CN108318460A - A kind of Lp-PLA2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology - Google Patents
A kind of Lp-PLA2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Download PDFInfo
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- CN108318460A CN108318460A CN201711271894.8A CN201711271894A CN108318460A CN 108318460 A CN108318460 A CN 108318460A CN 201711271894 A CN201711271894 A CN 201711271894A CN 108318460 A CN108318460 A CN 108318460A
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01N2333/914—Hydrolases (3)
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Abstract
The present invention provides a kind of Lp PLA2 detection kits, methods of preparation and use based on bimolecular fluorescence complementary technology.The kit includes：The fluorescin N-terminal segment of anti-Lp PLA2 antibody couplings, the fluorescin C-terminal segment of anti-Lp PLA2 antibody couplings；The invention also discloses a kind of preparation method of the Lp PLA2 diagnostic kits based on bimolecular fluorescence complementary technology, this method includes：The preparation of the fluorescin N-terminal segment of anti-Lp PLA2 antibody couplings, the preparation of the fluorescin C-terminal segment of anti-Lp PLA2 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, the range of linearity is wide, specificity is good, free of cleaning, accuracy is high, it is used convenient for clinical detection, the generation of predictable atherosclerosis has considerable meaning to preventing coronary heart disease accident, has great market value.
Description
Technical field
The present invention relates to a kind of bimolecular fluorescence complementary technologies to contain for Lp-PLA2 in ion vitro immunization diagnosis detection human body
Amount, belongs to medical diagnosis on disease detection field.
Background technology
Human lipoprotein associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), point
Son amount is 45.4kD.Lp-PLA2 is mainly synthesized and is secreted by ripe macrophage and lymphocyte, and is adjusted by inflammatory mediator
Section, the Lp-PLA2 during people recycles exists in the form of being combined with hdl particle, wherein 2/3 is combined with LDL, 1/3 and HDL,
VLDL is combined.
Lp-PLA2 is to cause extensive concern and the closely related new Inflammation Marker of atherosclerosis (AS) in recent years.
The detection of Lp-PLA2 can directly and accurately reflect the degree of intravascular inflammation, and can be used as a dynamic indicator.Lp-PLA2
It is independent cardiovascular and cerebrovascular disease risk profile index, is not influenced by other biological index, is also inflammation, atherosclerosis
Contacting between cardiovascular and cerebrovascular disease provides new evidence.Lp-PLA2 levels are in notable linear phase with cardiovascular and cerebrovascular disease
It closes, by detecting the Lp-PLA2 in blood, can effectively understand the degree of inflammation and its stabilization of atherosclerotic plaque
Property.
The method of detection Lp-PLA2 mainly has enzyme-linked immunosorbent assay, immunochromatographic method and chemiluminescence immunoassay at present
Analytic approach etc., has that detection sensitivity is relatively low, reagent is unstable, less reproducible, it is difficult to carry out quantitative disadvantage.
To solve the above problems, using bimolecular fluorescence complementary technology, with the antibody construction technology platform of Lp-PLA2, grind
Send out a kind of easy, intuitive, sensitive Lp-PLA2 quick detection reagents.It is pre- to be applied to atherosclerosis disease risk
It surveys, the pathogenetic accuracy rate of early warning coronary disease can be improved, there is great market value.
Invention content
For the technical problems in the prior art, the present invention provides a kind of inspection can be used for quantitative detection Lp-PLA2
Test agent box, its method of preparation and use.
The invention is realized by the following technical scheme：
A method of it prepares based on bimolecular fluorescence complementary technology Lp-PLA2 detection kits, includes the following steps：
1) anti-Lp-PLA2 antibody couplings fluorescin N-terminal segment；
2) anti-Lp-PLA2 antibody couplings fluorescin C-terminal segment；
In the above-mentioned technical solutions, the anti-Lp-PLA2 antibody be for Lp-PLA2 different epitopes monoclonal antibody or
Polyclonal antibody.
In the above scheme, in the step of anti-Lp-PLA2 antibody couplings fluorescin N-terminal segment, fluorescin N-terminal
The mass ratio of segment and anti-Lp-PLA2 antibody is 1: 1-10.
In the above scheme, in the step of anti-Lp-PLA2 antibody couplings fluorescin C-terminal segment, fluorescin C-terminal
The mass ratio of segment and anti-Lp-PLA2 antibody is 1: 1-10.
Examination is detected according to the Lp-PLA2 based on bimolecular fluorescence complementary technology prepared described in any of the above technical solution
Agent box.It is mainly formed：
1) the fluorescin N-terminal segment of anti-Lp-PLA2 antibody couplings；
2) the fluorescin C-terminal segment of anti-Lp-PLA2 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-Lp-PLA2 antibody couplings and anti-are added in the reacting hole of kit
The fluorescin C-terminal segment of Lp-PLA2 antibody couplings, hybrid reaction 5-60min；
2) exciting light irradiates 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 Lp-PLA2 detection kit principles provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Schematic diagram, wherein the anti-Lp-PLA2 antibody of 1-, 2- bridging agents, 3- fluorescin N-terminal segments, 4- determinands (Lp-PLA2), 5- are anti-
Lp-PLA2 antibody, 6- fluorescin C-terminal segments, 7- bridging agents.
Fig. 2 is the Lp-PLA2 detection kits detection provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Range of linearity figure.
Fig. 3 is the Lp-PLA2 detection kit results provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Correlation compares.
Specific implementation mode
The Lp-PLA2 detection kits based on bimolecular fluorescence complementary technology, preparation below with reference to attached drawing to the present invention
And its application method is described in detail.
Embodiment 1
Anti- Lp-PLA2 antibody couplings fluorescin N-terminal segment, with the segment of yellow fluorescence protein (YFP) 1-154 amino acid
For 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) desalting column Sephadex G-25 or 0.05mol/LpH9.5CB dialysis is used to remove excessive glutaraldehyde.
5) the anti-Lp-PLA2 antibody of 0.1mg is taken, 1mg/ml antibody is prepared with 0.05mol/L pH9.5CB, 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, room temperature closes 2h, to close remaining aldehyde radical, terminates anti-
It answers.
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 pH7.2PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 2
Anti- Lp-PLA2 antibody couplings fluorescin C-terminal segment, with the piece of yellow fluorescence protein (YFP) 155-238 amino acid
For section YFPC, specific implementation process is：
1) 0.1mgYFPC albumen is added in centrifuge tube, is prepared with 0.05mol/LpH9.5 carbonate buffer solutions (CB)
YFPC albumen is diluted to 1mg/mL.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) desalting column Sephadex G-25 or 0.05mol/LpH9.5CB dialysis is used to remove excessive glutaraldehyde.
5) the anti-Lp-PLA2 antibody of 0.1mg is taken, 1mg/mL antibody is prepared with 0.05mol/L pH9.5CB, by activation
YFPC albumen and antibody mixing.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ L0.2mol/L lysine solutions are added, room temperature closes 2h, to close remaining aldehyde radical, terminates anti-
It answers.
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 pH7.2PBS dialysis purification conjugates, are diluted to required concentration before use.
Case study on implementation 3
Kit mainly forms：
1) the fluorescin N-terminal segment of anti-Lp-PLA2 antibody couplings；
2) the fluorescin C-terminal segment of anti-Lp-PLA2 antibody couplings.
Embodiment 4
Kit application method, includes the following steps：
1) the fluorescin N-terminal piece Lp-PLA2 of sample, anti-Lp-PLA2 antibody couplings is added in the reacting hole of kit
The fluorescin C-terminal segment of antibody coupling, hybrid reaction 5-60min；
2) exciting light irradiates 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：
1. linear
Compound concentration is the Lp- of 0ng/ml, 25ng/ml, 100ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml
PLA2 standard solutions.The fluorescence for being separately added into 20 μ l standard items in reacting hole, the anti-Lp-PLA2 antibody couplings of 50 μ l being added
The fluorescin C-terminal segment of the anti-Lp-PLA2 antibody couplings of 50 μ l, 37 DEG C of incubation 10min are added in albumen n end segment.After incubation,
Exciting light irradiates 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 dense after being added with known quantity Lp-PLA2 standard items
Angle value is compared with the theoretical value of addition, calculates the rate of recovery of Lp-PLA2.Testing result is as follows：
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：It 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 2ng/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 Lp- in right amount
In PLA2 positive serum samples, it is respectively 0.5mg/ml, 1.0mg/ml to make the content of hemoglobin in serum.Triglycerides is molten
Liquid takes respectively to be added in Lp-PLA2 positive serum samples in right amount, make the content of Triglycerides in Serum be respectively 0.5mg/ml,
1.0 mg/ml.Bilirubin solution is taken respectively and is added in Lp-PLA2 positive serum samples in right amount, serum mesobilirubin is made
Content is respectively 25 μ g/ml, 50 μ g/ml.To adding the Lp-PLA2 positive samples of hemoglobin, triglycerides and bilirubin
It is measured.Using the ratio of theoretical concentration and measured concentration as the rate of recovery, the rate of recovery is between 95.2%-103.6%.Show
Lp-PLA2 reagents based on bimolecular fluorescence complementary technology are red not by hemoglobin, triglycerides, courage when detecting serum sample
The interference of element.
6. correlation
As shown in figure 3, the correlation of the Lp-PLA2 kit high with Weihai prestige is：Y=0.990x+6.993, 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 low advantage of instrument requirements.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art within the technical scope disclosed by the invention, the change or replacement that can be readily occurred in, all
It is covered by the protection scope of the present invention.
Claims (7)
1. a kind of Lp-PLA2 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-Lp-PLA2 antibody couplings and anti-Lp-PLA2 antibody couplings
Fluorescin C-terminal segment；
2) application method：The fluorescin N-terminal segment of sample, anti-Lp-PLA2 antibody couplings is added in the reacting hole of kit
With the fluorescin C-terminal segment of Lp-PLA2 body antibody couplings, hybrid reaction 5-60min；
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 Lp-PLA2 detection kits based on bimolecular fluorescence complementary technology, which is characterized in that including
Following steps：
1) preparation of anti-Lp-PLA2 antibody couplings fluorescin N-terminal segment；
2) preparation of anti-Lp-PLA2 antibody couplings fluorescin C-terminal segment.
3. anti-Lp-PLA2 antibody according to claim 1 is for the monoclonal antibody of Lp-PLA2 different epitopes or more grams
Grand 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 Lp-PLA2 detection kits based on bimolecular fluorescence complementary technology according to claim 2
Method, which is characterized in that in the anti-Lp-PLA2 antibody couplings fluorescin N-terminal segment step, fluorescin N-terminal segment
Mass ratio with Lp-PLA2 antibody is 1: 1-10.
6. method prepared by the Lp-PLA2 detection kits according to claim 2 based on bimolecular fluorescence complementary technology,
It is characterized in that, in the anti-Lp-PLA2 antibody couplings fluorescin C-terminal segment step, fluorescin C-terminal segment and Lp-
The mass ratio of PLA2 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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| Application Number | Priority Date | Filing Date | Title |
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| CN201711271894.8A CN108318460A (en) | 2017-11-27 | 2017-11-27 | A kind of Lp-PLA2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711271894.8A CN108318460A (en) | 2017-11-27 | 2017-11-27 | A kind of Lp-PLA2 detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105542010A (en) * | 2015-12-31 | 2016-05-04 | 武汉华美生物工程有限公司 | Lipoprotein-associated phospholipase A2 monoclonal antibodies, antibody pairs, preparation method and application |
-
2017
- 2017-11-27 CN CN201711271894.8A patent/CN108318460A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105542010A (en) * | 2015-12-31 | 2016-05-04 | 武汉华美生物工程有限公司 | Lipoprotein-associated phospholipase A2 monoclonal antibodies, antibody pairs, preparation method and application |
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: 20180724 |