CN106290926A - A kind of apolipoprotein B immunoturbidimetry detection kit - Google Patents
A kind of apolipoprotein B immunoturbidimetry detection kit Download PDFInfo
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- 102100040202 Apolipoprotein B-100 Human genes 0.000 title claims abstract description 31
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 101710095342 Apolipoprotein B Proteins 0.000 title claims abstract description 23
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- 238000012360 testing method Methods 0.000 claims abstract description 32
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- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 8
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 8
- 108010027006 Apolipoproteins B Proteins 0.000 claims description 12
- 102000018616 Apolipoproteins B Human genes 0.000 claims description 12
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- 238000003556 assay Methods 0.000 description 5
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- 102000007592 Apolipoproteins Human genes 0.000 description 2
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- 102000057248 Lipoprotein(a) Human genes 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
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- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
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- 238000012795 verification Methods 0.000 description 1
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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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
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Abstract
The invention discloses a kind of apolipoprotein B immunoturbidimetry detection kit, belong to clinical vitro detection reagent technique field.Test kit of the present invention includes reagent R1, reagent R2, calibration object.By adding the magnetic nanoparticle of a certain amount of coated with silica in reagent R1 and adding a certain amount of bovine serum albumin and Kathon CG in reagent R2, effectively raise stability and the sensitivity for analysis of test kit, its range of linearity is preferable, the accuracy of reagent is high, is conducive to the most further promoting the use of.
Description
Technical field
The present invention relates to clinical vitro detection reagent technique field, particularly to a kind of apolipoprotein B immunoturbidimetry inspection
Test agent box.
Background technology
Protein part in lipoprotein is referred to as apolipoprotein (Apo)；Apolipoprotein has important in lipoprotein metabolism
Physiological function；Apo is to use ABC nomenclature, it has now been found that many types, is generally divided into 5～7 classes, mainly measures it
ApoAI, ApoB two kinds；ApoAI is mainly synthesized by liver, and small intestinal also can synthesize, and it is HDL-C (HDL-
CHOL) major structural protein, accounts for the 60%～70% of HDL-CHOL total protein, and the mensuration of ApoAI can directly reflect HDL-CHOL
Level；ApoB is also synthesized by liver, is the major structural protein of low-density lipoprotein cholesterol (LDL-CHOL), accounts for LDL-
The 97% of CHOL total protein content, the mensuration of ApoB can directly reflect the level of LDL-CHOL.
Apolipoprotein B (Apo B) is the protein that a class has polymorphism in molecular weight, immunity and metabolism, according to it
Molecular weight and percentage can be divided into B100, B48, B74, B26 and a small amount of B50；Under normal circumstances, with Apo B100 and
Apo B48 is the most important；Apo B100 is distributed mainly in blood plasma VLDL, IDL and LDL, accounts for albumen in this three classes lipoprotein and contains
25%, 60%, the 95% of amount.ApoB48 is then distributed in CM, accounts for the 5% of its protein content.Human serum or blood plasma under normal circumstances
Middle ApoB term of reference is women: 0.75～1.50g/L, male: 0.60～1.00g/L.
Atherosclerosis main damage Wall of Artery film, seriously involving middle film is that blood vessel wall cholesteryl ester bulk deposition becomes
Atherosclerotic plaque, makes blood vessel wall fiber thicken and narrow a kind of pathological change；Main infringement large artery trunks and medium-sized artery, as
Aorta, coronary artery and cerebral arteries, cause the local blood supply insufficiency of some internal organs, cardio-cerebrovascular disorder often occur, even have
Mortality is damaged；In recent years, cardiovascular patient average annual growth, every day, the whole world about 5000 people died from cardiac disorder,
Wherein many people are determined by inherited genetic factors；At present, ApoAI and ApoB measure as coronary heart disease risk index research and
Epidemiological study is gradually increased, and each hospital laboratory is the most in succession carried out this and measured；ApoAI, ApoB are considered as moving
The protection factor of pulse atherosclerosis and risk factor；Measure its content and ratio to atherosclerosis, sentencing of cardiovascular disease
Disconnected and prediction provides valuable index, has important diagnosis and prevention meaning.
ApoB is the major structural protein of LDL, and ApoB concentration level is relevant with atherosclerosis；Using total gallbladder
Sterin and triglyceride sieve coronary heart disease dangerous time, in addition to detection lipoprotein (a) with Apolipoprotein A1, detect simultaneously
ApoB can provide more information to various lipoprotein disorders, it is also possible to substitutes low-density lipoprotein cholesterol detection.
In human serum or blood plasma, apolipoprotein B goat-anti people's ApoB polyclonal antibody corresponding thereto combines in a liquid,
Form insolubilized immune complexes, make reactant liquor produce muddiness, the content of apolipoprotein B in turbidity height reflection sample；The latter
Dosage/the response curve can made by calibration object calculates；The method is a kind of without pretreatment sample, and technology and equipment requires not
Height, and precision and specificity higher analysis method.Owing to the method need not the equipment of costliness, it is possible to achieve automatization,
And a large amount of specimen can be measured, therefore suffer from clinic and be widely popularized；But common apolipoprotein B immunoturbidimetry detectable is steady
Qualitative bad, sensitivity is the highest, thus limits its popularization and application clinically.
Summary of the invention
The problem existed for prior art, the invention provides a kind of apolipoprotein B immunoturbidimetry detection kit,
This test kit is compared with conventional test kit, and stability is better than conventional detection kit, and sensitivity for analysis is high, is conducive to facing
Popularization and application on bed.
The present invention is achieved by the following measures:
A kind of apolipoprotein B immunoturbidimetry detection kit, it is characterised in that it comprises reagent R1, reagent R2 and calibration object,
Wherein reagent R1 consists of: 100mmol/L pH7.5 Tris buffer, 0.1% sodium azide (w/v), 0.1%-2% silicon dioxide bag
The magnetic nanoparticle (w/v) covered；Reagent R2 consists of: 100mmol/L pH7.5Tris buffer, 30ml/L goat-anti people ApoB
Antibody, 20-40g/L bovine serum albumin, 0.05% Kathon-CG (v/v).
The magnetic nanoparticle of described coated with silica is Fe3O4/SiO2Composite nanoparticle, particle diameter is 20-50nm.
Described reagent R1 and reagent R2 volume ratio in use are R1:R2=3:1.
The magnetic nanoparticle of coated with silica used in the present invention uses following methods to prepare:
Polyol process is used to prepare Fe3O4Nanoparticle.Take certain ferric acetyl acetonade and 2,2'-ethylenedioxybis(ethanol). joins and is heated at reflux instead
Answer in device, under conditions of magnetic agitation and Ar gas shielded, device is slowly heated to boiling, and when maintaining the reflux for one section
Between.Cooling down addition ethyl acetate in backward reaction solution makes the ferriferrous oxide nano-particle of generation produce flocculation, Magnetic Isolation
Black product, cleans for several times, is distributed in ethanol, i.e. obtains stable Fe3O4Nanoparticle alcohol colloidal solution.Afterwards by institute
Obtain Fe3O4Nanoparticle uses Stober Hydrolyze method to prepare SiO2The Fe of cladding3O4Nanoparticle.Gained Fe3O4/SiO2It is combined and receives
The particle diameter of rice corpuscles is 20-50nm.
Calibration object used in the present invention is the APOB calibration object that Jiu Fengrunda Bioisystech Co., Ltd produces.
The test kit of the present invention is carried out on the automatic biochemistry analyzer with double reagent function, and its specifically used method is such as
Shown in Fig. 4:
Add normal saline, sample or calibration object 4 μ l, after adding R1 reagent 450 μ l preincubate 5min afterwards, read absorbance
A1, after adding the reagent R2 reaction 5min of 150 μ l afterwards, reads absorbance A 2, and calculates Δ A.
Beneficial effects of the present invention:
The apolipoprotein B immunoturbidimetry detection kit that the present invention provides, by adding coated with silica in reagent R1
Magnetic nanoparticle, this magnetic nanoparticle under the effect of Electrostatic Absorption with goat-anti people's APOB antibodies so that anti-
Body forms coagulation on magnetic nanoparticle, is greatly improved the sensitivity for analysis of reagent；In reagent R2, add cattle simultaneously
Serum albumin, solves antibody this difficult problem unstable in weak solution, and it can make antibody stabilization in test, and relatively
Neutrality, but do not interfere with the character of antibody, and Kathon-CG is a kind of novel high-efficiency environment friendly type wide-spectrum bactericide, preservative,
This test kit uses Kathon-CG efficiently solve BSA and preserve the most mouldy shortcoming, therefore BSA and Kathon-CG for a long time
Common effect effectively enhances the stability of test kit, without the accuracy generation impact on reagent, is conducive to this reagent
The most further promote.
Accompanying drawing explanation
Fig. 1 embodiment 2 accuracy validation laboratory test results and matched group testing result dependency；
Fig. 2 embodiment 3 accuracy validation laboratory test results and matched group testing result dependency；
Fig. 3 embodiment 4 accuracy validation laboratory test results and matched group testing result dependency；
The specifically used method of Fig. 4；
Fig. 5 embodiment 1-4 linear correlation confirmatory experiment testing result；
Fig. 6 stability confirmatory experiment testing result；
Fig. 7 sensitivity for analysis experimental result.
Detailed description of the invention
In order to be better understood from the present invention, further describe below in conjunction with specific embodiment.
Embodiment 1
The apolipoprotein B immunoturbidimetry detection kit of a kind of routine, it includes reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH 7.5 Tris buffer 100 mmol/L
Sodium azide 0.1%(w/v)
Polyethylene glycol 6000 0.5%(v/v)；
Reagent R2 consists of:
PH7.5 Tris buffer 100 mmol/L
Goat-anti people APOB antibody 30ml/L.
The test kit that the present embodiment describes, in use, its assay method is that the Toshiba 120 using and having double reagent function is automatic
Analyser, operates as follows:
Add normal saline, sample or calibration object 4 μ l, after adding R1 reagent 450 μ l preincubate 5min afterwards, read absorbance
A1, after adding the reagent R2 reaction 5min of 150 μ l afterwards, reads absorbance A 2, and calculates Δ A.
The calibration object that the present embodiment is used is the APOB calibration object that Jiu Fengrunda Bioisystech Co., Ltd produces.
Embodiment 2
A kind of apolipoprotein B immunoturbidimetry detection kit, it includes reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH 7.5 Tris buffer 100 mmol/L
Sodium azide 0.1%(w/v)
Polyethylene glycol 6000 0.5%(v/v)
The magnetic nanoparticle 0.1%(w/v of coated with silica)；
Reagent R2 consists of:
PH7.5 Tris buffer 100 mmol/L
Goat-anti people APOB antibody 30ml/L
Bovine serum albumin 20g/L
Kathon-CG 0.05%(v/v).
Concrete assay method is with embodiment 1.
Embodiment 3
A kind of apolipoprotein B immunoturbidimetry detection kit, it includes reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH 7.5 Tris buffer 100 mmol/L
Sodium azide 0.1%(w/v)
Polyethylene glycol 6000 0.5%(v/v)
The magnetic nanoparticle 1%(w/v of coated with silica)
Reagent R2 consists of:
PH7.5 Tris buffer 100 mmol/L
Goat-anti people APOB antibody 30ml/L
Bovine serum albumin 30g/L
Kathon-CG 0.05%(v/v).
Concrete assay method is with embodiment 1.
Embodiment 4
A kind of apolipoprotein B immunoturbidimetry detection kit, it includes reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH 7.5 Tris buffer 100 mmol/L
Sodium azide 0.1%(w/v)
Polyethylene glycol 6000 0.5%(v/v)
The magnetic nanoparticle 2%(w/v of coated with silica)；
Reagent R2 consists of:
PH7.5 Tris buffer 100 mmol/L
Goat-anti people APOB antibody 30ml/L
Bovine serum albumin 40g/L
Kathon-CG 0.05%(v/v).
Concrete assay method is with embodiment 1.
The kit assay performance prepared in above-described embodiment 1-4 is carried out experimental verification.
Accuracy validation is tested:
Using the test kit of embodiment 2,3,4 as experimental group, market obtains the apolipoprotein B that a kind of accuracy of accreditation is excellent
Test kit (production of Changchun company) carries out contrast experiment as a control group, detects 40 samples, and the result of detection is such as
Fig. 1-Fig. 3.
Detection by the detection data of Fig. 1-Fig. 3, embodiment 2,3,4 detection kit and control test test kit
Results relevance is respectively 0.9970,0.9967,0.9963, and dependency is relatively good, shows on test kit and the market of the present invention
The apolipoprotein B detection kit with excellent accuracy obtaining accreditation has high consistency, it was demonstrated that test kit of the present invention adds
Other the various compositions added do not result in impact to its accuracy, and test kit still keeps preferable accuracy.
Linear dependence confirmatory experiment:
Finding apolipoprotein B high level sample is 2.50g/L, carries out serial dilution, the sample of 6 variable concentrations of preparation with normal saline
This, be followed successively by the sample of 2.5g/L, 2.0g/L, 1.5g/L, 1.0g/L, 0.5g/L, 0g/L concentration, each sample of each concentration level
Measuring three times respectively, take its meansigma methods respectively, the reagent being utilized respectively embodiment 1,2,3,4 detects；Testing result such as Fig. 5
Shown in.
Testing result shows, embodiment 1-4 testing result dependency is all higher than 0.999, and this illustrates that reagent of the present invention has
Preferably linear dependence.
Stability confirmatory experiment
2 DEG C～8 DEG C, store reagents in the light protected environment of non-corrosiveness gas, the stability of four kinds of embodiment reagent of detection, four
Kind reagent is monthly chosen same sample and is measured its absorbance three times, averages, enters with fresh embodiment 1 reagent testing result
Row contrast, so that it is determined that the stabilization time of reagent；Detection data such as Fig. 6.
Experimental result shows, embodiment 1 reagent 2 DEG C～8 DEG C, the light protected environment of non-corrosiveness gas is stored 15 months
Stable, and embodiment 2,3,4 reagent 2 DEG C～8 DEG C, the light protected environment of non-corrosiveness gas is stored 24 months stable, explanation
Adding bovine serum albumin and Kathon-CG in reagent, the two jointly acts on and effectively raises apolipoprotein B detectable
The stability of box.
Sensitivity for analysis confirmatory experiment
By apolipoprotein B test kit of the present invention test concentration known at the sample of 0.92g/L, record absorbance difference；Profit respectively
Detect with the reagent of embodiment 1,2,3,4.Testing result is as shown in Figure 7.
By detection data, the absorbance difference of embodiment 2,3,4 detection kit all high than embodiment 1, say
The bright magnetic nanoparticle adding coated with silica in reagent is tied with goat-anti people's APOB antibody under the effect of Electrostatic Absorption
Close, so that antibody forms coagulation on magnetic nanoparticle, expand response signal, be greatly improved the analysis spirit of reagent
Sensitivity.
Comprehensive above analysis, the apolipoprotein B immunoturbidimetry detection kit that the present invention provides, pass through in reagent R1
Add a certain amount of coated with silica magnetic nanoparticle and add in reagent R2 a certain amount of bovine serum albumin and
Kathon-CG can effectively improve stability and the sensitivity for analysis of test kit, and the range of linearity is preferable, the accuracy of reagent
Preferable；Therefore, the apolipoprotein B immunoturbidimetry detection kit that the present invention provides is conducive to the most further pushing away
Wide use.
Claims (3)
1. an apolipoprotein B immunoturbidimetry detection kit, it is characterised in that it comprises reagent R1, reagent R2 and calibration
Product, wherein reagent R1 consists of: 100mmol/L pH7.5 Tris buffer, 0.1% sodium azide (w/v), 0.1%-2% titanium dioxide
The magnetic nanoparticle (w/v) of silicon cladding；Reagent R2 consists of: 100mmol/L pH7.5Tris buffer, 30ml/L goat-anti people
ApoB antibody, 20-40g/L bovine serum albumin, 0.05% Kathon-CG (v/v).
Test kit the most according to claim 1, it is characterised in that the magnetic nanoparticle of described coated with silica is
Fe3O4/SiO2Composite nanoparticle, particle diameter is 20-50nm.
Test kit the most according to claim 1, it is characterised in that described reagent R1 and reagent R2 volume ratio in use
Example is R1:R2=3:1.
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|---|---|---|---|
| CN201610661812.XA CN106290926A (en) | 2016-08-13 | 2016-08-13 | A kind of apolipoprotein B immunoturbidimetry detection kit |
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