CN108169152A - A kind of angiotensin converting enzyme detection kit and its application method - Google Patents
A kind of angiotensin converting enzyme detection kit and its application method Download PDFInfo
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Abstract
The invention belongs to clinical vitro detection reagent technique fields, and in particular to a kind of angiotensin converting enzyme detection kit and its application method；Kit of the present invention includes reagent R and calibration object, by adding in the compound stabilizer being made of 4 FPBA, glucomannans, NaCl, polyethylene glycol, Triton X-100, thimerosal in reagent R, effectively raise the stability of kit, its range of linearity is also preferable, the accuracy of reagent is high, is conducive to further promote the use of in the market.
Description
Technical field
The present invention relates to clinical vitro detection reagent technique field, more particularly to a kind of angiotensin converting enzyme detection examination
Agent box and its application method.
Background technology
ACE is the rate-limiting enzyme in renin-angiotensin system, and important work is played in renin-angiotensin system
With.ACE acts on the carboxyl terminal of Ang I, sloughs histidyl- leucine, forms Ang II.In addition to this, ACE can also degrade
With the vasoactive bradykinin of strong expandable, the effect for adjusting antiotasis, stabilizing blood pressure is played.Human body includes ACE highests
Tissue site be brain choroid plexus, nigro-striatal pathway and basal ganglia region.The active difference of crowd's Different Individual ACE is larger,
But activity change is smaller between individual tissue, is not influenced by general environment factor.
ACE may act on beta-amyloid protein (amyloidbeta-protein, A β) and influence Alzheimer disease
Generation, the development of (Alzheimerdisease, AD).Hemming etc. is the study found that ACE can reduce the A β of active somatic cell secretion
Level, the result support ACE that can adjust the neurological susceptibility of AD and evolution this hypothesis by degrading A β.The discoveries such as Zou,
ACE can make A β1~42It is converted into the smaller A β of neurotoxicity1~40, reduce A β1~42 /Aβ1~40Ratio reduces the exception of A β
Deposition, so as to inhibit the pathologic process of AD.Current study show that AD patient's intracerebral ACE activity increases.
Numerous studies show that ACE inhibits (angiotensin-convertingenzymeinhibitors, ACEI) can
Improve cognitive function.The experiments such as Hirawa find that the ACEI of low dosage can make salt density value mouse Hippocampal CA 1 nerve cell number, hair
Thin vessel density significantly increases, and generates dose-dependent memory improvement function.Some researches show that ACEI can delay gently in recent years
The process that degree cognition dysfunction develops to AD can also slow down the tempo of AD.ACEI improve cognitive function may with it is following
Mechanism is related:1. improving, central nervous system bradykinin is horizontal, the neurotransmitters such as triggering acetylcholine, excitatory amino acid
Release, can improve learning and memory function to some extent;2. improving the activity of nitricoxide synthase, increase the blood of brain tissue
Liquid is supplied, and improves learning and memory function;3. inhibiting the generation of Ang II, improve cognitive function;4. reduce releasing for intracerebral deltorphin delta
Put, promote release of corticotropin etc..
Human serum or blood plasma Angiotensin-Converting invertase term of reference are 12 ~ 68U/L.The side of serum ACE is measured at present
Method has radionuclide method, spectrophotometry, fluorescence method, Capillary Micellar Electrokinetic Chromatography.Radionuclide method is easily radiated
Property nucleic pollution, be unsuitable for hospital routinely apply.Spectrophotometry is cumbersome, is not used to clinical daily measure.Fluorescence
Method, Capillary Micellar Electrokinetic Chromatography, can not extensive uses due to by equipment limit.With high effective liquid chromatography for measuring group
ACE enzymatic activitys are knitted, sensibility and specificity is much higher than common ultraviolet spectrophotometry.But due to by equipment limit,
It is larger to promote difficulty.
Angiotensin converting enzyme detection kit（Colorimetric method）Testing principle is under ACE catalysis, and angiotensin I turns
Angiotensin II is turned to, and synthesis substrate cleavage is made to be amino acid and dipeptides.340nm detects cracking reaction, it is seen that absorbance
It is corresponding to lower.
Angiotensin converting enzyme detection kit（Colorimetric method）It is that one kind need not pre-process sample, technology and equipment requirement
It is not high, and precision and the higher analysis method of specificity, since this method does not need to expensive equipment, can realize automatic
Change, and a large amount of samples can be measured, therefore be widely popularized by clinic.
Applicant proposed a kind of entitled patent application of reproducible angiotensin converting enzyme detection kit, Shens
Please be number for 201510169134.0, detection kit that this patent application technology provides includes the component of following content：
FAPGG, 0.5mmol/L；Sodium chloride, 300mmol/L；Borate buffer solution, PH 8.2,80mM；AEO-9；0.1%-1%；Using
The defects of kit detection angiotensin converting enzyme overcomes poor repeatability in the prior art.The offer of page 5 of its specification
Stability test data illustrate in the time range for preserving 15 months, stable reagent, testing result is accurate.
But the detection kit that above-mentioned patent application technology provides, it is simple due to forming, be still not suitable for depositing for a long time
It puts, stability is still poor, is not suitable for clinically a wide range of promote and apply.
Invention content
To solve the above problems, the object of the present invention is to provide a kind of angiotensin converting enzyme detection kit,
The kit has the characteristics that stability is high, accuracy is high, the range of linearity is wide, sensitivity for analysis is high, is conducive to reagent in clinic
On popularization and application.
The present invention is achieved by the following technical solutions：A kind of angiotensin converting enzyme detection kit, it includes
Reagent R and calibration object, wherein reagent R include the component of following content：
FAPGG 0.5mmol/L
Borate buffer solution（PH 8.2） 80mM
Thimerosal 0.5%（W/V）
4- formylphenyl boronic acids (4-FPBA) 0.01%（W/V）
Glucomannans 0.5-1 mg/mL
NaCl 5mmol/L
Polyethylene glycol 20mg/mL
Triton X-100 1%（V/V）.
The application method of above-mentioned kit is that the volume ratio of sample and reagent R when in use is S：R=20：200.
The angiotensin converting enzyme detection kit that the present invention describes（Colorimetric method）, when in use, assay method is
Using automatic biochemistry analyzer, such as 120 automatic analyzer of Toshiba, operation is as shown in Figure 1, add in physiological saline, sample or school
Quasi- 20 μ l of product add reagent 200 μ l preincubates 5min, start recording absorbance value A1 after 30 seconds later, after reacting 3min,
Absorbance A 2 is read, and calculates Δ A/min.Calibration object used in the present invention is the compound calibration of Landau company of Britain production
Product.
The beneficial effects of the present invention are：
1, angiotensin converting enzyme colorimetric determination kit provided by the invention, by being added in reagent by 4- formyls
Phenylboric acid（4-FPBA）, glucomannans, NaCl, polyethylene glycol, Triton X-100, thimerosal composition compound stabilizer, respectively
Component synergistic effect makes stable reagent performance excellent, effectively enhances the stability of kit, extends the storage life of reagent
Limit, has an impact without the accuracy to reagent and sensitivity for analysis, is conducive to the reagent and further promotes in the market.
2, the present invention is allow the detection kit shelf-life obtained to extend to 24 months, is kept by the proportioning of science
Stable reagent and testing result are accurate, are conducive to the reagent and clinically promote and apply on a large scale.
3, kit provided by the invention also has the characteristics that accuracy is high, the range of linearity is wide, sensitivity for analysis is high.
Description of the drawings
Fig. 1 is the application method figure of detection kit provided by the invention；
Fig. 2 is 1 accuracy validation laboratory test results of embodiment and 1 testing result dependency diagram of comparative example；
Fig. 3 is 2 accuracy validation laboratory test results of embodiment and 1 testing result dependency diagram of comparative example.
Specific embodiment
In order to better understand the present invention, it is further described with reference to specific embodiment.
Embodiment 1
A kind of angiotensin converting enzyme detection kit provided in this embodiment, it includes reagent R and calibration objects, wherein reagent
R includes the component of following content：
FAPGG 0.5mmol/L
Borate buffer solution（PH 8.2） 80mM
Thimerosal 0.5%（W/V）
4- formylphenyl boronic acids (4-FPBA) 0.01%（W/V）
Glucomannans 0.5mg/mL
NaCl 5mmol/L
Polyethylene glycol 20mg/mL
Triton X-100 1%（V/V）
The kit of the present embodiment description, when in use, assay method are using automatic biochemistry analyzer, such as Toshiba 120 certainly
Dynamic analyzer, operation are as follows：20 μ l of physiological saline, sample or calibration object are added in, add 200 μ l preincubates 5min of reagent later
Start recording absorbance value A1 after 30 seconds after reacting 3min, reads absorbance A 2, and calculate Δ A/min.The present embodiment is used
Calibration object be Landau company of Britain production compound calibration object.
Embodiment 2
A kind of angiotensin converting enzyme detection kit provided in this embodiment, it includes reagent R and calibration objects, wherein reagent
R includes the component of following content：
FAPGG 0.5mmol/L
Borate buffer solution（PH 8.2） 80mM
Thimerosal 0.5%（W/V）
4- formylphenyl boronic acids (4-FPBA) 0.01%（W/V）
Glucomannans 1mg/mL
NaCl 5mmol/L
Polyethylene glycol 20mg/mL
Triton X-100 1%（V/V）
Specific assay method is the same as embodiment 1.
Comparative example 1
This comparative example is using a kind of excellent angiotensin converting enzyme kit of the accuracy for obtaining accreditation in the market.It includes
Reagent R and calibration object, wherein reagent R include the component of following content：
FAPGG 0.5mmol/L
300 mmol/L of sodium chloride
Borate buffer solution（PH 8.2） 80mM
Sodium azide 0.5%（W/V）
Specific assay method is the same as embodiment 1.
Comparative example 2
A kind of angiotensin converting enzyme detection kit that this comparative example provides, it includes reagent R and calibration objects, wherein reagent
R includes the component of following content：
FAPGG 0.5mmol/L
Borate buffer solution（PH 8.2） 80mM
Thimerosal 0.5%（W/V）
Glucomannans 0.5mg/mL
NaCl 5mmol/L
Polyethylene glycol 20mg/mL
Triton X-100 1%（V/V）
Specific assay method is the same as embodiment 1.
Comparative example 3
A kind of angiotensin converting enzyme detection kit that this comparative example provides, it includes reagent R and calibration objects, wherein reagent
R includes the component of following content：
FAPGG 0.5mmol/L
Borate buffer solution（PH 8.2） 80mM
Thimerosal 0.5%（W/V）
4- formylphenyl boronic acids (4-FPBA) 0.1%（W/V）
Glucomannans 1mg/mL
NaCl 5mmol/L
Polyethylene glycol 20mg/mL
Triton X-100 1%（V/V）
Specific assay method is the same as embodiment 1.
Experimental verification is carried out to kit assay performance obtained in above-described embodiment 1-3 below.
Experiment one
Accuracy validation is tested：Using the kit of embodiment 1,2 as experimental group, the standard of accreditation is obtained in comparative example 1 in the market
The excellent angiotensin converting enzyme kit of exactness carries out contrast experiment as a control group, and 40 samples are detected, inspection
The result of survey such as Fig. 2-Fig. 3.
By the detection data of Fig. 2-Fig. 3 it is found that 1 control test kit of embodiment 1,2 detection kits and comparative example
Testing result linearly dependent coefficient r be respectively 0.9992,0.9990, correlation is relatively good, show the present invention kit with
The angiotensin converting enzyme detection kit with excellent accuracy for obtaining accreditation in the market has high consistency, it was demonstrated that this
Other various compositions of invention kit addition will not impact its accuracy, and kit still keeps preferable accurate
Degree.
Experiment two
Linear dependence confirmatory experiment：Angiotensin converting enzyme high level sample is found as 150 U/L, with physiological saline system
Row dilution, prepares the sample of 6 various concentrations, is followed successively by 150 U/L, 120 U/L, 90 U/L, 60 U/L, 30 U/L, 0 U/L
The sample of concentration, each concentration level various kinds originally measure three times respectively, take its average value respectively.It is utilized respectively embodiment 1,2 and
The reagent of comparative example 1 is detected.It is as shown in the table for testing result：
The linearly related confirmatory experiment testing result of table 1
| Theoretical concentration（U/L） | Embodiment 1（U/L） | Embodiment 2（U/L） | Comparative example 1（U/L） |
| 0 | 0.17 | 0.5 | 0.47 |
| 30 | 29 | 30.31 | 29.33 |
| 60 | 59.67 | 60.2 | 59.92 |
| 90 | 88.37 | 88.77 | 91.47 |
| 120 | 119 | 123.04 | 123.31 |
| 150 | 152.94 | 149.21 | 149.56 |
| Correlation coefficient r | 0.9996 | 0.9994 | 0.9995 |
Above-mentioned testing result shows that embodiment 1,2 and 1 testing result correlation of comparative example are all higher than 0.990, reach product standard
It is required that illustrating that compound stabilizer is added in reagent will not reduce the linear dependence of reagent detection, the range of linearity is wide.
Experiment three
Stability confirmatory experiment：The store reagents in 2 DEG C~8 DEG C, the light protected environment of non-corrosive gas detect embodiment 1,2
With the stability of comparative example 1-3 reagents.5 kinds of reagents monthly choose same sample measures its absorbances three times, are averaged, and new
Fresh 1 reagent testing result of comparative example is compared, so that it is determined that the stabilization time of reagent.Detection data such as table 2.
2 stability confirmatory experiment testing result of table
| Time（Month） | Fresh comparative example 1（U/L） | Embodiment 1（U/L） | Embodiment 2（U/L） | Comparative example 1（U/L） | Comparative example 2（U/L） | Comparative example 3（U/L） |
| 12 | 41 | 41 | 40 | 40 | 42 | 18 |
| 13 | 31 | 32 | 33 | 31 | 31 | 15 |
| 14 | 37 | 40 | 40 | 41 | 38 | 17 |
| 15 | 38 | 38 | 37 | 37 | 37 | 18 |
| 16 | 35 | 34 | 35 | 33 | 35 | 17 |
| 17 | 37 | 38 | 38 | 35 | 36 | 18 |
| 18 | 39 | 39 | 38 | 33 | 32 | 17 |
| 19 | 38 | 39 | 38 | 27 | 38 | 18 |
| 20 | 42 | 40 | 43 | 21 | 25 | 15 |
| 21 | 37 | 38 | 37 | 15 | 20 | 15 |
| 22 | 37 | 37 | 38 | 6 | 16 | 9 |
| 23 | 40 | 39 | 39 | 3 | 13 | 5 |
| 24 | 34 | 34 | 35 | 1 | 10 | 1 |
Experimental result shows, comparative example 1 and 2 reagent of comparative example in 2 DEG C~8 DEG C, the light protected environment of non-corrosive gas respectively
18 and 19 months stabilizations are stored, the 19th and 20 months detection numerical value begin to decline 29% or so, and comparative example 3 detects numerical value one and opens
Begin, with regard to relatively low 58% or so, to illustrate that 3 accuracy of comparative example is bad；Embodiment 1 provided by the invention, 2 reagents are in 2 DEG C~8 DEG C, nothing
24 months stabilizations are stored in the light protected environment of corrosive gas, detection numerical value is still relatively stablized, and deviation illustrates within 5% in reagent
In by adding in the compound stabilizer of scientific matching can effectively improve the stabilization of angiotensin converting enzyme detection kit
Property.
In summary it analyzes, angiotensin converting enzyme detection kit provided by the invention passes through addition in reagent R
Compound stabilizer can effectively improve the stability of kit, and the range of linearity is preferable, and the accuracy of reagent is also preferable.Therefore,
Angiotensin converting enzyme detection kit provided by the invention is conducive to further promote the use of in the market.
Claims (2)
1. a kind of angiotensin converting enzyme detection kit, it is characterised in that：It includes reagent R and calibration objects, wherein reagent R
Include the component of following content：
FAPGG 0.5mmol/L
Borate buffer solution（PH 8.2） 80mM
Thimerosal 0.5%（W/V）
4- formylphenyl boronic acids (4-FPBA) 0.01%（W/V）
Glucomannans 0.5-1 mg/mL
NaCl 5mmol/L
Polyethylene glycol 20mg/mL
Triton X-100 1%（V/V）.
2. the application method of kit according to claim 1, it is characterised in that：The body of sample and reagent R when in use
Product ratio is S：R=20：200.
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| CN201711441523.XA CN108169152A (en) | 2017-12-27 | 2017-12-27 | A kind of angiotensin converting enzyme detection kit and its application method |
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| CN201711441523.XA CN108169152A (en) | 2017-12-27 | 2017-12-27 | A kind of angiotensin converting enzyme detection kit and its application method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109837270A (en) * | 2018-06-19 | 2019-06-04 | 深圳市安帝宝科技有限公司 | A method of keep myo-Inositol dehydrogenase, Ketoamine oxidase and sphingomyelinase steady in a long-term in a liquid |
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Application publication date: 20180615 |