CN106383239A - Apolipoprotein E detection kit - Google Patents
Apolipoprotein E detection kit Download PDFInfo
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- CN106383239A CN106383239A CN201610790535.2A CN201610790535A CN106383239A CN 106383239 A CN106383239 A CN 106383239A CN 201610790535 A CN201610790535 A CN 201610790535A CN 106383239 A CN106383239 A CN 106383239A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses an apolipoprotein E quantitative detection kit. The kit contains a reagent R1 and a reagent R2; the reagent R1 comprises HEPES, NaCl, dodecyl phenol and sodium azide, and the pH value of the reagent R1 is 6.9-7.9; and the reagent R2 comprises the HEPES, NaCl, BSA, sodium azide and apolipoprotein E monoclonal antibody coated emulsion particles, and the pH value of the reagent R2 is 6.9-7.9. The detection kit has excellent sensitivity and linear range, so the detection kit can be widely used for clinic assisted diagnosis and screening of cardiovascular and cerebrovascular diseases.
Description
Technical field
The present invention relates to in-vitro diagnosis field of medical examination.Specifically, the present invention relates to a kind of apo E is quantitatively examined
Test agent box.
Background technology
Apo E (apolipoprotein E, ApoE) is that one kind is rich in arginic basic protein, is present in blood plasma
In chylomicron (CM), Plasma Very Low Density Lipoprotein (VLDL) and its remnant, it is body LDL receptor
Aglucon, is also liver plasma membrane and the aglucon of Plasma Very Low Density Lipoprotein remains and part HDL receptor.A part
ApoE can form complex with ApoA II (Apo A2) in blood.Shore found ApoE, Rall first in 1973
Measure the protein of ApoE equal to nineteen eighty-two with primary structure.Calculate according to previous scholars expert and measure, find ApoE in media as well
There are 62% alpha-helix, 9% beta sheet structure, and 11% β-corner and 18% random coil structure.In fibrin ferment
Under effect, ApoE molecule can be hydrolyzed to two regions, N- petiolarea and C- petiolarea, and N- petiolarea is more stable, belongs to Heparin-binding area,
Rich in basic amino acid.C- petiolarea helix degree is higher, and this region is unstable, is mainly combined with lipoprotein.
Recently research finds that ApoE can synthesize in Various Tissues, wherein mainly synthesizes in liver and brain tissue, at other
Tissue includes monocyte (containing macrophage) also synthesis capability.In brain, the mRNA expression total amount of ApoE is the 1/3 of liver, star
Shape cell is its main synthesising part.The effect of the ApoE generating in brain possibly makes intracellular lipid redistribute and keep
Cholesterol in brain environment.Find the ApoE having high concentration in brain tumor, infer it possibly as neurogliocytoma
Mark.
The major function of ApoE is mediation chylomicron, VLDL and its remains, part HDL
Metabolic process, simultaneously apo E also assist in nervous system normal growth and damage after repair process.In blood plasma
ApoE level is related to myocardial infarction, hyperlipoprotememia, atherosclerotic etc.；And the level of the ApoE in brain then with old age
Dementia and cerebral infarction are waited indefinitely correlation.And the concentration of ApoE is relevant with the content of plasma triglyceride level, therefore ApoE is in human body
Content can be used as the important indicator of cardiovascular and cerebrovascular disease.
The detection method of clinical conventional ApoE is common immunoturbidimetry at present, and for example, CN104198730 discloses one
Plant apolipoprotein E detection kit, but the insufficient sensitivity of kit.CN103399160 discloses a kind of immunoturbidimetry and carries
Lipoprotein E detection kit, solves specific problem by adding surfactant and preservative.But these technical schemes
The repeatability of sensitivity and low value region all cannot be ensured simultaneously.In addition find according to Market Feedback, be often subject in clinical sample test
To the inaccurate interference of low concentration region pattern detection, additionally due to being subject to individual difference, apo E immue quantitative detection reagent box
Also tend to there is the problems such as correlation is bad, and insufficient sensitivity is high and limit its application.
Therefore, this area urgent need is a kind of easy to operate, and specificity, sensitivity and low value region repeatability are satisfied by wanting simultaneously
The apolipoprotein E detection kit asked.
Content of the invention
It is an object of the invention to provide a kind of apo E measures kit, it is more convenient to use, and can improve and criticize
Between precision, sensitivity and correlation the features such as.
In a first aspect, the present invention provides a kind of apolipoprotein E detection kit, described kit is equipped with reagent R1, examination
Agent R2；Wherein reagent R1 is made up of HEPES, NaCl, dodecylphenol, Sodium azide, and pH is 7.4 ± 0.5；Reagent R2 by HEPES,
NaCl, BSA, Sodium azide and apo E monoclonal antibody coated latex particle composition, pH is 7.4 ± 0.5.
In a preferred embodiment, in reagent R1, HEPES, NaCl, dodecylphenol, the content of Sodium azide are respectively
40-50mM, 0.3-0.8% (W/V), 2-3% (W/V) and 0.05-0.15% (W/V), preferably respectively 40mM, 0.5% (W/
V), 2.5% (W/V), 0.1% (W/V).
In a preferred embodiment, HEPES, NaCl, BSA, Sodium azide, apo E monoclonal antibody in reagent R2
The content of coated latex particle is respectively 20-50mM, 0.3-0.8% (W/V), 0.05-0.20% (W/V), 0.05-0.15%
(W/V), 1/80-1/10 (V/V) is preferably respectively 40mM, 0.5% (W/V), 0.1% (W/V), 0.1% (W/V), 1/20 (V/
V).
In a particular embodiment, it is also equipped with calibration object, cleaning solution, operation instructions in described kit and be used for moving
The device of liquid.
In a preferred embodiment, described calibration object utilizes ApoE high level human serum/blood plasma to prepare, and obtains final concentration of
The mannitol of the BSA of 50mg/ml, 20mg/ml, 0.9% sodium chloride, the trypsin inhibitor of 1mg/L.
In a particular embodiment, described latex particle is the polystyrene latex particles of surface band carboxyl.
In a particular embodiment, the particle diameter of described particle is 60-300nm, preferably 80nm.
In second aspect, the present invention provide a kind of for the reagent R1 in apolipoprotein E detection kit, described reagent R1
It is made up of HEPES, NaCl, dodecylphenol, Sodium azide.
In a preferred embodiment, in reagent R1, HEPES, NaCl, dodecylphenol, the content of Sodium azide are respectively
20-50mM, 0.3-0.8% (W/V), 2-3% (W/V) and 0.05-0.15% (W/V), preferably respectively 40mM, 0.5% (W/
V), 2.5% (W/V), 0.1% (W/V).
In the third aspect, the present invention provides the reagent R1 described in second aspect present invention preparing apo E detection examination
Purposes in agent box.
In fourth aspect, the present invention provides the preparation method of the detection kit described in first aspect present invention, described side
Method includes for described reagent R1, reagent R2 and optional calibration object, cleaning solution and operation instructions making apo E detection
Kit.
At the 5th aspect, the present invention provides a kind of method of detection apo E, and methods described is included using the present invention the
On the one hand the detection kit described in detects the apo E in vitro samples.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, thus constituting new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description
Fig. 1 shows the linear verification experimental result of the detection kit of the present invention.
Specific embodiment
Inventor is through extensively in-depth study, it was unexpectedly found that the reagent R1 in apo E kit
Adding dodecylphenol in preparation process, adding latex particle during being coated of R2, thus significantly improving detection simultaneously
Specificity, sensitivity and low value region repeatability and betweenrun precision.Complete the present invention on this basis.
The apo E kit of the present invention
In order to achieve the above object, the present inventor with the addition of specific surface-active in the preparation process of reagent R1 again
Agent-dodecylphenol, adds latex particle during being coated of R2 simultaneously, effectively increases the sensitivity of kit.
Therefore, the present invention is that it is by HEPES, NaCl, dodecylphenol, Sodium azide based on such a reagent R1 first
Composition.Based on the teachings of the present invention and routine techniques means of the prior art, those skilled in the art can determine reagent R1
In each composition concrete content.But in a preferred embodiment, HEPES, NaCl, dodecylphenol, Sodium azide in reagent R1
Content be respectively 20-50mM, 0.3-0.8% (W/V), 2-3% (W/V) and 0.05-0.15% (W/V) and be preferably respectively
40mM, 0.5% (W/V), 2.5% (W/V), 0.1% (W/V).
Further, the present invention provides such a apo E kit, in described kit equipped with mentioned reagent R1 and
Reagent R2；Wherein reagent R2 is by HEPES, NaCl, BSA, Sodium azide and the coated latex particle of apo E monoclonal antibody
Composition, pH is 7.4 ± 0.5.
Based on the teachings of the present invention and routine techniques means of the prior art, those skilled in the art can also determine examination
The concrete content of each composition in agent R2.But in a preferred embodiment, HEPES, NaCl, BSA, Sodium azide, load in reagent R2
The content of the coated latex particle of lipoprotein E monoclonal antibody is respectively 20-50mM, 0.3-0.8% (W/V), 0.05-0.20%
(W/V), 0.05-0.15% (W/V), 1/80-1/10 (V/V), preferably respectively 40mM, 0.5% (W/V), 0.1% (W/V),
0.1% (W/V), 1/20 (V/V).
Those skilled in the art know, can further be provided with calibration object, cleaning solution and operation instructions in the kit of the present invention.
In a particular embodiment, described calibration object utilizes ApoE high level human serum/blood plasma to prepare, and obtains final concentration of 50mg/ml's
The mannitol of BSA, 20mg/ml, 0.9% sodium chloride, the trypsin inhibitor of 1mg/L.
The present invention adds latex particle during being coated of R2, therefore, based in the teachings of the present invention and prior art
Routine techniques means, those skilled in the art can determine specific latex particle and its particle diameter.But in preferred embodiment party
In formula, described latex particle is the polystyrene latex particles of surface band carboxyl；The particle diameter of described particle is 60-300nm, preferably
80nm.
Based on the technology contents of the present invention, those skilled in the art could be aware that the preparation side of the detection kit of the present invention
Method, for example, make apo E detection by mentioned reagent R1, reagent R2 and optional calibration object, cleaning solution and operation instructions
Kit.
On the basis of the apolipoprotein E detection kit that the present invention provides, the present invention also provides a kind of detection to carry fat egg
The method of white E, including the apo E being detected using the detection kit of the present invention in vitro samples.
Those skilled in the art know, the method detecting apo E using the apolipoprotein E detection kit of the present invention
Can be used for diagnostic purpose, but be not limited to diagnostic purpose, such as it can also be used to scientific research purpose etc..
Advantages of the present invention
1. the sensitivity height of the apolipoprotein E detection kit of the present invention, good stability, easy to use；
2. the apolipoprotein E detection kit of the present invention possesses excellent betweenrun precision, linear and correlation；With
3. the apo E monoclonal antibody of the present invention is coated particle after 37 DEG C of preservations 3 days, and calibration has good stability.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number are percentage by weight and weight
Number.
Unless otherwise defined, all specialties used in literary composition and scientific words and meaning familiar to one skilled in the art institute
Justice is identical.Additionally, any method similar or impartial to described content and material all can be applicable in the inventive method.Wen Zhong
Described preferable implementation is only presented a demonstration with material and is used.
Material
Reagent used by following examples is all commercially available conventional reagent, and such as Chinese Medicine group chemical reagent has
Limit company.
The preparation of embodiment 1.ApoE quantitative test kit detection agent
1.1 ApoE detection kit R1 agent prescriptions
Table 1 ApoE detection kit R1 agent prescription
Take the purified water of 700mL first, be separately added into thereto 4- HEPES (HEPES), NaCl, 12
Alkyl phenol and Sodium azide, so as to final concentration is respectively 40mM, 0.5% (W/V), 2.5% (W/V) and 0.1% (W/V), put into magnetic
In power agitator, stirring is so as to be completely dissolved.Then use sodium hydrate regulator solution pH to 7.4 ± 0.5, be settled to 1000mL,
Last again pH is adjusted to 7.4 ± 0.2, this is R1 solution.
2.ApoE detection kit R2 agent prescription
The formula of table 2 ApoE detection kit R2 reagent dilutions
Table 3 washs formula of liquid
The preparation of 80nm latex particle monoclonal antibody reagent in 2.1 detection agents：
(1) take the polystyrene latex particles of 10mg 80nm, with the MES buffer by centrifugation of 25mM pH 6.0, ultrasonic wash
Wash 1-2 time；
(2) add the EDC solution (10mg/ml) of 0.5ml and NHS (100mg/ml) solution of 0.75ml, be suspended in 37 DEG C
Reaction 1h；
(3) centrifugation removes supernatant, with MES buffer solution 3 times, then be washed once with HEPES buffer solution and adds 1ml
HEPES buffer solution is fully dispersed；
(4) add in anti-apo E monoclonal antibody and the latex suspended emulsion of 1.0mg/ml, the lower crosslinking of room temperature concussion
90min, adds 0.1% BSA, room temperature concussion closing 30min afterwards；
(5) use the MES buffer solution 2 times of 25mM pH 6.0；Be washed once with the MOPSO of 50mM pH7.4 again；
(6) add the HEPES that 4ml contains 50mM pH 7.4, in 100mM 0.1%BSA solution, 50mM0.5%NaCl,
30mM 0.1%NaN3, preserve in 2-8 DEG C.
3. apo E reagent calibration object preparation：
Take apo E high level human blood to spend fat diluted plasma by a certain percentage, and add the BSA, 20mg/ of 50mg/ml
Ml mannitol, 0.9% sodium chloride, 1mg/L trypsin inhibitor, finally it is diluted to 4 concentration gradients, respectively 1.46mg/
Ml, 4.38mg/ml, 8.76mg/ml, 14.6mg/ml.
4. it is measured using apo E quantitative determination reagent kit：
Method of testing is as follows：
Kit used in the present invention is carried out on the automatic biochemistry analyzer with double reagent function, and it is specifically used
Method is：
Add sample or calibration object 3 μ l, add R1 reagent 240 μ l preincubate 5min afterwards, then read absorbance A 1,
After adding the reagent R2 reaction 5min of 60 μ l afterwards, read absorbance A 2, then calculate △ A.
The sample measures repeatability of embodiment 2. low concentration region compares
The present inventor compares the particle of the anti-apo E antibody labeling of the present invention sample to low concentration region further
Measure repeatability.
In experiment, sample 1 is the clinical sample that apo E concentration is about (1.9mg/dl), and sample 2 is (5.5mg/
Dl the sample about).Each sample retest 10 times, calculates CV value.
The repeated comparative result of low concentration region sample measures is as shown in the table：
Table 4. low concentration region sample measures result
The above results show：When detecting low value concentration samples, conventional method can not detect specific numerical value substantially, sensitive
Degree is relatively low；And when utilizing the particle detections low concentration of anti-apo E antibody labeling of the present invention, be coated on latex particle
Apo E particle reaction in antibody and low concentration clinical sample, the immune complex signal of formation is amplified by latex particle,
Compared with conventional method, greatly improve the sensitivity for analysis of kit.
Embodiment 3. precision confirmatory experiment：
In the present embodiment, inventor determines and determines apo E using the different kit that this is invented
Betweenrun precision, be compared with the kit of main flow on market, result is as shown in the table simultaneously：
Betweenrun precision (mg/dl)
Table 5. detection kit betweenrun precision test result
The clinical sample that wherein sample 1 acts on for 7.52mg/dl for apo E concentration, sample 2 is that apo E is dense
Spend the sample for 13.35mg/dl effect.
By the detection data of table 5, good kit phase is sold in the betweenrun precision of this kit and market
When illustrating that this kit can preferably ensure overall the stablizing of kit after with the addition of dodecyl nonionic surfactant
Property.
Embodiment 4. linear verification is tested
It is 14.6mg/dl using apo E high level human serum/blood plasma sample, carries out gradient dilution with physiological saline, prepare
Become 14 different samples, carry out Linear Experiment checking with kit of the present invention, result such as Fig. 1.
Can just find out from figure, kit of the present invention is linearly good, illustrate to adopt latex enhancing immune turbidimetry, add
The sensitivity of kit after latex particle, can not only be improved, do not affect other performances of kit simultaneously.
Embodiment 5. relevance verification is tested
It is 14mg/dl using apo E high level sample, carries out gradient dilution with sterilized water, prepare 11 variable concentrations
Sample, respectively 14mg/dl, 12.6mg/dl, 11.2mg/dl, 9.8mg/dl, 8.4mg/dl, 7mg/dl, 5.6mg/dl,
Each sample of 4.2mg/dl, 2.8mg/dl, 1.4mg/dl, 0mg/dl is each to be surveyed three times, takes its mean value respectively.Use the present invention respectively
Detection kit detect, testing result is as shown in table 6 with the carrying out of the kit of main flow on market.
Table 6. relevance verification result
Above-mentioned testing result shows：Compared with the kit of main flow on market, more preferably, this says correlation kit of the present invention
Bright kit, after with the addition of dodecylphenol nonionic surfactant, improves the correlation of kit, is conducive to this
Reagent further genralrlization commercially.
Comprehensive above example understands, the apolipoprotein E detection kit of the present invention, by the spy adding in reagent R1
Fixed nonionic surface active agent dodecylphenol can make microballoon be distributed with colloid sample after polymerisation.This specific nonionic
Type surfactant can effectively dissolve VLDL cell coat, make the lipoprotein E of inside exposed, decrease the antigen particles of absorption
Otherness, makes test result relatively reliable.Add latex particle can expand reaction signal simultaneously in R2, greatly improve reagent
Sensitivity for analysis.By Analysis of test results, the range of linearity of this kit is good.Therefore, the load fat that invention provides
Albumen E detection kit is conducive to commercially further genralrlization to use.
Discuss：
It is not desired to be bound by any theory, inventors believe that dodecylphenol is as a kind of specific non-ionic surface
Activating agent, does not ionize in water, but and has preferable dissolubility in organic solvent in water, is difficult inorganic by strong electrolyte
Salt and acid, the impact of alkali.Due to by individual difference, the exposed difference in outside of the ApoE particle in VLDL, therefore tying
The antigen levels closing also have very big difference, which results in the difference of correlation in test.The present invention with the addition of this in R1
After specific nonionic surfactant, dodecylphenol can effectively dissolve VLDL cell coat, makes the lipoprotein E of inside naked
Dew, decreases the otherness of the antigen particles of absorption, makes test result relatively reliable.Add breast during being coated of R2 simultaneously
Micelle, such as polystyrene particle, thus effectively increase the sensitivity of kit.
Comparative example 1.
The present inventor has attempted other surfactants in above-mentioned R1 reagent, is Tween-20, Tween-40, Qula respectively
Logical -100, take human blood sample 6.2mg/dl and tested, the surfactant adding with this has carried out degree of accuracy contrast verification,
Test 10 times respectively, result is as shown in table 7.
Table 7 accuracy validation is tested
From the above results, after adding surfactant sodium dodecyl base phenol, the degree of accuracy of kit can be effectively improved, because
This this kit is expected to further in marketing.And in addition to dodecylphenol, all cannot be obtained using other surfactants
Obtain the technique effect that the present invention obtains.The solution have the advantages that in R1 reagent using specific surfactant and be combined
Latex particle obtains.
The all documents referring in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned instruction content having read the present invention, those skilled in the art can
To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (8)
1. a kind of apolipoprotein E detection kit, described kit is equipped with reagent R1, reagent R2；Wherein reagent R1 by HEPES,
NaCl, dodecylphenol, Sodium azide composition, pH is 7.4 ± 0.5；Reagent R2 by HEPES, NaCl, BSA, Sodium azide and carries fat
The coated latex particle of albumen E monoclonal antibody forms, and pH is 7.4 ± 0.5.
2. detection kit as claimed in claim 1 it is characterised in that be also equipped with described kit calibration object, cleaning solution,
Device for liquid relief and operation instructions.
3. detection kit as claimed in claim 1 or 2 is it is characterised in that described latex particle is the poly- of surface band carboxyl
Styrene latex particle.
4. detection kit as claimed in claim 3 is it is characterised in that the particle diameter of described particle is 60-300nm, preferably
80nm.
5. a kind of for the reagent R1 in apolipoprotein E detection kit, described reagent R1 is by HEPES, NaCl, dodecyl
Phenol, Sodium azide composition.
6. purposes in preparing apolipoprotein E detection kit for the reagent R1 described in claim 5.
7. the preparation method of the detection kit any one of claim 1-4, methods described is included described reagent
Apo E detection examination made by R1, reagent R2 and optional calibration object, cleaning solution, the device for liquid relief and operation instructions
Agent box.
8. a kind of method of detection apo E, methods described is included using the detection examination any one of claim 1-4
Agent box detects the apo E in vitro samples.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109187997A (en) * | 2018-09-20 | 2019-01-11 | 苏州普瑞斯生物科技有限公司 | A kind of kit and preparation method of the concentration measuring Remnant lipoprotein cholesterol |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187997A (en) * | 2018-09-20 | 2019-01-11 | 苏州普瑞斯生物科技有限公司 | A kind of kit and preparation method of the concentration measuring Remnant lipoprotein cholesterol |
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