CN108226526A - A kind of SAA detection kits, method of preparation and use based on bimolecular fluorescence complementary technology - Google Patents
A kind of SAA detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Download PDFInfo
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- CN108226526A CN108226526A CN201711272610.7A CN201711272610A CN108226526A CN 108226526 A CN108226526 A CN 108226526A CN 201711272610 A CN201711272610 A CN 201711272610A CN 108226526 A CN108226526 A CN 108226526A
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Abstract
The present invention provides a kind of SAA diagnostic kits based on bimolecular fluorescence complementary technology.The kit includes：The fluorescin N-terminal segment of anti-SAA antibody couplings, the fluorescin C-terminal segment of anti-SAA antibody couplings；The invention also discloses a kind of preparation method of the SAA diagnostic kits based on bimolecular fluorescence complementary technology, this method includes：The preparation of the fluorescin N-terminal segment of anti-SAA antibody couplings, the preparation of the fluorescin C-terminal segment of anti-SAA 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, it is applied to the monitoring of clinical virus and bacterium infection, can improve virus and the accuracy rate of bacterium infection monitoring, have great market value.
Description
Technical field
The content of SAA 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
Serum amyloid A protein (serum amyloid A, SAA) is a kind of acute time limit reactive protein, belongs to and carries fat egg
Heterogeneous proteinoid in white family, relative molecular weight about 12 000.In the acute time limit reacts, pierced through IL-1, IL-6 and TNF
Swash, SAA is synthesized in liver by the macrophage and fibroblast that are activated, can be increased to the 100-1000 of initial concentration
Times, but half-life period is extremely short, and only 50 minutes or so.SA A are related with high-density lipoprotein (HDL), it can be adjusted during inflammation
The metabolism of high-density lipoprotein.Mono- especially important characteristic of SAA is that its catabolite can be with amyloid A (AA) fibrinogen
Mode be deposited in different organs, this is a kind of serious complication in chronic inflammatory diseases.
Similar with c reactive protein (CRP), the content concn of SAA is the sensitive indicator for reflecting infectious diseases Earlier period of inflammation,
Help to diagnose inflammation, assess its activity, monitor its activity and treatment.But in diagnosis virus infection, kidney occur for SAA detections
The patient (patient for particularly carrying out immunosuppressive therapy) of graft-rejection and the capsule with adrenocortical hormones in treating
Property fibrosis patients in terms of, it is more conclusive than CRP.The study found that in the case for suffering from inflammatory arthritis, SAA and Disease Activity
Relationship it is most close.Diagnostic sensitivity to infection can be improved by detecting CRP and SAA simultaneously.For SAA amyloidosis patients,
SAA levels are returned back to normally as the treatment of objective, the state of an illness can be improved.
At present, the method for detecting SAA mainly has enzyme-linked immunosorbent assay, immunochromatographic method and chemiluminescence immune assay
Method.Enzyme-linked immunosorbent assay uses horseradish peroxidase or alkali phosphatase enzyme mark antibody, and is catalyzed substrate and generates face
Color change has the characteristics of easy to operate, the stable reagent phase is long, but the detection sensitivity of enzyme-linked immunosorbent assay is relatively low,
It is mainly used in communicable disease screening etc. at present and requires detection sensitivity relatively low project；Immunochromatographic method has operation letter
The advantages that list, detection speed is fast, but it is low there is also sensitivity, and reagent is unstable, less reproducible, it is difficult to carry out quantitative lack
Point.Chemiluminescence immunoassay has many advantages, such as easy to operate, and detection speed is fast, high-throughput detection, but there is also heterogeneous
Reaction, detection time is long, criticizes the shortcomings that interior batch variation is big.
To solve the above problems, if the antibody of SAA, using bimolecular fluorescence complementary technology as platform, research and development one can be utilized
Kind is for thrombotic diseases diagnosis and the SAA quick detection reagents of thromboembolism treatment effect monitoring.Make itself and existing detection reagent phase
Than having many advantages, such as that easy to operate, high sensitivity, free of cleaning, precision is high；It is applied to clinical virus and bacterium infection
Monitoring can improve virus and the accuracy rate of bacterium infection monitoring, then can be will be widely welcomed by market and have great city
Field 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 SAA
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 SAA detection kits, include the following steps：
1) anti-SAA antibody couplings fluorescin N-terminal segment；
2) anti-SAA antibody couplings fluorescin C-terminal segment；
In the above-mentioned technical solutions, the anti-SAA antibody is the monoclonal antibody or Anti-TNF-α for SAA different epitopes
Body.
In said program, the step of the anti-SAA antibody couplings fluorescin N-terminal segment in, fluorescin N-terminal segment
Mass ratio with anti-SAA antibody is 1: 1-10.
In said program, the step of the anti-SAA antibody couplings fluorescin C-terminal segment in, fluorescin C-terminal segment
Mass ratio with anti-SAA antibody is 1: 1-10.
The prepared SAA 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-SAA antibody couplings；
2) the fluorescin C-terminal segment of anti-SAA 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-SAA antibody couplings and anti-SAA 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 SAA detection kits principle signal provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Figure；Reference sign：The anti-SAA antibody of 1-, 2- bridging agents, 3- fluorescin N-terminal segments, 4- determinands (SAA), the anti-SAA of 5-
Antibody, 6- fluorescin C-terminal segments, 7- bridging agents.
Fig. 2 is that the SAA 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 SAA 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 SAA detection kits based on bimolecular fluorescence complementary technology, prepare and its
Application method is described in detail.
Embodiment 1
Anti- SAA 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/LpH9.5 CB and removes excessive glutaraldehyde.
5) the anti-SAA 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 SephadexG-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- SAA 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-SAA 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 SephadexG-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 3
Kit mainly forms：
1) the fluorescin N-terminal segment of anti-SAA antibody couplings；
2) the fluorescin C-terminal segment of anti-SAA antibody couplings.
Embodiment 4
Kit application method, includes the following steps：
1) sample, the fluorescin N-terminal segment of anti-SAA antibody couplings and anti-SAA 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 0 μ g/mL, 10 μ g/mL, 30 μ g/mL, 80 μ g/mL, 150 μ g/mL SAA standard solutions.Anti-
Ying Kongzhong is separately added into 20 μ L standard items, the fluorescin N-terminal segment for adding in the anti-SAA antibody couplings of 50 μ L, adds in 50 μ L and resists
The fluorescin C-terminal segment of SAA antibody couplings, 37 DEG C incubate 10 minutes.After incubation, exciting light irradiation reacting hole measures each
Reacting hole luminous quantity obtains fluorescence signal value.
Using fluorescence signal value as ordinate, standard concentration is abscissa, draws standard working curve (see Fig. 2).
2. accuracy
Recovery test：Be added in normal human serum sample with known quantity SAA standard items, measure add in after concentration value with
The theoretical value of addition is compared, and calculates the rate of recovery of SAA.Testing result is as follows：
| Sample number | Add in SAA concentration (μ g/mL) | Measure the concentration (μ g/mL) of SAA | The rate of recovery (%) |
| 1 | 5 | 5.2 | 104 |
| 2 | 20 | 21.2 | 106 |
| 3 | 100 | 105.6 | 105.6 |
| 4 | 140 | 138.7 | 99.1 |
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 0.5 μ g/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 SAA 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 SAA 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 SAA positive serum samples in right amount, the content for making serum mesobilirubin is respectively 25
μg/mL、50μg/mL.The SAA 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 97.0%-101.9%.Show mutual based on bimolecular fluorescence
The SAA reagents of benefit technology are not interfered when detecting serum sample by hemoglobin, triglycerides, bilirubin.
6. correlation
As shown in figure 3, the correlation with Siemens SAA kits is：Y=0.985x-0.258, 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 SAA 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-SAA antibody couplings and anti-SAA antibody couplings
End fragment；
2) application method：Sample, the fluorescin N-terminal segment of anti-SAA antibody couplings and anti-are added in the reacting hole of kit
The fluorescin C-terminal segment of SAA 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 SAA detection kits based on bimolecular fluorescence complementary technology, which is characterized in that including as follows
Step：
1) preparation of anti-SAA antibody couplings fluorescin N-terminal segment；
2) preparation of anti-SAA antibody couplings fluorescin C-terminal segment.
3. anti-SAA antibody according to claim 1 is the monoclonal antibody or polyclonal antibody for SAA 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 SAA detection kits based on bimolecular fluorescence complementary technology according to claim 2,
It is characterized in that, in the anti-SAA antibody couplings fluorescin N-terminal segment step, fluorescin N-terminal segment and SAA antibody
Mass ratio be 1: 1-10.
6. method prepared by the SAA detection kits according to claim 2 based on bimolecular fluorescence complementary technology, special
Sign is, in the anti-SAA antibody couplings fluorescin C-terminal segment step, fluorescin C-terminal segment and the matter of SAA 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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| CN201711272610.7A CN108226526A (en) | 2017-11-27 | 2017-11-27 | A kind of SAA detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
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| CN201711272610.7A CN108226526A (en) | 2017-11-27 | 2017-11-27 | A kind of SAA 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 |
|---|---|---|---|---|
| CN103076455A (en) * | 2012-12-26 | 2013-05-01 | 上海奥普生物医药有限公司 | Kit for quickly and quantificationally detecting serum amyloid A, and preparation and application thereof |
-
2017
- 2017-11-27 CN CN201711272610.7A patent/CN108226526A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103076455A (en) * | 2012-12-26 | 2013-05-01 | 上海奥普生物医药有限公司 | Kit for quickly and quantificationally detecting serum amyloid A, and preparation and application thereof |
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 |