CN108226520A - A kind of CK-MB detection kits, method of preparation and use based on bimolecular fluorescence complementary technology - Google Patents
A kind of CK-MB detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Download PDFInfo
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- CN108226520A CN108226520A CN201711271924.5A CN201711271924A CN108226520A CN 108226520 A CN108226520 A CN 108226520A CN 201711271924 A CN201711271924 A CN 201711271924A CN 108226520 A CN108226520 A CN 108226520A
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Abstract
The present invention provides a kind of CK MB diagnostic kits based on bimolecular fluorescence complementary technology.The kit includes：The fluorescin N-terminal segment of anti-CK MB antibody couplings, the fluorescin C-terminal segment of anti-CK MB antibody couplings；The invention also discloses a kind of preparation method of the CK MB diagnostic kits based on bimolecular fluorescence complementary technology, this method includes：The preparation of the fluorescin N-terminal segment of anti-CK MB antibody couplings, the preparation of the fluorescin C-terminal segment of anti-CK MB 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, can early warning myocardial infarction and cerebral thrombus generation, there is considerable meaning to prevention cardiovascular and cerebrovascular accident, there is great market value.
Description
Technical field
Contain the present invention relates to a kind of bimolecular fluorescence complementary technology for CK-MB in ion vitro immunization diagnosis detection human body
Amount, belongs to medical diagnosis on disease detection field.
Background technology
Creatine kinase (CK) is one and is transported with intracellular energy that contraction of muscle, the regeneration of ATP has the important of direct relation
Kinases is a dimer, with the presence of the form of 3 kinds of isodynamic enzymes：2 B monomers (CK-BB), 2 M monomers (CK-MM), MB's
Monomer mixture (CK-MB).CK-MB CK-MB in cardiac muscle, cardiac muscle account for the 15%~25% of CK total amounts.So in cardiac muscle
Damage, during particularly acute myocardial infarction AMI (AMI), measure CK-MB has great clinical meaning to the diagnosis of AMI.CK-MB is
The index that AMI clinical diagnosises play an important role, can be as the Index for diagnosis of unstable angina pectoris (UA), clinic doctor
Life can quantify testing result according to Serum CK-MB to judge the prognosis situation of UA patient, facilitate and select best therapeutic scheme, with
Phase reaches best therapeutic effect.It, can in order to will appreciate that the process of disease condition for ischemia myocardial damage, particularly AMI
Risk stratification is carried out to the disease using CK-MB mass raised degree, clinician to be facilitated to be established using the risk stratification
Rational therapeutic scheme mitigates patient suffering and financial burden.
The measure of conventional CK-MB is mainly using immunodepression and enzyme linked immunosorbent assay, immunodepression
It detects rapid, time saving, has higher sensibility, but influence factor and defect are numerous.Enzyme linked immunosorbent assay has operation letter
The characteristics of list, the stable reagent phase is long, but detection sensitivity is relatively low.
To solve the above problems, using bimolecular fluorescence complementary technology, with the antibody construction technology platform of CK-MB, research and development
Go out a kind of easy, intuitive, sensitive CK-MB quick detection reagents.It is applied to the monitoring of acute myocardial infarction AMI, inspection can be improved
The accuracy rate of survey has great market value.
Invention content
For the technical problems in the prior art, the present invention provides a kind of detection that can be used for quantitatively detecting CK-MB
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 CK-MB detection kits, include the following steps：
1) anti-CK-MB antibody couplings fluorescin N-terminal segment；
2) anti-CK-MB antibody couplings fluorescin C-terminal segment；
In the above-mentioned technical solutions, the anti-CK-MB antibody is for the monoclonal antibody of CK-MB different epitopes or more grams
Grand antibody.
In said program, the step of the anti-CK-MB antibody couplings fluorescin N-terminal segment in, fluorescin N-terminal piece
The mass ratio of Duan Yukang CK-MB antibody is 1: 1-10.
In said program, the step of the anti-CK-MB antibody couplings fluorescin C-terminal segment in, fluorescin C-terminal piece
The mass ratio of Duan Yukang CK-MB antibody is 1: 1-10.
The prepared CK-MB 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-CK-MB antibody couplings；
2) the fluorescin C-terminal segment of anti-CK-MB 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-CK-MB antibody couplings and anti-CK- are added in the reacting hole of kit
The fluorescin C-terminal segment of MB 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 shows for the CK-MB detection kit principles provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
It is intended to, wherein, the anti-CK-MB antibody of 1-, 2- bridging agents, 3- fluorescin N-terminal segments, 4- determinands (CK-MB), the anti-CK-MB of 5-
Antibody, 6- fluorescin C-terminal segments, 7- bridging agents.
Fig. 2 is the CK-MB detection kit detection lines provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Property areal map.
Fig. 3 is the CK-MB detection kit result phases provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology
Closing property compares.
Specific embodiment
Below with reference to attached drawing to the present invention the CK-MB detection kits based on bimolecular fluorescence complementary technology, prepare and
Its application method is described in detail.
Embodiment 1
Anti- CK-MB 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) it is dialysed with desalting column Sephadex G-25 or 0.05mol/L pH9.5 CB and removes excessive glutaraldehyde.
5) the anti-CK-MB antibody of 0.1mg is taken, 1mg/mL antibody is prepared with 0.05mol/L pH9.5 CB, by the YFPN of activation
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
0.01 mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 2
Anti- CK-MB 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-CK-MB antibody of 0.1mg is taken, 1mg/mL antibody is prepared with 0.05mol/L pH9.5 CB, by the YFPC of activation
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
0.01 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-CK-MB antibody couplings；
2) the fluorescin C-terminal segment of anti-CK-MB antibody couplings.
Embodiment 4
Kit application method, includes the following steps：
1) sample, the fluorescin N-terminal segment of anti-CK-MB antibody couplings, anti-CK-MB are added in the reacting hole of kit
The fluorescin C-terminal segment of antibody 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, 1ng/mL, 5ng/mL, 25ng/mL, 100ng/mL, 250ng/mL, 500ng/mL's
CK-MB standard solutions.20 μ L standard items, the fluorescence egg for adding in the anti-CK-MB 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-CK-MB antibody couplings of 50 μ L, 37 DEG C of incubation 10min.After incubation, excitation
Photo-irradiation reaction 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 with known quantity CK-MB standard items, measures concentration after adding in
Value is compared with the theoretical value added in, calculates the rate of recovery of CK-MB.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：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 0.2ng/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 CK- in right amount
In MB positive serum samples, the content for making hemoglobin in serum is respectively 0.5mg/mL, 1.0mg/mL.By triglycerides solution
It takes and is added in CK-MB positive serum samples in right amount respectively, the content for making Triglycerides in Serum is respectively 0.5mg/mL, 1.0
mg/mL.Bilirubin solution is taken respectively and is added in CK-MB positive serum samples in right amount, makes the content point of serum mesobilirubin
It Wei not 25 μ g/mL, 50 μ g/mL.The CK-MB positive samples for adding hemoglobin, triglycerides and bilirubin are measured.
Using the ratio of theoretical concentration and measured concentration as the rate of recovery, the rate of recovery is between 98.9%-101.1%.Show based on double points
The CK-MB reagents of sub- fluorescence complementary 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 CK-MB kits：Y=1.028x-0.518, R2=0.999.
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 CK-MB detection kits, method of preparation and use based on bimolecular fluorescence complementary technology, it is characterised in that：
1) kit mainly forms：The fluorescence of the fluorescin N-terminal segment of anti-CK-MB antibody couplings and anti-CK-MB antibody couplings
PROTEIN C end fragment；
2) application method：In the reacting hole of kit add in sample, anti-CK-MB antibody couplings fluorescin N-terminal segment and
The fluorescin C-terminal segment of CK-MB 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 CK-MB detection kits based on bimolecular fluorescence complementary technology, which is characterized in that including such as
Lower step：
1) preparation of anti-CK-MB antibody couplings fluorescin N-terminal segment；
2) preparation of anti-CK-MB antibody couplings fluorescin C-terminal segment.
3. anti-CK-MB antibody according to claim 1 is the monoclonal antibody or Anti-TNF-α for CK-MB different epitopes
Body.
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. side prepared by a kind of CK-MB detection kits based on bimolecular fluorescence complementary technology according to claim 2
Method, which is characterized in that in the anti-CK-MB antibody couplings fluorescin N-terminal segment step, fluorescin N-terminal segment and CK-
The mass ratio of MB antibody is 1: 1-10.
6. method prepared by the CK-MB detection kits according to claim 2 based on bimolecular fluorescence complementary technology,
It is characterized in that, in the anti-CK-MB antibody couplings fluorescin C-terminal segment step, fluorescin C-terminal segment resists with CK-MB
The mass ratio of body 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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| CN201711271924.5A CN108226520A (en) | 2017-11-27 | 2017-11-27 | A kind of CK-MB detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
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| CN201711271924.5A CN108226520A (en) | 2017-11-27 | 2017-11-27 | A kind of CK-MB detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349168A (en) * | 2018-12-20 | 2020-06-30 | 东莞市朋志生物科技有限公司 | Anti-human CKMB antibody and application thereof |
| CN112778419A (en) * | 2021-02-01 | 2021-05-11 | 重庆中元汇吉生物技术有限公司 | anti-CK-MB antibodies or antigen-binding portions thereof and uses thereof |
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| CN101620233A (en) * | 2009-05-27 | 2010-01-06 | 华中科技大学 | Method for detecting interaction of proteins |
| CN103290091A (en) * | 2012-03-22 | 2013-09-11 | 华中科技大学 | Protein interaction detection method with low false positive rate |
| CN104569429A (en) * | 2015-01-04 | 2015-04-29 | 深圳市艾瑞生物科技有限公司 | Homogeneous immunometric fluorescent compound set for quickly and quantificationally detecting brain natriuretic peptide (BNP) and preparation method of homogeneous immunometric fluorescent compound set |
| CN105132384A (en) * | 2015-07-25 | 2015-12-09 | 大庆麦伯康生物技术有限公司 | Hybridomas capable of producing anti-CK (creatine kinase)-MB monoclonal antibodies |
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2017
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Patent Citations (4)
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|---|---|---|---|---|
| CN101620233A (en) * | 2009-05-27 | 2010-01-06 | 华中科技大学 | Method for detecting interaction of proteins |
| CN103290091A (en) * | 2012-03-22 | 2013-09-11 | 华中科技大学 | Protein interaction detection method with low false positive rate |
| CN104569429A (en) * | 2015-01-04 | 2015-04-29 | 深圳市艾瑞生物科技有限公司 | Homogeneous immunometric fluorescent compound set for quickly and quantificationally detecting brain natriuretic peptide (BNP) and preparation method of homogeneous immunometric fluorescent compound set |
| CN105132384A (en) * | 2015-07-25 | 2015-12-09 | 大庆麦伯康生物技术有限公司 | Hybridomas capable of producing anti-CK (creatine kinase)-MB monoclonal antibodies |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349168A (en) * | 2018-12-20 | 2020-06-30 | 东莞市朋志生物科技有限公司 | Anti-human CKMB antibody and application thereof |
| CN112778419A (en) * | 2021-02-01 | 2021-05-11 | 重庆中元汇吉生物技术有限公司 | anti-CK-MB antibodies or antigen-binding portions thereof and uses thereof |
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Application publication date: 20180629 |