CN108226107A - A kind of BNP detection kits, method of preparation and use based on bimolecular fluorescence complementary technology - Google Patents
A kind of BNP detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Download PDFInfo
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- CN108226107A CN108226107A CN201711271921.1A CN201711271921A CN108226107A CN 108226107 A CN108226107 A CN 108226107A CN 201711271921 A CN201711271921 A CN 201711271921A CN 108226107 A CN108226107 A CN 108226107A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The present invention provides a kind of BNP diagnostic kits based on bimolecular fluorescence complementary technology.The kit includes：The fluorescin N-terminal segment of anti-BNP antibody couplings, the fluorescin C-terminal segment of anti-BNP antibody couplings；The invention also discloses a kind of preparation method of the BNP diagnostic kits based on bimolecular fluorescence complementary technology, this method includes：The preparation of the fluorescin N-terminal segment of anti-BNP antibody couplings, the preparation of the fluorescin C-terminal segment of anti-BNP antibody couplings；Finally also disclose the application method of the kit；Kit of the present invention has many advantages, such as that easy to operate, free of cleaning, precision is good, accuracy is high, is used convenient for clinical detection, is applied to the monitoring of heart failure, can improve the accuracy rate of heart failure medical diagnosis on disease, has great market value.
Description
Technical field
The content of BNP in human body is detected for ion vitro immunization diagnosis the present invention relates to a kind of bimolecular fluorescence complementary technology,
Belong to medical diagnosis on disease detection field.
Background technology
Brain natriuretic peptide (Brain Natriuretic Peptide, BNP) is also known as B-typeNatriuretic Peptide (B-type Natriuretic
Peptide), brain natriuretic peptide is the another member after atrial natriuretic peptide (ANP) Natriuretic Peptide System Played afterwards, mainly by the diuresis of heart secretion
A member of Na Tai families, the polypeptide being made of 32 amino acid residues.Since it is to be equal to 1988 by Japanese scholars Sudoh first
Year separates thus gains the name from pig brain.The certainly steady balance of blood pressure and blood volume can be adjusted, and has diuresis, actually it leads
To derive from ventricle.BNP has important Pathological Physiology meaning, it can promote to arrange sodium, urination, have stronger vasodilator
Effect, can fight the contracting blood vessel function of renin-angiotensin-aldosterone system (RAAS), equally be that human body resists appearance with ANP
Measure a principal endocrine system of overload and hypertension.Cardiac dysfunction can greatly activate Natriuretic Peptide System Played, the heart
Room load increase causes BNP to discharge.
BNP is mainly synthesized and is secreted by ventricular muscle cell, and the change of ventricle load and wall tension is that stimulation BNP secretes
Essential condition.The removing of BNP has two approach：When it is receptor-mediated by the c-type of natriuretic peptide family, it inside swallows after intracellular by molten
Enzyme body is degraded；Second is that it degrades through neutral endopeptidase (NEP).Natriuretic Peptide System Played shares tri- receptor of A, B, C, is transmembrane receptor,
The removing of BNP mainly passes through two approach：First：Intracellular will be swallowed in BNP, then degraded by lysosomal enzyme by the way that C is receptor-mediated；
Second：It is degraded by neutral endopeptidase to BNP, this enzyme concentration in lungs and kidney is higher.ANP is compared with BNP to neutral endopetidase
The affinity of restriction endonuclease wants greatly more, but main path of second of approach still for BNP metabolism, then since C receptors are to the parent of ANP
BNP is also higher than with power, the biological half-life (20 minutes) of BNP is caused to be longer than ANP (about 3 minutes) in this way.
At present in terms of the clinical research of BNP is concentrated mainly on Left Ventricular Global Dysfunction (LVD), left chamber function here refers to
Contractile function.Normal person or LVD patient, BNP mainly synthesize secretion by left room cardiac muscle cell, flow back into veinlet
Cycle is entered by coronary sinus to interventricular septum vein, secretion is mainly adjusted by left wall tension, the severity of LVD with
It secretes positive correlation, and peripheral blood B NP levels can reflect ventricle secretion rate and LVD degree.
Selvais when thinking that BNP diagnosing CHF and its during severity better than ANP, they by normal person, have it is normal left
The patients with coronary heart disease of room ejection fraction (LVEF), ANP, BNP concentration of different degrees of CHF patient are compared, and find the severe heart
Grade BNP concentration that declines is apparently higher than slight heart failure concentration, BNP difference CHF and normal person and the energy of the normal patients with coronary heart disease of LVEF
Power is better than ANP, and BNP concentration and the correlation of LVEF are better than ANP, are better than LVEF again when judging CHF degree, it is believed that BNP
Available for being diagnosed to outpatient service cardiovascular patient.
Common BNP detection methods mainly have Magnetism particulate immuno chemistry luminescence method.The method improves the sensitivity of detection and accurate
Degree, but heterogeneous reaction is belonged to, operating process needs to clean, and reduces the precision of reagent.
To solve the above problems, if the antibody of BNP can be utilized, using bimolecular fluorescence complementary technology as platform, develop
A kind of BNP quick detection reagents for kit for diagnosing heart failure.Make it compared with existing detection reagent, have and operate quick, sensitivity
The advantages that high, detection is quickly, precision is high；It is applied to the monitoring of heart failure, the accuracy rate of heart failure medical diagnosis on disease can be improved,
It will then be will be widely welcomed by market and there is great market value.
Invention content
For the technical problems in the prior art, the present invention provides a kind of detection examination that can be used for quantitatively detection BNP
Agent box, its method of preparation and use.
The invention is realized by the following technical scheme：
It is a kind of to prepare the method based on bimolecular fluorescence complementary technology BNP detection kits, include the following steps：
1) anti-BNP antibody couplings fluorescin N-terminal segment；
2) anti-BNP antibody couplings fluorescin C-terminal segment；
In the above-mentioned technical solutions, the anti-BNP antibody is the monoclonal antibody or Anti-TNF-α for BNP different epitopes
Body.
In said program, the step of the anti-BNP antibody couplings fluorescin N-terminal segment in, fluorescin N-terminal segment
Mass ratio with anti-BNP antibody is 1: 1-10.
In said program, the step of the anti-BNP antibody couplings fluorescin C-terminal segment in, fluorescin C-terminal segment
Mass ratio with anti-BNP antibody is 1: 1-10.
The prepared BNP detection reagents based on bimolecular fluorescence complementary technology according to any of the above technical solution
Box.Its mainly form including：
1) the fluorescin N-terminal segment of anti-BNP antibody couplings；
2) the fluorescin C-terminal segment of anti-BNP antibody couplings.
The application method of kit described in any of the above technical solution, it is characterised in that：Include the following steps：
1) sample, the fluorescin N-terminal segment of anti-BNP antibody couplings and anti-BNP is added in the reacting hole of kit to resist
The fluorescin C-terminal segment of body coupling, hybrid reaction 5-60 minutes；
2) exciting light irradiation reacting hole measures each reacting hole luminous quantity and obtains fluorescence signal value.
The reacting hole of this kit can be microwell plate, micro-fluidic reagent disc, reaction cup, reaction tube etc..
Description of the drawings
Fig. 1 is the BNP detection kits principle signal provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Figure, wherein, the anti-BNP antibody of 1-, 2- bridging agents, 3- fluorescin N-terminal segments, 4- determinands (BNP), the anti-BNP antibody of 5-, 6-
Fluorescin C-terminal segment, 7- bridging agents.
Fig. 2 is that the BNP detection kits detection provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology is linear
Areal map.
Fig. 3 is that the BNP detection kits result provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology is related
Property compares.
Specific embodiment
Below with reference to attached drawing to the present invention the BNP detection kits based on bimolecular fluorescence complementary technology, prepare and its
Application method is described in detail.
Embodiment 1
Anti- BNP antibody couplings fluorescin N-terminal segment, with the segment YFPN of yellow fluorescence protein (YFP) 1-154 amino acid
For, specific implementation process is：
1) 0.1mg YFPN albumen is added in centrifuge tube, is matched with 0.05mol/L pH9.5 carbonate buffer solutions (CB)
YFPN albumen is diluted to 1mg/ml by system.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) it is dialysed with desalting column Sephadex G-25 or 0.05mol/L pH9.5 CB and removes excessive glutaraldehyde.
5) the anti-BNP antibody of 0.1mg is taken, 1mg/ml antibody is prepared with 0.05mol/L pH9.5 CB, by the YFPN eggs of activation
The mixing of white and antibody.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ l 0.2mol/L lysine solutions are added in, room temperature closing 2h to close remaining aldehyde radical, is terminated anti-
It should.
8) 4 DEG C of placement 2h.
9) polymer insoluble matter is removed by reactant by Sephadex G-200 gel columns or with 0.45 μm of filter membrane, used
0.01 mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 2
Anti- BNP antibody couplings fluorescin C-terminal segment, with the segment of yellow fluorescence protein (YFP) 155-238 amino acid
For YFPC, specific implementation process is：
1) 0.1mg YFPC albumen is added in centrifuge tube, is matched with 0.05mol/L pH9.5 carbonate buffer solutions (CB)
YFPC albumen is diluted to 1mg/ml by system.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) it is dialysed with desalting column Sephadex G-25 or 0.05mol/L pH9.5 CB and removes excessive glutaraldehyde.
5) the anti-BNP antibody of 0.1mg is taken, 1mg/ml antibody is prepared with 0.05mol/L pH9.5 CB, by the YFPC eggs of activation
The mixing of white and antibody.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ l 0.2mol/L lysine solutions are added in, room temperature closing 2h to close remaining aldehyde radical, is terminated anti-
It should.
8) 4 DEG C of placement 2h.
9) polymer insoluble matter is removed by reactant by Sephadex G-200 gel columns or with 0.45 μm of filter membrane, used
0.01mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 3
Kit mainly forms：
1) the fluorescin N-terminal segment of anti-BNP antibody couplings；
2) the fluorescin C-terminal segment of anti-BNP antibody couplings.
Embodiment 4
Kit application method, includes the following steps：
1) sample, the fluorescin N-terminal segment of anti-BNP antibody couplings and anti-BNP is added in the reacting hole of kit to resist
The fluorescin C-terminal segment of body coupling, hybrid reaction 5-60 minutes；
2) exciting light irradiation reacting hole measures each reacting hole luminous quantity and obtains fluorescence signal value.
The reacting hole of this kit can be microwell plate, micro-fluidic reagent disc, reaction cup, reaction tube etc..
Embodiment 5
Kit method evaluation of the present invention：
It is 1. linear
Compound concentration is that the BNP of 0pg/ml, 10pg/ml, 50pg/ml, 250pg/ml, 1000pg/ml, 4000pg/ml are marked
Quasi- product solution.20 μ l standard items, the fluorescin N-terminal segment for adding in the anti-BNP antibody couplings of 50 μ l are separately added into reacting hole,
The fluorescin C-terminal segment of the anti-BNP antibody couplings of 50 μ l is added in, 37 DEG C incubate 10 minutes.After incubation, photo-irradiation reaction is excited
Hole measures each reacting hole luminous quantity and obtains fluorescence signal value.
Using fluorescence signal value as ordinate, standard concentration is abscissa, draws standard working curve (see attached drawing 2).
2. accuracy
Recovery test：It is added in the plasma specimen of normal person with known quantity BNP standard items, measures concentration value after adding in
It is compared with the theoretical value of addition, calculates the rate of recovery of BNP.Testing result is as follows：
| Sample number | Add in BNP concentration (pg/ml) | Measure the concentration (pg/ml) of BNP | The rate of recovery (%) |
| 1 | 50 | 51.2 | 102.4 |
| 2 | 300 | 312.3 | 104.1 |
| 3 | 800 | 791.3 | 98.9 |
| 4 | 2000 | 1989.1 | 99.4 |
3. precision
Choose the sample of 3 parts of various concentrations, respectively duplicate measurements 20 times according to the method described in the present invention.According to 20 times
Measurement result calculates average deviation CV values.
4. sensitivity for analysis
The definition of sensitivity for analysis is：Refer to the amount that can be distinguished statistically with zero-dose.It is repeated 20 times measurement
Zero-dose point, calculates its average value (X) and standard deviation (SD), and the concentration value with the calculating of X+2SD is the analysis of the kit
Sensitivity.The sensitivity for analysis of kit of the present invention is 2pg/ml.
5. anti-interference
The immunological assay reagents based on bimolecular fluorescence complementary technology of the present invention are detected in interference substance (haemolysis, height
Blood fat, high bilirubin) in the presence of detect plasma specimen accuracy.Hemoglobin solutions are taken respectively and are added in right amount
In BNP positive plasma specimens, the content for making hemoglobin in plasma specimen is respectively 0.5mg/ml, 1.0mg/ml.By glycerine three
Ester solution takes respectively to be added in BNP positive plasma specimens in right amount, and the content for making triglycerides in plasma specimen is respectively
0.5mg/ml、 1.0mg/ml.Bilirubin solution is taken respectively and is added in BNP positive plasma specimens in right amount, is made in plasma specimen
The content of bilirubin is respectively 25 μ g/ml, 50 μ g/ml.It is positive to the BNP for adding hemoglobin, triglycerides and bilirubin
Plasma specimen is measured.Using the ratio of theoretical concentration and measured concentration as the rate of recovery, the rate of recovery is in 96.8%-101.4%
Between.Show the BNP reagents based on bimolecular fluorescence complementary technology when detecting plasma sample not by hemoglobin, glycerine three
The interference of ester, bilirubin.
6. correlation
As shown in figure 3, the correlation with Abbott BNP kits is：Y=0.996x+4.964, R2=0.998.
The present invention is compared with existing method and product, and with detection sensitivity height, specificity is good, cost is relatively low, to detection
The advantages of instrument requirements are low.
The above description is merely a specific embodiment, but protection scope of the present invention is not limited thereto, any
Those familiar with the art disclosed herein technical scope in, the change or replacement that can readily occur in, all
It is covered by the protection scope of the present invention.
Claims (7)
1. a kind of BNP detection kits, method of preparation and use based on bimolecular fluorescence complementary technology, it is characterised in that：
1) kit mainly forms：The fluorescin C of the fluorescin N-terminal segment of anti-BNP antibody couplings and anti-BNP antibody couplings
End fragment；
2) application method：Sample, the fluorescin N-terminal segment of anti-BNP antibody couplings and anti-are added in the reacting hole of kit
The fluorescin C-terminal segment of BNP antibody couplings, hybrid reaction 5-60 minutes；
3) detection method：Exciting light irradiates reacting hole, measures each reacting hole luminous quantity and obtains fluorescence signal value.
2. a kind of preparation method of the BNP detection kits based on bimolecular fluorescence complementary technology, which is characterized in that including as follows
Step：
1) preparation of anti-BNP antibody couplings fluorescin N-terminal segment；
2) preparation of anti-BNP antibody couplings fluorescin C-terminal segment.
3. anti-BNP antibody according to claim 1 is the monoclonal antibody or polyclonal antibody for BNP different epitopes.
4. fluorescin according to claim 1, including but not limited to green fluorescent protein, blue fluorescent protein, cyan
Fluorescin, yellow fluorescence protein, red fluorescent protein.
5. method prepared by a kind of BNP detection kits based on bimolecular fluorescence complementary technology according to claim 2,
It is characterized in that, in the anti-BNP antibody couplings fluorescin N-terminal segment step, fluorescin N-terminal segment and BNP antibody
Mass ratio be 1: 1-10.
6. method prepared by the BNP detection kits according to claim 2 based on bimolecular fluorescence complementary technology, special
Sign is, in the anti-BNP antibody couplings fluorescin C-terminal segment step, fluorescin C-terminal segment and the matter of BNP antibody
Amount is than being 1: 1-10.
7. reacting hole according to claim 1, including but not limited to microwell plate, micro-fluidic reagent disc, reaction cup, reaction
Pipe.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101477127A (en) * | 2008-11-03 | 2009-07-08 | 山东省医药生物技术研究中心 | Human brain natriuretic non-competitive double-antibody sandwich method ELISA detection reagent kit |
-
2017
- 2017-11-27 CN CN201711271921.1A patent/CN108226107A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101477127A (en) * | 2008-11-03 | 2009-07-08 | 山东省医药生物技术研究中心 | Human brain natriuretic non-competitive double-antibody sandwich method ELISA detection reagent kit |
Non-Patent Citations (2)
| Title |
|---|
| CLIFF I. STAINS ET AL.: "A General Approach for Receptor and Antibody-Targeted Detection of Native Proteins utilizing Split-Luciferase Reassembly", 《ACS CHEMICAL BIOLOGY》 * |
| 黄欣媛等: "蛋白片段互补分析技术研究进展", 《中国生物工程杂志》 * |
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Application publication date: 20180629 |