CN109946465A - A kind of the magnetic microparticle chemiluminescence detection kit and preparation method of people's corticotropin - Google Patents
A kind of the magnetic microparticle chemiluminescence detection kit and preparation method of people's corticotropin Download PDFInfo
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- CN109946465A CN109946465A CN201711039876.7A CN201711039876A CN109946465A CN 109946465 A CN109946465 A CN 109946465A CN 201711039876 A CN201711039876 A CN 201711039876A CN 109946465 A CN109946465 A CN 109946465A
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Abstract
The invention discloses a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin and preparation methods.The kit includes: corticotropin detection antibody, corticotropin calibration object, chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B and the cleaning solution that coupling has the magnetic particle of corticotropin 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.Direct chemoluminescence method high sensitivity that the present invention establishes, high specificity, accurate quickly detection time is short, and testing result has higher accuracy and repeatability, which is applicable to various luminometer devices.
Description
Technical field
The invention belongs to technical field of immune assay, the immune magnetic of specifically a kind of people's corticotropin is micro-
Grain chemiluminescence detection kit and preparation method.
Background technique
Corticotropin is abbreviated as ACTH, is a kind of peptide hormone of vertebrate pituitary secretion.ACTH
Containing 39 amino acid, molecular weight 4500.It can promote adrenocortical hyperblastosis and the generation of cortin and divide
It secretes.Direct regulation and control of its generation and secretion by hypothalamus cortico-trophin-releasing factor (CRF) (CRF).Secreted the skin contained
Matter hormone can also influence hypophysis and hypothalamus in turn, weaken their activity.It has stimulation adrenal gland cortical development and machine
The effect of energy.Adrenal cortex zona fasciculata is mainly acted on, the secretion of glucocorticosteroid is stimulated.By the excessive skin of hormone
When matter hormone, the secretion of ACTH will receive inhibition, and atrophy occurs for adrenal cortex.
ACTH increase be found in primary adrenal cortical hypofunction, ectopic ACTH syndrome, Cushing disease,
Nelson syndrome, congenital adrenal cortical hyper plasia, heredity adrenal cortex are to ACTH not response syndrome, periodicity
ACTH, ADH secretion increase that syndrome, (such as operation, wound, shock, low blood are warded off, using SU4885 ACTH can divide for other
It secretes and increases).ACTH reduction is found in hypofunction of anterior pituitary, adrenal cortical adenoma, pure ACTH and lacks synthesis
Sign, iatrogenic ACTH reduction etc..
Up to the present, the method for detecting people's corticotropin mainly has: radio immunoassay
(Radioimmunoassay, RIA) and enzyme-linked immunosorbent assay (ELISA).But although radioimmunoassay has
The advantages of high sensitivity, high specific, but its operating procedure is more, reagent price is expensive, special detection device need to be used, and
And make operator vulnerable to radiological hazard, while the decay of radioactive substance that the service life of kit can also be marked
The limitation of phase, therefore the original drugs using RIA measurement many in recent years have used other immunoassay methods instead more.Enzyme-linked immunization
Although detection is cheap, quick, sensitivity is also inadequate, is only applicable to the detection and identification of micro substance, CN
202033370 U(2011 November) a kind of enzyme-linked immunosorbent assay kit of people's corticotropin is disclosed,
Kit polystyrene agent plate (PS) as solid phase carrier, be coated in advance on agent plate capillary strip it is certain density with
Sample to be tested is added in the ACTH of OVA carrier conjugation, and the ACTH competitive binding formed on ELISA Plate in coated ACTH and sample is anti-
The situation of ACTH antibody, then by Avidin horseradish peroxidase (HRP) amplified signal, with 3,3 ', 5,5 '-tetramethyls connection
Aniline (TMB) and sulfuric acid complete the preparation of the ELISA kit of people ACTH, the master of this method respectively as substrate and terminate liquid
It wants the disadvantage is that sensitivity is low.Therefore, a kind of detection rush adrenal gland skin effective, quick, simple, sensitive, anti-interference is high is established
The method of matter hormone has a very important significance.
The present invention uses method for direct chemoluminescence method, using acridinium ester as chemiluminescent labels with obvious excellent
Gesture is mainly manifested in: reaction does not need catalyst, as long as alkaline environment can carry out, is swift in response, can complete catching reaction
The photon of generation, background luminescence is low, and signal-to-noise ratio is high, and disturbing factor is few, and reagent stability is good, and system is simple, and exciting liquid is at low cost,
Easily and protein bind, and photon yield is not reduced acridinium ester after being coupled.The Magnetism particulate immuno chemistry luminescence method that the present invention establishes is sensitive
Spend height, high specificity, it is accurate quickly, detection time is short, testing result have higher accuracy with it is repeated.
Summary of the invention
The rush 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 cortex hormone of aadrenaline.
To achieve the above object, the invention provides the following technical scheme:
A kind of the magnetic microparticle chemiluminescence detection kit and preparation method of corticotropin, magnetic provided by the present invention
Particulate chemistry luminescent method detects the kit of corticotropin, is caught using magnetic particle coupling corticotropin
Antibody is obtained, acridinium ester label corticotropin detects antibody.Kit further include corticotropin calibration object,
Chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B and the cleaning solution of above-mentioned acridinium ester effect.
As a further solution of the present invention, the magnetic particle can be directly even with corticotropin capture antibody
Magnetic particle and Streptavidin can also be coupled, while capture antibody using biotin labeling corticotropin by connection.
As a further solution of the present invention, the surface modification group of the magnetic particle is one or more active function groups
Group, including but not limited to carboxyl, amino, p-toluenesulfonyl or Streptavidin-biotin.
As a further solution of the present invention, the chemiluminescent labels be acridinium ester, as NSP-DMAE-NHS,
NSP-SA-NHS etc..
As a further solution of the present invention, the acridinium ester label is corticotropin detection antibody.
As a further solution of the present invention, the buffering of acridinium ester label corticotropin detection antibody
Liquid is pH 8.0-11.0, the Na that concentration is 0.01-0.20 mol/L2CO3-NaHCO3Buffer.
As a further solution of the present invention, the capture antibody and detection antibody are monoclonal antibody.
As a further solution of the present invention, the calibration object be with the Tris containing 0.5-5.0% BSA or PBS or
HEPES buffer solution is matrix, and corticotropin sterling is added and configures, and calibration object form is liquid.
As a further solution of the present invention, the corticotropin calibration object solution concentration is respectively as follows: 0
pg/mL、0.5 pg/mL、3.0 pg/mL、18.0 pg/mL、108.0 pg/mL、650.0 pg/mL。
As a further solution of the present invention, the chemiluminescence preexciting liquid A is H2O2And HNO3Mixed liquor,
Middle H2O2Mass fraction be 0.01-5.0%, HNO3Concentration be 0.01-1.0 mol/L.
As a further solution of the present invention, the chemiluminescence exciting liquid B is the mixed of Triton X-100 and NaOH
Liquid is closed, wherein the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1.0 mol/L.
As a further solution of the present invention, the cleaning solution are as follows: pH 7.0-9.0, concentration are 5.0-50.0 mmol/
The Tris or HEPES or other solution of L, wherein the Tween-20 for being 0.01-0.25% containing mass fraction.
The principle of the present invention is the high degree of specificity by antibody-antigene reaction in conjunction with the high sensitivity that acridinium ester shines
Get up, the photon generated using acridinium ester catching reaction is to detect production concentration.
The advantage of the invention is that combining magnetic microparticle chemiluminescence technology using double antibody sandwich method, measurement people promotees adrenal gland
Cortin content.Acridinium ester has a clear superiority as the direct chemiluminescence of marker, is mainly manifested in: reaction does not need
Catalyst is swift in response as long as alkaline environment can carry out, can completely catching reaction generate photon, background luminescence is low, letter
Make an uproar than high, disturbing factor is few, and reagent stability is good, and system is simple, and exciting liquid is at low cost, acridinium ester easily and protein bind, and
Photon yield is not reduced after connection.
Specific embodiment
The present invention provides the magnetic microparticle chemiluminescence detection kit and preparation method of a kind of people's corticotropin,
For make the purpose of the present invention, technical solution and effect definitely, it is clear, the present invention is described in more detail below.
The present invention provides the magnetic microparticle chemiluminescence detection kit and preparation method of a kind of people's corticotropin,
Wherein, the kit of magnetic microparticle chemiluminescence method detection corticotropin provided by the present invention, using magnetic particle
It is coupled corticotropin and captures antibody, acridinium ester label corticotropin detects antibody.Kit further includes
Corticotropin calibration object, above-mentioned acridinium ester effect chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B and
Cleaning solution.
Specifically, magnetic particle coupling suspension of the present invention is captured the magnetic of antibody coupling by corticotropin
Particle and reagent buffer composition, wherein the concentration of corticotropin capture antibody is 0.1-2.0 mg/mL.
Specifically, the kernel of magnetic bead of the present invention be ferroso-ferric oxide, the magnetic particle can directly with promote adrenal gland
Cortin captures antibody coupling, can also be coupled magnetic particle and Streptavidin, while promoting adrenal gland using biotin labeling
Cortin captures antibody.Magnetic bead will affect accuracy using the endless bulk deposition of preceding solid phase, therefore should select dispersibility when selection
It is good, the slow magnetic bead of sinking speed.
Specifically, for the present invention when preparing magnetic particle suspension, the coupled antibody buffer is pH 5.0-6.0, concentration
For the MES buffer of 20-200 mmol/L.
Specifically, for the present invention when preparing magnetic particle suspension, the Block buffer is the buffer containing 1%BSA.
Specifically, for the present invention when preparation marks liquid-phase reagent, the label buffer is that pH 8.0-11.0, concentration are
The Na of 0.01-0.20 mol/L2CO3-NaHCO3Buffer.
Specifically, calibration object of the present invention is to be with the Tris containing 0.5-5.0% BSA or PBS or HEPES buffer solution
Matrix is added corticotropin sterling and configures, and calibration object form is liquid.
Specifically, chemiluminescence preexciting liquid A of the present invention is H2O2And HNO3Mixed liquor, wherein H2O2Quality
Score is 0.01-5.0%, HNO3Concentration be 0.01-1.0 mol/L.
Specifically, chemiluminescence exciting liquid B of the present invention is the mixed liquor of Triton X-100 and NaOH, wherein
The mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1.0 mol/L.
Specifically, cleaning solution of the present invention are as follows: pH 7.0-9.0, concentration be 5.0-50.0 mmol/L Tris or
HEPES or other solution, wherein the Tween-20 for being 0.01-0.25% containing mass fraction.
Below by embodiment, the present invention is described in detail.
A kind of embodiment 1: the establishment of the magnetic microparticle chemiluminescence kit of detection people corticotropin
And preparation method, comprising the following steps:
1. the establishment of kit:
A kind of magnetic microparticle chemiluminescence kit for detecting corticotropin is set up, it is made to contain following component:
The corticotropin monoclonal antibody of magnetic particle coupling；
Another plant of corticotropin monoclonal antibody of acridinium ester label；
Corticotropin serial standards solution；
Chemiluminescence preexciting liquid A and chemiluminescence exciting liquid B；
Cleaning solution.
2. the preparation that coupling has the magnetic particle suspension of corticotropin monoclonal antibody
(1) it takes 1 mg carboxyl magnetic particle in 0.5 mL centrifuge tube, the MES buffer that 200 μ L concentration are 0.1 mol/L is added,
It is vortexed and mixes, be placed on magnetic frame, standing 5 min separates magnetic particle with liquid, discards supernatant, and washs 3 times, adds 200
The MES(pH of μ L is 6.0) buffer, is vortexed.
(2) the corticotropin monoclonal antibody of 15 μ g is added, makes the molar ratio 150:1 of carboxyl and antibody,
It is vortexed, revolving reaction pipe is 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.
3. the preparation of the corticotropin monoclonal antibody liquid-phase reagent of acridinium ester label
(1) it purifies corticotropin monoclonal antibody: 100 μ g corticotropin monoclonal antibodies is placed in
In bag filter, and bag filter is placed in the label buffer not less than 1 L and is dialysed, during which buffered fluid exchange 5 times, last time
Dialysed overnight, label buffer are pH 9.8, the Na that concentration is 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) the corticotropin monoclonal antibody solution after through dialysing is placed in 500 μ L centrifuge tubes, is added
Enter 200 μ L label buffer, the NSP-DMAE-NHS DMF solution (being protected from light) of 759 μ L, 6.5 mmol/L is then added
Make the molar ratio 7.4:1 of acridinium ester Yu corticotropin monoclonal antibody, oscillation mixes, reacts 1 h at room temperature, add
Enter the lysine that 100 μ L concentration are 10 g/L, stand 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, the chemiluminescence intensity and 280nm for measuring efflux respectively are inhaled
Shading value.
(6) eluent that shading value is high and absorbance is big is collected, dispenses stored frozen after 1% BSA is added.
4. the preparation of corticotropin calibration object
It is 0 pg/ that corticotropin sterling, which is configured to mark concentration, with the Tris-HCl buffer containing 2% BSA
The calibration object ladder of mL, 0.5 pg/mL, 3.0 pg/mL, 18.0 pg/mL, 108.0 pg/mL, 650.0 pg/mL totally 6 concentration
Angle value.
5. the preparation of chemiluminescence exciting liquid A, B
(1) chemiluminescence preexciting liquid A is by H2O2And HNO3Composition.Wherein, wherein H2O2Mass fraction be 1.5%, HNO3It is dense
Degree is 0.1 mol/L, is distributed into 20 mL/ branch with brown bottle, 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 be 1.0%, NaOH concentration be 0.4 mol/L, be distributed into 20 mL/ branch with brown bottle, 2-8 DEG C saves backup.
6. the preparation of cleaning solution
3.05g Tris is weighed, in the beaker of 8.775g NaCl to 1000 mL, additions 1mL mass fraction is 0.05% Tween-
20, it stirs and evenly mixs, constant volume, is saved backup after pH is adjusted to 7.6.
Embodiment 2: it is detected using the magnetic microparticle chemiluminescence detection kit of the corticotropin
The step of are as follows:
(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, cleans 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 corticotropin luminous intensity proportion relation corresponding thereto in sample.
Embodiment 3: the performance indicator of kit
(1) sensitivity for analysis
0 pg/mL 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), RLU value corresponding to M+2SD is obtained, according to zero-dose school
Concentration-RLU value result between quasi- product and adjacent calibration object carries out two o'clock regression fit and obtains linear function, by M+2SD's
RLU value is brought into above-mentioned equation, and corresponding concentration value is found out.By minimum detection limit detection method in detection scheme, it is repeated 3 times
Experiment, finally finding out this kit is 0.3 pg/mL to the sensitivity for analysis of corticotropin.
(2) precision
It is the sample of (50.0 ± 5.0) pg/mL and (500.0 ± 50.0) pg/mL, Mei Genong with ACTH kit test concentrations
Degree retest 10 times calculates kit precision, the results showed that kit CV is respectively less than 5%.
(3) stability
Corticotropin detection kit is taken to carry out normal storage stability test, 2-8 DEG C of placement distinguishes temporally 1,
It is detected within 3,5,7,9,11,12,13,14,15 months；Uncap stability test respectively by 2-8 DEG C place 0 day, 7 days, 14 days,
It is measured within 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days.Corticotropin as the result is shown
Detection 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, have
The effect phase 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. the magnetic microparticle chemiluminescence detection kit and its composition of a kind of people's corticotropin, it is characterised in that: packet
Include the corticotropin inspection that coupling has the magnetic particle suspension of corticotropin capture antibody, acridinium ester label
Survey antibody, calibration object, chemiluminescence preexciting liquid A, chemiluminescence exciting liquid B and cleaning solution.
2. a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin according to claim 1 and its
Composition, it is characterised in that: the kernel of the magnetic bead is ferroso-ferric oxide or di-iron trioxide, the surface modification group of magnetic particle
For one or more activity functional groups, including but not limited to carboxyl, amino, p-toluenesulfonyl or Streptavidin-biology
Element.
3. a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin according to claim 1 and its
Composition, it is characterised in that: the magnetic particle can directly with corticotropin capture antibody coupling, or by magnetic particle with
Streptavidin coupling, while antibody is captured using biotin labeling corticotropin.
4. a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin according to claim 1 and its
Composition, it is characterised in that: the chemiluminescent labels are acridinium ester, such as NSP-DMAE-NHS, NSP-SA-NHS.
5. a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin according to claim 1 and its
Composition, it is characterised in that: the acridinium ester label is corticotropin detection antibody.
6. a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin according to claim 1 and its
Composition, it is characterised in that: the capture antibody and detection antibody is monoclonal antibody.
7. a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin according to claim 1 and its
Composition, it is characterised in that: the calibration object is that it is pure that corticotropin is added using the buffer containing BSA as matrix
The calibration object solution of series of concentrations gradient made of product configuration.
8. a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin according to claim 1 and its
Composition, it is characterised in that: the chemiluminescence preexciting liquid A is by H2O2And HNO3Mixed liquor composition, chemiluminescence exciting liquid
B is made of the mixed liquor of Triton X-100 and NaOH.
9. a kind of magnetic microparticle chemiluminescence detection kit of people's corticotropin according to claim 1 and its
Composition, comprising the following steps: a certain amount of sample to be tested is added in reaction cup, adds magnetic particle coupling suspension, mixes,
37 DEG C of incubations are added cleaning solution and remove supernatant；Acridinium ester label is added according to a certain percentage later, 37 DEG C of incubations are added
Cleaning solution removes supernatant, and chemiluminescence preexciting liquid A and exciting liquid B is added, measures corresponding luminous intensity RLU/s.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112816715A (en) * | 2020-12-30 | 2021-05-18 | 北京联众泰克科技有限公司 | Composition for detecting adrenocorticotropic hormone, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
| CN117417446A (en) * | 2022-07-18 | 2024-01-19 | 东莞市朋志生物科技有限公司 | Anti-corticotropin antibodies or functional fragments thereof, reagents and kits for detecting corticotropin |
-
2017
- 2017-10-31 CN CN201711039876.7A patent/CN109946465A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112816715A (en) * | 2020-12-30 | 2021-05-18 | 北京联众泰克科技有限公司 | Composition for detecting adrenocorticotropic hormone, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
| CN117417446A (en) * | 2022-07-18 | 2024-01-19 | 东莞市朋志生物科技有限公司 | Anti-corticotropin antibodies or functional fragments thereof, reagents and kits for detecting corticotropin |
| CN117417446B (en) * | 2022-07-18 | 2024-04-19 | 东莞市朋志生物科技有限公司 | Anti-corticotropin antibodies or functional fragments thereof, reagents and kits for detecting corticotropin |
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