CN109444431A - A kind of quantitative detecting method and detection kit of cardiac myosin binding protein C - Google Patents
A kind of quantitative detecting method and detection kit of cardiac myosin binding protein C Download PDFInfo
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- CN109444431A CN109444431A CN201811530495.3A CN201811530495A CN109444431A CN 109444431 A CN109444431 A CN 109444431A CN 201811530495 A CN201811530495 A CN 201811530495A CN 109444431 A CN109444431 A CN 109444431A
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
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Abstract
The invention discloses the quantitative detecting methods of cardiac myosin binding protein C a kind of, it takes antibody coupling superparamagnetic particles that specific binding occurs with sample to be tested first to react, the first reactant is obtained, wherein antibody coupling superparamagnetic particles are the product that superparamagnetic particles and anti-cMyBP-C monoclonal antibody A are coupled;Then it takes the first reactant that specific binding occurs with enzyme mark object to react, obtains the second reactant, wherein enzyme mark object is the product that enzyme and anti-cMyBP-C monoclonal antibody B are coupled;It finally takes chemiluminescent substrate and the second reactant that enzymatic reaction occurs, measures luminous signal, obtain the concentration value of cMyBP-C.Methodology mode of the invention is sandwich method, and compared with cMyBP-C ELISA kit, the kit of detection method of the invention and preparation can greatly promote its detection sensitivity and the range of linearity.By carrying out evaluation of methodology, sensitivity for analysis 0.05ng/ml, detection range 0.1-50ng/ml, with clinical AMI patient correlation with higher and coincidence rate to kit prepared by the present invention.
Description
Technical field
The present invention relates to biological species, and vitro diagnostic techniques are immunized, more particularly, to a kind of cardiac myosin binding protein C
Quantitative detecting method, the invention further relates to the immue quantitative detection reagent boxes of cardiac myosin binding protein C.
Background technique
Acute myocardial infarction AMI (acute myocardial infarction, AMI) is severe cardiovascular disease, morbidity
Reason is complicated, its disease incidence increases year by year in recent years, and has rejuvenation trend, seriously jeopardizes patient health, therefore carry out to it
Early stage diagnosis and treatment are of great significance.The measurement of serum cardiac necrosis marker plays a significant role in the diagnosis of AMI.Flesh calcium egg
White (cardiac troponin, cTn) has become the preferred biomarker of diagnosis AMI.But for myocarditis, lung
The patients such as arterial embolism, cTn also can be increased non-specifically, and patient or ischemia symptom for disease time less than 6h
Patient is sent out again, needs to repeat to detect cTn.Cardiac myosin binding protein C (cardiac myosin binding
Protein-C, cMyBP-C) it is the distinctive thick filament structural proteins of cardiac muscle cell, it cMyBP-C dephosphorylation and releases when AMI
It is put into blood, haemoconcentration sharply increases, and detection serum cMyBP-C concentration can provide help for the diagnosis of AMI.
The research prompt cMyBP-C of Govindan etc. can become a potential myocardial necrosis marker, and its serum-concentration
Compared with cTn high several times or even dozens of times, it is easy to detect.
Currently, cMyBP-C ELISA kit is mostly used to the detection of cMyBP-C, but, line long there are the reaction time
Property narrow range, the low defect of sensitivity.CMyBP-C immunochemiluminescence kit is not reported then, therefore it provides a kind of heart
The quantitative detecting method of cardiac myosin binding protein-C and to prepare a kind of cMyBP-C for meeting Clinical Acute myocardial infarction accurately fixed
The kit of amount detection has important practical significance.
Summary of the invention
The purpose of the present invention is to provide the methods of energy accurate quantitative analysis detection cardiac myosin binding protein C a kind of, originally
Invention also provides the immue quantitative detection reagent box of cardiac myosin binding protein C simultaneously.
To achieve the above object, the present invention can take following technical proposals:
The quantitative detecting method of cardiac myosin binding protein C of the present invention includes the following steps:
The first step takes antibody coupling superparamagnetic particles that specific binding occurs with sample to be tested and reacts, obtains the first reactant;Its
Described in antibody coupling superparamagnetic particles be product that superparamagnetic particles and anti-cMyBP-C monoclonal antibody A are coupled;
Second step takes the first reactant that specific binding occurs with enzyme mark object and reacts, obtains the second reactant;The wherein enzyme
Mark object is the product that enzyme and anti-cMyBP-C monoclonal antibody B are coupled;
Third step takes chemiluminescent substrate and the second reactant that enzymatic reaction occurs, measures luminous signal, obtain cMyBP-C's
Concentration value.
The temperature that specific binding reaction occurs for the first step, second step is 37 DEG C, and the reaction time is 5~15min.
The temperature that enzymatic reaction occurs for the third step is 37 DEG C, and the reaction time is 5~15min.
The anti-cMyBP-C monoclonal antibody A and anti-cMyBP-C monoclonal antibody B can identify cMyBP-C antigen table
Position, but the epitope of anti-cMyBP-C monoclonal antibody A identification antigen and the epitope of anti-cMyBP-C monoclonal antibody B identification antigen are not
Together.
Testing principle of the invention are as follows: when containing antigen cMyBP-C in sample, Paramagnetic particles coupled antibody and antigen exist
It is reacted in 37 DEG C, forms superparamagnetic particles coupled antibody-antigen im-mune complexes;It is removed by washing and does not participate in reaction
Substance, then add the enzyme conjugates of antibody coupling, reacted in 37 DEG C, removed by washing and do not participate in the anti-of reaction
Corresponding with enzyme chemiluminescent substrate is added in the enzyme conjugates of body coupling, the enzyme effect on immune complex in luminous substrate,
It shines under enzymatic catalysis, luminous signal is detected by Full-automatic chemiluminescence analyzer.
In conjunction with above-mentioned testing principle, the present invention is prepared for the reagent of quantitative detection cardiac myosin binding protein C a kind of
Box:
Kit of the present invention includes antibody coupling enzyme conjugates, chemiluminescent substrate and antibody coupling superparamagnetic particles, cMyBP-C
Calibration product;Antibody coupling superparamagnetic particles therein are the production that superparamagnetic particles and anti-cMyBP-C monoclonal antibody A are coupled
Object.
Superparamagnetic particles used in the present invention are common magnetic particle in immunodiagnosis, and granularity is 500nm-1um,
Its reaction density is 1~10mg/ml.
The active group on the superparamagnetic particles surface that the present invention uses is carboxyl (- COOH), amino (- NH2) or benzene first
Sulfonyl.When practical preparation, active group preferably uses carboxyl (- COOH), and the magnetic particle of carboxylic group is in chemical cross-linking agent
EDC(1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride) and NHS(N- HOSu NHS) effect under, with
The amino group of anti-cMyBP-C monoclonal antibody A forms amido bond and covalent bond.
Wherein for being coupled the anti-cMyBP-C monoclonal antibody of enzyme conjugates and the anti-cMyBP-C Dan Ke of coupling magnetic particle
Grand antibody is the antibody for cMyBP-C different epitopes.
When the enzyme conjugates that the present invention uses is horseradish peroxidase (HRP), chemiluminescent substrate using luminol or
Different luminol;If enzyme conjugates uses alkaline phosphatase (ALP), chemiluminescent substrate uses 3-(2- spiral adamantane) -4-
Methoxyl group-(3- phosphorus oxygen acyl)-phenyl -1,2- dioxane disodium salt (AMPPD).
It is respectively 0ng/mL, 0 .05ng/mL, 0.5ng/mL, 2ng/mL, 10ng/mL that the MyBP-C calibration product, which are concentration,
With the solution of the MyBP-C of 50ng/mL.
Methodology mode of the invention is sandwich method, the advantage is that: compared with cMyBP-C ELISA kit, this hair
The kit of bright detection method and preparation can greatly promote its detection sensitivity and the range of linearity.By being prepared to the present invention
Kit carry out evaluation of methodology, sensitivity for analysis 0.05ng/ml, detection range 0.1-50ng/ml, with clinical AMI
Patient's correlation with higher and coincidence rate.
Detailed description of the invention
Fig. 1 is the canonical plotting of kit calibration product of the present invention.
Specific embodiment
More detailed explanation is done to the present invention below by specific embodiment, in order to the reason of those skilled in the art
Solution, but following embodiments are not intended to limit the scope of the present invention.
(enzyme conjugates uses horseradish mistake to the kit of the preparation of embodiment 1 quantitative detection cardiac myosin binding protein C
Oxide enzyme)
1, antibody coupling superparamagnetic particles are prepared
Take the superparamagnetic particles (diameter is 1um~3um) that surface active groups are carboxylic group, with coating buffer (
The phosphate buffer of 50mmol/L, pH 7.6) washing 5 times, it is glutaraldehyde activated to be added 10%;After washing be diluted to
The cMyBP-C antibody of 100-500 μ g/mL is coated with, concussion reaction 2h;Washing, then again with containing 50m mol/L acetic acid
The buffer blind 1h of pH4.4,0.02mol/L;Confining liquid is added after removing contains 0.5% Tween-20,1% BSA, 0.1%
The pH7.4 of Procline300,0.02 mol/L phosphate buffer are stored, and 2~8 DEG C save backup.
2, antibody coupling horseradish peroxidase (HRP) conjugate is prepared
It weighs 5mg HRP to be dissolved in 1ml distilled water, 0.06mol/L NaIO is added4Aqueous solution (bis- distilled water+the 128mg of 10ml
NaIO4) 0.5ml, it mixes, sets 4 DEG C of 30min.Above-mentioned solution is fitted into bag filter, to the sodium acetate buffer of 1mM PH4.4
Liquid dialysis, 4 DEG C overnight;The aqueous solution 1ml of the purifying cMyBP-C antibody containing 5mg is added, mixes, and be packed into bag filter, uses
The carbonate buffer solution of 0.05mol/L pH 9.5, slowly stirring dialysis 6h, is allowed to combine;It is slowly added to NaBH4 Solution
(5mg/ml) 0.2ml mixes, is placed in 4 DEG C of 2h;It is slowly added to isometric saturated ammonium sulfate solution in the above solution, mixes
Even, supernatant is removed in 4 DEG C of 30min, centrifugation, and precipitating is redissolved with the PBS solution of a little 0.02mol/L pH7.4, is packed into dialysis
Bag is stayed overnight with same liquid in 4 DEG C of dialysis desalinations;Next day takes out centrifugation, to remove insoluble matter to get enzyme-antibody conjugates,
After the dilution of 7.4 PBS solution of 0.02mol/L pH, 1mL/ bottles are distributed into, cryo-conservation.
3, preparation cMyBP-C calibrates product
Humanization MyBP-C albumen is configured with standard items buffer (10mM phosphate buffer, 7 .4 of 1%Casein, pH)
It is 0ng/mL, 0 .05ng/mL, 0.5ng/mL, 2ng/mL, 10ng/mL and 50ng/mL at concentration, every bottle of 1mL packing is freeze-dried
After processing, 4 DEG C are saved backup.
4, chemiluminescent substrate is prepared
Luminous substrate A liquid: contain 0 .1M luminol;
Luminous substrate B liquid: contain 0 .5%H2O2。
The detection method of the kit of the present invention of embodiment 2
Using Full-automatic chemiluminescence immunoassay analysis meter, the kit prepared using embodiment 1 is detection instrument: instrument is sequentially added
The cMyBP- of the horseradish peroxidase-labeled of the sample of 25 μ L, the coated magnetic particle of cMyBP-C antibody of 20 μ L and 100 μ L
C antibody after reacting 15min, carries out Magneto separate, and reaction mixture is sent into darkroom by instrument, sequentially adds luminous substrate A liquid (containing 0
.1M luminol) and B liquid (containing 0 .5%H2O2) progress luminescence-producing reaction, luminous intensity is finally recorded, is calculated from standard curve
The cMyBP-C content of sample.
CMyBP-C calibration product are detected using the above method, the standard curve drawn out is as shown in Figure 1.
The performance test of the kit of the present invention of embodiment 3
1, sensitivity technique
Referring to CLSI EP17-A file recommendation experimental program, the sensitivity of cMyBP-C chemiluminescence immunoassay kit is calculated, is asked
The sensitivity obtained is 0 .05ng/mL.
2, linearity test
It is 0ng/mL to concentration, 0 .05ng/mL, 0.5ng/mL, 2ng/mL, 10ng/mL and 50ng/mL standard items do linear point
Analysis calculates linearly dependent coefficient, r2=0 .99, the kit are 0.1-50ng/ to the range of linearity of cMyBP-C sample detection
ml。
3, precision detects
Taking concentration is two cMyBP-C samples of 0 .1ng/mL and 20ng/mL, and each each concentration of sample respectively does 5 parallel, use
Three batches of kits are detected, and are calculated kit and are criticized interior and difference between batch, as a result such as the following table 1, table 2, table 3.
Accuracy in 1 0.1ng/ml sample of table batch
Accuracy in 2 20ng/ml sample of table batch
3 batches of accuracies of table
Show in the kit batch and difference between batch is respectively less than 10%.
Claims (10)
1. a kind of quantitative detecting method of cardiac myosin binding protein C, it is characterised in that: include the following steps:
The first step takes antibody coupling superparamagnetic particles that specific binding occurs with sample to be tested and reacts, obtains the first reactant;Its
Described in antibody coupling superparamagnetic particles be product that superparamagnetic particles and anti-cMyBP-C monoclonal antibody A are coupled;
Second step takes the first reactant that specific binding occurs with enzyme mark object and reacts, obtains the second reactant;The wherein enzyme
Mark object is the product that enzyme and anti-cMyBP-C monoclonal antibody B are coupled;
Third step takes chemiluminescent substrate and the second reactant that enzymatic reaction occurs, measures luminous signal, obtain cMyBP-C's
Concentration value.
2. the quantitative detecting method of cardiac myosin binding protein C according to claim 1, it is characterised in that: described
The temperature that specific binding reaction occurs for the first step, second step is 37 DEG C, and the reaction time is 5~15min.
3. the quantitative detecting method of cardiac myosin binding protein C according to claim 1, it is characterised in that: described
The temperature that enzymatic reaction occurs for third step is 37 DEG C, and the reaction time is 5~15min.
4. the quantitative detecting method of cardiac myosin binding protein C according to claim 1, it is characterised in that: described
Anti- cMyBP-C monoclonal antibody A and anti-cMyBP-C monoclonal antibody B can identify cMyBP-C epitope, but anti-
CMyBP-C monoclonal antibody A identifies that the epitope of antigen is different from the anti-cMyBP-C monoclonal antibody B identification epitope of antigen.
5. a kind of kit of quantitative detection cardiac myosin binding protein C, it is characterised in that: combined including antibody coupling enzyme
Object, chemiluminescent substrate and antibody coupling superparamagnetic particles, cMyBP-C calibrate product;The antibody coupling superparamagnetic particles are super
The product that Paramagnetic particles and anti-cMyBP-C monoclonal antibody A are coupled.
6. the kit of quantitative detection cardiac myosin binding protein C according to claim 5, it is characterised in that: institute
The granularity for stating superparamagnetic particles is 500nm-1um, and reaction density is 1~10mg/ml.
7. the kit of quantitative detection cardiac myosin binding protein C according to claim 5, it is characterised in that: institute
The active group for stating superparamagnetic particles surface is carboxyl, amino or benzene mesyl.
8. the kit of quantitative detection cardiac myosin binding protein C according to claim 5, it is characterised in that: institute
The anti-cMyBP-C monoclonal antibody for stating anti-cMyBP-C monoclonal antibody and the coupling magnetic particle for being coupled enzyme conjugates is needle
To the antibody of cMyBP-C different epitopes.
9. the kit of quantitative detection cardiac myosin binding protein C according to claim 5, it is characterised in that: institute
State enzyme conjugates be horseradish peroxidase when, chemiluminescent substrate use luminol or different luminol;If enzyme conjugates uses
Alkaline phosphatase, then chemiluminescent substrate uses 3-(2- spiral adamantane) -4- methoxyl group-(3- phosphorus oxygen acyl)-phenyl -1,2- bis-
Oxygen cyclohexane disodium salt (AMPPD).
10. the kit of quantitative detection cardiac myosin binding protein C according to claim 5, it is characterised in that: institute
It is respectively 0ng/mL, 0 .05ng/mL, 0.5ng/mL, 2ng/mL, 10ng/mL and 50ng/mL that MyBP-C calibration product, which are stated, as concentration
MyBP-C solution.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109946453A (en) * | 2019-04-02 | 2019-06-28 | 珠海丽珠试剂股份有限公司 | A kind of coupling method of proliferating cell nuclear antigen |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103869085A (en) * | 2014-04-04 | 2014-06-18 | 郑州安图生物工程股份有限公司 | Kit for detecting anthropogenic N terminal-b-type natriuretic peptide precursor |
-
2018
- 2018-12-14 CN CN201811530495.3A patent/CN109444431A/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103869085A (en) * | 2014-04-04 | 2014-06-18 | 郑州安图生物工程股份有限公司 | Kit for detecting anthropogenic N terminal-b-type natriuretic peptide precursor |
Non-Patent Citations (1)
Title |
---|
SURESH GOVINDAN ET AL: "Cardiac myosin binding protein-C is a potential diagnostic biomarker for myocardial infarction", 《J MOL CELL CARDIOL》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109946453A (en) * | 2019-04-02 | 2019-06-28 | 珠海丽珠试剂股份有限公司 | A kind of coupling method of proliferating cell nuclear antigen |
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Application publication date: 20190308 |