CN108690869A - A kind of high-density lipoprotein cholesterol detection kit and its detection method - Google Patents
A kind of high-density lipoprotein cholesterol detection kit and its detection method Download PDFInfo
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- CN108690869A CN108690869A CN201710217986.1A CN201710217986A CN108690869A CN 108690869 A CN108690869 A CN 108690869A CN 201710217986 A CN201710217986 A CN 201710217986A CN 108690869 A CN108690869 A CN 108690869A
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- density lipoprotein
- cholesterol
- lipoprotein cholesterol
- morpholines
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- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 108010023302 HDL Cholesterol Proteins 0.000 title claims abstract description 41
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 95
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- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims abstract description 23
- 239000007853 buffer solution Substances 0.000 claims abstract description 22
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims abstract description 22
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 14
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- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
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- 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
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
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- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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- 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
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- 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
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/90—Developer
- C12Q2326/96—4-Amino-antipyrine
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Abstract
The present invention provides a kind of high-density lipoprotein cholesterol assay kit, including reagent R1 and reagent R2, the reagent R1 include following component:2- (N- morpholines) ethanesulfonic acid buffer, N- ethyls-N- (2- hydroxyl -3- sulfopropyls) -3'5- dimethoxyanilines sodium, naphthalene sulfonic acid formaldehyde polymer;The reagent R2 includes following component:2- (N- morpholines) ethanesulfonic acid buffer, 4-AA, cholesterol esterase, cholesterol oxidase, peroxidase, magnesium chloride or Magnesium dichloride hexahydrate.The invention belongs to technical field of biological, the stability of kit provided by the invention is good, and without carrying out complicated processing to enzyme, cost is relatively low, and the accurate detection to high-density lipoprotein cholesterol may be implemented.
Description
Technical field
The invention belongs to technical field of biological more particularly to a kind of high-density lipoprotein cholesterol detection kit and
Its detection method.
Background technology
Cholesterol can be used as the important indicator of human body lipid metabolism detection.Cholesterol and triglycerides in blood generally with
Protein combines, and exists in the form of lipoprotein.Lipoprotein includes:It is chylomicron (CM), very low density lipoprotein (VLDL), low
Density lipoprotein (LDL) and high-density lipoprotein (HDL), wherein low-density lipoprotein are mainly responsible for cholesterol transport to periphery
Tissue, and high-density lipoprotein be remove arterial wall on cholesterol to liver transport.
Epidemiology and clinical research prove that the diseases Probability such as HDL-C and coronary heart disease, atherosclerosis is in negative
It closes, one of the risk factor as Abnormal Lipid Metabolism.According to China"Dyslipidemia remedial proposal"The criterion of proposition,
The ideal range of HDL-C is:>1.04mmol/L(>40mg/dL).It is cardiovascular when high-density lipoprotein cholesterol concentration reduces
The dangerous of disease increases.The higher reason of high-density lipoprotein cholesterol concentration may be due to primary high-density lipoprotein
Mass formed by blood stasis, it is also possible to due to manual labor overdraw, injection estrogen insulin, take contraceptive etc., it is also possible to due to chronic
Hepatitis, hepatic sclerosis, alcoholic-toxic hepatic injury, fatty liver diseases.Therefore, high-density lipoprotein cholesterol is one important
Intermediary outcomes clinically can be used for the auxiliary diagnosis of hypercholesterolemia, coronary heart disease and atherosclerosis.
The traditional detection method of high-density lipoprotein cholesterol includes centrifugal process, electrophoresis and precipitation method etc., but there is behaviour
Make the shortcomings of cumbersome, detection structure is not accurate enough.With the fast development of detection technique, high-density lipoprotein cholesterol is real
Full-automatic detection is showed, high-density lipoprotein cholesterol detection kit is developed and is widely used.Chinese patent
Application CN 105137098 discloses a kind of measure reagent of cholesterol in serum high-density LP, including high-affinity enzyme
Object, stabilizer, chromogen, surfactant, bovine serum albumin(BSA) and preservative are closed, the combination of EDTA-Na and beta-cyclodextrin are used
Object is improved the activity of enzyme by specific preparation method, improves the sensitivity of measure reagent, but the processing of enzyme as stabilizer
Process is cumbersome.Chinese patent application CN 105255993 discloses a kind of determination sample middle-high density lipoprotein cholesterol content
Method and kit, which includes reagent 1, reagent 2 and specification, and reagent 1 includes:Polyvinyl sulfuric acid tripotassium, poly- second
The only methyl ether of glycol, alpha-cyclodextrin sulfate;Reagent 2 includes:Cholesterol esterase, cholesterol oxidase, peroxidase, HDAOS,
At least one surfactant;The testing result of the kit has good correlation with contrast agents.
The activity for the enzymatic reagent used in high-density lipoprotein cholesterol detection kit is easy by pH, temperature, chemistry
There is stability difference in the interference of reagent, influence clinical stablizing and use so as to cause detection reagent.Therefore it provides one
The stable high-density lipoprotein cholesterol detection kit of kind detection is of great significance.
Invention content
To solve problems of the prior art (stability of reagent is poor, enzyme processing procedure is cumbersome etc.), this
Invention provides a kind of high-density lipoprotein cholesterol detection kit, and the stability of reagent is good, without carrying out complicated place to enzyme
Reason, cost is relatively low, the accurate detection to high-density lipoprotein cholesterol may be implemented, the testing result with commercialized product is without apparent
Difference.
The purpose of the present invention will further illustrate by the following detailed description.
The present invention provides a kind of high-density lipoprotein cholesterol detection kit, including reagent R1 and reagent R2, the examination
Agent R1 includes following component and its concentration:5~20g/L of 2- (N- morpholines) ethanesulfonic acid (MES) buffer solution, N- ethyls-N- (2- hydroxyls
Base -3- sulfopropyls) -3'5- dimethoxyanilines sodium (DAOS) 0.05~0.2g/L, naphthalene sulfonic acid formaldehyde polymer (DEMOL T-
45):1~10g/L;The reagent R2 includes following component and its concentration:2- (N- morpholines) ethanesulfonic acid (MES) buffer solution 5~
20g/L, 4-AA (4AAP) 0.1~1g/L, 1~10KU/L of cholesterol esterase, cholesterol oxidase (CO) 1~
10KU/L, peroxidase (POD) 1~10KU/L, 1~10g/L of magnesium chloride or Magnesium dichloride hexahydrate.
Preferably, the reagent R1 includes following component and its concentration:2- (N- morpholines) 10~15g/ of ethanesulfonic acid buffer
L, N- ethyls-N- (2- hydroxyl -3- sulfopropyls) -3'0.08~0.15g/L of 5- dimethoxyaniline sodium, naphthalene sulfonic acid formaldehyde polymer
4~8g/L;The reagent R2 includes following component and its concentration:2- (N- morpholines) 10~15g/L of ethanesulfonic acid buffer, 4- amino
0.2~0.8g/L of antipyrine, 1~5KU/L of cholesterol esterase, 1~5KU/L of cholesterol oxidase, 4~8KU/ of peroxidase
L, 4~10g/L of Magnesium dichloride hexahydrate.
It is highly preferred that the reagent R1 includes following component and its concentration:2- (N- morpholines) ethanesulfonic acid buffer 12g/L,
N- ethyls-N- (2- hydroxyl -3- sulfopropyls) -3'5- dimethoxyaniline sodium 0.1g/L, naphthalene sulfonic acid formaldehyde polymer 5g/L;It is described
Reagent R2 includes following component and its concentration:2- (N- morpholines) ethanesulfonic acid buffer 12g/L, 4-AA 0.5g/L,
Cholesterol esterase 3KU/L, cholesterol oxidase 3KU/L, peroxidase 6KU/L, Magnesium dichloride hexahydrate 5g/L.
Preferably, the reagent R1 further includes:1~5g/L of NaOH, 0.5~2g/L of potassium sorbate;The reagent R2 is also wrapped
It includes:1~5g/L of NaOH, 0.5~2g/L of potassium sorbate, 5~20g/L of polyoxyethylene hardened castor oil.
Preferably, the reagent R1 further includes:NaOH 2g/L, potassium sorbate 1g/L;The reagent R2 further includes:
NaOH2g/L, potassium sorbate 1g/L, polyoxyethylene hardened castor oil 10g/L.
Preferably, further include human serum standard items.
In addition, the present invention also provides the detection method of high-density lipoprotein cholesterol detection kit, include the following steps:
Blood serum sample is detached, reagent R2 is added into blood serum sample, mixing is examined after being incubated 3~5min under 540nm~600nm wavelength
Absorbance value A1 is surveyed, reagent R1 is then added, mixing detects absorbance after being incubated 3~5min under 540nm~600nm wavelength
Value A2 calculates the content of high-density lipoprotein cholesterol by serum standard panel data.
In detection process, two steps of reaction point carry out:In serum containing apolipoprotein B (APOB) lipoprotein (LDL, IDL,
VLDL, CM and lpa) first with the enzyme reaction in reagent R2 and be eliminated, due to a lack of chromogen without develop the color;Then reagent is added
R1, the specific surfactant in reagent discharge the cholesterol in high-density lipoprotein cholesterol (HDL-C), make with enzymatic reagent
It is developed the color with complete Trinder reactions are formed.
Compared with prior art, beneficial effects of the present invention include:Inventor first changes existing detection reagent
Into obtaining the good formula one of testing result accuracy, and find not ideal enough (after the destructive test enzyme of the stability of formula one
Activity and testing result significantly reduce, the inactivation due to cholesterol oxidase is the discovery that by AD Experiment), and then pass through
A large amount of realize finds that a certain amount of magnesium chloride is added in reagent R2 can significantly increase the activity of cholesterol oxidase, to real
The stable detection of existing high-density lipoprotein cholesterol, in 2~8 degree conditions long shelf-life up to 18 months, and will not be made to detecting
At interference, cost is relatively low, the testing result no significant difference with existing commercialized product.
Description of the drawings
The response curve figure of Fig. 1 formulas one.
The response curve figure of Fig. 2 formulas two.
The response curve figure of Fig. 3 formulas six.
The response curve figure of Fig. 4 formulas seven.
Specific implementation mode
The present invention is described in further details with reference to the accompanying drawings and examples.
In the present invention, involved component is conventional commercial product, or can be obtained by ordinary skill in the art means
.
One high-density lipoprotein cholesterol detection kit of embodiment (formula two)
High-density lipoprotein cholesterol detection kit includes reagent R1 and reagent R2, the reagent R1 include following component
And its concentration:2- (N- morpholines) ethanesulfonic acid buffer 12g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -3'5- dimethoxys
Aniline sodium 0.1g/L, naphthalene sulfonic acid formaldehyde polymer 5g/L, NaOH 2g/L, potassium sorbate 1g/L;The reagent R2 includes such as the following group
Point and its concentration:2- (N- morpholines) ethanesulfonic acid buffer 12g/L, 4-AA 0.5g/L, cholesterol esterase 3KU/L,
Cholesterol oxidase 3KU/L, peroxidase 6KU/L, Magnesium dichloride hexahydrate 5g/L, NaOH 2g/L, potassium sorbate 1g/L, gather
Ethylene oxide hardened castor oil 10g/L.
Two high-density lipoprotein cholesterol detection kit of embodiment
High-density lipoprotein cholesterol detection kit includes reagent R1, reagent R2 and human serum standard items, the reagent
R1 includes following component and its concentration:2- (N- morpholines) ethanesulfonic acid buffer 15g/L, N- ethyl-N- (2- hydroxyl -3- sulphurs third
Base) -3'5- dimethoxyaniline sodium 0.15g/L, naphthalene sulfonic acid formaldehyde polymer 8g/L, NaOH 2g/L, potassium sorbate 1g/L;Institute
It includes following component and its concentration to state reagent R2:2- (N- morpholines) ethanesulfonic acid buffer 15g/L, 4-AA 0.8g/
L, cholesterol esterase 5KU/L, cholesterol oxidase 5KU/L, peroxidase 8KU/L, Magnesium dichloride hexahydrate 8g/L, NaOH 2g/
L, potassium sorbate 1g/L, polyoxyethylene hardened castor oil 20g/L.
Three high-density lipoprotein cholesterol detection kit of embodiment
High-density lipoprotein cholesterol detection kit includes reagent R1 and reagent R2, the reagent R1 include following component
And its concentration:2- (N- morpholines) ethanesulfonic acid buffer 10g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -3'5- dimethoxys
Aniline sodium 0.08g/L, naphthalene sulfonic acid formaldehyde polymer 4g/L, NaOH 2g/L, potassium sorbate 1g/L;The reagent R2 includes as follows
Component and its concentration:2- (N- morpholines) ethanesulfonic acid buffer 10g/L, 4-AA 0.2g/L, cholesterol esterase 1KU/
L, cholesterol oxidase 1KU/L, peroxidase 4KU/L, Magnesium dichloride hexahydrate 4g/L, NaOH 2g/L, potassium sorbate 1g/L,
Polyoxyethylene hardened castor oil 5g/L.
1 high-density lipoprotein cholesterol detection kit of comparative example (formula one)
High-density lipoprotein cholesterol detection kit includes reagent R1 and reagent R2, the reagent R1 include following component
And its concentration:2- (N- morpholines) ethanesulfonic acid buffer 12g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -3'5- dimethoxys
Aniline sodium 0.1g/L, naphthalene sulfonic acid formaldehyde polymer 5g/L, NaOH 2g/L, potassium sorbate 1g/L;The reagent R2 includes such as the following group
Point and its concentration:2- (N- morpholines) ethanesulfonic acid buffer 12g/L, 4-AA 0.5g/L, cholesterol esterase 3KU/L,
Cholesterol oxidase 3KU/L, peroxidase 6KU/L, NaOH 2g/L, potassium sorbate 1g/L, polyoxyethylene hardened castor oil
10g/L。
Formula one with formula two difference lies in:Not magnesium chloride containing.
The sample detection comparison of the different detection reagents of test example one
Experiment reagent:Above-mentioned formula one and formula two and the HDL-C of Guangzhou Ke Fang Bioisystech Co., Ltd production are surveyed
Determine kit (including reagent R1 and R2, but ingredient is different from the present invention, hereinafter referred to as commercially available group).Above-mentioned experiment is tried respectively
Agent is used for the detection of high-density lipoprotein cholesterol, is calibrated simultaneously with Olympus AU400 Biochemical Analyzers while measuring 40
Blood serum sample, the results are shown in Table 1.
The different detection reagents of table 1 measure the result (unit of 40 blood serum samples:mmol/L)
Through statistics, correlation coefficient r=0.9991 of one measurement result of commercially available group of measurement result and formula, commercially available group of measurement knot
Fruit with formula two measurement result correlation coefficient r=0.9991, formula one measurement result with formula two measurement result correlation coefficient rs=
0.9998, illustrate that correlation is good, 5g/L Magnesium dichloride hexahydrates are added in reagent R2 to interfere testing result, be formulated
One and formula two with through State Food and Drug Administration approval listing product no significant difference, as a result accurately and reliably.
The accelerated stability of the different detection reagents of test example two is investigated
37 degree of accelerated shelf life tests are carried out to formula one and formula two, to investigate the stability of its component, as a result such as table 2
It is shown.
Sample detection result (the unit of the formula of table 2 one and two accelerated destructive test of formula after 1 week:mmol/L)
37 degree of accelerated shelf life tests:Refer to and reagent is mounted in bottle and is sealed, is placed on inside 37 degree of water baths, into
Row accelerated shelf life test, 1 week time of 37 degree of destructive tests are equivalent to (2~8 degree) of general storage temperature and preserve 1 year.
As known from Table 2, when not accelerating, 40 sample detection result average values for being formulated one are 1.49, are formulated two detection
As a result average value is 1.50, substantially quite with formula one;After 37 degree of accelerated shelf life tests 1 week, it is formulated one testing result
Average value is 0.74, and the testing result average value for being formulated two is 1.48.
It is formulated the sample detection result after destructive test 1 week to be remarkably decreased, average fall reaches
50%, and be formulated two since a certain amount of magnesium chloride of content is as protective agent, the sample detection result after destructive test 1 week
Significant change does not occur, average fall is only 1.2%, belongs to zone of reasonableness.Average fall=(when not accelerating
Mean value-mean value of acceleration 1 week)/mean value when not accelerating.
In addition, kit (formula two) provided by the invention carries out 40 samples after 2~8 degree of conditions preserve 18 months
The average result of detection only declines 1.9% compared with preserving 0 month average result, belongs to zone of reasonableness, and it is accurate to remain to realize
Detection.
To investigate the concrete reason that the testing result being formulated after accelerating 1 week once 37 degree is remarkably decreased, following experiment is carried out:
Its key component DEMOL T-45 or cholesterol oxygen are added respectively in the R1 and R2 in formula one after 37 degree accelerate 1 week respectively
Change enzyme, addition is respectively the 50% of corresponding initial concentration, and then the results are shown in Table 3 to reagent 4 for detection reagent 1 respectively.
Reagent 1:R1 and R2 is not accelerated;
Reagent 2:R1 and R2 accelerates 1 week through 37 degree;
Reagent 3:Equal 37 degree of R1 and R2 accelerates 1 week, and is added into 2.5g/L DEMOL T-45 in R1;
Reagent 4:Equal 37 degree of R1 and R2 accelerates 1 week, and is added into 1.5KU/L cholesterol oxidases in R2.
3 reagent 1 of table to reagent 4 sample detection result (unit:mmol/L)
As known from Table 3:Measured value and examination after reagent 2 1. (not adding DEMOL T-45 and cholesterol oxidase) accelerated experiment
Agent 1 has notable difference;Measured value has notable difference with reagent 1 after reagent 3 2. (individually adding DEMOL T-45) accelerated experiment,
It is almost the same with 2 measured value of reagent, illustrate that DEMOL T-45 are not inactivated in reagent R1;3. reagent 4 (is individually added into cholesterol oxygen
Change enzyme) measured value and 1 no significant difference of reagent after accelerated experiment, it is significantly increased compared with the measured value of reagent 2, illustrates reagent R2
Middle cholesterol oxidase has apparent inactivation.
Further, the R2 reagents of the R2 reagents and formula two that detect formula one respectively by activation measurement not accelerating and
37 degree accelerate 1 week when cholesterol oxidase vigor, respectively detect 20 times, as a result as shown in table 4 and table 5.
CO vigor (the units of the R2 reagents detection of the formula of table 4 one:KU/L)
The CO vigor of the R2 reagents detection of the formula of table 5 two
From table 4 and table 5 as can be seen that accelerated 1 week of formula one, seriously, inactivation ratio has reached 48.8% to CO inactivations, and
Formula two is only 2.0% since a certain amount of Magnesium dichloride hexahydrate is added as CO inactivations after protective agent, illustrates six chloride hydrates
Magnesium has preferable protective effect to CO.
Dosage of the three protective agent Magnesium dichloride hexahydrate of test example in reagent R2 is investigated
Formula one is as follows:R1:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, DAOS 0.1g/L, DEMOL T-45
5g/L;R2:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, 4AAP 0.5g/L, cholesterol esterase 3KU/L, cholesterol oxygen
Change enzyme (CO) 3KU/L, POD 6KU/L, EMANON CH-40 10g/L.
Formula two is as follows:R1:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, DAOS 0.1g/L, DEMOL T-45
5g/L;R2:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, 4AAP 0.5g/L, cholesterol esterase 3KU/L, cholesterol oxygen
Change enzyme (CO) 3KU/L, POD 6KU/L, EMANON CH-40 10g/L, Magnesium dichloride hexahydrate 5g/L.
Formula three is as follows:R1:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, DAOS 0.1g/L, DEMOL T-45
5g/L;R2:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, 4AAP 0.5g/L, cholesterol esterase 3KU/L, cholesterol oxygen
Change enzyme (CO) 3KU/L, POD 6KU/L, EMANON CH-40 10g/L, Magnesium dichloride hexahydrate 1g/L.
Formula four is as follows:R1:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, DAOS 0.1g/L, DEMOL T-45
5g/L;R2:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, 4AAP 0.5g/L, cholesterol esterase 3KU/L, cholesterol oxygen
Change enzyme (CO) 3KU/L, POD 6KU/L, EMANON CH-40 10g/L, Magnesium dichloride hexahydrate 2g/L.
Formula five is as follows:R1:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, DAOS 0.1g/L, DEMOL T-45
5g/L;R2:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, 4AAP 0.5g/L, cholesterol esterase 3KU/L, cholesterol oxygen
Change enzyme (CO) 3KU/L, POD 6KU/L, EMANON CH-40 10g/L, Magnesium dichloride hexahydrate 10g/L.
Formula six is as follows:R1:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, DAOS 0.1g/L, DEMOL T-45
5g/L;R2:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, 4AAP 0.5g/L, cholesterol esterase 3KU/L, cholesterol oxygen
Change enzyme (CO) 3KU/L, POD 6KU/L, EMANON CH-40 10g/L, Magnesium dichloride hexahydrate 15g/L.
Formula seven is as follows:R1:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, DAOS 0.1g/L, DEMOL T-45
5g/L;R2:MES 12g/L, NaOH 2g/L, potassium sorbate 1g/L, 4AAP 0.5g/L, cholesterol esterase 3KU/L, cholesterol oxygen
Change enzyme (CO) 3KU/L, POD 6KU/L, EMANON CH-40 10g/L, Magnesium dichloride hexahydrate 20g/L.
To investigate dosage of the protective agent Magnesium dichloride hexahydrate in reagent R2, formula one is detected respectively by activation measurement
To the vigor of the CO when not accelerating to accelerate 1 week with 37 degree of formula seven, the results are shown in Table 6.
Table 6 is formulated one to the CO when not accelerating to accelerate 1 week with 37 degree of formula seven vigor (unit:KU/L)
As can be seen from Table 6, be formulated one CO inactivations it is serious, inactivation ratio has reached 49.0%, is separately added into protective agent
It is respectively after Magnesium dichloride hexahydrate 1g/L, 2g/L, 5g/L, 10g/L, 15g/L, 20g/L:35.4%, 21.2%, 1.0%,
1.7%, 2.7%, 2.4%.Illustrate play when sample-adding amount 1~20g/L of the protective agent Magnesium dichloride hexahydrate in reagent R2
Certain protective effect, but sample-adding amount be 1~2g/L when, protecting effect is less desirable.
In addition, being detected respectively to the formula response curve of formula one to formula seven, formula one is fast to five reaction of formula
Speed, and be formulated six and react slower with formula seven, one response curve is formulated as shown in Figure 1, being formulated two response curve such as Fig. 2 institutes
Show, is formulated six response curve as shown in figure 3, the response curve of formula seven is as shown in Figure 4.Illustrate the addition of Magnesium dichloride hexahydrate
Certain inhibition can be generated to composition in formula when amount is more than 10g/L, cause reaction that cannot carry out rapidly.
Therefore, the resultant effect of formula two (addition of magnesium chloride is 5g/L) is best.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the present invention's
Protection domain.
Claims (7)
1. a kind of high-density lipoprotein cholesterol detection kit, it is characterised in that:Including reagent R1 and reagent R2, the reagent
R1 includes following component and its concentration:2- (N- morpholines) 5~20g/L of ethanesulfonic acid buffer, N- ethyls-N- (2- hydroxyl -3- sulphurs third
Base) -3'0.05~0.2g/L of 5- dimethoxyaniline sodium, 1~10g/L of naphthalene sulfonic acid formaldehyde polymer;The reagent R2 includes such as
Lower component and its concentration:2- (N- morpholines) 5~20g/L of ethanesulfonic acid buffer, 0.1~1g/L of 4-AA, cholesterol
1~10KU/L of esterase, 1~10KU/L of cholesterol oxidase, 1~10KU/L of peroxidase, magnesium chloride or Magnesium dichloride hexahydrate 1
~10g/L.
2. high-density lipoprotein cholesterol detection kit according to claim 1, it is characterised in that:The reagent R1 packets
Include following component and its concentration:2- (N- morpholines) 10~15g/L of ethanesulfonic acid buffer, N- ethyls-N- (2- hydroxyl -3- sulphurs third
Base) -3'0.08~0.15g/L of 5- dimethoxyaniline sodium, 4~8g/L of naphthalene sulfonic acid formaldehyde polymer;The reagent R2 includes such as
Lower component and its concentration:2- (N- morpholines) 10~15g/L of ethanesulfonic acid buffer, 0.2~0.8g/L of 4-AA, courage are solid
1~5KU/L of alcohol esterase, 1~5KU/L of cholesterol oxidase, 4~8KU/L of peroxidase, 4~10g/L of Magnesium dichloride hexahydrate.
3. high-density lipoprotein cholesterol detection kit according to claim 2, it is characterised in that:The reagent R1 packets
Include following component and its concentration:2- (N- morpholines) ethanesulfonic acid buffer 12g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -3'
5- dimethoxyaniline sodium 0.1g/L, naphthalene sulfonic acid formaldehyde polymer 5g/L;The reagent R2 includes following component and its concentration:2-
(N- morpholines) ethanesulfonic acid buffer 12g/L, 4-AA 0.5g/L, cholesterol esterase 3KU/L, cholesterol oxidase
3KU/L, peroxidase 6KU/L, Magnesium dichloride hexahydrate 5g/L.
4. high-density lipoprotein cholesterol detection kit according to any one of claim 1 to 3, it is characterised in that:
The reagent R1 further includes:1~5g/L of NaOH, 0.5~2g/L of potassium sorbate;The reagent R2 further includes:NaOH1~5g/L,
0.5~2g/L of potassium sorbate, 5~20g/L of polyoxyethylene hardened castor oil.
5. high-density lipoprotein cholesterol detection kit according to claim 4, it is characterised in that:The reagent R1 is also
Including:NaOH 2g/L, potassium sorbate 1g/L;The reagent R2 further includes:NaOH 2g/L, potassium sorbate 1g/L, polyoxyethylene
Hardened castor oil 10g/L.
6. high-density lipoprotein cholesterol detection kit according to claim 1, it is characterised in that:It further include human serum
Standard items.
7. the detection method of high-density lipoprotein cholesterol detection kit according to claim 1, it is characterised in that:Packet
Include following steps:Detach blood serum sample, into blood serum sample be added reagent R2, mixing, be incubated 3~5min after, in 540nm~
Absorbance value A1 is detected under 600nm wavelength, and reagent R1, mixing, after being incubated 3~5min, in 540nm~600nm waves is then added
Long lower detection absorbance value A2, passes through the content that serum standard panel data calculate high-density lipoprotein cholesterol.
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| 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 |
| CN109613282A (en) * | 2019-01-24 | 2019-04-12 | 浙江夸克生物科技有限公司 | A kind of high-density lipoprotein cholesterol assay kit and its application |
| 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 |
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