CN107449748A - HDL-C detection kit and its application method - Google Patents
HDL-C detection kit and its application method Download PDFInfo
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- 108010023302 HDL Cholesterol Proteins 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 42
- 108010089254 Cholesterol oxidase Proteins 0.000 claims abstract description 12
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- 108010055297 Sterol Esterase Proteins 0.000 claims abstract description 4
- 102000000019 Sterol Esterase Human genes 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 239000006173 Good's buffer Substances 0.000 claims description 15
- 238000002835 absorbance Methods 0.000 claims description 15
- 239000012530 fluid Substances 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 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 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 2
- 108010024636 Glutathione Proteins 0.000 claims description 2
- 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 2
- 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 2
- -1 dimethyl betaine Chemical compound 0.000 claims description 2
- 235000013922 glutamic acid Nutrition 0.000 claims description 2
- 239000004220 glutamic acid Substances 0.000 claims description 2
- 229960003180 glutathione Drugs 0.000 claims description 2
- 239000000787 lecithin Substances 0.000 claims description 2
- 235000010445 lecithin Nutrition 0.000 claims description 2
- 229940067606 lecithin Drugs 0.000 claims description 2
- 229920000136 polysorbate Polymers 0.000 claims description 2
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims 1
- 229960003237 betaine Drugs 0.000 claims 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 abstract description 9
- 239000004094 surface-active agent Substances 0.000 abstract description 9
- 102000003992 Peroxidases Human genes 0.000 abstract description 7
- 108040007629 peroxidase activity proteins Proteins 0.000 abstract description 7
- 150000001875 compounds Chemical class 0.000 abstract description 5
- 239000003381 stabilizer Substances 0.000 abstract description 5
- 102000016938 Catalase Human genes 0.000 abstract description 4
- 108010053835 Catalase Proteins 0.000 abstract description 4
- 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 abstract description 3
- 229930006000 Sucrose Natural products 0.000 abstract description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical class OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 abstract description 3
- 229920000447 polyanionic polymer Polymers 0.000 abstract description 3
- 239000003755 preservative agent Substances 0.000 abstract description 3
- 239000005720 sucrose Substances 0.000 abstract description 3
- 229920002554 vinyl polymer Polymers 0.000 abstract description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 abstract description 2
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 abstract description 2
- 102000004316 Oxidoreductases Human genes 0.000 abstract description 2
- 108090000854 Oxidoreductases Proteins 0.000 abstract description 2
- 229960005070 ascorbic acid Drugs 0.000 abstract description 2
- 235000010323 ascorbic acid Nutrition 0.000 abstract description 2
- 239000011668 ascorbic acid Substances 0.000 abstract description 2
- 238000007796 conventional method Methods 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- YCSMVPSDJIOXGN-UHFFFAOYSA-N CCCCCCCCCCCC[Na] Chemical compound CCCCCCCCCCCC[Na] YCSMVPSDJIOXGN-UHFFFAOYSA-N 0.000 abstract 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 abstract 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 abstract 1
- 125000001118 alkylidene group Chemical group 0.000 abstract 1
- 239000001110 calcium chloride Substances 0.000 abstract 1
- 229910001628 calcium chloride Inorganic materials 0.000 abstract 1
- 229920001983 poloxamer Polymers 0.000 abstract 1
- 229910052938 sodium sulfate Inorganic materials 0.000 abstract 1
- 235000011152 sodium sulphate Nutrition 0.000 abstract 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 16
- 239000007993 MOPS buffer Substances 0.000 description 12
- 239000002872 contrast media Substances 0.000 description 12
- 108010010234 HDL Lipoproteins Proteins 0.000 description 9
- 235000012000 cholesterol Nutrition 0.000 description 8
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 108010004103 Chylomicrons Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 108010007622 LDL Lipoproteins Proteins 0.000 description 4
- 102000007330 LDL Lipoproteins Human genes 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 108090001030 Lipoproteins Proteins 0.000 description 2
- 102000004895 Lipoproteins Human genes 0.000 description 2
- 239000007990 PIPES buffer Substances 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 241001044369 Amphion Species 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- QCCKPZOPTXCJPL-UHFFFAOYSA-N dodecyl(dimethyl)azanium;hydroxide Chemical compound [OH-].CCCCCCCCCCCC[NH+](C)C QCCKPZOPTXCJPL-UHFFFAOYSA-N 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 108010022197 lipoprotein cholesterol Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000007039 two-step reaction Methods 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
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
HDL-C detection kit and its application method of the present invention are related to biochemistry detection field.Its purpose is to a kind of its application method of the HDL-C detection kit that provides that stability is high, toxicity is low, the degree of accuracy and precision are high.The kit of the present invention includes reagent 1:Surfactant;Polyvinyl sulfuric acid salt;Lauryl sodium sulfate;Compound stabilizer;Calcium chloride;Cholesterol oxidase;Cholesterol esterase;Ascorbic acid oxidase;Catalase;4 amino-antipyrines;Proclin series preservatives;Ethylene glycol;Sucrose;Remaining is buffer solution;Reagent 2:Surfactant;Polyoxyethylene alkylidene tribenzyl;Ethylene glycol;Sucrose;Peroxidase etc..Kit of the present invention uses a variety of mixed surfactants, including Families of poloxamers, polyvinyl sulfuric acid salt, lauryl sodium sulfate etc., and the polyanion used than conventional method compares, and it has preferably specificity and selectivity.
Description
Technical field
The present invention relates to biochemistry detection field, is surveyed more particularly to a kind of by means of determining the chemical physical property of material
Determine the kit of material.
Background technology
HDL-C assay method has a variety of, mostly even phase determination method, including PEG modifications in the market
Enzyme process, immune partition method, direct assay, removing method.Wherein, removing method is to be presently considered to ideal and effective survey
Determine method, this method includes two types, first, reaction promoter peroxidase removes method (SPD methods), second, hydrogen peroxide
Enzyme removes method (CAT methods).
SPD method measuring principles are the affinity differences based on lipoprotein and surfactant.In reagent 1, promote in reaction
In the presence of agent, chylomicron (CM), VLDL (VLDL) and low-density lipoprotein (LDL) are formed solvable in serum
Property compound, the free cholesterol on its top layer is in the lower generation H of cholesterol oxidase (CHOD) effect2O2, in peroxidase
(POD) finally removed in the presence of.Reagent 2 includes special surface activating agent, acts on HDL (HDL), makes it
Crack, discharge the cholesterol in HDL, react and develop the color with enzymatic reagent.
CAT methods will not form soluble complex during the course of the reaction, but in the presence of special surface activating agent,
Cholesterol and enzymatic reagent reaction generation H in CM, VLDL and LDL2O2, H2O2H is decomposed into by catalase (CAT)2O and O2And
It is eliminated, Sodium azide should be added in reagent 2, suppresses CAT activity, meanwhile, another surfactant solves HDL particles
From exposing its cholesterol, with cholesterol enzyme reagent reacting and developing the color.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of stability is high, toxicity is low, does not generate precipitation, the degree of accuracy and essence
High HDL-C detection kit its application method of density.
The present invention relates to a kind of HDL-C detection kit, the kit includes reagent 1;The examination
Agent 1 includes:Cholesterol oxidase and cholesterol esterase.
Preferably, the kit also includes reagent 2;
The reagent 1 includes:
Remaining is GOOD ' S buffer solutions MOPS (3- (N- morpholines) propane sulfonic acid) main component of buffer solution (MOPS be), institute
PH of cushioning fluid is stated as 7.0;
The reagent 2 includes:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0.
Preferably, the compound stabilizer includes 10~13g/L of Tween 80,20~30g/L of glutamic acid, and glutathione 1~
3g/L, 50~60g/L of trehalose, 2~6g/L of dodecyldimethylammonium hydroxide inner salt, 3~6g/L of lecithin, buffer solution 100~
120mmol/L;Answering in its compound stabilizer and its application method for the Multiple Antibodies of Application No. 201610166107.2
Close stabilizer.
Preferably, the reagent 1 includes:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0;
The reagent 2 includes:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0.
Assay method involved by this detection kit removes method for SPD, reacts parent using surfactant and lipoprotein
With power difference, reach directly measure HDL~C purpose, this method is a kind of even phase detection technique, reacts and is formed without precipitation, no
Biochemical Analyzer pipeline can be blocked.This method mainly includes two-step reaction:Reacting the first step is:Using there is special parent to HDL~C
Low-density lipoprotein (LDL), extra-low density fat in surfactant, polyanion, bivalent cation and serum sample with power
Albumen (VLDL), chylomicron (CM), form soluble complex, in the presence of CHOD and CAT, surface free cholesterol
With H2O and O2Form be eliminated;React second step:Another surfactant interacts with HDL, brings it about cracking,
Hydrogen peroxide is generated in the presence of CHOD, CHER, so that in the presence of peroxidase (POD) and developer, it is anti-by developing the color
HDL~C should be determined.
The invention further relates to a kind of application method of HDL-C detection kit, methods described include with
Lower step:
The assay method of the detection kit is Two point end assay;
(1) 300 parts of reagents 1 and 3 parts of testing samples are well mixed, reaction solution 1 is obtained after 37 DEG C of incubations;
(2) and then 100 parts of reagents 2 of addition are well mixed, obtain reaction solution 2, determine the absorbance A 1 of reaction solution 2;
(3) absorbance A 2 of reaction solution 2 is surveyed after 5min again, measure dominant wavelength is 600nm, a length of 700nm of complementary wave;
(4) absorbance of HDL-C titer is measured with the operation of step (1)-(3), respectively
Absorbance A 3 and A4 are obtained, measure dominant wavelength is 600nm, a length of 700nm of complementary wave;
(5) calculated with absorbance difference, HDL-C concentration is calculated according to formula 1;
Formula 1:
HDL-C concentration=△ A samples/Δ A titers × concentration of standard solution;
△ A samples=A2-A1;
Δ A titers=A4-A3;
Above-mentioned number is volume parts.
Its application method difference from prior art of HDL-C detection kit of the present invention is:
1st, HDL-C detection kit of the present invention uses a variety of mixed surfactants, including pool Lip river sand
Nurse series, polyvinyl sulfuric acid salt, lauryl sodium sulfate etc., the polyanion used than conventional method compare, and it has more
Good is specific and selective, and sample can be kept limpid in detection process, avoids turbidity interference, and without blocking analytical instrument
The risk of pipeline.
2nd, GOOD ' S buffer solutions MOPS is a kind of amphion in HDL-C detection kit of the present invention
Buffer solution, its major advantage are to be not involved in and do not disturb biochemistry to reflect process, and the unrestraints such as enzymeization reaction are acted on.Reagent
Each composition in the ethylene glycol of middle addition, sucrose and compound stabilizer can play the stability action for keeping enzyme in reagent.Add
The protease inhibitors Sodium azide entered, can effectively suppress the activity of catalase, and the proclin series preservatives of addition are
A kind of new biological preservative, there is good compatibility with a variety of enzymes, there is preferable stability and hypotoxicity.
3rd, HDL-C is made up of apolipoprotein, phosphatide, cholesterol and a small amount of aliphatic acid.The courage of the present invention
Sterol oxidizing ferment (CHOD) combines catalase in this reaction first step, and free cholesterol is reacted and ultimately generates H2O and
O2, during this, CHOD plays scavenging action, and the reaction for next step HDL-C (HDL-C) provides premise, removal
Non- HDL-C interference, and then measurement result accuracy is improved, it is more crucial to next step reaction;Second step is reacted to develop the color instead
Should, the hydrogen peroxide (H needed for the reaction2O2) need under CHOD and CHER collective effects, gained is reacted by HDL-C.To sum up institute
State, cholesterol oxidase and cholesterol esterase have vital effect in this course of reaction.
Brief description of the drawings
Fig. 1 is the linear dependence figure of HDL-C detection kit of the present invention and contrast agents box;
Fig. 2 is contrast agents box and the linear dependence figure of HDL-C detection kit of the present invention;
Fig. 3 is the test OD value scatter diagrams of contrast agents box;
Fig. 4 is the test OD value scatter diagrams of HDL-C detection kit of the present invention.
Embodiment
By following examples and checking test to its use of the HDL-C detection kit of the present invention
Method is further described.
Embodiment 1
The HDL-C detection kit raw material and proportioning of the present embodiment are as follows:
Kit is made up of reagent 1 and reagent 2;
Reagent 1 is as follows:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0;
Reagent 2 is as follows:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0.
Embodiment 2
The HDL-C detection kit raw material and proportioning of the present embodiment are as follows:
Kit is made up of reagent 1 and reagent 2;
Reagent 1 is as follows:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0;
Reagent 2 is as follows:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0.
Embodiment 3
The HDL-C detection kit raw material and proportioning of the present embodiment are as follows:
Kit is made up of reagent 1 and reagent 2;
Reagent 1 is as follows:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0;
Reagent 2 is as follows:
Remaining is GOOD ' S buffer solutions MOPS, and the pH of cushioning fluid is 7.0.
Embodiment 4
Sample middle-high density lipoprotein cholesterol is detected using product made from embodiment 1-3, step is as follows:
(1) 300 parts of reagents 1 and 3 parts of testing samples are well mixed, reaction solution 1 is obtained after 37 DEG C of incubations;
(2) and then 100 parts of reagents 2 of addition are well mixed, obtain reaction solution 2, determine the absorbance A 1 of reaction solution 2;
(3) absorbance A 2 of reaction solution 2 is surveyed after 5min again, measure dominant wavelength is 600nm, a length of 700nm of complementary wave;
(4) absorbance of HDL-C titer is measured with the operation of step (1)-(3), respectively
Absorbance A 3 and A4 are obtained, measure dominant wavelength is 600nm, a length of 700nm of complementary wave;
(5) calculated with absorbance difference, HDL-C concentration is calculated according to formula 1;
Formula 1:
HDL-C concentration=△ A samples/Δ A titers × concentration of standard solution;
△ A samples=A2-A1;
Δ A titers=A4-A3;
Above-mentioned number is volume parts.
Checking test
Product in above-described embodiment 1~3 is tested, and contrasted with other company's similar-type products, as a result
As shown in table 1.
1 kit of the present invention of table and contrast agents box parameter comparison
The range of linearity (mmol/L) | The degree of accuracy | Sensitivity for analysis (mmol/L) | |
Contrast agents box | 0.05~3.88 | ≤ ± 10% | 2.58 |
Kit of the present invention | 0~4 | ≤ ± 10% | 0.9 |
Contrast agents box is domestic commercially available HDL~C detection kits, and its composition and formula are:Reagent 1 includes
PIPES buffer solutions, TOOS, ascorbic acid oxidase, Tween 80;Reagent 2 include PIPES buffer solutions, 4~AAP, CHOD, CHER,
POD。
Test 1
Prepare HDL-C various concentrations master sample, respectively 0,0.5,1.0,1.5,2.0,2.5,
3.0th, 3.5,4.0mmol/L, determines its concentration with kit of the present invention and contrast agents box respectively.As a result such as table 2.
The range of linearity of table 2 contrasts
As a result it can be seen that:During concentration of specimens Wei≤3.0mmol/L, contrast agents box is smaller due to the range of linearity, it is impossible to
Detection, it can be seen that the kit range of linearity of the present invention is wider.
Test 2
Using contrast agents box and kit of the present invention as material, calibrated using Landau calibration object, with Roche high level and
Low value Quality Control is measured, as a result such as table 3.
The degree of accuracy of table 3 contrasts
As a result show:The test result of kit of the present invention is higher than control kit closer to target value, the degree of accuracy.
Test 3
Withinrun precision:Using Roche high level and low value quality-control product as sample, replication 10 times (n=10), is calculated respectively
The coefficient of variation (CV%) value.Specific such as table 4.
The withinrun precision of table 4 contrasts
It can be seen that by the result of upper table:The withinrun precision of kit of the present invention is higher than contrast agents box.
Test 4
It is measured using Beckman AU480 Biochemical Analyzers, using kit of the present invention and contrast agents box as material, is used
Landau calibration object is calibrated, and determines the HDL-C of 40 clinical samples content, the results showed that kit test knot of the present invention
Fruit has good correlation with contrast agents acquired results, such as table 5.
The linear dependence of table 5 contrasts
Although the foregoing describing the embodiment of the present invention, it will be appreciated by those of skill in the art that these
It is merely illustrative of, protection scope of the present invention is defined by the appended claims.Those skilled in the art is not carrying on the back
On the premise of principle and essence from the present invention, various changes or modifications can be made to these embodiments, but these are changed
Protection scope of the present invention is each fallen within modification.
Claims (5)
- A kind of 1. HDL-C detection kit, it is characterised in that:The kit includes reagent 1;The examination Agent 1 includes:Cholesterol oxidase and cholesterol esterase.
- 2. HDL-C detection kit according to claim 1, it is characterised in that:The kit is also Including reagent 2;The reagent 1 includes:Remaining is GOOD ' S buffer solutions, and the pH of cushioning fluid is 7.0;The reagent 2 includes:Remaining is GOOD ' S buffer solutions, and the pH of cushioning fluid is 7.0.
- 3. HDL-C detection kit according to claim 1, it is characterised in that:The stable composition Agent includes 10~13g/L of Tween 80,20~30g/L of glutamic acid, 1~3g/L of glutathione, 50~60g/L of trehalose, dodecane Base 2~6g/L of dimethyl betaine, 3~6g/L of lecithin, 100~120mmol/L of buffer solution.
- 4. HDL-C detection kit according to claim 1, it is characterised in that:The reagent 1 includes:Remaining is GOOD ' S buffer solutions, and the pH of cushioning fluid is 7.0;The reagent 2 includes:Remaining is GOOD ' S buffer solutions, and the pH of cushioning fluid is 7.0.
- A kind of 5. application method of HDL-C detection kit, it is characterised in that:Methods described includes following Step:(1) 300 parts of reagents 1 and 3 parts of testing samples are well mixed, reaction solution 1 is obtained after 37 DEG C of incubations;(2) and then 100 parts of reagents 2 of addition are well mixed, obtain reaction solution 2, determine the absorbance A 1 of reaction solution 2;(3) absorbance A 2 of reaction solution 2 is surveyed after 5min again, measure dominant wavelength is 600nm, a length of 700nm of complementary wave;(4) absorbance of HDL-C titer is measured with the operation of step (1)-(3), respectively obtained Absorbance A 3 and A4, measure dominant wavelength are 600nm, a length of 700nm of complementary wave;(5) calculated with absorbance difference, HDL-C concentration is calculated according to formula 1;Formula 1:HDL-C concentration=△ A samples/Δ A titers × concentration of standard solution;△ A samples=A2-A1;Δ A titers=A4-A3;Above-mentioned number is volume parts.
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