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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- CN107449748A CN107449748A CN201710681745.2A CN201710681745A CN107449748A CN 107449748 A CN107449748 A CN 107449748A CN 201710681745 A CN201710681745 A CN 201710681745A CN 107449748 A CN107449748 A CN 107449748A
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- 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
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- 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
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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Cited By (10)
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|---|---|---|---|---|
| CN108627510A (en) * | 2018-06-06 | 2018-10-09 | 临安卡尔生物技术有限公司 | High-density lipoprotein cholesterol detection kit |
| CN108982383A (en) * | 2018-07-19 | 2018-12-11 | 江西维瑞生物科技有限公司 | lipoprotein cholesterol detection system |
| CN109580511A (en) * | 2018-12-06 | 2019-04-05 | 潍坊泽成生物技术有限公司 | A kind of detection method and detection kit of high-density lipoprotein cholesterol |
| CN110736846A (en) * | 2018-07-20 | 2020-01-31 | 上海练佰生物技术中心 | Reagent, method and kit for measuring small and dense lipoproteins |
| CN111337440A (en) * | 2018-12-18 | 2020-06-26 | 上海底物生化科技有限公司 | Cardiovascular and cerebrovascular event risk screening kit and detection method thereof |
| CN111595658A (en) * | 2020-06-06 | 2020-08-28 | 曹季红 | Lysate for extracting protein in cells and preparation method thereof |
| CN111893163A (en) * | 2020-08-06 | 2020-11-06 | 武汉生之源生物科技股份有限公司 | High-density lipoprotein 3 cholesterol detection kit, preparation method and application |
| CN112695071A (en) * | 2020-12-16 | 2021-04-23 | 浙江伊利康生物技术有限公司 | High-density lipoprotein 3 determination reagent, method and kit |
| CN113308513A (en) * | 2021-05-27 | 2021-08-27 | 宁波瑞源生物科技有限公司 | Small and dense low-density lipoprotein cholesterol detection kit and detection method thereof |
| CN114134202A (en) * | 2021-11-26 | 2022-03-04 | 深圳市雷诺华科技实业有限公司 | Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower |
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|---|---|---|---|---|
| CN108627510A (en) * | 2018-06-06 | 2018-10-09 | 临安卡尔生物技术有限公司 | High-density lipoprotein cholesterol detection kit |
| CN108982383A (en) * | 2018-07-19 | 2018-12-11 | 江西维瑞生物科技有限公司 | lipoprotein cholesterol detection system |
| CN110736846A (en) * | 2018-07-20 | 2020-01-31 | 上海练佰生物技术中心 | Reagent, method and kit for measuring small and dense lipoproteins |
| CN110736846B (en) * | 2018-07-20 | 2023-07-14 | 上海微鸿企业管理有限公司 | Small and dense lipoprotein determination reagent, method and kit |
| CN109580511A (en) * | 2018-12-06 | 2019-04-05 | 潍坊泽成生物技术有限公司 | A kind of detection method and detection kit of high-density lipoprotein cholesterol |
| CN111337440A (en) * | 2018-12-18 | 2020-06-26 | 上海底物生化科技有限公司 | Cardiovascular and cerebrovascular event risk screening kit and detection method thereof |
| CN111595658A (en) * | 2020-06-06 | 2020-08-28 | 曹季红 | Lysate for extracting protein in cells and preparation method thereof |
| CN111595658B (en) * | 2020-06-06 | 2023-11-14 | 宾傲 | Lysate for extracting proteins in cells and preparation method thereof |
| CN111893163A (en) * | 2020-08-06 | 2020-11-06 | 武汉生之源生物科技股份有限公司 | High-density lipoprotein 3 cholesterol detection kit, preparation method and application |
| CN112695071A (en) * | 2020-12-16 | 2021-04-23 | 浙江伊利康生物技术有限公司 | High-density lipoprotein 3 determination reagent, method and kit |
| CN113308513A (en) * | 2021-05-27 | 2021-08-27 | 宁波瑞源生物科技有限公司 | Small and dense low-density lipoprotein cholesterol detection kit and detection method thereof |
| CN114134202A (en) * | 2021-11-26 | 2022-03-04 | 深圳市雷诺华科技实业有限公司 | Method for measuring high-density lipoprotein cholesterol by using inorganic hybrid nanoflower |
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