CN109946461A - A kind of B-typeNatriuretic Peptide precursor chemical luminescence detection kit and preparation method thereof - Google Patents
A kind of B-typeNatriuretic Peptide precursor chemical luminescence detection kit and preparation method thereof Download PDFInfo
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- CN109946461A CN109946461A CN201711039877.1A CN201711039877A CN109946461A CN 109946461 A CN109946461 A CN 109946461A CN 201711039877 A CN201711039877 A CN 201711039877A CN 109946461 A CN109946461 A CN 109946461A
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- peptide precursor
- typenatriuretic peptide
- chemiluminescence
- typenatriuretic
- detection kit
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Abstract
The invention discloses a kind of chemiluminescence detection kits and preparation method thereof of B-typeNatriuretic Peptide precursor.The kit includes: B-typeNatriuretic Peptide precursor the detection antibody, B-typeNatriuretic Peptide precursor serial standards, chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B, cleaning solution that coupling has the magnetic particle of B-typeNatriuretic Peptide precursor capture antibody, acridinium ester label.Kit of the present invention is using Magneto separate chemiluminescence as detection means, in combination with acridinium ester label technology.Compared with the technology of existing detection B-typeNatriuretic Peptide precursor, this kit has high sensitivity, and signal-to-noise ratio is high, quickly the advantages of detection.
Description
Technical field
The invention belongs to technical field of immune assay, specifically a kind of B-typeNatriuretic Peptide precursor chemical electrochemiluminescent immunoassay inspection
Test agent box and preparation method thereof.
Background technique
BNP is one of identical natriuretic peptide family in structure, and natriuretic peptide system includes ANP, BNP and CNP.ANP
It is the product of atrium and ventricle secretion with BNP, most BNP is mainly secreted by left ventricle, it is by precursor protein
(proBNP) it secretes, proBNP is cracked into active BNP and inactive NT-proBNP.The secretion of BNP can produce
The effects of sharp sodium, diuresis, vasodilation and smooth muscle relaxation.ANP has function identical with BNP, but is mainly secreted by atrium.
Plasma ANP and BNP can cause to increase under a variety of pathological states, and especially under the situation that wall tension increases, circulation is held
Amount increases (such as: heart failure, renal failure, aldosterone increase) or the removing of natriuretic peptide reduces (such as renal failure).ANP is previously stored atrium
Little particle and be atrial stretch secretion product, however the secretion of BNP is controlled by metaboilic level, it usually needs compared with
Prolonged stimulation.The plasma BNP concentrations of Patients with Cardiac Failure will appear raising, and the raised degree of BNP and New York Heart disease
Meeting (NYHA) classification is corresponding.Furthermore correlative study shows the plasma BNP concentrations of many patients relevant to myocardial damage
There is certain raising.Therefore, BNP making a definite diagnosis with critically important clinical meaning to early stage heart failure.
ProBNP can further be cracked into N-terminal 1-76 BNP and C-terminal BNP.N-terminal Natriuretic Peptide (NT-proBNP) be by
A kind of polypeptide hormone containing 32 amino acid that the heart, brain are secreted, when ventricle tension increases, heart overload promotes its secretion,
The row's of rising sodium, diuresis, the physiological action for expanding blood vessel in body.Its elevated concentrations and heart failure (acute heart failure AHF chronic heart failure CHF)
Severity is consistent.When doubtful heart failure (HF), the detection of Ying Shouxuan N-terminal Natriuretic Peptide (NT-proBNP).N-terminal brain benefit
Pro-BNP (NT-proBNP) feminine gender has very high predictive value, can exclude the presence of heart failure.In expiratory dyspnea patient, N-terminal brain
Natriuretic peptide precursor (NT-proBNP) is the relatively strong indication factor that (HF) will occur, and can effectively identify chronic obstructive and exhale
Inhale difficult and cardiac dyspnea.The Hazard degree assessment especially after screening left chamber function bad (LVD) and myocardial infarction
Aspect can show obvious superiority.
Currently, the method for detection B-typeNatriuretic Peptide precursor has colloidal gold method, radio immunoassay, Enzyme-multiplied immune technique, change
Learn electrochemiluminescent immunoassay technology etc..Wherein, radioimmunoassay technique, although radioimmunoassay is with highly sensitive, high specific
Advantage, but its operating procedure is more, needs special detection device, and reagent price is expensive, need to use matched instrument and presence
Radioactive pollution makes operator by radiological hazard；In addition, radioactive isotope is easily decayed, validity period is short, is not easy to protect
It deposits.Enzyme-linked immunoassay method, using horseradish peroxidase or alkaline phosphatase marker, enzyme marker easy in inactivation, chromogenic substrate
Light-exposed easy decomposition, sensitivity is low, and the compound similar to structure has certain cross reaction, causes test result inaccurate.
102323418 A(2016.02 of CN) disclose a kind of chemiluminescence detection B-typeNatriuretic Peptide precursor agents box, the examination
Agent box uses alkali phosphatase enzyme mark B-typeNatriuretic Peptide precursor monoclonal antibody, using mainly enzymatic the shortcomings that alkaline phosphatase
It is affected by environment big: if thimerosal, pH value, ion can all impact it, to influence measurement result, and substrate reaches platform
The time of phase is long, and substrate is at high cost, leads to testing cost height.And a kind of B-typeNatriuretic Peptide precursor chemical of our company's exploitation shines
Kit has the advantages that detection is stablized, measurement is quick, high sensitivity using acridinium ester as marker.
Chemiluminescence method is the simplicity detected compared with the advantages of other methods.
The system uses Magneto separate system, and the chemiluminescence of acridinium ester label is detected, it has the advantage of:
Acridinium ester is not required to the presence of catalyst as luminous agent, can shine in the dilute alkaline soln for having hydrogen peroxide；Acridine ester molecule
Measure it is small, avoid masking antibody combining site, system overall sensitivity can be improved, be swift in response；Background is low, signal-to-noise ratio is high, is one
The effective chemiluminescent labels of class.
Summary of the invention
The B that the purpose of the present invention is to provide a kind of sensitivity is higher, the reaction time is short, easy to operate, anti-interference is high
The magnetic microparticle chemiluminescence detection kit and preparation method of type natriuretic peptide precursor.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of the magnetic microparticle chemiluminescence detection kit and preparation method of B-typeNatriuretic Peptide precursor, magnetic provided by the present invention are micro-
The kit of grain chemiluminescence method detection B-typeNatriuretic Peptide precursor captures antibody using magnetic particle coupling B-typeNatriuretic Peptide precursor,
Acridinium ester label B-typeNatriuretic Peptide precursor detects antibody.Kit further includes B-typeNatriuretic Peptide precursor calibration object, above-mentioned acridinium ester work
Chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B and cleaning solution.
The kernel of magnetic bead of the present invention is ferroso-ferric oxide or di-iron trioxide, and the surface modification group of the magnetic bead is
Carboxyl, amino, p-toluenesulfonyl or Streptavidin-biotin.
Magnetic particle coupling suspension in the present invention is delayed by the magnetic particle and reagent of B-typeNatriuretic Peptide precursor capture antibody coupling
Fliud flushing composition, wherein the concentration of B-typeNatriuretic Peptide precursor capture antibody is 0.1-2.0 mg/mL.
Chemiluminescent labels in the present invention are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
Acridinium ester label in the present invention is acridinium ester label B-typeNatriuretic Peptide precursor detection antibody-solutions, wherein acridine
The concentration range of ester solution is 2-85 mmol/L.
In the present invention and before the B-typeNatriuretic Peptide of the B-typeNatriuretic Peptide precursor capture antibody of magnetic particle coupling and acridinium ester label
It is monoclonal antibody that antibody is surveyed in physical examination.
Coupling buffer is pH 5.0-6.0 in the present invention, and concentration is the MES buffer of 20-200 mmol/L.
In the present invention when preparing magnetic particle suspension, the Block buffer is the buffer of the BSA containing 1-5%.
Heretofore described B-typeNatriuretic Peptide precursor series of calibration product concentration is respectively as follows:: 0.0 ng/L, 10.0 ng/L,
50.0 ng/L, 250 ng/L, 1200ng/L, 3000 ng/L, buffer are Tris or PBS containing 0.5-5.0 % BSA
Or HEPES.
The buffer of acridinium ester label B-typeNatriuretic Peptide precursor detection antibody in the present invention is that pH 8.0-11.0, concentration are
The Na of 0.01-0.20 mol/L2CO3-NaHCO3。
Chemiluminescence preexciting liquid A in the present invention are as follows: H2O2And HNO3Mixed liquor, wherein H2O2Mass fraction be
0.01-5.0%, HNO3Concentration is 0.01-1.0 mol/L.
Chemiluminescence exciting liquid B in the present invention are as follows: the mixed liquor of Triton X-100 and NaOH, wherein Triton X-
100 mass fraction is 0.01-2.0 %, and the concentration of NaOH is 0.05-1.0 mol/L.
Cleaning solution in the present invention are as follows: pH 7.0-9.0, concentration be 5.0-50.0 mmol/L Tris or HEPES or its
Its buffer, wherein the Tween-20 for being 0.01-0.25% containing mass fraction.
Compared with the prior art, the advantages of the present invention are as follows: stabilization of kit of the invention, detection is fast, high sensitivity and
Specificity is good.
Specific embodiment
The technical solution in the present invention will clearly and detailedly be illustrated below.Experimental method is such as without special in example
Illustrate, is conventional method.
Embodiment: the establishment of kit and its preparation of concrete component
1. the establishment of kit
A kind of chemiluminescence detection kit of B-typeNatriuretic Peptide precursor, makes it contain following component:
Carboxyl magnetic bead is coupled B-typeNatriuretic Peptide precursor monoclonal antibody；
The B-typeNatriuretic Peptide precursor monoclonal antibody of acridinium ester label；
B-typeNatriuretic Peptide precursor serial standards solution
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
Cleaning solution.
Embodiment 2: the preparation of concrete component
1. the preparation of magnetic bead coupled antibody
(1) take 1 mg carboxyl magnetic particle in 0.5 mL centrifuge tube, 0.1 mol/L MES buffer of addition is vortexed and mixes, sets
Magnetic particle is separated with liquid on magnetic frame, standing 5 min, is discarded supernatant liquid, is washed 3 times, add the MES of 200 μ L
(pH value 6.0) buffer is vortexed.
(2) 15 μ g B-typeNatriuretic Peptide precursor monoclonal antibodies are added, make the molar ratio 150:1: whirlpool of carboxyl and antibody
Rotation, revolving reaction pipe are incubated at room temperature 30 min.
(3) the coupling reagent EDC that 10 μ L concentration are 10 mg/mL is added, in being vortexed on rotatable reactor, is incubated at room temperature 2
h。
(4) glycine buffer (concentration be 50 mmol/Ls) of the 200 μ L containing 1%BSA is taken to be closed, time 1h.
(5) supernatant is removed, the cleaning buffer solution (TBS+0.05%Tween-20) of 200 μ L is added, is washed 3 times.
(6) the above-mentioned magnetic particle suspension prepared is placed in 500 μ L to save liquid (300 mmol/L glycine, 2% sweet
Oil, 5% sucrose) in, 2-8 DEG C of preservation.
1.1. the preparation (0.1 mol/L) of MES buffer
The MES solid for taking 19.52 g, is dissolved in the deionized water of 500 mL, and being adjusted with the NaOH of 1 mol/L to pH is 6.0；?
The solution mixed up is transferred in the volumetric flask of 1 L, constant volume.
1.2. the preparation of coupling reagent EDC (concentration is 10 mg/mL)
It takes the EDC of 1 g in the beaker of 50 mL, the MES buffer of pre-cooling is added, stir, to its solution transfer to 100 mL's
In volumetric flask, constant volume.
2. the preparation of acridinium ester label antibody
(1) the B-typeNatriuretic Peptide precursor monoclonal antibody of 100 μ g is placed in bag filter, and bag filter is placed in not less than 1 L
Label buffer in dialyse, during which buffer is at least replaced 5 times, last time dialysed overnight, label buffer be pH 9.8,
Concentration is the Na of 50 mmol/L2CO3-NaHCO3Buffer.
(2) the acridinium ester NSP-DMAE-NHS for weighing 1.7 mg is dissolved in 447 μ L anhydrous dimethyl formamides (DMF),
It is made into the NSP-DMAE-NHS DMF solution of 6.5 mmol/L.
(3) monoclonal antibody solution after dialysis is placed in 500 μ L centrifuge tubes, the label buffer of 200 μ L is added,
The NSP-DMAE-NHS DMF solution (being protected from light) of 759 μ L, 6.5 mmol/L is added, makes acridinium ester and monoclonal antibody
Molar ratio is 7.4:1, and concussion mixes, and reacts at room temperature 1 h, and 100 μ L concentration of addition are 10 g/L lysines, stands 15
Min makes to mark reaction terminating.
(4) marker NSP-DMAE-NHS-Ab and free NSP-DMAE-NHS by Sephadex G-50 column (1 ×
It 25cm) separates, is balanced with the PBS that purification buffer pH 6.3, concentration are 0.1mol/L and elute chromatographic column.
(5) protein peak is detected with chromatograph in separation process, measures the chemiluminescence intensity and 280 nm of efflux respectively
Absorbance value.
(6) eluent that shading value is high and absorbance is big is collected, dispenses stored frozen after 1%BSA is added.
3. the preparation of standard items
The preparation of B-typeNatriuretic Peptide precursor serial standards is pure by B-typeNatriuretic Peptide precursor with the Tris-HCl buffer containing 2% BSA
Product are configured to mark concentration are as follows: 0.0 ng/L, 10.0 ng/L, 50.0 ng/L, 250 ng/L, 1200ng/L, 3000 ng/L,
The calibration object gradient value of totally 6 concentration.
4. the preparation of chemiluminescence exciting liquid A, B
(1) chemiluminescence preexciting liquid A is by H2O2And HNO3Composition.Wherein, H2O2Mass fraction is 1.5%, HNO3Concentration is 0.1
Mol/L is distributed into 20 mL/ branch with brown bottle, and 2-8 DEG C saves backup.
(2) chemiluminescence exciting liquid B is made of the mixed liquor of Triton X-100 and NaOH.Wherein, Triton X-100
Mass fraction is 1.0%, and NaOH concentration is 0.4 mol/L, is distributed into 20 mL/ branch with brown bottle, 2-8 DEG C saves backup.
5. the preparation of cleaning solution
Weigh 3.05 g Tris, in the beaker of 8.775 g NaCl to 1000 mL, it is 0.05% that 1mL mass fraction, which is added,
Tween-20 is stirred and evenly mixed, constant volume, and pH is adjusted to 7.6, is saved backup.
Embodiment 3: the implementation of specific kit
(1) 50 μ L of sample to be tested is added in reaction cup, adds magnetic particle coupling 150 μ L of suspension, oscillation mixes, and 37 DEG C
It is incubated for 8 min.
(2) it separates, wash 3 times, the reaction vessel after washing, which is sufficiently vibrated, makes magnetic particle scatter.
(3) 150 μ L of acridinium ester label is added into reaction cup, oscillation mixes, 37 DEG C of 7 min of incubation.
(4) it separates, cleans 3 times, the reaction vessel after washing, which is sufficiently vibrated, makes magnetic particle scatter.
(5) 100 μ L chemiluminescence exciting liquid B are added after 100 μ L chemiluminescence preexciting liquid A, 1 s being added, measure it
Relative luminous intensity, the content of B-typeNatriuretic Peptide precursor luminous intensity proportion relation corresponding thereto in sample.
Embodiment 4: the performance indicator of kit
(1) sensitivity for analysis
0 ng/L calibration object in kit is used replication 20 times, to obtain the RLU value of 20 measurement results as sample to be tested
(relative light unit) calculates its average value (M) and standard deviation (SD), obtains RLU value corresponding to M+2SD, calibrated according to zero-dose
Concentration-RLU value result between product and adjacent calibration object carries out two o'clock regression fit and obtains linear function, by the RLU value of M+2SD
It brings into above-mentioned equation, finds out corresponding concentration value.By minimum detection limit detection method in detection scheme, 3 repeated experiments, most
Finding out this kit afterwards is 8.0 ng/L to the sensitivity for analysis of B-typeNatriuretic Peptide precursor.
(2) precision
It is (100.0 ± 20.0) ng/L's and (500.0 ± 100.0) ng/L with B-typeNatriuretic Peptide precursor agents box test concentrations
Sample, each concentration retest 10 times calculate kit precision, the results showed that kit CV is respectively less than 5%.
(4) stability
B-typeNatriuretic Peptide precursor detection kit is taken to carry out normal storage stability test, 2-8 DEG C of placement distinguishes temporally 1,3,5,
It is detected within 7,9,11,12,13,14,15 months；Stability test uncap respectively by 2-8 DEG C of placement 0 day, 7 days, 14 days, 16
It, be measured within 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days.B-typeNatriuretic Peptide precursor detects as the result is shown
Kit is stored in 2-8 DEG C, in light protected environment, and validity period is 12 months.It uncaps and is stored in 2-8 DEG C, in light protected environment, validity period
It is 30 days.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (9)
1. a kind of chemiluminescence detection kit and its composition of B-typeNatriuretic Peptide precursor characterized by comprising coupling has Type B
Natriuretic peptide precursor captures the magnetic particle suspension of antibody, the B-typeNatriuretic Peptide precursor of acridinium ester label detects antibody, B-typeNatriuretic Peptide
Precursor serial standards, chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B, cleaning solution.
2. the chemiluminescence detection kit and its composition of a kind of B-typeNatriuretic Peptide precursor according to claim 1, feature
Be: the kernel of the magnetic bead is ferroso-ferric oxide or di-iron trioxide, and the surface modification group of magnetic particle is a kind of or more
Kind activity functional groups, including but not limited to carboxyl, amino, p-toluenesulfonyl or Streptavidin-biotin.
3. a kind of chemiluminescence detection kit of B-typeNatriuretic Peptide precursor according to claim 1, which is characterized in that institute
The chemiluminescent labels stated are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
4. a kind of chemiluminescence detection kit of B-typeNatriuretic Peptide precursor according to claim 1, which is characterized in that a word used for translation
Pyridine ester label is B-typeNatriuretic Peptide precursor detection antibody.
5. a kind of chemiluminescence detection kit of B-typeNatriuretic Peptide precursor according to claim 1, which is characterized in that institute
The magnetic particle stated can directly capture antibody coupling with B-typeNatriuretic Peptide precursor, or magnetic particle and Streptavidin are coupled, and adopt simultaneously
Antibody is captured with biotin labeling B-typeNatriuretic Peptide precursor.
6. a kind of chemiluminescence detection kit of B-typeNatriuretic Peptide precursor according to claim 1, which is characterized in that institute
The capture antibody and detection antibody stated are monoclonal antibody.
7. a kind of chemiluminescence detection kit of B-typeNatriuretic Peptide precursor according to claim 1, which is characterized in that institute
The chemiluminescence preexciting liquid A stated is by H2O2And HNO3 Mixed liquor composition, chemiluminescence exciting liquid B by Triton X-100 and
The mixed liquor of NaOH forms.
8. a kind of chemiluminescence detection kit of B-typeNatriuretic Peptide precursor according to claim 1, which is characterized in that institute
The calibration object stated is that series of concentrations gradient made of the configuration of B-typeNatriuretic Peptide precursor is added using the buffer containing BSA as matrix
Calibration object solution.
9. a kind of chemiluminescence detection kit of B-typeNatriuretic Peptide precursor according to claim 1, comprising the following steps:
A certain amount of sample to be tested is added in reaction cup, adds magnetic particle coupling suspension, mixes, cleaning is added in 37 DEG C of incubations
Liquid removes supernatant；Acridinium ester label is added according to a certain percentage later, 37 DEG C of incubations are added cleaning solution and remove supernatant, add
Enter chemiluminescence preexciting liquid A and exciting liquid B, measures corresponding luminous intensity RLU/s.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111007266A (en) * | 2019-11-20 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma |
| CN111912838A (en) * | 2020-06-24 | 2020-11-10 | 宁波瑞源生物科技有限公司 | BNP chemical light-emitting detection kit and application thereof |
-
2017
- 2017-10-31 CN CN201711039877.1A patent/CN109946461A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111007266A (en) * | 2019-11-20 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma |
| CN111912838A (en) * | 2020-06-24 | 2020-11-10 | 宁波瑞源生物科技有限公司 | BNP chemical light-emitting detection kit and application thereof |
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