CN108267595A - A kind of Myo detection kits, method of preparation and use based on bimolecular fluorescence complementary technology - Google Patents
A kind of Myo detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Download PDFInfo
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- CN108267595A CN108267595A CN201711271893.3A CN201711271893A CN108267595A CN 108267595 A CN108267595 A CN 108267595A CN 201711271893 A CN201711271893 A CN 201711271893A CN 108267595 A CN108267595 A CN 108267595A
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- myo
- fluorescin
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The present invention provides a kind of Myo detection kits, method of preparation and use based on bimolecular fluorescence complementary technology.The kit includes：The fluorescin N-terminal segment of anti-Myo antibody couplings, the fluorescin C-terminal segment of anti-Myo antibody couplings；The invention also discloses a kind of preparation method of the Myo diagnostic kits based on bimolecular fluorescence complementary technology, this method includes：The preparation of the fluorescin N-terminal segment of anti-Myo antibody couplings, the preparation of the fluorescin C-terminal segment of anti-Myo 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 acute myocardial infarction AMI, can improve the accuracy rate of Diagnosis of Acute Myocardial Infarction, has great market value.
Description
Technical field
The content of Myo 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
Acute myocardial infarction AMI (Acute Myocardial Infarction, AMI) be in the world morbidity and mortality compared with
One of high disease.It is shown according to nearest American Heart Association's data, there are 7,200,000 AMI patients in the U.S. within 2003.But AMI simultaneously
It is one of disease of high misdiagnosis rate again, some patients for suffering from AMI really do not obtain appropriate processing, lead to higher death
Rate；And other non-AMI patients receive unnecessary treatment, cause unnecessary cost.World health organisation recommendations：Allusion quotation
There are two to meet in abnormal three indexs of pectoralgia, ECG change and myocardium enzyme of type, you can diagnosis AMI.But many AMI patients
Early stage is without typical clinical symptoms and the abnormal change of electrocardiogram, therefore the detection of myocardial injury markers just seems particularly heavy
It will.Application of the Applications of Cardiac Markers in clinic in recent years has obtained more and more extensive attention, and development is also quite rapid, is facing
There are many Applications of Cardiac Markers in bed.
Myoglobins (Myoglobin, Myo) is a kind of oxygen combination hemoprotein, is distributed mainly on cardiac muscle and skeletal muscle
Tissue.Nineteen sixty is illustrated by Kendrew with X-ray diffraction method, this is first tertiary protein structure being described in the world.
Myo is made of a globin polypeptide chain and a prosthetic heme group, reversible can be combined with oxygen, there is the energy of storage transport oxygen
Power, Myo contents are considerably less in normal human serum, quick release during AMI, see raising after paresthesia epilepsy in 1h blood, in 2~4h
Up to 10 times or so of normal upper limit, 5~10h reaches peak, and about 30h returns to baseline, and compared with CK-MB, time of occurrence earlier, and restores
It is then relatively slow, there is important value in early diagnosis of acute myocardial infarction.Research shows that Myo is to the AMI sensibility diagnosed and spy
The opposite sex is respectively 100%, 86.7%, Myo feminine gender desired value highests (100%), although Myo Cardiac-specifics are not high, due to
Sensibility is high, and Myo feminine genders are particularly helpful to exclude the diagnosis of AMI.Due to half-life short in the blood of Myo, so contribute to see again
Examine whether there is in the AMI courses of disease again infarct occur and infarct whether there is expansion.
Common Myo detection methods have gold mark qualitative test, fluorescent immune method, linked immunosorbent adsorption test (ELISA) and magnetic
Particulate chemistry luminescence method (CMIA).Gold mark qualitative experiment is quick, but this method sensitivity it is low, can not dynamic monitoring Myo contents
Variation.Fluorescent immune method is complicated for operation, there is higher requirement to operating environment and operating personnel.The detection sensitivity of ELISA method
It is relatively low, be mainly used in communicable disease screening etc. at present and require detection sensitivity relatively low project, and the reaction time compared with
It is long.CMIA methods are improved from ELISA method, have the features such as easy to operate, detection speed is fast compared with ELISA method, still
CMIA methods are heterogeneous reactions, and operating process needs to clean, and it reduce the repeatabilities of detection.
To solve the above problems, if the antibody of Myo can be utilized, using bimolecular fluorescence complementary technology as platform, develop
A kind of Myo quick detection reagents for myocardial infarction medical diagnosis on disease.Make it compared with existing detection reagent, there is operation side
Just, high sensitivity, it is free of cleaning, precision is high the advantages that；It is applied to the monitoring of acute myocardial infarction AMI, the acute heart can be improved
The accuracy rate of flesh infarct diagnosis, then can 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 detection examination that can be used for quantitatively detection Myo
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 Myo detection kits, include the following steps：
1) anti-Myo antibody couplings fluorescin N-terminal segment；
2) anti-Myo antibody couplings fluorescin C-terminal segment；
In the above-mentioned technical solutions, the anti-Myo antibody is the monoclonal antibody or Anti-TNF-α for Myo different epitopes
Body.
In said program, the step of the anti-Myo antibody couplings fluorescin N-terminal segment in, fluorescin N-terminal segment
Mass ratio with anti-Myo antibody is 1: 1-10.
In said program, the step of the anti-Myo antibody couplings fluorescin C-terminal segment in, fluorescin C-terminal segment
Mass ratio with anti-Myo antibody is 1: 1-10.
The prepared Myo 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-Myo antibody couplings；
2) the fluorescin C-terminal segment of anti-Myo 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-Myo antibody couplings and anti-Myo 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 Myo detection kits principle signal provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Figure, wherein：The anti-Myo antibody of 1-, 2- bridging agents, 3- fluorescin N-terminal segments, 4- determinands (Myo), the anti-Myo antibody of 5-, 6-
Fluorescin C-terminal segment, 7- bridging agents.
Fig. 2 is that the Myo 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 Myo 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 Myo detection kits based on bimolecular fluorescence complementary technology, prepare and its
Application method is described in detail.
Embodiment 1
Anti- Myo 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-Myo 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
0.01mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 2
Anti- Myo 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-Myo 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-Myo antibody couplings；
2) the fluorescin C-terminal segment of anti-Myo antibody couplings.
Embodiment 4
Kit application method, includes the following steps：
1) sample, the fluorescin N-terminal segment of anti-Myo antibody couplings and anti-Myo 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 0ng/ml, 20ng/ml, 50ng/ml, 100ng/ml, 500ng/ml, 1000ng/ml, 3000ng/
The Myo standard solutions of ml.20 μ l standard items, the fluorescence egg for adding in the anti-Myo antibody couplings of 50 μ l are separately added into reacting hole
White N-terminal segment adds in the fluorescin C-terminal segment of the anti-Myo antibody couplings of 50 μ l, and 37 DEG C incubate 10 minutes.After incubation, exciting light
Reacting hole is irradiated, each reacting hole luminous quantity is measured 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 with known quantity Myo standard items, measures concentration value after adding in
It is compared with the theoretical value of addition, calculates the rate of recovery of Myo.Testing result is as follows：
| Sample number | Add in Myo concentration (ng/ml) | Measure the concentration (ng/ml) of Myo | The rate of recovery (%) |
| 1 | 150 | 147.5 | 98.3 |
| 2 | 500 | 524.1 | 104.8 |
| 3 | 1000 | 1026.3 | 102.6 |
| 4 | 2000 | 1987.7 | 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 3ng/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 Myo in right amount
In positive serum sample, the content for making hemoglobin in serum is respectively 0.5mg/ml, 1.0mg/ml.By triglycerides solution point
It does not take and is added in Myo positive serum samples in right amount, the content for making Triglycerides in Serum is respectively 0.5mg/ml, 1.0mg/
ml.Bilirubin solution is taken respectively and is added in Myo positive serum samples in right amount, the content for making serum mesobilirubin is respectively 25
μg/ml、50μg/ml.The Myo positive samples for adding hemoglobin, triglycerides and bilirubin are measured.It will be theoretical dense
Degree and the ratio of measured concentration are as the rate of recovery, and the rate of recovery is between 96.3%-101.9%.Show mutual based on bimolecular fluorescence
The Myo reagents of benefit technology are not interfered when detecting serum sample by hemoglobin, triglycerides, bilirubin.
6. correlation
As shown in figure 3, it is with the correlation of Roche Myo electrochemistry kits：Y=1.001x+0.436, R2=0.997.
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 Myo 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-Myo antibody couplings and anti-Myo antibody couplings
End fragment；
2) application method：Sample, the fluorescin N-terminal segment of anti-Myo antibody couplings and anti-are added in the reacting hole of kit
The fluorescin C-terminal segment of Myo 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 Myo detection kits based on bimolecular fluorescence complementary technology, which is characterized in that including as follows
Step：
1) preparation of anti-Myo antibody couplings fluorescin N-terminal segment；
2) preparation of anti-Myo antibody couplings fluorescin C-terminal segment.
3. anti-Myo antibody according to claim 1 is the monoclonal antibody or polyclonal antibody for Myo 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 Myo detection kits based on bimolecular fluorescence complementary technology according to claim 2,
It is characterized in that, in the anti-Myo antibody couplings fluorescin N-terminal segment step, fluorescin N-terminal segment and Myo antibody
Mass ratio be 1: 1-10.
6. method prepared by the Myo detection kits according to claim 2 based on bimolecular fluorescence complementary technology, special
Sign is, in the anti-Myo antibody couplings fluorescin C-terminal segment step, fluorescin C-terminal segment and the matter of Myo 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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| CN201711271893.3A CN108267595A (en) | 2017-11-27 | 2017-11-27 | A kind of Myo detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070254373A1 (en) * | 1998-02-02 | 2007-11-01 | Odyssey Pharmaceuticals, Inc. | Fragments of fluorescent proteins for protein fragment complementation assays |
| CN101365805A (en) * | 2005-10-27 | 2009-02-11 | 波士顿大学董事会 | Real time nucleic acid detection in vivo using protein complementation |
| WO2009111073A2 (en) * | 2008-03-06 | 2009-09-11 | Odyssey Thera, Inc. | High-content and high throughput assays for identification of lipid-regulating pathways, and novel therapeutic agents for lipid disorders |
| CN103033627A (en) * | 2011-09-29 | 2013-04-10 | 北京源德生物医学工程有限公司 | Myoglobin enzymatic chemiluminescence immunodetection method and kit |
-
2017
- 2017-11-27 CN CN201711271893.3A patent/CN108267595A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070254373A1 (en) * | 1998-02-02 | 2007-11-01 | Odyssey Pharmaceuticals, Inc. | Fragments of fluorescent proteins for protein fragment complementation assays |
| CN101365805A (en) * | 2005-10-27 | 2009-02-11 | 波士顿大学董事会 | Real time nucleic acid detection in vivo using protein complementation |
| WO2009111073A2 (en) * | 2008-03-06 | 2009-09-11 | Odyssey Thera, Inc. | High-content and high throughput assays for identification of lipid-regulating pathways, and novel therapeutic agents for lipid disorders |
| CN103033627A (en) * | 2011-09-29 | 2013-04-10 | 北京源德生物医学工程有限公司 | Myoglobin enzymatic chemiluminescence immunodetection method and 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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