description 2
- 239000011654 magnesium acetate Substances 0.000 claims description 2
- 239000000594 mannitol Substances 0.000 claims description 2
- 235000010355 mannitol Nutrition 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 125000000647 trehalose group Chemical group 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims 1
- -1 polyoxyethylene Polymers 0.000 claims 1
- 239000003223 protective agent Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 24
- 239000000203 mixture Substances 0.000 abstract description 11
- 108090001060 Lipase Proteins 0.000 description 19
- 239000004367 Lipase Substances 0.000 description 18
- 102000004882 Lipase Human genes 0.000 description 18
- 235000019421 lipase Nutrition 0.000 description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000003908 quality control method Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 5
- 206010033645 Pancreatitis Diseases 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000005515 coenzyme Substances 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 3
- 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 3
- 238000012360 testing method Methods 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 2
- 206010033647 Pancreatitis acute Diseases 0.000 description 2
- BQODPTQLXVVEJG-UHFFFAOYSA-N [O].C=C Chemical compound [O].C=C BQODPTQLXVVEJG-UHFFFAOYSA-N 0.000 description 2
- 201000003229 acute pancreatitis Diseases 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- DVDUMIQZEUTAGK-UHFFFAOYSA-N p-nitrophenyl butyrate Chemical compound CCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 DVDUMIQZEUTAGK-UHFFFAOYSA-N 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- VMPHSYLJUKZBJJ-UHFFFAOYSA-N trilaurin Chemical compound CCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCC)COC(=O)CCCCCCCCCCC VMPHSYLJUKZBJJ-UHFFFAOYSA-N 0.000 description 2
- 238000004879 turbidimetry Methods 0.000 description 2
- 208000007743 Acute Abdomen Diseases 0.000 description 1
- 108010051152 Carboxylesterase Proteins 0.000 description 1
- 102000013392 Carboxylesterase Human genes 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 108010058834 acylcarnitine hydrolase Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 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
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Substances [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of preparation method of fatty enzyme detection kit, comprise the following steps：Compound concentration is 80mmol/L 160mmol/L cushioning liquid; pH value is adjusted to 8.8 as reaction dissolvent; first add solvent composition; it is stirring while adding; rotating speed is controlled in 120r/pm; it is continuous to rotate 15min; a certain amount of enzymatic protective reagent is added afterwards; substrate glycerol trilaurin 100mmol/L 300mmol/L, 0.2mmol/L 0.4mmol/L of oxidized coenzyme I, glycerol dehydrogenase (GDH) 0.5KU/L 3KU/L are separately added into until completely dissolved; enzymatic protective reagent adds before oxidized form cozymase is added, and is eventually adding Liquid BPF aN30.6g/L 1g/L, 28 DEG C are placed in after being mixed thoroughly and is sealed.This method can prepare fatty enzyme detection kit.
Description
Technical field
The invention belongs to biological reagent field, in particular to a kind of preparation method of fatty enzyme detection kit.
Background technology
Lipase (Lipase, glycerol ester hydrolase) is under the jurisdiction of carboxylic ester hydrolase class, can be progressively by triglycerides
It is hydrolyzed into glycerine and aliphatic acid.Pancreas is mainly derived from, is secondly stomach and small intestine, can hydrolyze and a variety of contain long-chain (8-18 carbochains) fat
The glyceride of fat acid.Lipase synthesis has a higher organ specificity in the acinar tissue of pancreas, pancreatitis morbidity early stage,
The rise of i.e. detectable serum lipase concentration, acute pancreatitis are that a kind of clinic is common, morbidity is anxious, the state of an illness is dangerous, dies of illness
The high acute abdomen of rate, serum lipase of taking a blood sample in time, to diagnosing, treating and prognosis is significant.Early detection serum
Lipase can preferably distinguish acute and non-acute pancreatitis, and its serum expression and pancreatitis disease progression degree are in notable
Positive correlation, there is higher sensitiveness, be the main monitoring index of the Diagnosis of Pancreatic inflammation state of an illness.
The method of detection lipase (LPS) mainly has PNPB methods, pH-Stat methods, titration and turbidimetry etc. at present.PNPB
Method, pH-Stat methods, titration can not coordinate automatic clinical chemistry analyzer, not be suitable for clinical diagnosis；Most of LPS inspections at present
Test agent box uses turbidimetry, is the defects of the method larger to the dependence of substrate and the degree of accuracy of reaction is not high, repeats
Property is poor, and the range of linearity is narrower.
Therefore, it is necessary to this is improved.
The content of the invention
The invention aims to overcome shortcoming and defect existing for prior art, and provide a kind of lipase detection examination
The preparation method of agent box.Fatty enzyme detection kit can be prepared by this method, detection kit is using coenzyme instruction system
Continuous monitoring method, without Sample pretreatment, directly it can carry out high-volume pattern detection, operation letter using automatic clinical chemistry analyzer
List, the degree of accuracy is high, reproducible, the range of linearity is wide, is adapted to clinical practice to promote.
To achieve the above object, the technical scheme is that compound concentration is molten for 80mmol/L-160mmol/L buffering
Liquid, pH value is adjusted to 8.8 as reaction dissolvent, first adds solvent composition, it is stirring while adding, rotating speed control in 120r/pm,
It is continuous to rotate 15min, a certain amount of enzymatic protective reagent is added afterwards, is separately added into substrate glycerol trilaurin until completely dissolved
100mmol/L-300mmol/L, the 0.2mmol/L-0.4mmol/L of oxidized coenzyme I, glycerol dehydrogenase (GDH) 0.5KU/L-
3KU/L, enzymatic protective reagent add before oxidized form cozymase is added, and are eventually adding Liquid BPF aN30.6g/L-1g/L, treat to mix completely
2-8 DEG C is placed in after closing uniformly to be sealed.
Further setting is the addition of enzymatic protective reagent in terms of 5g/L-10g/L.
Further set is that solvent addition is using the solvent as developing liquid agent
0.4mL-1.6mL/100mL；Or the solvent is solid solvent 0.4g-1.6g/100mL.
Further set is that the cushioning liquid is citric acid-magnesium acetate buffer solution, glycine-magnesium acetate buffer solution, miaow
Azoles-magnesium acetate buffer solution, acetic acid-magnesium acetate buffer solution or boric acid-magnesium acetate buffer solution.
Further set is described enzymatic protective reagent for one or more groups in trehalose, sucrose, mannitol, fructose
Close.
It is that described enzymatic protective reagent is trehalose and sucrose 1 further to set:1 mass ratio combines.
Further set is that the solvent gathers for Tween-20, Tween-80, aliphatic alcohol polyethenoxy (7) ether, laurate
One or more combinations in oxygen ethene (9) ester, Macrogol 6000, PEG 8000, alkylphenol-polyethenoxy (10) ether.
It is that the solvent is Tween-80 and aliphatic alcohol polyethenoxy (7) ether 1 further to set:3 volume ratios combine.
Fatty enzyme detection kit can be configured by the above method of the present invention, the fatty enzyme detection kit is applicable
In the continuous monitoring method using coenzyme instruction system, it may be determined that the linear phase simultaneously calculates change (the △ A/ of absorbance per minute
Min), enzymatic activity is calculated according to this value exactly again, can be detected using automatic clinical chemistry analyzer；Enzyme and substrate reactions
It is specific good, the testing result degree of accuracy is high.
It is of the invention mainly to be detected using the continuous monitoring method of coenzyme instruction system.Its Cleaning Principle is：LPS catalysis point
Trilaurin is solved, generates glycerine and aliphatic acid.In (the NAD of oxidized coenzyme I+) participation under, glycerol dehydrogenase (GDH)
Hydrogen on glycerine is gone into reduced coenzyme Ⅰ (NADH), because NADH has characteristic absorption at 340nm, so NADH rising
Speed and LPS vigor are directly proportional.Reaction equation is as follows：
The present invention is applied to the preparation of fatty enzyme detection kit, with method is simple to operate, result precision is high, is made
Reagent is reproducible, the characteristics of range of linearity is wide.
Beneficial effect：The preparation method of a kind of fatty enzyme detection kit involved in the present invention, using acetic acid-magnesium acetate
Buffer solution, can not only improve the stability of reagent, and can to greatly improve its as LPS activator accurate by Mg2+ therein
Degree and sensitivity；Solvent is added simultaneously, strengthens the surface tension of oil-water interfaces, improves the degree of reaction of reagent, increase examination
Agent can linear scope.
The present invention is described further with reference to specification drawings and specific embodiments.
Brief description of the drawings
Fig. 1 is the linear result figure for the reagent prepared using the embodiment of the present invention 1；
Fig. 2 is the linear result figure for the reagent prepared using the embodiment of the present invention 2；
Fig. 3 is the linear result figure for the reagent prepared using the embodiment of the present invention 3.
Embodiment
The present invention is specifically described below by embodiment, is served only for that the present invention is further described, no
It is understood that for limiting the scope of the present invention, the technician in the field can be according to the content of foregoing invention to the present invention
Make some nonessential modifications and adaptations.
Embodiment 1
Compound concentration is 80mmol/L acetic acid-magnesium acetate buffer solution, and pH value is adjusted into 8.8 as reaction dissolvent, first added
Enter solvent composition, i.e., 0.1% Tween-80 (Tween-80) and 0.3% aliphatic alcohol polyethenoxy (7) ether, side edged
Stirring, rotating speed are controlled in 120r/pm, continuously rotate 15min, add enzymatic protective reagent composition, i.e. 2.5g/L trehalose afterwards
With 2.5g/L sucrose composition, it is auxiliary that substrate glycerol trilaurin 100mmol/L, oxidized form are separately added into until completely dissolved
(the NAD of enzyme I+) 0.2mmol/L, glycerol dehydrogenase (GDH) 0.5KU/L, enzymatic protective reagent must add before enzyme-added, finally plus
Enter Liquid BPF aN30.6g/L, after being mixed thoroughly being placed in 2-8 DEG C is sealed.
Embodiment 2
Compound concentration is 120mmol/L acetic acid-magnesium acetate buffer solution, and pH value is adjusted into 8.8 as reaction dissolvent, first added
Enter solvent composition, i.e., 0.25% Tween-80 (Tween-80) and 0.75% aliphatic alcohol polyethenoxy (7) ether, Bian Jia
Side is stirred, and rotating speed is controlled in 120r/pm, continuously rotates 15min, adds enzymatic protective reagent composition, i.e. 3.5g/L marine alga afterwards
The sucrose composition of sugar and 3.5g/L, is separately added into substrate glycerol trilaurin 200mmol/L, oxidized form until completely dissolved
Cozymase (NAD+) 0.3mmol/L, glycerol dehydrogenase (GDH) 1.8KU/L, enzymatic protective reagent must add before enzyme-added, finally
Add Liquid BPF aN30.8g/L, after being mixed thoroughly being placed in 2-8 DEG C is sealed.
Embodiment 3
Compound concentration is 160mmol/L acetic acid-magnesium acetate buffer solution, and pH value is adjusted into 8.8 as reaction dissolvent, first added
Enter solvent composition, i.e., 0.4% Tween-80 (Tween-80) and 1.2% aliphatic alcohol polyethenoxy (7) ether, side edged
Stirring, rotating speed control continuously rotate 15min in 120r/pm, add enzymatic protective reagent composition afterwards, i.e., 5g/L trehalose and
5g/L sucrose composition, substrate glycerol trilaurin 300mmol/L, oxidized coenzyme I are separately added into until completely dissolved
(NAD+) 0.4mmol/L, glycerol dehydrogenase (GDH) 3KU/L, enzymatic protective reagent must add before enzyme-added, be eventually adding anti-
Rotten agent NaN31g/L, after being mixed thoroughly being placed in 2-8 DEG C is sealed.
Embodiment 4
The liquid single-reagent prepared using 1-3 of embodiment of the present invention method is subjected to performance evaluation
1. accuracy validation：
From Landau is low, high level quality-control product (1530, lot number：1045UN；1532, lot number：801UE), it is complete using BS-420
Automatic biochemistry analyzer is detected, and concrete operation method is as described in table one, after being calibrated using One point standard method, LPS measured values
And Blank absorbance values can be directly read from instrument, in order to reduce accidental error, it is repeated three times and takes average, calculates measure
The relative deviation of value and target value.
Reagent measure low value Quality Control (1530) degree of accuracy that table 2 is prepared using embodiment 1-3 is compared
Reagent measure high level Quality Control (1532) degree of accuracy that table 3 is prepared using embodiment 1-3 is compared
From result above, the average that the liquid single-reagent prepared using the method for the present invention determines quality-control product is located at target
In the range of value, relative deviation is obvious<10%, illustrate higher using the liquid single-reagent degree of accuracy of method preparation of the invention.
2. Precision Experiment：
From routine clinical sample, the liquid single-reagent replication prepared with 1-3 of embodiment of the present invention method 20 times,
The coefficient of variation is calculated, the results are shown in Table 4.
The reagent repeatability measure that table 4 is prepared using embodiment 1-3
| Determine number | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| 1 | 76.3 | 75.3 | 76.8 |
| 2 | 74.3 | 74.1 | 75.4 |
| 3 | 76.3 | 75.3 | 75.3 |
| 4 | 76.3 | 76.8 | 76.4 |
| 5 | 76.3 | 76.3 | 76.1 |
| 6 | 74.6 | 75.6 | 76.2 |
| 7 | 76.3 | 76.3 | 76.6 |
| 8 | 75.4 | 76.4 | 75.4 |
| 9 | 76.3 | 76.8 | 77.8 |
| 10 | 75.2 | 75.2 | 76.6 |
| 11 | 76.2 | 76.7 | 76.1 |
| 12 | 76.2 | 76.2 | 76.1 |
| 13 | 75.0 | 75.5 | 75.0 |
| 14 | 76.3 | 76.5 | 76.8 |
| 15 | 76.3 | 76.1 | 76.4 |
| 16 | 76.1 | 76.1 | 76.7 |
| 17 | 77.2 | 77.2 | 77.1 |
| 18 | 76.2 | 77.6 | 76.5 |
| 19 | 77.2 | 76.8 | 76.5 |
| 20 | 76.2 | 75.2 | 76.2 |
| Average | 76.01 | 76.10 | 76.30 |
| Standard deviation (SD) | 0.7276 | 0.8085 | 0.6423 |
| The coefficient of variation (CV/%) | 0.96% | 1.06% | 0.84% |
From result above, the coefficient of variation (CV) that the liquid single-reagent prepared using the method for the present invention is determined is respectively
For 0.96%, 1.06%, 0.84%, substantially<8%, the liquid single-reagent for illustrating to use the method for the present invention to prepare has good
Good precision.
3. the range of linearity detects：
Lipase concentration 350U/L sample is produced, is diluted to 7 by 1,1/2,1/4,1/8,1/16,1/32,1/64 respectively
Individual various concentrations (Xi), the liquid single-reagent prepared with 1-3 of embodiment of the present invention method determine 3 times according to detection method and taken
Average (Yi), using Xi as independent variable, Yi is that dependent variable obtains equation of linear regression, calculates linear regression coefficient R2, will be dilute
Release concentration (xi) and substitute into equation of linear regression, calculate yi estimated value and the relative deviation of yi and estimated value.
The reagent range of linearity measure that table 5 is prepared using embodiment 1
The reagent range of linearity measure that table 6 is prepared using embodiment 2
The reagent range of linearity measure that table 7 is prepared using embodiment 3
It was found from data above, the liquid single-reagent that 1-3 of embodiment of the present invention method is prepared determines linearly dependent coefficient
Respectively：0.9998th, 0.9999,0.9999,0.9900 is all higher than, there is extraordinary linear, and linear wider range of institute.
To sum up performance evaluation is understood, the liquid single-reagent prepared using 1-3 of embodiment of the present invention method has the degree of accuracy
It is high, reproducible, measure the range of linearity it is wide the advantages of, clinical demand can be met, have in terms of the Diagnosis of Pancreatic inflammation state of an illness very well
Application value.
Application Example
Lipase (LPS) detection kit of the present invention is applied to all kinds of automatic clinical chemistry analyzers, now in full-automatic biochemical
Application on analyzer BS-420, its specifically used method are as follows：
The pattern detection operation sequence of table 1
Computational methods：LPS contents (U/L)=Δ AT/min × F in sample
ΔAT/min：Pipe average minute clock is determined with respect to blank tube absorbance change
Note：TV：Reagent sample cumulative volume, SV are sample volume；6.22：NAD+MM molecule extinction at 340nm
Coefficient；
1.0：Cuvette optical path (cm)；1000：U/mL to U/L conversion coefficient.
Quality-control product used in the present invention is the high and low value quality-control product of Landau；Institute's test sample product are not haemolysis serum.
Claims (8)
1. a kind of preparation method of fatty enzyme detection kit, it is characterised in that comprise the following steps：Compound concentration is 80mmol/
L-160mmol/L cushioning liquid, pH value is adjusted to 8.8 as reaction dissolvent, first adds solvent, it is stirring while adding, by exhibition
Open untill agent stirs, add enzymatic protective reagent afterwards, be separately added into substrate glycerol trilaurin until completely dissolved
100mmol/L-300mmol/L, the 0.2mmol/L-0.4mmol/L of oxidized coenzyme I, glycerol dehydrogenase 0.5KU/L-3KU/L, enzyme
Protective agent adds before oxidized form cozymase is added, and is eventually adding Liquid BPF aN30.6g/L-1g/L, after being mixed thoroughly
2-8 DEG C is placed in be sealed.
A kind of 2. preparation method of fatty enzyme detection kit according to claim 1, it is characterised in that：Enzymatic protective reagent
Addition is in terms of 5g/L-10g/L.
A kind of 3. preparation method of fatty enzyme detection kit according to claim 1, it is characterised in that：The solvent is
Developing liquid agent 0.4mL-1.6mL/100mL；Or the solvent is solid solvent 0.4g-1.6g/100mL.
A kind of 4. preparation method of fatty enzyme detection kit according to claim 1, it is characterised in that：The buffering is molten
Liquid is citric acid-magnesium acetate buffer solution, glycine-magnesium acetate buffer solution, imidazoles-magnesium acetate buffer solution, acetic acid-magnesium acetate buffering
Liquid or boric acid-magnesium acetate buffer solution.
A kind of 5. preparation method of fatty enzyme detection kit according to claim 1, it is characterised in that：Described enzyme is protected
Shield agent is one or more combinations in trehalose, sucrose, mannitol, fructose.
A kind of 6. preparation method of fatty enzyme detection kit according to claim 5, it is characterised in that：Described enzyme is protected
It is trehalose and sucrose 1 to protect agent:1 mass ratio combines.
A kind of 7. preparation method of fatty enzyme detection kit according to claim 1, it is characterised in that：The solvent
For Tween-20, Tween-80, aliphatic alcohol polyethenoxy (7) ether, laurate polyoxyethylene (9) ester, Macrogol 6000, poly- second two
One or more combinations in alcohol 8000, alkylphenol-polyethenoxy (10) ether.
A kind of 8. preparation method of fatty enzyme detection kit according to claim 7, it is characterised in that：The solvent
For Tween-80 and aliphatic alcohol polyethenoxy (7) ether 1:3 volume ratios combine.
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| CN109490227A (en) * | 2018-10-18 | 2019-03-19 | 闫玮钰 | A kind of highly sensitive fatty enzyme reagent kit |
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| CN106501514A (en) * | 2016-10-03 | 2017-03-15 | 王贤俊 | Detection reagent for alanine aminotransferase box |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106501514A (en) * | 2016-10-03 | 2017-03-15 | 王贤俊 | Detection reagent for alanine aminotransferase box |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109490227A (en) * | 2018-10-18 | 2019-03-19 | 闫玮钰 | A kind of highly sensitive fatty enzyme reagent kit |
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Application publication date: 20171208 |