CN109297954A - A kind of insulin nano magnetic microparticle chemiluminescence assay kit and preparation method thereof and detection method - Google Patents
A kind of insulin nano magnetic microparticle chemiluminescence assay kit and preparation method thereof and detection method Download PDFInfo
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- CN109297954A CN109297954A CN201811474188.8A CN201811474188A CN109297954A CN 109297954 A CN109297954 A CN 109297954A CN 201811474188 A CN201811474188 A CN 201811474188A CN 109297954 A CN109297954 A CN 109297954A
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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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- 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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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Abstract
The invention belongs to medical science and disclose a kind of insulin nano magnetic microparticle chemiluminescence assay kit and preparation method thereof and detection method；The kit includes: M reagent, R reagent, insulin calibration object, increased response agent and Chemoluminescent substrate, and the M reagent includes the Tris buffer that concentration is the magnetic particle of 2 μ g/mL coating INS- biotinylated antibody, concentration is 0.2mol/L；The R reagent includes the Tris buffer that concentration is the INS-AP labelled antibody of 1 μ g/mL, concentration is 0.1mol/L；The insulin calibration object includes the insulin sample of two kinds of various concentrations and the Tris buffer of 0.1mol/L concentration；The beneficial effect that kit of the present invention has high specificity, stability good improves the sensitivity and specificity of detection, is suitable for automatic operation；Preparation method of the present invention is simple, is easy to realize in industrialized production, is beneficial to improve the preparation efficiency；Meanwhile detection method detection process is simple, enormously simplifies existing detecting step and program.
Description
Technical field
The present invention relates to technical field of medical detection more particularly to a kind of measurement examinations of insulin nano magnetic microparticle chemiluminescence
Agent box and preparation method thereof and detection method.
Background technique
Insulin is the polypeptide hormone of islet β cell, is made of 51 amino acid.In body after insulin secretion people's blood
Interior half-life period is usually 3-5 minutes, is mainly absorbed and is degraded by liver, reabsorption and degradation in proximal convoluted tubule on a small quantity.Portugal
Grape sugar is the most strong stimulation factor for promoting insulin secretion, and insulin is in diphasic pulse formula in healthy human body under glucose effect
Secretion.Many other factors, as metabolic, incretion, neural sexual factor and drug can influence the synthesis of insulin
And secretion.Play a significant role in the anabolism of insulin in vivo, in vivo it is almost all of tissue have directly or
Indirectly influence.Insulin generates insulin-like effects in conjunction with insulin receptor in metabolism, promote body to sugar, fat and
The synthesis and storage of protein.Increase: seeing cirrhosis, diabetes B, hyperthyroidism, acromegalia, nutrition not
Hypoglycemia caused by good figure myotonia, pancreatic regeneration；It partial amino-acid, glucagon, testosterone, growth hormone and oral keeps away
Pregnant medicine can be such that insulin in blood increases.Lower: seeing type 1 diabetes, part diabetes B, primary pituitary hypofunction disease, adrenal gland
Cortex hormone function is low, secondary injury of pancreas and chronic pancreatitis；Catecholamine, beta-blocker and diuretics can make pancreas islet
Plain reduced levels.
Currently, the laboratory testing method of insulin mainly has radio-immunity (RIA), enzyme linked immunological (ELISA), chemistry
Electrochemiluminescent immunoassay (CLIA) etc..Wherein RIA and ELISA occur more early, but that there are pollutions is strong, sensitivity is low due to it, detection when
Between it is long the disadvantages of, no longer according to the principal status of public economy.And Chemiluminescence Immunoassay is due to high sensitivity, dynamics range is wide, is suitable for
The advantages that automatic operation is generally acknowledged at present specificity and with higher sensitivity detection method.But it is applicable in the prior art
More complicated in Kit components of the chemiluminescence determination for insulin detection, stability is not good enough, and the spirit of its detection
Sensitivity and specificity need to be further increased.In consideration of it, how to provide, a kind of high specificity, stability is good, can improve detection
The insulin nano magnetic microparticle chemiluminescence assay kits and preparation method thereof of sensitivity and specificity be with detection method
Those skilled in the art need the technical problem solved.
Summary of the invention
More complicated for Kit components in the prior art, stability is not good enough, and the sensitivity of its detection and special
Property needs the shortcoming further increased, and the present invention provides a kind of high specificity, stability is good, can improve the spirit of detection
Insulin nano magnetic microparticle chemiluminescence assay kit of sensitivity and specificity and preparation method thereof and detection method.
The present invention using following technical scheme in order to solve the above technical problems, realized:
Design a kind of insulin nano magnetic microparticle chemiluminescence assay kit, the kit include: M reagent, R reagent,
Insulin calibration object, increased response agent and Chemoluminescent substrate, the M reagent include that concentration is that 2 μ g/mL are coated with INS- life
The magnetic particle of object element antibody, the Tris buffer that concentration is 0.2mol/L；The R reagent includes that concentration is 1 μ g/mL
INS-AP labelled antibody, the Tris buffer that concentration is 0.1mol/L；The insulin calibration object includes two kinds of various concentrations
Insulin sample and 0.1mol/L concentration Tris buffer.
Preferably, in the M reagent, R reagent and insulin calibration object also containing surfactant, stabilizer with
And pH adjusting agent.
Preferably, the mixed pH value range of each reagent in the kit is 7-9.
Preferably, the concentration range of the Chemoluminescent substrate are as follows: 5-20g/L, and its ingredient is mainly cleaned by concentration
Liquid, excitation cleaning solution and preexciting cleaning solution composition.
Preferably, concentrated cleaning solutions in the Chemoluminescent substrate, excite the composition of cleaning solution and preexciting cleaning solution
Ratio is 1:1-2:1-5.
Preferably, the magnetic particle of the coating INS- biotinylated antibody is the paramagnetism of anti-Human Insulin's monoclonal antibody
Particle.
Preferably, the increased response agent is in PEG4000, PEG6000, PEG8000, PEG10000 and PEG20000
It is one or more.
Preferably, the concentration range of the increased response agent are as follows: 10-30g/L.
A kind of preparation method of the nano magnetic particulate chemistry luminescent assay kit of insulin of the invention, including walk as follows
It is rapid:
Step 1: preparation M reagent: Tris buffer is first prepared, then by INS- biotinylated antibody and Streptavidin magnetic
Pearl mixes well the appropriate Tris buffer of addition and mixes well, reacts at room temperature after constant volume, obtains M reagent；
Step 2: preparation R reagent first prepares Tris buffer, then INS-AP antibody is added in Tris buffer, sufficiently
It mixes, obtains R reagent after constant volume；
Step 3: it prepares insulin calibration object: preparing Tris buffer, the insulin sample of two kinds of various concentrations is added
It in Tris buffer, mixes well, insulin calibration object is obtained after constant volume；
Step 4: respectively to Step 1: Step 2: being added in the M reagent of step 3 preparation, R reagent, insulin calibration object
Increased response agent and Chemoluminescent substrate remix uniformly.
A kind of detection method of insulin nano magnetic microparticle chemiluminescence assay kit of the invention, including walk as follows
It is rapid:
Step 1: being added in reaction tube sample to be detected, and sequentially adds M reagent, R reagent, increased response agent mixing,
30min is incubated at 20-40 DEG C, obtains double antigens sandwich compound, and double antigens sandwich compound is fixed on magnetic microsphere；
Step 2: adding cleaning solution, concussion, settle magnetic microsphere in magnetic field, remove supernatant, dispose not over to
The antigen that antibody forms the linkage flag tracer of double antigens sandwich compound is surveyed, is repeated step 3-5 times；
Step 3: being added in reaction tube Chemoluminescent substrate, mixes well, and is detected and is shone by luminous tester
Value, is calculated the content of anti-insulin antibody.
A kind of insulin nano magnetic microparticle chemiluminescence assay kit proposed by the present invention and preparation method thereof and detection
Method, beneficial effect are:
(1) content of the detection kit for insulin of the present invention for insulin in Quantitative in vitro measurement human serum, and this
It is actrapid monotard's monoclonal antibody that magnetic particle is coated in the kit of invention, and more existing Chemiluminescence immunoassay is compared, had
The good beneficial effect of high specificity, stability, substantially increases the sensitivity and specificity of detection；
(2) detection kit for insulin of the present invention is matched in main raw material(s) and has been carried out very in the when selection of surfactant
It is attempt more, the combination for being suitable for the magnetic particle enzyme labelled antibody of this system is finally obtained, thus high in measurement rapid sensitive, specificity
Under the premise of, the good box of whole system stability is easy to operate, is very suitable for that clinical Full-automatic chemiluminescence analyzer is mating to be made
With；
(3) detection kit for insulin of the present invention uses chemiluminescence immunoassay sandwich method for determining insulin level in serum；
Firstly, adding the samples to containing paramagnetic particles, buffer solution and the INS-AP for being coated with anti-Human Insulin's monoclonal antibody
In the reaction tube of labelled antibody.Different antigen sites reaction on INS-AP labelled antibody and human insulin molecule.Actrapid monotard
In conjunction with the monoclonal anti-human insulin being fixed in paramagnetic particles, to form sandwich complex；It is incubated in reaction tube
After the completion, the substance being incorporated in solid phase, which is placed in magnetic field, to be adsorbed, and unbonded substance is rinsed removing, then will be chemical
Luminous substrate is added in reaction tube, is then measured by full-automatic illumination analyzer to light generated in reaction；It is produced
The amount of the third contact of a total solar or lunar eclipse is directly proportional to the concentration of people INS in sample, has the advantages that high specificity, stability are good, improves detection
Sensitivity and specificity；It can be widely applied to the content of insulin in Quantitative in vitro measurement human serum；
(4) detection kit for insulin of the present invention uses Chemiluminescence Immunoassay, with high sensitivity, stability
Good, high specificity, dynamics range are wide, are suitable for the advantages that automatic operation；
(5) preparation method of the invention is simple, is easy to realize in industrialized production, is beneficial to improve the preparation efficiency；Together
When, detection method detection process of the invention is simple, enormously simplifies existing detecting step and program.
Detailed description of the invention
The present invention is described in further detail for embodiment in reference to the accompanying drawing, but does not constitute to of the invention
Any restrictions.
It is detected using zero-dose calibration object as sample when Fig. 1 is product experimental verification shown in the embodiment of the present invention two
Minimum detectability result figure；
First is carried out using the accurate sample of national standard concentration when Fig. 2 is product experimental verification shown in inventive embodiments two
Secondary testing result schematic diagram；
Second is carried out using the accurate sample of national standard concentration when Fig. 3 is product experimental verification shown in inventive embodiments two
Secondary testing result schematic diagram；
First is carried out using the accurate sample of national standard concentration when Fig. 4 is product experimental verification shown in inventive embodiments two
Secondary testing result schematic diagram；
Dependence test result in range of linearity when Fig. 5 is product first time experimental verification shown in inventive embodiments two
Figure；
Dependence test result in range of linearity when Fig. 6 is second of experimental verification of product shown in inventive embodiments two
Figure；
Dependence test result in range of linearity when Fig. 7 is product third time experimental verification shown in inventive embodiments two
Figure；
Precision CV value test result figure in being analyzed when Fig. 8 is product experimental verification shown in inventive embodiments two；
The betweenrun precision CV value that different zones are set as high when Fig. 9 is product experimental verification shown in inventive embodiments two is surveyed
Test result figure；
Different zones set low betweenrun precision CV value test when Figure 10 is product experimental verification shown in inventive embodiments two
Result figure；
Figure 11 is the specific outcome figure of product every batch of test result shown in the embodiment of the present invention two；
Figure 12 is test result figure of the product shown in inventive embodiments two to interfering substance (such as bilirubin, triglycerides).
Specific embodiment
With reference to the attached drawing in the embodiment of the present invention, technical solution in the embodiment of the present invention carries out clear, complete
Ground description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on this
The embodiment of invention, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, belongs to protection scope of the present invention.
Embodiment one
A kind of insulin nano magnetic microparticle chemiluminescence assay kit of the invention, the kit include: M reagent, R examination
Agent, insulin calibration object, increased response agent and Chemoluminescent substrate, the M reagent include that concentration is coated with for 2 μ g/mL
The magnetic particle of INS- biotinylated antibody, the Tris buffer that concentration is 0.2mol/L；The R reagent includes that concentration is 1 μ g/
The INS-AP labelled antibody of mL, the Tris buffer that concentration is 0.1mol/L；The insulin calibration object includes two kinds of differences
The insulin sample of concentration and the Tris buffer of 0.1mol/L concentration；M reagent, R reagent and the insulin calibration object
In also contain surfactant, stabilizer and pH adjusting agent, the mixed pH value of each reagent in the kit is 7,
The concentration of the Chemoluminescent substrate are as follows: 50g/L, and its ingredient is mainly by concentrated cleaning solutions, excitation cleaning solution and preexciting
Cleaning solution forms, the composition ratio of concentrated cleaning solutions, excitation cleaning solution and preexciting cleaning solution in the Chemoluminescent substrate
For 1:1:1, the magnetic particle of the coating INS- biotinylated antibody is the paramagnetic particles of anti-Human Insulin's monoclonal antibody, institute
The increased response agent stated is PEG4000, the concentration of the increased response agent are as follows: 10g/L.
Embodiment two
A kind of insulin nano magnetic microparticle chemiluminescence assay kit of the invention, the kit include: M reagent, R examination
Agent, insulin calibration object, increased response agent and Chemoluminescent substrate, the M reagent include that concentration is coated with for 2 μ g/mL
The magnetic particle of INS- biotinylated antibody, the Tris buffer that concentration is 0.2mol/L；The R reagent includes that concentration is 1 μ g/
The INS-AP labelled antibody of mL, the Tris buffer that concentration is 0.1mol/L；The insulin calibration object includes two kinds of differences
The insulin sample of concentration and the Tris buffer of 0.1mol/L concentration；M reagent, R reagent and the insulin calibration object
In also contain surfactant, stabilizer and pH adjusting agent, the mixed pH value of each reagent in the kit is 8,
The concentration of the Chemoluminescent substrate are as follows: 10g/L, and its ingredient is mainly by concentrated cleaning solutions, excitation cleaning solution and preexciting
Cleaning solution forms, the composition ratio of concentrated cleaning solutions, excitation cleaning solution and preexciting cleaning solution in the Chemoluminescent substrate
For 1:2:3, the magnetic particle of the coating INS- biotinylated antibody is the paramagnetic particles of anti-Human Insulin's monoclonal antibody, institute
The increased response agent stated is PEG4000, PEG6000 and PEG8000, the concentration of the increased response agent are as follows: 20g/L.
Embodiment three
A kind of insulin nano magnetic microparticle chemiluminescence assay kit of the invention, the kit include: M reagent, R examination
Agent, insulin calibration object, increased response agent and Chemoluminescent substrate, the M reagent include that concentration is coated with for 2 μ g/mL
The magnetic particle of INS- biotinylated antibody, the Tris buffer that concentration is 0.2mol/L；The R reagent includes that concentration is 1 μ g/
The INS-AP labelled antibody of mL, the Tris buffer that concentration is 0.1mol/L；The insulin calibration object includes two kinds of differences
The insulin sample of concentration and the Tris buffer of 0.1mol/L concentration；M reagent, R reagent and the insulin calibration object
In also contain surfactant, stabilizer and pH adjusting agent, the mixed pH value of each reagent in the kit is 9,
The concentration of the Chemoluminescent substrate are as follows: 20g/L, and its ingredient is mainly by concentrated cleaning solutions, excitation cleaning solution and preexciting
Cleaning solution forms, the composition ratio of concentrated cleaning solutions, excitation cleaning solution and preexciting cleaning solution in the Chemoluminescent substrate
For 1:2:5, the magnetic particle of the coating INS- biotinylated antibody is the paramagnetic particles of anti-Human Insulin's monoclonal antibody, institute
The increased response agent stated be PEG4000, PEG6000, PEG8000, PEG10000 and PEG20000, the increased response agent it is dense
Degree are as follows: 30g/L.
Example IV
The ingredient of insulin nano magnetic microparticle chemiluminescence assay kit insulin identical as embodiment two, of the invention
Nano magnetic particulate chemistry luminescent assay kit preparation method, include the following steps:
Step 1: preparation M reagent: Tris buffer is first prepared, then by INS- biotinylated antibody and Streptavidin magnetic
Pearl mixes well the appropriate Tris buffer of addition and mixes well, reacts at room temperature after constant volume, obtains M reagent；
Step 2: preparation R reagent: Tris buffer is first prepared, then INS-AP antibody is added in Tris buffer, sufficiently
It mixes, obtains R reagent after constant volume；
Step 3: it prepares insulin calibration object: preparing Tris buffer, the insulin sample of two kinds of various concentrations is added
It in Tris buffer, mixes well, insulin calibration object is obtained after constant volume；
Step 4: respectively to Step 1: Step 2: being added in the M reagent of step 3 preparation, R reagent, insulin calibration object
Increased response agent and Chemoluminescent substrate remix uniformly.
Embodiment five
The ingredient of plain nano magnetic particulate chemistry luminescent assay kit insulin nano identical as embodiment two, of the invention
The detection method of magnetic microparticle chemiluminescence assay kit, includes the following steps:
Step 1: being added in reaction tube sample to be detected, and sequentially adds M reagent, R reagent, increased response agent mixing,
30min is incubated at 30 DEG C, obtains double antigens sandwich compound, and double antigens sandwich compound is fixed on magnetic microsphere；
Step 2: adding cleaning solution, concussion, settle magnetic microsphere in magnetic field, remove supernatant, dispose not over to
The antigen that antibody forms the linkage flag tracer of double antigens sandwich compound is surveyed, step 4 time is repeated；
Step 3: being added in reaction tube Chemoluminescent substrate, mixes well, and is detected and is shone by luminous tester
Value, is calculated the content of anti-insulin antibody.
Performance detection test
It is carried out respectively using product, preparation method and detection method shown in embodiment two, example IV and embodiment five
Following test item is improved to verify the performance of product shown in the embodiment of the present invention
One, minimum detectability
Zero calibration object of replication 10 times.Calculate the mean value of chemiluminescence (RLU) valueWith standard deviation (SD).?
OutAccording to the concentration and chemiluminescence (RLU) value result progress two between zero-dose calibration object and adjacent calibration object
Point regression fit obtains linear function, willCorresponding RLU value is brought into equation, and corresponding concentration value is found out,
As a result as shown in Fig. 1, by attached drawing 1 it is found that insulin nano magnetic microparticle chemiluminescence assay kit of the present invention is to minimum inspection
Rising limit is 0.01 μm of ol/L.
Two, accuracy
National standard is configured to 5 concentration corresponding with kit internal calibration product by the kit shown in embodiment two
Point, every is measured in parallel 3 times, calculates the slope and potency ratio of two dose-response curves, test result such as attached drawing 2 to attached drawing 4
It is shown；By attached drawing 2 to attached drawing 4 it is found that insulin nano magnetic microparticle chemiluminescence assay kit testing result of the present invention and state
Family's normal concentration sample compares, and the accuracy of detection is very high, complies with the national standard requirements.
Three, linear
Linearly interval is diluted to 6 concentration with the high concentration sample close to the linearly interval upper limit by a certain percentage, wherein low
The sample for being worth concentration must be close to the lower limit of linearly interval.It is operated by kit specification, by the sample of each diluted concentration
Retest 3 times, average value is calculated, result average value and dilution ratio are subjected to straight line fitting with least square method, and calculate
The Xiang Guan Xi Shuo ∣ r ∣ of linear regression, as a result as shown in attached drawing 5 to attached drawing 7, the results showed that every batch of insulin nano magnetic particle
Related property is good in the linear range for the test result of luminescent assay kit.
Four, precision in analyzing
The different zones setting of dose-response curve it is high (Bole's life medical product Co., Ltd quality-control product, target value:
177.46mIU/L), the Quality Control of low (Bole's life medical product Co., Ltd quality-control product, target value: 64.32mIU/L) two concentration
Product measurement, in same primary analysis, each quality-control product replication 10 times, the coefficient of variation (CV) of measurement result, as a result such as
Shown in attached drawing 8, the results showed that every batch of insulin nano magnetic microparticle chemiluminescence measures precision in the analysis of reagent, that is,
The coefficient of variation (CV) meets prescribed requirement.
Five, betweenrun precision
The different zones setting of dose-response curve it is high (Bole's life medical product Co., Ltd quality-control product, target value:
177.46mIU/L), the Quality Control of low (Bole's life medical product Co., Ltd quality-control product, target value: 64.32mIU/L) two concentration
Product measurement, carries out independent analysis with the product of 3 different batches, every batch of replication 10 times, calculates each concentration samples 30
The average value (M) and standard deviation (SD) of secondary measurement result, as a result as shown in attached drawing 9- attached drawing 10, the above result shows that every batch of pancreas
The betweenrun precision of island element nano magnetic particulate chemistry luminescent assay kit test result, that is, the coefficient of variation (CV) meet rule
Provisioning request.
Six, specific
The proinsulin human that measurement concentration is 10ng/mL and actrapid monotard's sample that concentration is 500 μ IU/mL respectively, each 1
It is secondary, as a result as shown in Fig. 11, the results showed that the specificity of every batch of insulin nano magnetic microparticle chemiluminescence assay kit
Meet prescribed requirement.
Seven, anti-interference
The serum sample of 2 parts of Healthy Peoples is taken, every part of sample individually does a kind of interfering substance (such as hemoglobin, bilirubin)
Test, is allocated as ten parts for every part of sample again, the interfering components of various concentration (as shown in the table) is added respectively, then in reagent
The sample of the common interference substance containing various concentration gradient is measured under (box) regulation parameter, measurement result with it is not plus dry
The sample for disturbing substance compares, and jamming rate≤± 5% item can determine whether to be evaluated chaff interferent noiseless to method is evaluated.It is on the contrary then recognize
Effect is significantly interfered with to be evaluated chaff interferent to the method for being evaluated.As a result it should meet the requirement of kit quality standard.
Jamming rate (%) judges that calculation formula is as follows:
Interference value=disturbed specimen measured value-basis sample measurements
Jamming rate (%)=interference value/basic value × 100
Test result is as shown in Fig. 12, can be obtained by attached drawing 12: detection kit for insulin is in bilirubin≤90mg/
Jamming rate will not generate interference to test less than 5% when dL, triglycerides≤1800mg/dL.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Within the technical scope of the present disclosure, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claims
Subject to enclosing.
Claims (10)
1. a kind of insulin nano magnetic microparticle chemiluminescence assay kit, which is characterized in that the kit includes: M reagent, R
Reagent, insulin calibration object, increased response agent and Chemoluminescent substrate, the M reagent include that concentration is coated with for 2 μ g/mL
The magnetic particle of INS- biotinylated antibody, the Tris buffer that concentration is 0.2mol/L；The R reagent includes that concentration is 1 μ g/
The INS-AP labelled antibody of mL, the Tris buffer that concentration is 0.1mol/L；The insulin calibration object includes two kinds of differences
The insulin sample of concentration and the Tris buffer of 0.1mol/L concentration.
2. a kind of insulin nano magnetic microparticle chemiluminescence assay kit according to claim 1, which is characterized in that institute
Also contain surfactant, stabilizer and pH adjusting agent in M reagent, R reagent and the insulin calibration object stated.
3. a kind of insulin nano magnetic microparticle chemiluminescence assay kit according to claim 1 or 2, feature exist
In the mixed pH value range of each reagent in the kit is 7-9.
4. a kind of insulin nano magnetic microparticle chemiluminescence assay kit according to claim 1, which is characterized in that institute
State the concentration range of Chemoluminescent substrate are as follows: 5-20g/L, and its ingredient is mainly by concentrated cleaning solutions, excitation cleaning solution and pre-
Excite cleaning solution composition.
5. a kind of insulin nano magnetic microparticle chemiluminescence assay kit according to claim 4, which is characterized in that institute
The composition ratio for stating concentrated cleaning solutions in Chemoluminescent substrate, excitation cleaning solution and preexciting cleaning solution is 1:1-2:1-5.
6. a kind of insulin nano magnetic microparticle chemiluminescence assay kit according to claim 1, which is characterized in that institute
The magnetic particle for stating coating INS- biotinylated antibody is the paramagnetic particles of anti-Human Insulin's monoclonal antibody.
7. a kind of insulin nano magnetic microparticle chemiluminescence assay kit according to claim 1, which is characterized in that institute
The increased response agent stated is one of PEG4000, PEG6000, PEG8000, PEG10000 and PEG20000 or a variety of.
8. a kind of insulin nano magnetic microparticle chemiluminescence assay kit according to claim 1, which is characterized in that institute
State the concentration range of increased response agent are as follows: 10-30g/L.
9. the preparation side of the nano magnetic particulate chemistry luminescent assay kit of any one insulin as described in claim 1-8
Method, which comprises the steps of:
Step 1: preparation M reagent: Tris buffer is first prepared, then INS- biotinylated antibody is filled with Streptavidin MagneSphere
Divide mixing that appropriate Tris buffer is added to mix well, react after constant volume at room temperature, obtains M reagent；
Step 2: preparation R reagent first prepares Tris buffer, then INS-AP antibody is added in Tris buffer, mixes well,
R reagent is obtained after constant volume；
Step 3: it prepares insulin calibration object: preparing Tris buffer, Tris is added in the insulin sample of two kinds of various concentrations
It in buffer, mixes well, insulin calibration object is obtained after constant volume；
Step 4: respectively to Step 1: Step 2: reaction is added in the M reagent of step 3 preparation, R reagent, insulin calibration object
Reinforcing agent and Chemoluminescent substrate remix uniformly.
10. the detection method of any one insulin nano magnetic microparticle chemiluminescence assay kit as described in claim 1-8,
It is characterized by comprising the following steps:
Step 1: being added in reaction tube sample to be detected, and sequentially adds M reagent, R reagent, increased response agent mixing, 20-
30min is incubated at 40 DEG C, obtains double antigens sandwich compound, and double antigens sandwich compound is fixed on magnetic microsphere；
Step 2: adding cleaning solution, and concussion settles magnetic microsphere in magnetic field, removes supernatant, disposes not over to be measured anti-
Body forms the antigen of the linkage flag tracer of double antigens sandwich compound, repeats step 3-5 times；
Step 3: being added in reaction tube Chemoluminescent substrate, mixes well, and detects luminous value, meter by luminous tester
Calculation obtains the content of anti-insulin antibody.
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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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| CN1963505A (en) * | 2006-11-10 | 2007-05-16 | 郑州安图绿科生物工程有限公司 | Chemiluminescent examining method for insulin content and C-peptide content in human blood serum |
| CN102435737A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Quantitative insulin (INS) determination kit and assay method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1963505A (en) * | 2006-11-10 | 2007-05-16 | 郑州安图绿科生物工程有限公司 | Chemiluminescent examining method for insulin content and C-peptide content in human blood serum |
| CN102435737A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Quantitative insulin (INS) determination kit and assay method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN116466097B (en) * | 2023-06-14 | 2023-08-29 | 天津市协和医药科技集团有限公司 | Human insulin detection kit |
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Application publication date: 20190201 |