CN108548924A - AngiotensinⅡ Magnetism particulate immuno chemistry luminescence method detection kit and detection method - Google Patents
AngiotensinⅡ Magnetism particulate immuno chemistry luminescence method detection kit and detection method Download PDFInfo
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- CN108548924A CN108548924A CN201810297607.9A CN201810297607A CN108548924A CN 108548924 A CN108548924 A CN 108548924A CN 201810297607 A CN201810297607 A CN 201810297607A CN 108548924 A CN108548924 A CN 108548924A
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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
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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Abstract
The invention discloses Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kits, including Streptavidin magnetic particle Magneto separate reagent, biotinylated Angiotensin II monoclonal antibody, alkaline phosphatase target Angiotensin II derivative, calibration object.The kit has the advantages that high sensitivity, specificity, specificity.The invention also discloses the detection method of Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit, this method is simple, avoids radioactive pollution, extends the reagent term of validity.
Description
Technical field
The invention belongs to chemical diagnosis reagent technique field, more particularly to a kind of Magnetism particulate immuno chemistry luminescence method angiotensins
II assay kits and detection method.
Background technology
Feritin generates angiotensin I by shearing the proangiotensin of liver synthesis.Angiotensin I can be further
Angiotensin II is cut by angiotensin converting enzyme (AngiotensinConVertingEnzyme, ACE)
(Angiotensin II)。
Angiotensin II is most important component part in angiotensins.Vascular smooth machine, the adrenal gland skin of human body
There are angiotensin receptors on the cell at some positions of matter glomerular zone cell and brain, heart and kidney organ.Blood vessel
Angiotensin Converting Enzyme II is combined with angiotensin receptor, causes corresponding physiological effect.Angiotensins vasoactive smooth muscle, can
Whole body arteriole is set to shrink, arterial pressure increases.Angiotensin II is one of known strongest Vasoconstrictor.Effect
In peripheral blood vessel, vein is made to shrink, returned blood volume increases, and acts on maincenter, causes to feel yearningly.The measurement of Angiotensin II can answer
For in the treatment and detection of hypertension.
Angiotensin II is the direct working substance that renin-angiotensin-aldosterone system adjusts blood pressure.Together
When Angiotensin II can be used as the hyperplasia that somatomedin directly facilitates vascular endothelial cell.It is nearest the study found that
Angiotensin II is directly and vascular endothelial proliferation, hemadostewnosis and arteries resistance increase have direct relation.Therefore,
The detection of Angiotensin II is increasingly paid attention to, and the detection of the auxiliary diagnosis and therapeutic effect of hypertension is clinically used for.
The detection method of detection Angiotensin II is radiating immuning analysis technology and chemoluminescence method at present, wherein radiating
Immuno analytical method has radioactive pollution, the defect more than operating procedure, and chemoluminescence method has high sensitivity, specificity
Well, the advantages of being simple to operate and friendly to environment, but chemoluminescence method includes Streptavidin magnetic microparticle chemiluminescence immune assay
Technology and fluorescein isothiocynate magnetic microparticle chemiluminescence immune assay technology, though the two all have high sensitivity, specificity it is good,
The advantage being simple to operate and friendly to environment, but fluorescein isothiocynate magnetic particle chemiluminescence immunoassay technology is detecting
Its sensitivity and specificity still needs to further increase when Angiotensin II.
Invention content
In view of the problems of the above-mentioned prior art, the purpose of the present invention is to propose to the good one kind of high sensitivity, specificity
Magnetism particulate immuno chemistry luminescence method Angiotensin II assay kit and detection method.
The purpose of the present invention will be achieved by the following technical programs：
Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit, including Magneto separate reagent, biotinylated blood vessel
Angiotensin Converting Enzyme II monoclonal antibodies, alkaline phosphatase target Angiotensin II derivative, calibration object；The Magneto separate reagent is chain
Mould Avidin magnetic particle.
Preferably, a concentration of 0.5-2.0ug/ml of the biotinylated Angiotensin II monoclonal antibody, it is described
A concentration of 0.1-1.0ug/ml of alkaline phosphatase target Angiotensin II derivative.
The preferably described biotinylated Angiotensin II monoclonal antibody is prepared by following steps：1) in blood vessel
Biotin-NHS is added in Angiotensin Converting Enzyme II monoclonal antibodies, reacts at room temperature 1.5-2.5h after mixing well, obtains reaction product；2) make
The reaction product dialysed described in step 1) by bag filter with elution buffer, obtains dialysate；It 3) will with anti-reagent buffer
Dialysate described in step 2) be diluted to 0.5ug/ml to get.
Preferably, the molecular weight of biotinylated Angiotensin II monoclonal antibody described in step 1) is 30000, institute
The molecular weight for stating biotin-NHS is 587；
The biotinylated Angiotensin II monoclonal antibody and the molar ratio of the biotin-NHS are 1: 18-
22；
Biotin-the NHS is the biotin-NHS that a concentration of 5mg/ml is dissolved to by DMSO.
Preferably, elution buffer described in step 2) is the 0.15M PBS buffer solution of pH=7.5,
The molecular cut off of the bag filter is 500-1000；
Anti- reagent buffer described in step 3) is pure by three (methylol) aminomethanes, sodium chloride, Sodium azide, ox blood
Albumen, cow's serum, horse serum composition.
Preferably, the preparation method of the alkaline phosphatase target Angiotensin II derivative is as follows：1) blood vessel is tight
It opens element II derivatives to be dialysed with 0.1M PBS, then be activated with 2IT；2) alkali phosphorus enzyme is activated with SMCC；3)
By after activation Angiotensin II derivative and alkaline phosphatase mix, 2-8 ° react 18 hours, obtain reactant；4) will
Reactant is purified with chromatographic column, and the peaks I and the peaks II are collected by collection of illustrative plates；5) blood for being prepared step 4) with anti-reagent buffer
The dilution of angiotensin II derivatives-alkaline phosphatase to get.
Preferably, the calibration object is prepared by following steps：By Angiotensin II sterling Angiotensin II
Calibration object buffer solution be diluted concentration be respectively 0pg/ml, 25pg/ml, 100pg/ml, 250pg/ml, 500pg/ml and
The Angiotensin II solution of 1000pg/ml to get.
Preferably, the Angiotensin II calibration object buffer solution by pH=7.4 0.05M Tris buffer solutions, 0.5%
Bovine serum albumin(BSA), 0.2% preservative composition.
A kind of detection method of kit, includes the following steps：First by calibration object or sample, biotinylated vasotonia
Plain II monoclonal antibodies, the mixing of alkaline phosphatase target Angiotensin II derivative, are incubated 15-30min；Add strepto- parent
With biscuit porcelain pearl, it is incubated 5-10min；Magneto separate 1-2min, removes supernatant；Wash liquid 3 times；Add substrate, measured value.
Preferably, the sample-adding amount of the calibration object or sample is 30 μ l, biotinylated Angiotensin II monoclonal resists
Body sample-adding amount is 30 μ l, the sample-adding amount of alkaline phosphatase target Angiotensin II derivative is 30 μ l；
The sample-adding amount of the Streptavidin MagneSphere is 30 μ l；The sample-adding amount of the washing lotion is 300 μ l；The substrate adds
Sample amount is 200 μ l.
Compared with prior art, a kind of Magnetism particulate immuno chemistry luminescence method Angiotensin II assay kit provided by the invention
And preparation method, what is reached has the technical effect that：1) preparation method of kit of the present invention is simple and convenient, and the detection of kit is sensitive
Degree is higher, has a good application prospect；2) kit of the present invention is for when detecting, entire reaction system to avoid radioactivity dirt
Dye, extends the reagent term of validity, simplifies experimental implementation flow.3) since the combination between Avidin and biotin is with high
Therefore its reaction of affinity while improving sensitivity, does not increase nonspecific interference, Er Qiejie in high specificity
Closing characteristic will not be impacted because of the high dilution of reaction reagent, and reaction examination can be reduced to the maximum extent by making it in practical applications
The non-specific effect of agent.
Description of the drawings
Fig. 1 is that strepto- is affine for 2 biotinylated Angiotensin II monoclonal antibody-of embodiment in test example 1 of the present invention
The curve matching figure of biscuit porcelain pearl detection method；
Fig. 2 is the magnetic particle of the 1 coated carboxylated of Angiotensin II monoclonal antibody of comparative example in test example 1 of the present invention
The curve matching figure of detection method.
Below just in conjunction with the embodiments, the embodiment of the present invention is described in further detail, so that technical solution is more
It should be readily appreciated that, grasp.
Specific implementation mode
Below by specific embodiment, the present invention will be described, but the present invention is not limited thereto.In following embodiments
The experimental method is unless otherwise specified conventional method；The reagent and material unless otherwise specified can be from business
Approach obtains, and following example is not to limit the scope of the claims of the present invention, all equivalence enforcements without departing from carried out by the present invention
Or change, it is intended to be limited solely by the scope of this patent.
1 Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit of embodiment
The kit of the present embodiment includes the Magneto separate reagent of Streptavidin magnetic particle, biotinylated angiotensins
II monoclonal antibodies, alkaline phosphatase target Angiotensin II derivative, calibration object.
The Angiotensin II antibody used in the present embodiment is monoclonal antibody, and antibody is anti-for mouse, calibration object and sample
In include antigen to be checked.
1, biotinylated Angiotensin II monoclonal antibody preparation process is as follows：
1) 1mg Angiotensin II monoclonal antibodies are measured；
2 carried out by Angiotensin II monoclonal antibody molecule amount (30000) and biotin-NHS molecular weight (587) in terms of
It calculates, biotin-NHS is weighed according to the inventory of molar ratio 1: 20；
3) biotin-NHS for weighing 0.4mg or so is used in combination DMSO to carry out being dissolved to final concentration of 5mg/ml；
4) biotin-NHS of DMSO dissolvings is added in 1mg Angiotensin II monoclonal antibodies, mixes well rear chamber
Temperature reaction 2h；
5) reaction product in the bag filter dialysis step 4) that molecular cut off is 500, elution buffer pH=7.5 are used
0.15M PBS buffer solution；
6) it uses anti-reagent buffer that mixture is diluted to 0.5ug/ml to use as working solution.
Wherein, anti-reagent buffer is by three (methylol) aminomethanes, sodium chloride, Sodium azide, bovine serum albumin(BSA), ox blood
Clearly, horse serum forms.
2, Angiotensin II derivative enzyme marking reagent preparation process is as follows：
Angiotensin II derivative is dialysed with 0.1M PBS, is then activated with 2IT；
Alkaline phosphatase is activated with SMCC；
By after activation Angiotensin II derivative and alkaline phosphatase mix, 2-8 ° react 18 hours；
Reactant after Angiotensin II derivative is connected with alkaline phosphatase is purified with chromatographic column, is passed through
Collection of illustrative plates collects the peaks I and the peaks II；
Angiotensin II derivative-alkaline phosphatase prepared by step 4) will be diluted to anti-reagent buffer
0.1ug/ml to get.
3, calibration object preparation process is as follows：
1) Angiotensin II calibration object buffer solution is prepared：It is added 0.5% N in the 0.05M Tris buffer solutions of pH=7.4
Seralbumin, 0.2% preservative；
2) by Angiotensin II sterling with Angiotensin II calibration object buffer solution be diluted to concentration be respectively 0pg/ml,
The Angiotensin II solution of 25pg/ml, 100pg/ml, 250pg/ml, 500pg/ml and 1000pg/ml, should to get calibration object
Calibration object form is liquid and with good stability.
The detection method of kit prepared by 2 embodiment 1 of embodiment
First by calibration object or sample 30ul, biotinylated Angiotensin II monoclonal antibody 30ul, alkaline phosphatase
The 30ul mixing of target Angiotensin II derivative enzyme marking reagent, is incubated 30min；
Add Magneto separate reagent (Streptavidin MagneSphere) 30ul, is incubated 5min；
Magnetic separator detaches 2min, removes supernatant；
Washing 3 times, each 300ul washing lotions；
Add substrate 200ul, measured value.
The detection method of 1 fluorescein isothiocynate Magnetism particulate immuno chemistry luminescence method detection kit of comparative example
First by calibration object or the coated Angiotensin II monoclonal antibody 30ul of sample 30ul, FITC, alkaline phosphatase
The Angiotensin II derivative enzyme marking reagent 30ul mixing of label, is incubated 30min；
Add Magneto separate reagent (carboxyl magnetic bead) 30ul, is incubated 5min；
Magnetic separator detaches 2min, removes supernatant；
Washing 3 times, each 300ul washing lotions；
Add substrate 200ul, measured value.
1 Streptavidin magnetic microparticle chemiluminescence immune assay technology of test example
This test example 1 is the detection method of the detection method and the comparative example 1 that use embodiment 2 simultaneously respectively to a series of
Concentration：The Angiotensin II calibration object of 0pg/ml, 25pg/ml, 100pg/ml, 250pg/ml, 500pg/ml and 1000pg/ml
It is detected, then obtains respectively to value, and be compared, curve matching such as Fig. 1, Fig. 2；
Wherein, the detection method of embodiment 2 is named as biotinylated Angiotensin II monoclonal antibody-strepto- is affine
Biscuit porcelain pearl detection method；The magnetic that the detection method of comparative example 1 is named as the coated carboxylated of Angiotensin II monoclonal antibody is micro-
Grain detection method.
The comparison of the sensitivity (table 1) of embodiment 2 and comparative example 1, precision (table 2) and serologic test (table 3).
The sensitivity of table 1 embodiment 2 and comparative example 1
The precision of table 2 embodiment 2 and comparative example 1
3 embodiment 1 of table and 1 measured value of comparative example and to value comparison and measured value between compare
It can be seen from the results that different system measured values, all 0.9 or more, illustrate that correlation is good with to value correlation, and
Measured value correlation is more than 0.9 between different systems, illustrates that system 1 is consistent with 2 measured value of system.
Several preferred embodiments of the present invention have shown and described in above description, but as previously described, it should be understood that the present invention
Be not limited to form disclosed herein, be not to be taken as excluding other embodiments, and can be used for various other combinations,
Modification and environment, and the above teachings or related fields of technology or knowledge can be passed through in the scope of the invention is set forth herein
It is modified.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then it all should be in this hair
In the protection domain of bright appended claims.
Claims (10)
1. Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit, which is characterized in that including Magneto separate reagent, biotin
The Angiotensin II monoclonal antibody of change, alkaline phosphatase target Angiotensin II derivative, calibration object；The Magneto separate
Reagent is Streptavidin magnetic particle.
2. Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit according to claim 1, which is characterized in that institute
State a concentration of 0.5-2.0ug/ml of biotinylated Angiotensin II monoclonal antibody, the alkaline phosphatase target blood vessel
A concentration of 0.1-1.0ug/ml of Angiotensin Converting Enzyme II derivatives.
3. Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit according to claim 2, which is characterized in that institute
Biotinylated Angiotensin II monoclonal antibody is stated to be prepared by following steps：1) anti-in Angiotensin II monoclonal
Biotin-NHS is added in body, reacts at room temperature 1.5-2.5h after mixing well, obtains reaction product；2) passed through using elution buffer
The reaction product that bag filter is dialysed described in step 1), obtains dialysate；3) with anti-reagent buffer by the dialysis described in step 2)
Object be diluted to 0.5ug/ml to get.
4. Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit according to claim 3, which is characterized in that step
It is rapid 1) described in biotinylated Angiotensin II monoclonal antibody molecular weight be 30000, the biotin-NHS point
Son amount is 587；
The biotinylated Angiotensin II monoclonal antibody and the molar ratio of the biotin-NHS are 1: 18-22；
Biotin-the NHS is the biotin-NHS that a concentration of 5mg/ml is dissolved to by DMSO.
5. Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit according to claim 3, which is characterized in that step
It is rapid 2) described in elution buffer be pH=7.5 0.15M PBS buffer solution,
The molecular cut off of the bag filter is 500-1000；
Anti- reagent buffer described in step 3) is by three (methylol) aminomethanes, sodium chloride, Sodium azide, bovine serum albumin
In vain, cow's serum, horse serum composition.
6. Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit according to claim 2, which is characterized in that institute
The preparation method for stating alkaline phosphatase target Angiotensin II derivative is as follows：1) by Angiotensin II derivative 0.1M
PBS dialyses, and is then activated with 2IT；2) alkali phosphorus enzyme is activated with SMCC；3) by the angiotensins after activation
II derivatives and alkaline phosphatase are mixed, and 2-8 ° is reacted 18 hours, and reactant is obtained；4) reactant is carried out with chromatographic column pure
Change, the peaks I and the peaks II are collected by collection of illustrative plates；5) the Angiotensin II derivative-alkali for being prepared step 4) with anti-reagent buffer
Acid phosphatase dilution to get.
7. Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit according to claim 1, which is characterized in that institute
Calibration object is stated to be prepared by following steps：Angiotensin II sterling is carried out with Angiotensin II calibration object buffer solution dilute
Release the angiotensins that concentration is respectively 0pg/ml, 25pg/ml, 100pg/ml, 250pg/ml, 500pg/ml and 1000pg/ml
II solution to get.
8. Angiotensin II Magnetism particulate immuno chemistry luminescence method detection kit according to claim 7, which is characterized in that institute
Angiotensin II calibration object buffer solution is stated by the 0.05M Tris buffer solutions of pH=7.4,0.5% bovine serum albumin(BSA), 0.2%
Preservative forms.
9. a kind of detection method according to claim 1-8 any one of them kits, which is characterized in that including walking as follows
Suddenly：First by calibration object or sample, biotinylated Angiotensin II monoclonal antibody, alkaline phosphatase target angiotensins
II derivatives mix, and are incubated 15-30min；Streptavidin MagneSphere is added, 5-10min is incubated；Magneto separate 1-2min, goes
Clearly；Wash liquid 3 times；Add substrate, measured value.
10. the detection method of kit according to claim 9, which is characterized in that the sample-adding of the calibration object or sample
Amount is 30 μ l, biotinylated Angiotensin II monoclonal antibody sample-adding amount is 30 μ l, alkaline phosphatase target vasotonia
The sample-adding amount of plain II derivatives is 30 μ l；
The sample-adding amount of the Streptavidin MagneSphere is 30 μ l；The sample-adding amount of the washing lotion is 300 μ l；The sample-adding amount of the substrate
For 200 μ l.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175493A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Magnetic particle chemiluminescence method human cortisol determination kit and use method thereof |
| CN111208290A (en) * | 2020-02-27 | 2020-05-29 | 江苏泽成生物技术有限公司 | Magnetic particle chemiluminescence aldosterone determination kit and use method thereof |
| CN112816694A (en) * | 2020-12-31 | 2021-05-18 | 泰州泽成生物技术有限公司 | Abnormal prothrombin detection kit and preparation method thereof |
| CN114236115A (en) * | 2021-12-13 | 2022-03-25 | 泰州泽成生物技术有限公司 | Kit for determining alpha tumor necrosis factor by magnetic particle chemiluminescence method and preparation method thereof |
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| CN103513037A (en) * | 2013-08-09 | 2014-01-15 | 北京润诺思医疗科技有限公司 | chemiluminescence immunoassay kit and preparation method thereof |
| CN104614537A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Detection kit for angiotensin II and preparation method and application of detection kit |
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- 2018-03-30 CN CN201810297607.9A patent/CN108548924A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103513037A (en) * | 2013-08-09 | 2014-01-15 | 北京润诺思医疗科技有限公司 | chemiluminescence immunoassay kit and preparation method thereof |
| CN104614537A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Detection kit for angiotensin II and preparation method and application of detection kit |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175493A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Magnetic particle chemiluminescence method human cortisol determination kit and use method thereof |
| CN111208290A (en) * | 2020-02-27 | 2020-05-29 | 江苏泽成生物技术有限公司 | Magnetic particle chemiluminescence aldosterone determination kit and use method thereof |
| CN112816694A (en) * | 2020-12-31 | 2021-05-18 | 泰州泽成生物技术有限公司 | Abnormal prothrombin detection kit and preparation method thereof |
| CN114236115A (en) * | 2021-12-13 | 2022-03-25 | 泰州泽成生物技术有限公司 | Kit for determining alpha tumor necrosis factor by magnetic particle chemiluminescence method and preparation method thereof |
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Application publication date: 20180918 |