CN106324254A - Anti-insulin antibody detection kit and detection method thereof - Google Patents
Anti-insulin antibody detection kit and detection method thereof Download PDFInfo
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- CN106324254A CN106324254A CN201610672237.3A CN201610672237A CN106324254A CN 106324254 A CN106324254 A CN 106324254A CN 201610672237 A CN201610672237 A CN 201610672237A CN 106324254 A CN106324254 A CN 106324254A
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- insulin
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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
- G01N33/543—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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- 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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Abstract
The invention discloses an anti-insulin antibody magnetic particle chemiluminescence detection kit which contains a fluorescein labeled insulin antigen I solution, alkaline phosphatase labeled insulin antigen II solution, a fluorescein antibody coating magnetic particle suspension, an IAA calibration substance and a luminous substrate solution. A magnetic particle technology and a chemiluminescence technology are integrated, and a reaction system tending to be homogeneous is provided. The chemiluminescence technology and immune magnetic particles are combined, and a one-step reaction mode is adopted, so that the detection sensitivity and the precision are greatly improved, a detection range is widened, and quantitative detection of anti-insulin antibodies can be performed with higher accuracy, specificity, precision and lower cost.
Description
Technical field
The present invention relates to anti-insulin antibody field, be specifically related to one and combine immunity magnetic particle isolation technics and chemistry
Anti-insulin antibody magnetic microparticle chemiluminescence detection kit of luminescence immunoassay technology and preparation method thereof and detection method.
Background technology
Insulin human antibody is normally present in the diabetic serum accepting heterologous pancreas source extract for treating, this antibody energy
Bound insulin forms complex, makes insulin inactivate, and this is the diabetic one of the main reasons to insulin resistant.Existing
The anti-insulin antibody having proven to 5 kinds of Ig types all exists, but based on IgG class.Insulin antibody have two types,
A kind of occurring in accepted the patient that exogenous insulin is treated, the insulin system occurring mainly and using of its anti-insulin antibody
The purity of agent is relevant；Another kind occurs in the patients serum of never received insulin treatment, and this type of insulin antibody becomes pancreas
Island element autoantibody.Insulin antibody has important effect at aspects such as the diagnosis of the disease such as diabetes, hypoglycemia, treatments.
Therefore, anti-insulin antibody detection by quantitative is significant in patients serum clinically.
Anti-insulin antibody detection method many employings immunological method at present.Immunology detection technology utilizes antigen, antibody
The technology of test substance in specific reaction principle testing machine body.The detection of Placenta function, fluorescence immunoassay, enzyme linked immunosorbent detection
And chemiluminescence detection technology is developing progressively ripe, and progressively instead of the technology such as traditional immunoprecipitation, immune cohesion, immunity
The development learning detection technique makes Clinical detection inspection there occurs huge change: can detect project sharp increase, detection sensitivity and spy
Different Du Genggao, automaticity increases.The especially chemiluminescence immunoassay detection technique of Later development further increases detection
Sensitivity and the detection performance such as specificity, for disease detection by quantitative, diagnose, treat and provide data intuitively.
Anti-insulin antibody is the important detection project of hospital, and the diagnosis to the disease such as diabetes, hypoglycemia has
With or without alternative reference role.The anti-insulin antibody detectable of the listing of China's approval at present mostly is domestic, the inspection of employing
Survey methodology mostly is enzyme and exempts from and chemiluminescence.
Enzyme exempts from detection method at aspects such as detection sensitivity, the detection ranges of linearity far away from chemiluminescence detecting method.This alienation
Learn luminescence and also there is the advantages such as substrate harm is little, effect duration length, interference factor are few, automaticity is high.Therefore, in the face of composition
Complicated clinical testing sample, chemiluminescence performance is even better.
Magnetic particle immunoassay technology is that the Magnetic solid phases microgranule of the certain granules size utilizing synthesis of polymer material is made
Carrier, is coated the various immunity work such as antibody or antigen with specificity affinity with the method such as physical absorption, chemical coupling
Property material, there is separating rate block, efficiency is high, reproducible, simple to operate, do not affect and separated cell or other biological material
Shape and the feature such as function, orientable motion under additional the action of a magnetic field biology so that some special composition separated,
Concentrate or purification.
Summary of the invention
The technical problem to be solved in the present invention is the defect overcoming prior art, it is provided that a kind of anti-insulin antibody is quantitatively examined
Test agent box, magnetic separation technique and chemiluminescence are combined by this test kit, simple and quick, and result is accurate；The present invention's
Another object is for providing the preparation method of this test kit.
For achieving the above object, the present invention can use following technical proposals:
The anti-insulin antibody detection kit of the present invention, including solution, the alkalescence of fluorescein-labeled insulin antigens I
The solution of the insulin antigen II of phosphatase enzyme mark, it is coated the magnetic particle suspension of anti-fluorescein antibody, IAA calibration object and the luminous end
Thing solution.
Further, the insulin antigen II of described alkali phosphatase enzyme mark is passed through with insulin antigen by alkali phosphatase
Cross-linking agent disuccinimidyl suberate is formed by connecting.
Further, the working concentration of the solution of described fluorescein-labeled insulin antigens I is 0.8-1.2 μ g/ml, PH
It is 7～9；The working concentration of the insulin antigen II of described alkali phosphatase enzyme mark be 0.5-0.8 μ g/ml, PH be 7～9.
Further, the described preparation process containing the solution of fluorescein-labeled insulin antigens I is as follows:
(1) preparation Tris-HCl buffer that PH is 7-9 containing fluorescein, containing dense in described Tris-HCl buffer
Degree the sodium chloride of PEG-6000,0.7-0.9wt% of 4-8wt%, the sodium azide of 0.08-0.1wt%, the PC-300 of 0.6wt%,
0.8-1wt% bovine serum albumin and 20mM/L EDTA；
(2) it is the ratio of 150:1 according to fluorescein and insulin antigens I mol ratio, Tris-HCl in step (1) is buffered
Liquid mixes with insulin antigens I, fully mixes, room temperature standing and reacting；
(3) reactant liquor is separated unconjugated fluorescein by gel column, obtain resisting containing fluorescein-labeled insulin
Former 1 solution, degree of thickening and pH value, i.e. obtain the solution containing fluorescein-labeled insulin antigens I.
Further, the preparation process of the solution of the described insulin antigen II containing alkali phosphatase enzyme mark is as follows:
(1) use dimethyl sulfoxide solvent to be dissolved by insulin antigen II, keep the final concentration of 30-of insulin antigen II
60mg/ml, adds cross-linking agent disuccinimidyl suberate mix homogeneously, is placed in room temperature reaction 2-3 hour；Use dimethyl
Insulin antigen II is diluted by sulfoxide solvent with disuccinimidyl suberate junctional complex 1:10, saves backup at putting 2-8 DEG C；
(2) according to the amount that mol ratio is 1:2 of junctional complex in alkali phosphatase and (1) by alkaline phosphatase buffer with
(1) junctional complex mix homogeneously in, room temperature placing response 1.5h, generate the insulin antigen II of described alkali phosphatase enzyme mark；Its
Alkaline phosphatase buffer concentration is 1-2mg/ml；
(3) by reactant liquor by G-25 gel column desalination, concentration and pH value are adjusted.
Further, the preparation process of the magnetic particle suspension being coated anti-fluorescein antibody described in is as follows:
In the environment of coupling agent carbodiimide, will mix all with anti-fluorescein antibody containing the magnetic particle of carboxyl-reactive group
Even, room temperature placing response 10-20h, Magneto separate, except supernatant, adjust concentration and pH, i.e. obtain described in be coated anti-fluorescein antibody
Magnetic particle suspension；Wherein said magnetic particle has superparamagnetism, and the carboxyl-reactive group content that every gram of magnetic particle is carried is not
Less than 0.5mml；Anti-fluorescein antibody is monoclonal antibody, and dilution titer is more than 1:100 ten thousand.
Further, the preparation process of described luminous substrate solution is as follows:
Take TRIS 2g, NaCl 6g, Na2SO3 0.005g and Proclin-300 0.5ml in beaker, add 600ml
Purified water, is sufficiently stirred for mixing and extremely dissolves, regulate PH to 7.5；Add 200ml luminous substrate, filter with 0.2 μm filter, purified water
It is settled to 1000ml, mixes and i.e. obtain described substrate；Wherein, luminous substrate is selected from diamantane (obsolete), luminol and derivant, different Rumi
One or more in promise and derivant, acridinium ester.
The anti-insulin antibody detection kit of the present invention is for anti-insulin antibody quantitative detecting method, including as follows
Step:
(1) immunoreation: add sample to be detected in reaction tube, and be sequentially added into containing fluorescein-labeled insulin anti-
The solution of former I, the solution of the insulin antigen II containing alkali phosphatase enzyme mark, mixing, incubation 30min at 20-40 DEG C；Add bag
By the magnetic particle suspension of anti-fluorescein antibody, mix, incubation 30min at 20-40 DEG C, it is thus achieved that double antigens sandwich complex, and will
Double antigens sandwich complex is fixed on magnetic microsphere；
(2) washing: add cleanout fluid, concussion, make magnetic particle settle in magnetic field, except supernatant, dispose not over to be measured
Antibody forms the antigen of the linkage flag tracer of double antigens sandwich complex, repeats step 3-5 time；
(3) substrate detection is added: in reaction tube, add luminous substrate solution, fully mix, detect luminous value, be calculated
The content of anti-insulin antibody.
The present invention compared with prior art has the advantage that
One, immunity magnetic particle technology and chemiluminescence are combined by this test kit, it is provided that a kind of close to homogeneous
Reaction system, allows the insulin antigen of linkage flag tracer first react with detection antibody, forms double antigens sandwich structure
Complex.Owing to being formed during double antigens sandwich complex, with labelled antigen be all small-molecule substance, its space bit
Resistance is far smaller than the sterically hindered of magnetic microsphere, it is possible to promote that Ag-Ab association reaction is fully carried out so that sensitivity, essence
The detection such as density, detection range performance is greatly improved；
Two, use disuccinimidyl suberate to carry out insulin antigen as cross-linking agent and alkali phosphatase coupling be,
There is compared with other cross-linking agent higher coupling efficiency, and process stabilizing, while improving detection efficiency, reduce preparation
Cost.
Three, the insulin antigen of the calibration object of test kit fluorescein-labeled insulin antigens I, alkali phosphatase enzyme mark
II, the magnetic particle suspension luminous substrate etc. of anti-fluorescein antibody is all the optimization formula under this reaction system.
Four, the test kit of the present invention can (especially ZECEN CIA series be complete with Full-automatic chemiluminescence immunoassay analysis meter
Robotics luminescence immunoassay instrument) support the use, it is achieved that the full-automation of pattern detection so that insulin antibody concentration is examined
Survey easily and fast, simply, carry out in bulk can farthest reduce systematic error, for the use of test kit imitate the phase and
Detection performance provides safeguard.
Accompanying drawing explanation
Accompanying drawing is for providing a further understanding of the present invention, and constitutes a part for description, with the reality of the present invention
Execute example together for explaining the present invention, be not intended that limitation of the present invention.In the accompanying drawings:
Fig. 1 is detection calibration product standard curve；
Fig. 2 is serum sample testing result dependency.
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are illustrated, it will be appreciated that preferred reality described herein
Execute example be merely to illustrate and explain the present invention, be not intended to limit the present invention.
Embodiment
First, following methods is used to prepare the anti-insulin antibody detection kit of the present invention:
The first step, the preparation solution containing fluorescein-labeled insulin antigens I
The preparation Tris-HCl buffer that PH is 7-9 containing fluorescein, this is stated in buffer containing concentration 4-8wt%
The sodium chloride of PEG-6000,0.7-0.9wt%, the sodium azide of 0.08-0.1wt%, PC-300,0.8-1wt% cattle of 0.6wt%
Serum albumin and 20mM EDTA.It is the ratio of 150:1 according to fluorescein and insulin antigens I molecular proportion, by step (1)
Middle PH be 7-9 buffer be that 7-9 buffer mixes with insulin antigens I PH, fully mix, room temperature standing and reacting；By reactant liquor
Separate unconjugated fluorescein by gel column, obtain containing fluorescein-labeled insulin antigens I solution, degree of thickening and PH
Value, i.e. obtains the solution containing fluorescein-labeled insulin antigens I.
Second step, the solution of the preparation insulin antigen II containing alkali phosphatase enzyme mark
Use dimethyl sulfoxide solvent to be dissolved by insulin antigen II, keep final concentration of 30-60mg/ml,
Add cross-linking agent disuccinimidyl suberate mix homogeneously, be placed in room temperature reaction 2-3 hour；Use dimethyl
Insulin antigen is diluted by sulfoxide solvent with disuccinimidyl suberate junctional complex 1:10, saves backup at putting 2-8 DEG C；Press
Mix homogeneously with the amount that mol ratio is 1:2 of aforementioned junctional complex according to alkali phosphatase, room temperature placing response 1.5h, generate described alkali
The insulin antigen II of acid phosphatase labelling.Wherein alkaline phosphatase buffer concentration is 1-2mg/ml.Reactant liquor is passed through G-
25 gel column desalinations, adjust concentration and value, i.e. obtain the solution of insulin antigen II containing alkali phosphatase enzyme mark.
3rd step, preparation is coated the magnetic particle suspension of anti-fluorescein antibody
In the environment of coupling agent carbodiimide, will mix all with anti-fluorescein antibody containing the magnetic particle of carboxyl-reactive group
Even, room temperature placing response 10-20h, Magneto separate, except supernatant, adjust concentration and PH, i.e. obtain described in be coated anti-fluorescein antibody
Magnetic particle suspension.Wherein said magnetic particle has superparamagnetism, and the carboxyl-reactive group content that every gram of magnetic particle is carried is not
Less than 0.5mml；Anti-fluorescein antibody is monoclonal antibody, and dilution titer is more than 1:100 ten thousand.
4th step, the preparation of luminous substrate
Take TRIS 2g, NaCl 6g, Na2SO3 0.005g and Proclin-300 0.5ml in beaker, add 600ml
Purified water, is sufficiently stirred for mixing and extremely dissolves, regulate PH to 7.5；Add 200ml luminous substrate, filter with 0.2 μm filter, purified water
It is settled to 1000ml, mixes and i.e. obtain described substrate.
The quantitative detecting method of anti-insulin antibody immue quantitative detection reagent box of the present invention:
(1) detecting step:
(1) immunoreation: add sample to be detected in reaction tube, and be sequentially added into containing fluorescein-labeled insulin anti-
The solution of former I, the solution of the insulin antigen II containing alkali phosphatase enzyme mark, mixing, incubation 30min at 20-40 DEG C；Add bag
By the magnetic particle suspension of anti-fluorescein antibody, mixing, incubation 30min at 20-40 DEG C；
(2) washing: add cleanout fluid, concussion, make magnetic particle settle in magnetic field, except supernatant, dispose not over to be measured
Antibody forms the antigen of the linkage flag tracer of double antigens sandwich complex, repeats step 3-5 time；
(3) substrate detection is added: in reaction tube, add luminous substrate solution, fully mix, detect luminous value, be calculated
The content of anti-insulin antibody.
(4) use four parameter fitting modes, with calibration object concentration value as X-axis, with calibration object luminous intensity values as Y-axis, build
Day-mark directrix curve.Luminous intensity values according to sample to be tested is back-calculated corresponding concentration value.
Test kit of the present invention is identified according to methodology, and performance parameter can reach following index:
Standard curve is linear: R was more than for 0.999 (as shown in Figure 1)
Sensitivity: to zero calibration object duplicate detection 20 times, calculates meansigma methods (M) and the standard deviation (SD) of luminous intensity, and
Calculate M-2SD, according to concentration corresponding on standard curve, calculate corresponding concentration value, be sensitivity for analysis.Wherein, sensitive
Degree is not more than 0.18ng/ml.
Elaboration: between variation in analyzing, analysis, variation and batch variation were respectively less than for 10% (as shown in table 1)
A) make a variation in, analyzing and between analysis tables of data
Table 1 analyze interior and analyze between make a variation tables of data
B), batch variation tables of data
Table 2 batch variation tables of data
| Q1 | Q2 | Q3 | |
| Variation CV% | 8.09 | 6.25 | 5.06 |
Specificity: magnetic particle two step specific recognition of the present invention, relatively other detectable have preferably improvement.Use this
Bright test kit 200 parts of clinical negative serums of detection, this reagent false positive rate is 0%.
Accuracy: by adding recovery method, by reference material luminous value matching concentration value, matching concentration and sign concentration
Ratio meansigma methods between 0.85-1.15 (as shown in table 3)
The accuracy parameter of table 3 test kit of the present invention
Compare with like product: 120 parts are faced by this test kit with Elabscience insulin human antibody kit simultaneously
Bed serum sample is measured, and the two measurement result good relationship, coefficient R was 0.991 (as shown in Figure 2).
Finally it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention,
Although being described in detail the present invention with reference to previous embodiment, for a person skilled in the art, it still may be used
So that the technical scheme described in foregoing embodiments to be modified, or wherein portion of techniques feature is carried out equivalent.
All within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included in the present invention's
Within protection domain.
Claims (8)
1. an anti-insulin antibody detection kit, it is characterised in that include the molten of fluorescein-labeled insulin antigens I
Liquid, alkali phosphatase enzyme mark insulin antigen II solution, be coated the magnetic particle suspension of anti-fluorescein antibody, IAA calibration object
With luminous substrate solution.
Anti-insulin antibody detection kit the most according to claim 1, it is characterised in that described alkali phosphatase enzyme mark
Insulin antigen II be formed by connecting by cross-linking agent disuccinimidyl suberate with insulin antigen by alkali phosphatase.
Anti-insulin antibody detection kit the most according to claim 2, it is characterised in that described fluorescein-labeled pancreas
The working concentration of the solution of island element antigens I be 0.8-1.2 μ g/ml, PH be 7～9；The insulin of described alkali phosphatase enzyme mark resists
The working concentration of former II be 0.5-0.8 μ g/ml, PH be 7～9.
Anti-insulin antibody detection kit the most according to claim 3, it is characterised in that described containing fluorescein-labeled
The preparation process of the solution of insulin antigens I is as follows:
(1) preparation Tris-HCl buffer that PH is 7～9 containing fluorescein, containing concentration 4 in described Tris-HCl buffer
～the PC-300 of the sodium azide of the sodium chloride of the PEG-6000 of 8wt%, 0.7～0.9wt%, 0.08～0.1wt%, 0.6wt%,
0.8～1wt% bovine serum albumin and 20mM/L EDTA；
(2) be the ratio of 150:1 according to fluorescein and insulin antigens I mol ratio, by Tris-HCl buffer in step (1) with
Insulin antigens I mixes, and fully mixes, room temperature standing and reacting；
(3) reactant liquor is separated unconjugated fluorescein by gel column, obtain containing fluorescein-labeled insulin antigen 1 molten
Liquid, degree of thickening and pH value, i.e. obtain the solution containing fluorescein-labeled insulin antigens I.
Anti-insulin antibody detection kit the most according to claim 3, it is characterised in that described containing alkali phosphatase mark
The preparation process of the solution of the insulin antigen II of note is as follows:
(1) use dimethyl sulfoxide solvent to be dissolved by insulin antigen II, keep the final concentration of 30-of insulin antigen II
60mg/ml, adds cross-linking agent disuccinimidyl suberate mix homogeneously, is placed in room temperature reaction 2-3 hour；Use dimethyl
Insulin antigen II is diluted by sulfoxide solvent with disuccinimidyl suberate junctional complex 1:10, saves backup at putting 2-8 DEG C；
(2) according to the amount that mol ratio is 1:2 of junctional complex in alkali phosphatase and (1) by alkaline phosphatase buffer and (1)
Junctional complex mix homogeneously, room temperature placing response 1.5h, generate the insulin antigen II of described alkali phosphatase enzyme mark；Its neutral and alkali
Phosphatase buffer concentration is 1-2mg/ml；
(3) by reactant liquor by G-25 gel column desalination, concentration and pH value are adjusted.
Anti-insulin antibody detection kit the most according to claim 3, it is characterised in that described in be coated anti-fluorescein antibody
The preparation process of magnetic particle suspension as follows:
In the environment of coupling agent carbodiimide, will mix homogeneously with anti-fluorescein antibody containing the magnetic particle of carboxyl-reactive group,
Room temperature placing response 10-20h, Magneto separate, except supernatant, adjust concentration and pH, i.e. obtain described in be coated the magnetic of anti-fluorescein antibody
Microparticle suspending liquid；Wherein said magnetic particle has superparamagnetism, and the carboxyl-reactive group content that every gram of magnetic particle is carried is the least
In 0.5mml；Anti-fluorescein antibody is monoclonal antibody, and dilution titer is more than 1:100 ten thousand.
Anti-insulin antibody detection kit the most according to claim 1, it is characterised in that described luminous substrate solution
Preparation process is as follows:
Take TRIS 2g, NaCl 6g, Na2SO3 0.005g and Proclin-300 0.5ml in beaker, add 600ml purification
Water, is sufficiently stirred for mixing and extremely dissolves, regulate PH to 7.5；Add 200ml luminous substrate, filter with 0.2 μm filter, purified water constant volume
To 1000ml, mix and i.e. obtain described substrate；Wherein, luminous substrate selected from diamantane (obsolete), luminol and derivant thereof, different luminol and
One or more in its derivant, acridinium ester.
8. use any one of claim 1-7 the anti-insulin antibody detection kit described in claim to be used for resisting islets of langerhans
Element antibody quantitative detecting method, it is characterised in that comprise the steps:
(1) immunoreation: add sample to be detected in reaction tube, and be sequentially added into containing fluorescein-labeled insulin antigens I
Solution, the solution of insulin antigen II containing alkali phosphatase enzyme mark, mixing, incubation 30min at 20-40 DEG C；Addition is coated
The magnetic particle suspension of anti-fluorescein antibody, mixing, incubation 30min at 20-40 DEG C, it is thus achieved that double antigens sandwich complex, and will be double
Antigen sandwich complex is fixed on magnetic microsphere；
(2) washing: add cleanout fluid, concussion, make magnetic particle settle in magnetic field, except supernatant, dispose not over test antibodies
Form the antigen of the linkage flag tracer of double antigens sandwich complex, repeat step 3-5 time；
(3) substrate detection is added: in reaction tube, add luminous substrate solution, fully mix, detect luminous value, be calculated anti-pancreas
The content of island element antibody.
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| CN108152507A (en) * | 2017-11-28 | 2018-06-12 | 泰州泽成生物技术有限公司 | A kind of I type precollagen amino terminal peptides detection kit and its detection method |
| CN109725162A (en) * | 2018-12-21 | 2019-05-07 | 广州市进德生物科技有限公司 | A kind of detection kit and its method of complete homogeneous determination insulin |
| CN111175496A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Pregnancy-associated protein A magnetic particle chemiluminescence detection kit and use method thereof |
| CN111175497A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Magnetic particle chemiluminescence digoxin assay kit and application method thereof |
| CN116466097A (en) * | 2023-06-14 | 2023-07-21 | 天津市协和医药科技集团有限公司 | Human insulin detection kit |
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| CN116466097A (en) * | 2023-06-14 | 2023-07-21 | 天津市协和医药科技集团有限公司 | Human insulin detection kit |
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