CN107941790A - Magnetism particulate immuno chemistry luminescence method measures the kit and its detection method of feritin - Google Patents
Magnetism particulate immuno chemistry luminescence method measures the kit and its detection method of feritin Download PDFInfo
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- CN107941790A CN107941790A CN201711218385.9A CN201711218385A CN107941790A CN 107941790 A CN107941790 A CN 107941790A CN 201711218385 A CN201711218385 A CN 201711218385A CN 107941790 A CN107941790 A CN 107941790A
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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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Abstract
The present invention provides a kind of kit of Magnetism particulate immuno chemistry luminescence method measure feritin, including biotinylation renin antibody, enzyme-labeled antibody reagent, Streptavidin magnetic particle and calibration object；By the detection architecture for building biotinylation renin antibody+enzyme-labeled antibody reagent+calibration object, it is compared with the magnetic particle of the coated carboxylated of feritin monoclonal antibody, correlation between two systems is 0.9508, illustrate that the measured value of two kinds of systems is consistent, it was demonstrated that the feasibility of technical scheme.The kit of the present invention avoids radioactive pollution, extends the reagent effect phase, simplifies experimental implementation flow.Combination between Avidin and biotin has high affinity, its reaction is in high specificity.Therefore, while sensitivity is improved, nonspecific interference is not increased.And binding characteristic will not be impacted because of the high dilution of reaction reagent, it is set to reduce the non-specific effect of reaction reagent to greatest extent in practical applications.
Description
Technical field
The present invention relates to a kind of detection kit, be specially a kind of Magnetism particulate immuno chemistry luminescence method measure feritin kit and
Its detection method.
Background technology
Feritin (renin) is glomerular, a kind of proteolytic enzyme of juxtaglomerular cell release, mainly by thin by glomerulus
Intracrine, the effect for the Angiotensin-Converting being released into pulmonary circulation are converted into angiotensinⅡ.AngiotensinⅡ
Parteriole smooth muscle contraction can directly be made, peripheral resistance increase, so that tubular distal concetrated pipe sodium strengthens again, causes body
Interior water and sodium retention.AngiotensinⅡ and converting Enzyme etc. are frequently present in blood plasma, therefore the release of feritin is to determine blood plasma
The critical of Angiotensin-Converting concentration.
The detection of plasma renin content, available for diagnosis due to hypertension caused by renal artery stenosis or the high blood of renovascular
Pressure, helps clinician to decide whether to carry out the iconography research of Renal vascular, diagnosing primary aldosteronism, Yi Jiwei
The generation of the complication of Essential Hypertensive Patients cardiovascular system provides effective information etc..In addition for direction of medication usage and
The evaluation of therapeutic effect has very important significance, for example, being substituted lowly and using steroid hormone for some adrenal functions
The patient for the treatment of, when therapeutic effect is sufficient, renin level is normal, and when therapeutic effect is insufficient, renin level is excessive.
Hypertension is most common angiocardiopathy, its incidence rises year by year, and can cause the serious heart, brain, kidney simultaneously
Disease is sent out, is cerebral apoplexy and the Major Risk Factors of coronary heart disease.The detection of plasma renin content, available for office hypertension, diagnosis
Primary aldosteronism, and provide effective letter for the complication of Essential Hypertensive Patients cardiovascular system
Breath etc..In addition it can be additionally used in the prognosis evaluation of hyperpietic.
The common methods of clinical detection feritin have enzyme linked immunosorbent assay, radio immunoassay, enzymatic chemistry hair at present
Light method, but these methods, all there is some shortcomings, flow is relatively complicated, and sensitivity has much room for improvement.
The content of the invention
The technical problem to be solved in the present invention is overcoming, existing feritin testing process is relatively complicated, and sensitivity needs to be carried
The defects of high, there is provided a kind of kit and its detection method of Magnetism particulate immuno chemistry luminescence method measure feritin.
In order to solve the above technical problem, the present invention provides following technical solution：
A kind of kit of Magnetism particulate immuno chemistry luminescence method measure feritin, the kit include biotinylation renin antibody, resist
Body enzyme marking reagent, Streptavidin magnetic particle, calibration object；
Biotinylation renin antibody preparation process in the kit:
After biotin-NHS is dissolved with DMSO, renin antibody, mole of renin antibody and biotin-NHS are added
Than for 1:18~22, react at room temperature 2h；With bag filter hemodialysis reaction product, dialyzate is diluted to working solution concentration；
The enzyme-labeled antibody reagent preparation process：
Will detection renin antibody and after alkaline phosphatase activates respectively, by the two mix 2-8 DEG C of 14~20h of reaction it is small when；
Reactant is purified with chromatographic column, collects and will be configured to working solution concentration with buffer solution after I peaks II peaks are mixed；
Further, renin antibody is detected in the enzyme-labeled antibody reagent preparation process to dialyse using the PBS of 0.1M,
Then activated with 2IT, the antibody after being activated；The enzyme-labeled antibody reagent preparation process alkaline phosphatase is used
SMCC is activated, the alkaline phosphatase after being activated.
Further, the working concentration of biotinylation renin antibody is 0.5ug/mL in kit, enzyme-labeled antibody reagent
Working concentration is 0.1ug/mL.
Further, the preparation method of calibration object is：0.5% is added in the TRIS buffer solutions for being 7.4 to the pH of 0.05M
Bovine serum albumin(BSA) and 0.2% preservative, which are made, prepares feritin calibration object buffer solution；Then use feritin calibration object buffer solution will
The feritin that feritin sterling is diluted to 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml and 2000pg/ml is molten
Liquid.
The application method of the kit of the present invention, comprises the following steps：
1) this 15ul, biotinylation renin antibody 30ul and enzyme-labeled antibody examination 30ul are sampled, is incubated 15min
2) Streptavidin MagneSphere 30ul is added to 1) middle, is incubated 5min；
3) Magneto separate 2min, removes supernatant；
4) wash 3 times, each 300ul washing lotions；
5) substrate 200ul, measured value are added.
It is coated with the same time using biotinylated renin antibody-Streptavidin MagneSphere (system 1) and feritin monoclonal antibody
Carboxylated magnetic particle (system 2), two systems are to 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml
Detected with the feritin sample of 2000pg/ml, while test same group of sample, and be compared with to value, the correlation of system 1
Property be 0.9767, the correlation of system 2 is 0.9397, illustrates that the correlation of two systems is all good, between two kinds of measure systems
Correlation be 0.9508, illustrate that the measured value of two kinds of systems is consistent, it was demonstrated that the feasibility of technical scheme.The present invention
Kit avoid radioactive pollution, extend reagent effect the phase, simplify experimental implementation flow.Between Avidin and biotin
Combination there is high affinity, its reaction is in high specificity.Therefore, while sensitivity is improved, do not increase non-
Specificity interference.And binding characteristic will not be impacted because of the high dilution of reaction reagent, makes it in practical applications can be most
Reduce to limits the non-specific effect of reaction reagent.
Embodiment
The preferred embodiment of the present invention is illustrated below, it will be appreciated that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
Embodiment
First, biotinylation renin antibody preparation process:
1) 1mg renin antibodies are measured；
2) calculated by antigen molecular (30000) and biotin-NHS molecular weight (587), according to molar ratio 1：
20 inventory weighs biotin, weighs the biotin-NHS of 0.4mg or so, and with DMSO be dissolved to final concentration of
5mg/mL, obtains the biotin of DMSO dissolvings；
3) biotin of DMSO dissolvings is added in antibody, 2h is reacted at room temperature after fully mixing；
4) reaction product in step 3) of being dialysed with the bag filter that molecular cut off is 500, elution buffer PH=
The PBS buffer of 7.5 0.15M；
5) dialyzate is diluted to 0.5ug/ml by the use of anti-reagent buffer to use as working solution；
2nd, enzyme-labeled antibody reagent preparation process：
1) feritin mouse monoclonal antibody is dialysed with the PBS of 0.1M, is then activated with 2IT, it is anti-after being activated
Body；
2) alkaline phosphatase is activated with SMCC, the alkaline phosphatase after being activated；
3) antibody after activation is mixed with the alkaline phosphatase after activation, when 2-8 DEG C of reaction 18 is small；
4) reactant is purified with chromatographic column, collecting will be mixed after I peaks II peaks are mixed with anti-reagent buffer
Antibody-alkali phosphorus enzyme in liquid is diluted to 0.1ug/ml as working solution；
3rd, the preparation process of calibration object；
1) feritin calibration object buffer solution is prepared：The BSA of addition 5% in the PBS buffer of the PH=7.4 of 0.05M, 0.2%
Preservative；
2) feritin antigen is diluted with calibration object buffer solution, be followed successively by：0pg/ml、50pg/ml、250pg/ml、
The feritin solution of 500pg/ml, 1000pg/ml and 2000pg/ml.
Kit using the present invention is detected, and is comprised the following steps：
1) this 15ul, biotinylation renin antibody 30ul and enzyme-labeled antibody examination 30ul are sampled, is incubated 15min
2) Streptavidin MagneSphere 30ul is added to 1) middle, is incubated 5min；
3) Magneto separate 2min, removes supernatant；
4) wash 3 times, each 300ul washing lotions；
5) substrate 200ul, measured value are added.
It is coated with the same time using biotinylated renin antibody-Streptavidin MagneSphere (system 1) and feritin monoclonal antibody
Carboxylated magnetic particle (system 2), two systems are to 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml
Detected with the feritin sample of 2000pg/ml, while test same group of sample, and be compared with to value, data such as following table institute
Show：
Under the different systems of table 1 measured value and to value contrast and measured value between contrast
The correlation of system 1 is 0.9767, and the correlation of system 2 is 0.9397, illustrates that the correlation of two systems is all good
Good, the correlation between two kinds of measure systems is 0.9508, illustrates that the measured value of two kinds of systems is consistent, it was demonstrated that technology of the invention
The feasibility of scheme.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To modify to the technical solution described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent replacement, improvement and so on, should be included in the present invention's
Within protection domain.
Claims (6)
1. a kind of kit of Magnetism particulate immuno chemistry luminescence method measure feritin, it is characterised in that the kit includes biotinylation kidney
Plain antibody, enzyme-labeled antibody reagent, Streptavidin magnetic particle, calibration object；
Biotinylation renin antibody preparation process in the kit:
After biotin-NHS is dissolved with DMSO, renin antibody is added, the molar ratio of renin antibody and biotin-NHS is
1:18~22, react at room temperature 2h；With bag filter hemodialysis reaction product, dialyzate is diluted to working solution concentration；
The enzyme-labeled antibody reagent preparation process：
Will detection renin antibody and after alkaline phosphatase activates respectively, by the two mix 2-8 DEG C of 14~20h of reaction it is small when；Will be anti-
Answer thing to be purified with chromatographic column, collect and will be configured to working solution concentration with buffer solution after I peaks II peaks are mixed.
2. the preparation method of kit as claimed in claim 1, it is characterised in that in the enzyme-labeled antibody reagent preparation process
Detection renin antibody is dialysed using the PBS of 0.1M, is then activated with 2IT, the antibody after being activated.
3. the preparation method of kit as claimed in claim 1 or 2, it is characterised in that prepared by the enzyme-labeled antibody reagent
Journey alkaline phosphatase is activated with SMCC, the alkaline phosphatase after being activated.
4. the preparation method of kit as claimed in claim 1, it is characterised in that biotinylation renin antibody in kit
Working concentration is 0.5ug/mL, and the working concentration of enzyme-labeled antibody reagent is 0.1ug/mL.
5. the preparation method of kit as claimed in claim 1, it is characterised in that the preparation method of calibration object is：To 0.05M
PH be 7.4 TRIS buffer solutions in add 0.5% bovine serum albumin(BSA) and 0.2% preservative be made prepare feritin calibration object
Buffer solution；Then feritin sterling is diluted to 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ using feritin calibration object buffer solution
The feritin solution of ml, 1000pg/ml and 2000pg/ml.
6. a kind of application method of kit as claimed in claim 1, it is characterised in that comprise the following steps：
1) this 15ul, biotinylation renin antibody 30ul and enzyme-labeled antibody examination 30ul are sampled, is incubated 15min
2) Streptavidin MagneSphere 30ul is added to 1) middle, is incubated 5min；
3) Magneto separate 2min, removes supernatant；
4) wash 3 times, each 300ul washing lotions；
5) substrate 200ul, measured value are added.
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| CN201711218385.9A CN107941790A (en) | 2017-11-28 | 2017-11-28 | Magnetism particulate immuno chemistry luminescence method measures the kit and its detection method of feritin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187972A (en) * | 2018-08-17 | 2019-01-11 | 迪瑞医疗科技股份有限公司 | A kind of magnetic microparticle chemiluminescence kit of quantitative detection plasma renin content and preparation method thereof |
| CN113533734A (en) * | 2021-07-22 | 2021-10-22 | 泰州泽成生物技术有限公司 | Kit for detecting Golgi protein 73 by magnetic particle chemiluminescence method and preparation method thereof |
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Application publication date: 20180420 |