CN108226529A - A kind of NT-proBNP detection kits, method of preparation and use based on bimolecular fluorescence complementary technology - Google Patents
A kind of NT-proBNP detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Download PDFInfo
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- CN108226529A CN108226529A CN201711272731.1A CN201711272731A CN108226529A CN 108226529 A CN108226529 A CN 108226529A CN 201711272731 A CN201711272731 A CN 201711272731A CN 108226529 A CN108226529 A CN 108226529A
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Abstract
The present invention provides a kind of NT proBNP diagnostic kits based on bimolecular fluorescence complementary technology.The kit includes：The fluorescin N-terminal segment of anti-NT proBNP antibody couplings, the fluorescin C-terminal segment of anti-NT proBNP antibody couplings；The invention also discloses a kind of preparation method of the NT proBNP diagnostic kits based on bimolecular fluorescence complementary technology, this method includes：The preparation of the fluorescin N-terminal segment of anti-NT proBNP antibody couplings, the preparation of the fluorescin C-terminal segment of anti-NT proBNP antibody couplings；Finally also disclose the application method of the kit；Kit of the present invention has many advantages, such as that specificity is good, easy to operate, detection is quick, the range of linearity is wide, free of cleaning, accuracy is high, it is used convenient for clinical detection, it is applied to the monitoring of heart failure disease, can improve the accuracy rate of heart failure medical diagnosis on disease, has great market value.
Description
Technical field
The present invention relates to a kind of bimolecular fluorescence complementary technology for NT-proBNP in ion vitro immunization diagnosis detection human body
Content belongs to medical diagnosis on disease detection field.
Background technology
1988, by a kind of isolated profit with strength out of pig brain for the first time such as Japanese scholars Tetsuji Sudoh
Sodium, diuresis, the polypeptide for expanding blood vessel and antihypertensive effect, are named as brain natriuretic peptide or natriuretic peptide (BrainNatriuretic
Peptide, BNP), BNP is primarily present in interventricular septum particle, when the volume expansion of ventricle and pressure load increase,
Secretory volume increases.NT-proBNP and BNP belongs to natriuretic peptide family, and the biological origin of the two is identical, but biological effect
It is not exactly the same with clinical meaning.During ventricular muscle cell pressure load increase, the preceding brain natriuretic peptide of 134 amino acid residues is synthesized
Then original cuts off rapidly the signal peptide containing 26 amino acid residues, form the proBNP containing 108 amino acid
(proBNP), it is then cracked into containing 32 amino acid, active B-typeNatriuretic Peptide (BNP) and contains under the action of restriction endonuclease
The N-terminal brain natriuretic peptide for having 76 amino acid, inactive is former (NT-proBNP).
The factors such as vascular capacitance load, wall stress increase, myocardial cell injury are that NT-proBNP compensatories is caused to be secreted
The main reason for increase.When myocardial damage or uneven heart function, the secretion compensatory of NT-proBNP increases, joins in cycle
With expansion blood vessel, the dynamic equilibrium of maintenance blood pressure, promotion natruresis and diuresis, antagonism renin-angiotensin-aldosterone system
Etc. adjustment effects.It plays an important role, and the increasing of NT-proBNP levels in terms of maintaining Cardiac compensation state, delaying disease process
Add directly proportional to the degree that heart function damages.Therefore, NT-proBNP can reflect the degree of heart failure.Research report in recent years
Road：BNP or NT-proBNP detections can be dead or angiocarpy after patients with heart failure discharge with the prognosis of effective evaluation patients with heart failure
The strong prediction index one that event is hospitalized again.NT-proBNP has following characteristics compared with BNP：(1) half-life period is longer (120min)；
(2) vitro stability is strong；(3) concentration in heart failure patient is higher than BNP.In view of these features, it is more suitable convenient for detection
It closes and is applied to clinic.
Common NT-proBNP detection methods have gold mark qualitative test, fluorescent immune method, linked immunosorbent adsorption test
(ELISA) and Magnetism particulate immuno chemistry luminescence method (CMIA).Gold mark qualitative experiment is quick, but this method sensitivity is low, can not dynamically supervise
Survey the variation of NT-proBNP contents.Fluorescent immune method is complicated for operation, there is higher requirement to operating environment and operating personnel.
The detection sensitivity of ELISA methods is relatively low, and it is relatively low to detection sensitivity requirement to be mainly used in communicable disease screening etc. at present
Project, and the reaction time is longer.CMIA methods are improved from ELISA method, have easy to operate, inspection compared with ELISA method
The features such as degree of testing the speed is fast, but CMIA methods are heterogeneous reactions, operating process needs to clean, and it reduce the repeatable of detection
Property.
If to solve the above problems, can utilize NT-proBNP antibody, using bimolecular fluorescence complementary technology as platform,
Develop a kind of NT-proBNP quick detection reagents for heart failure medical diagnosis on disease.It is made to have compared with existing detection reagent
Easy to operate, high sensitivity, it is free of cleaning, precision is high the advantages that；It is applied to the monitoring of heart failure disease, heart failure can be improved
The accuracy rate of medical diagnosis on disease can then be will be widely welcomed by market and have great market value.
Invention content
For the technical problems in the prior art, the present invention provides a kind of available for quantitatively detection NT-proBNP's
Detection kit, 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 NT-proBNP detection kits, include the following steps：
1) anti-NT-proBNP antibody couplings fluorescin N-terminal segment；
2) anti-NT-proBNP antibody couplings fluorescin C-terminal segment；
In the above-mentioned technical solutions, the anti-NT-proBNP antibody resists for the monoclonal for NT-proBNP different epitopes
Body or polyclonal antibody.
In said program, the step of the anti-NT-proBNP antibody couplings fluorescin N-terminal segment in, fluorescin N
The mass ratio of end fragment and anti-NT-proBNP antibody is 1: 1-10.
In said program, the step of the anti-NT-proBNP antibody couplings fluorescin C-terminal segment in, fluorescin C
The mass ratio of end fragment and anti-NT-proBNP antibody is 1: 1-10.
The prepared detections of the NT-proBNP based on bimolecular fluorescence complementary technology according to any of the above technical solution
Kit.Its mainly form including：
1) the fluorescin N-terminal segment of anti-NT-proBNP antibody couplings；
2) the fluorescin C-terminal segment of anti-NT-proBNP 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-NT-proBNP antibody couplings and anti-are added in the reacting hole of kit
The fluorescin C-terminal segment of NT-proBNP antibody couplings, 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 that the NT-proBNP detection kits provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology are former
Schematic diagram is managed, wherein, the anti-NT-proBNP antibody of 1-, 2- bridging agents, 3- fluorescin N-terminal segments, 4- determinands (NT-
ProBNP), the anti-NT-proBNP antibody of 5-, 6- fluorescin C-terminal segments, 7- bridging agents.
Fig. 2 is the NT-proBNP detection kits inspection provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Linear areal map.
Fig. 3 is the NT-proBNP detection kit knots provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Fruit correlation compares.
Specific embodiment
Below with reference to NT-proBNP detection kit based on bimolecular fluorescence complementary technology of the attached drawing to the present invention, system
Standby and its application method is described in detail.
Embodiment 1
Anti- NT-proBNP antibody couplings fluorescin N-terminal segment, with the piece of yellow fluorescence protein (YFP) 1-154 amino acid
For section YFPN, 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-NT-proBNP antibody of 0.1mg is taken, 1mg/ml antibody is prepared with 0.05mol/L pH9.5 CB, by activation
YFPN albumen and antibody mixing.
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 2
Anti- NT-proBNP antibody couplings fluorescin C-terminal segment, with yellow fluorescence protein (YFP) 155-238 amino acid
For segment 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-NT-proBNP antibody of 0.1mg is taken, 1mg/ml antibody is prepared with 0.05mol/L pH9.5 CB, by activation
YFPC albumen and antibody mixing.
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-NT-proBNP antibody couplings；
2) the fluorescin C-terminal segment of anti-NT-proBNP antibody couplings.
Embodiment 4
Kit application method, includes the following steps：
1) sample, the fluorescin N-terminal segment of anti-NT-proBNP antibody couplings and anti-are added in the reacting hole of kit
The fluorescin C-terminal segment of NT-proBNP antibody couplings, 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 0pg/ml, 20pg/ml, 100pg/ml, 500pg/ml, 2500pg/ml, 10000pg/ml, 25000
The NT-proBNP standard solutions of pg/ml, 50000pg/ml.20 μ l standard items are separately added into reacting hole, add in 50 μ l
The fluorescin N-terminal segment of anti-NT-proBNP antibody couplings adds in the fluorescin C-terminal of the anti-NT-proBNP antibody couplings of 50 μ l
Segment, 37 DEG C incubate 10 minutes.After incubation, exciting light irradiation reacting 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 serum specimen of normal person, is measured after adding in known quantity NT-proBNP standard items
Concentration value is compared with the theoretical value added in, calculates the rate of recovery of NT-proBNP.Testing result is as follows：
| Sample number | Add in NT-proBNP concentration (pg/ml) | Measure the concentration (pg/ml) of NT-proBNP | The rate of recovery (%) |
| 1 | 50 | 50.9 | 101.8 |
| 2 | 750 | 734.1 | 97.9 |
| 3 | 2000 | 2094.2 | 104.7 |
| 4 | 20000 | 19806.0 | 99.0 |
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 3pg/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 sample accuracy.Hemoglobin solutions are taken respectively and are added to NT- in right amount
In proBNP positive serum samples, the content for making hemoglobin in serum is respectively 0.5mg/ml, 1.0mg/ml.By glycerine three
Ester solution takes respectively to be added in NT-proBNP positive serum samples in right amount, and the content for making Triglycerides in Serum is respectively
0.5mg/ml、 1.0mg/ml.Bilirubin solution is taken respectively and is added in NT-proBNP positive serum samples in right amount, makes serum
The content of mesobilirubin is respectively 25 μ g/ml, 50 μ g/ml.To adding hemoglobin, triglycerides and the NT- of bilirubin
ProBNP positive samples are measured.Using the ratio of theoretical concentration and measured concentration as the rate of recovery, the rate of recovery is in 95.9%-
Between 101.3%.Show the NT-proBNP reagents based on bimolecular fluorescence complementary technology when detecting serum sample not by blood red
The interference of albumen, triglycerides, bilirubin.
6. correlation
As shown in figure 3, it is with the correlation of Roche NT-proBNP electrochemistry kits：Y=1.004x-8.381, 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 NT-proBNP detection kits, method of preparation and use based on bimolecular fluorescence complementary technology, feature exist
In：
1) kit mainly forms：The fluorescin N-terminal segment of anti-NT-proBNP antibody couplings and anti-NT-proBNP antibody are even
The fluorescin C-terminal segment of connection；
2) application method：The fluorescin N-terminal piece of sample, anti-NT-proBNP antibody couplings is added in the reacting hole of kit
The fluorescin C-terminal segment of section and anti-NT-proBNP 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.
A kind of 2. preparation method of the NT-proBNP detection kits based on bimolecular fluorescence complementary technology, which is characterized in that packet
Include following steps：
1) preparation of anti-NT-proBNP antibody couplings fluorescin N-terminal segment；
2) preparation of anti-NT-proBNP antibody couplings fluorescin C-terminal segment.
3. anti-NT-proBNP antibody according to claim 1 be for NT-proBNP different epitopes monoclonal antibody or
Polyclonal antibody.
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. prepared by a kind of NT-proBNP detection kits based on bimolecular fluorescence complementary technology according to claim 2
Method, which is characterized in that in the anti-NT-proBNP antibody couplings fluorescin N-terminal segment step, fluorescin N-terminal
The mass ratio of segment and NT-proBNP antibody is 1: 1-10.
6. side prepared by the NT-proBNP detection kits according to claim 2 based on bimolecular fluorescence complementary technology
Method, which is characterized in that in the anti-NT-proBNP antibody couplings fluorescin C-terminal segment step, fluorescin C-terminal segment
Mass ratio with NT-proBNP antibody is 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110208347A (en) * | 2019-05-10 | 2019-09-06 | 上海大学 | A kind of brain natriuretic peptide colorimetric/electrochemical sensing detection method visited based on nano enzyme |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101819205A (en) * | 2010-06-03 | 2010-09-01 | 中国人民解放军第三军医大学 | Human N-terminal pro-B-type natriuretic peptide (NT-proBNP) immunoassay kit and preparation method thereof |
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2017
- 2017-11-27 CN CN201711272731.1A patent/CN108226529A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101819205A (en) * | 2010-06-03 | 2010-09-01 | 中国人民解放军第三军医大学 | Human N-terminal pro-B-type natriuretic peptide (NT-proBNP) immunoassay kit and preparation method thereof |
Non-Patent Citations (2)
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| 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》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110208347A (en) * | 2019-05-10 | 2019-09-06 | 上海大学 | A kind of brain natriuretic peptide colorimetric/electrochemical sensing detection method visited based on nano enzyme |
| CN110208347B (en) * | 2019-05-10 | 2021-09-03 | 上海大学 | Method for preparing nano enzyme probe |
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Application publication date: 20180629 |