CN109239329A - A kind of insulin immue quantitative detection reagent box - Google Patents
A kind of insulin immue quantitative detection reagent box Download PDFInfo
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- CN109239329A CN109239329A CN201811213292.1A CN201811213292A CN109239329A CN 109239329 A CN109239329 A CN 109239329A CN 201811213292 A CN201811213292 A CN 201811213292A CN 109239329 A CN109239329 A CN 109239329A
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- Prior art keywords
- insulin
- quantitative detection
- detection reagent
- reagent box
- specific antibody
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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
- G01N33/531—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
-
- 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
-
- 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
- G01N33/543—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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- Urology & Nephrology (AREA)
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- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
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- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of insulin immue quantitative detection reagent box, the magnetic particle suspension including insulin specific antibody label, the horseradish peroxidase of insulin specific antibody label, the standard items of gradient concentration；The insulin specific antibody and magnetic particle are covalently attached with two step cross-linking method of glutaraldehyde.The chemiluminescence immune assay that the present invention uses is the analytical technology for combining chemical luminescent detecting with immune response, the kit of preparation has high sensitivity, high specificity, the range of linearity is wide, reaction time is short, stable reagent nonhazardous, it is reproducible, it is fast to analyze speed, the features such as automatic detection, the methods of relative immunity electrophoresis, immuno-precipitation reduce the requirement to technical staff, the influence to result such as different technologies personnel, environmental change is reduced, reduces the pollution to environment relative to radioimmunology, there is good promotion prospect.
Description
Technical field
The present invention relates to the measurements of actrapid monotard, use chemiluminescent immunoassay method to pancreas islet more particularly, to a kind of
Element carries out the kit of quantitative detection.
Background technique
Actrapid monotard is a kind of peptide hormone by islet β cell, and molecular mass is about 6000 dalton, is to adjust
The critical hormone of glucose metabolism in human body is saved, on the one hand inhibits fat, protein hydrolysis, inhibits hepatic glycogenolytic and saccharic new
It is raw, on the other hand promote utilization, the lipid synthesis of glucose.
The detection of insulin is mainly used for: 1) early diabetes screening；2) diagnosis and parting of diabetes；3) islet function
Assessment；4) diagnosis etc. of insulinoma.Clinically have to the detection of insulin to diagnosis, the treatment detection etc. of glycometabolism disease
Important reference significance.
Receive the patient of exogenous insulin treatment, the immunology detection of serum insulin is vulnerable to the dry of exogenous insulin
It disturbs, causes results abnormity, need to analyze result according to patient medication situation at this time.
Currently used enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA)
Using macromolecular enzyme mark, and enzyme be easy inactivation, it is this by interference suffered by colorimetric method or the detection technique of polarized light approach because
Plain too many, sensitivity, linear and stability is not high.
Summary of the invention
The purpose of the present invention is to provide a kinds to carry out quantitative inspection to insulin using chemiluminescent immunoassay method
The kit of survey.
To achieve the above object, the present invention can take following technical proposals:
Insulin immue quantitative detection reagent box of the present invention, the magnetic particle suspension including insulin specific antibody label,
The horseradish peroxidase of insulin specific antibody label, the standard items of gradient concentration；The insulin specific antibody with
Magnetic particle is covalently attached with two step cross-linking method of glutaraldehyde.
The magnetic particle is the ferroferric oxide particle that surface is covered with carboxyl or amino active group, and particle size is
0.1μm-5μm。
Insulin antigen of the standard items of the gradient concentration respectively containing 0,5,20,50,100,300 μ IU/ml, it is used
Matrix is that PH7.35-7.45 contains 1%BSA, 50% calf serum, the Tris buffer of 0.1% Proclin-300.
The kit further includes alkaline rinse.
The kit further includes substrate A liquid and substrate B liquid；The substrate A liquid is luminol, and substrate B liquid is peroxidating
Hydrogen.
Using the method for kit measurement insulin of the present invention are as follows:
Step 1: by the insulin specific antibody being marked on magnetic particle, another plant of pancreas of horseradish peroxidase-labeled
Island element specific antibody, sample to be tested mixing, sufficiently react, form sandwich complex；
Step 2: it is washed, the ingredient that unreacted forms sandwich complex is eluted；
Step 3: substrate is added, horseradish peroxidase enzyme catalytic substrate shines, and acquires luminous signal value with photodetector；
Step 4: preparing the standard items of gradient concentration, dosage is drawn according to suitable mathematical model with luminous signal value and concentration value
Response curve determines insulin concentration in sample.
Measuring method of the present invention includes immune response and two parts of enzyme-catalyzed chemical luminescence, is resisted with one plant of insulin specificity
Body is marked on immobilization carrier, to be detected in another plant of insulin specific antibody label horseradish peroxidase, with sample
Insulin forms sandwich complex by immune response, and using isolation technics, sandwich complex is separated with free enzyme conjugates,
According to the horseradish peroxidase enzyme catalytic substrate in sandwich complex, luminous intensity quantifies insulin in sample to be tested
Detection.
The present invention selects magnetic particle as solid phase carrier, after the completion of reaction, can pass through washing with magnet attraction
It is quick and convenient and rapidly separate free enzyme conjugates.In kit of the present invention, the magnetic particle (solid phase carrier) is used
Surface is covered with the ferroso-ferric oxide of carboxyl or amino active group, and particle size is 0.1 μm -5 μm, its main feature is that reaction surface
For product than board-like 20-30 times of expansion, adsorption efficiency is high；And uniform suspension is formed in a liquid, reaction is participated in similar liquid phase,
Greatly speed up reaction speed.
Chemiluminescence basic principle of the present invention is: horseradish peroxidase enzyme catalytic hydrogen peroxide decomposes, and generates water
With single oxygen, luminol is transformed into excitation state after the oxidation of single oxygen, issues the photon for detection.
The chemiluminescence immune assay that the present invention uses is the analysis skill for combining chemical luminescent detecting with immune response
The kit of art, preparation has high sensitivity, high specificity, and the range of linearity is wide, and the reaction time is short, stable reagent nonhazardous, weight
The features such as renaturation is good, and analysis speed is fast, automatic detection, the methods of relative immunity electrophoresis, immuno-precipitation are reduced to skill
The requirement of art personnel reduces the influence to result such as different technologies personnel, environmental change, reduces relative to radioimmunology
Pollution to environment has good promotion prospect.
Detailed description of the invention
The dose-effect curve drawn when Fig. 1 is kit measurement of the present invention.
Specific embodiment
More detailed explanation is done to the present invention below by specific embodiment, in order to the reason of those skilled in the art
Solution.
Embodiment 1 prepares insulin chemiluminescence immunoassay kit
1, reagent 1: the coated magnetic particle of insulin specific antibody
(surface is covered with the ferroso-ferric oxide of carboxyl or amino active group, partial size to insulin specific antibody with magnetic particle
Size is 0.1 μm -5 μm) it is covalently attached with two step cross-linking method of glutaraldehyde: the first step is with 10% glutaraldehyde by magnetic particle table
The active group in face sufficiently activates, reaction time 1h；Insulin specific antibody, concussion reaction 2h, after closing is added in second step
Constant volume saves；The magnetic particle suspension of coated antibody is stored in PH7.4 ~ 7.6 containing 1%BSA, the phosphoric acid of 0.1% Proclin-300
In salt buffer；
2, reagent 2: the horseradish peroxidase of insulin specific antibody label
The horseradish peroxidase of insulin specific antibody label is stored in PH7.35-7.45 containing 3%BSA, and 0.1%
In the Tris buffer of Proclin-300；
3, reagent 3: the calibration object of the insulin antigen containing gradient concentration
Gradient concentration standard items contain the insulin antigen of 0,5,20,50,100,300 μ IU/ml, matrix PH7.35- respectively
7.45 contain 1%BSA, 50% calf serum, the Tris buffer of 0.1% Proclin-300；Calibration object is freeze-drying state, and when use is used
The dissolution of 1ml purified water；
4, alkaline rinse: the phosphate buffer of pH7.5,2mol/L containing 2% Tween20,1% Procline300；
5, substrate A liquid: luminol；Substrate B liquid: hydrogen peroxide.
The detection method of 2 kits of embodiment
Specific detecting step are as follows:
1. sample pre-treatments:
General blood collection tube after blood sampling, is stored at room temperature 60min or 37 DEG C of incubation 30min, and then 4000rpm is centrifuged
10min will carry out sample process in strict accordance with standard processing mode, eliminate influence of the fibrinogen to experiment.
2. detection
2.1, reagent 1, reagent 2 and sample to be tested, 37 DEG C of reaction 15min are added in reaction cup.
2.2, under magnet suction-operated, antigen-antibody sandwich complex is fixed, and the ingredient of not formed compound is through washing
It is removed after washing.
2.3, reaction cup is added in luminol, hydrogen peroxide, shines through horseradish peroxidase enzyme catalytic, is sent out by Instrument measuring
Optical signal value.
2.4, dose-effect curve is drawn, such as according to four parameter fittings according to the signal value of gradient calibration object and concentration value
Shown in Fig. 1, pass through the insulin concentration being calculated in sample to be tested.
3 kit performance test of embodiment
1, kit sensitivity
Referring to the sensitivity of the experimental program detection kit of CLSI EP-17A file recommendation, the minimum inspection of this kit is measured
Output is not higher than 0.05, μ IU/mL, and Functional Sensitivity is 0.5 μ IU/mL
2, the range of linearity
Between 0.03~300 μ IU/mL, the linearly dependent coefficient r of measured value and theoretical value is greater than this kit range of linearity
0.99。
3, stability
Kit is placed from producing from semi-finished product in 37 DEG C 10 days (validity period is 18 months)；Finished product then presses 37 DEG C and places 7
Its (validity period is 12 months) is detected, and as a result meets and requires as defined in projects.
4, specific
This kit detects interfering substance result such as the following table 1, in the following endogenous material of shown Concentration Testing, does not find significant
Interference.
Table 1
In the following cross-reacting material of shown Concentration Testing, testing result is as shown in table 2, with pork insulin have close to 100% intersect it is anti-
(there are different degrees of interference in other producers with pork insulin) is answered, there is 1.2% crossing-over rate with proinsulin (genetic recombination).
With other several analogues without obvious cross reaction.
Table 2
5. repeatability
This kit repeatability is good, and concentration of specimens is from 2.3-130.6 μ IU/mL, and imprecision is from 4.1%-9.3% between batch.
Claims (5)
1. a kind of insulin immue quantitative detection reagent box, it is characterised in that: the magnetic particle including insulin specific antibody label is mixed
Suspension, the horseradish peroxidase of insulin specific antibody label, the standard items of gradient concentration；The insulin specificity is anti-
Body and magnetic particle are covalently attached with two step cross-linking method of glutaraldehyde.
2. insulin immue quantitative detection reagent box according to claim 1, it is characterised in that: the magnetic particle covers for surface
It is stamped the ferroferric oxide particle of carboxyl or amino active group, particle size is 0.1 μm -5 μm.
3. insulin immue quantitative detection reagent box according to claim 1, it is characterised in that: the standard items of the gradient concentration
Insulin antigen respectively containing 0,5,20,50,100,300 μ IU/ml, matrix used be PH7.35-7.45 contain 1%BSA, 50%
Calf serum, the Tris buffer of 0.1% Proclin-300.
4. insulin immue quantitative detection reagent box according to claim 1, it is characterised in that: the kit further includes alkalinity
Washing lotion.
5. insulin immue quantitative detection reagent box according to claim 1, it is characterised in that: the kit further includes substrate
A liquid and substrate B liquid；The substrate A liquid is luminol, and substrate B liquid is hydrogen peroxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811213292.1A CN109239329A (en) | 2018-10-18 | 2018-10-18 | A kind of insulin immue quantitative detection reagent box |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811213292.1A CN109239329A (en) | 2018-10-18 | 2018-10-18 | A kind of insulin immue quantitative detection reagent box |
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| Publication Number | Publication Date |
|---|---|
| CN109239329A true CN109239329A (en) | 2019-01-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811213292.1A Pending CN109239329A (en) | 2018-10-18 | 2018-10-18 | A kind of insulin immue quantitative detection reagent box |
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| Country | Link |
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| CN (1) | CN109239329A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111077323A (en) * | 2020-01-12 | 2020-04-28 | 天津市宝坻区人民医院 | Insulin determination kit for eliminating insulin autoantibody interference |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545912A (en) * | 2008-03-25 | 2009-09-30 | 北京科美东雅生物技术有限公司 | Trypsin micropore plate type magnetic granule chemoluminescence immunoassay measuring kit and preparation method thereof |
-
2018
- 2018-10-18 CN CN201811213292.1A patent/CN109239329A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545912A (en) * | 2008-03-25 | 2009-09-30 | 北京科美东雅生物技术有限公司 | Trypsin micropore plate type magnetic granule chemoluminescence immunoassay measuring kit and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111077323A (en) * | 2020-01-12 | 2020-04-28 | 天津市宝坻区人民医院 | Insulin determination kit for eliminating insulin autoantibody interference |
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Application publication date: 20190118 |
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