CN109655626A - A kind of detection kit and its method of complete homogeneous determination glucagon - Google Patents
A kind of detection kit and its method of complete homogeneous determination glucagon Download PDFInfo
- Publication number
- CN109655626A CN109655626A CN201811579133.3A CN201811579133A CN109655626A CN 109655626 A CN109655626 A CN 109655626A CN 201811579133 A CN201811579133 A CN 201811579133A CN 109655626 A CN109655626 A CN 109655626A
- Authority
- CN
- China
- Prior art keywords
- glucagon
- reagent
- antibody
- detection kit
- agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/74—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
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to field of biotechnology, disclose the detection kit and its method of a kind of complete homogeneous determination glucagon.The present invention realizes completely homogeneous detection glucagon using spatial neighbor chemiluminescence, it is based on double sandwich techniques, two monoclonal antibodies are bonded respectively on the antigenic determinant of glucagon molecule, by calibration object or sample to be tested, the anti-human glucagon antibody of horseradish peroxidase-labeled, 9, the anti-human glucagon antibody of 10- acridan label is added in microwell plate together, without coating, antigen after incubation in two monoclonal antibodies and sample forms antibody-antigen-antibody sandwich complex, washing process is not needed, after antioxidant mixing is added, triggering agent is added, the luminous intensity values in a period of time are continuously detected immediately, and calculate the content of glucagon in sample to be tested.Detection kit of the invention has the characteristics that high stability, detection rapid and convenient, high sensitivity and high specificity.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of detection kit of complete homogeneous determination glucagon
And its method.
Background technique
Glucagon (glucagon, Glu) is also known as glucagon, anti-insulin or insulin B.It is with pancreas islet
A kind of hormone that element is secreted by the alpha Cell of islet of vertebrate pancreas.It is opposite with insulin anti-, play a part of increasing blood glucose.
In nineteen fifty-three, crystallization is obtained by precipitation and separation.It is using N- terminal Histidin as starting point, and C-terminal threonine is the 29 of terminal
One single-stranded peptide (molecular weight is about 3500) of a amino acid residue composition, intramolecular does not have S -- S, at this point, complete
It is different from insulin entirely.The structure of the compound is by nearest chemical synthesis institute affirmative.The effect initial stage mistake of glucagon
Journey is specifically bound with the receptor being present on target cell film, adenyl cyclase is activated, cyclic AMP becomes
The activated phosphorylated enzyme of second messenger promotes decomposition of glycogen.
Glucagon immue quantitative detection reagent box provides a kind of serum, edta plasma, pancreas hyperglycemia in cell culture fluid
The quantitative detecting method of element, can be used for clinical detection, auxiliary as blood sugar disorders (such as diabetes, hyperglycemia skin disease syndrome)
Help the reference of diagnosis.
The glucagon detection of the registration certificate found in the general bureau of state food pharmaceuticals administration at present before the deadline
Kit only has one, and detection method is enhanced chemiluminescence immunoassay.This method has certain limitation, wherein most main
Wanting any is: reinforcing agent is only effective to luminol (class) hydrogen peroxide (or perborate) system of Catalyzed Synthesis By Peroxidase.Together
When certain reinforcing agents be not easy to obtain, this allows for the technology and is clinically widely applied to be restricted.Therefore, searching is easy to get
The good and applied widely reinforcing agent of reinforcing effect, so that it is the needing to solve at present of the task that this technology is more practical.Together
When, which is heterogeneous reaction, and needing will be on antibody coating and reaction plate.This has the antibody being coated on plate
It is likely to occur part to be fixed in conjunction with epitope, the variation of space structure occurs.The detection method is complex for operation step, detection time
It is long, it is easy to cause the reduction of antigen-antibody reaction specificity, affects the sensitivity of kit and linear.To glucagon
In detection, since glucagon sample is easy to happen the characteristic of agglutination, heterogeneous reaction is easy to cause antigen-antibody that can not fill
The combination divided, testing result are easy to appear wild effect.
There are also enzyme linked immunological double-antibody method, rocket immunoelectrophoresis, radiation to exempt from for the method for existing detection glucagon
Epidemic disease method etc..Enzyme linked immunological double-antibody method (ELISA) be known specific antibody is packaged in solid phase carrier (plastic plate shrinkage pool or
On the scraps of paper), sample to be checked is added, the antigen in sample can add after washing away unbonded material in conjunction with the antibody on carrier
The enzymic-labelled antibody for entering the antigen, washes away unbonded enzyme labelled antibody, and reagent adding R3 colour developing measures color with enzyme immune detector
Optical density, antigen can be quantitative determined.Rocket immunoelectrophoresis (rocket immunoelectrophoresis, RIEP) is by list
A kind of quantitative measurement technology combined to Immune proliferation and electrophoresis.When electrophoresis, do not moved contained in the antibody in agar gel
It is dynamic, and promote antigen in sample to positive swimming under the action of electric field.When antigen and antibody molecule reach proper proportion,
The insolubilized immune complexes precipitating peak of a shape such as rocket is formed, the antigen concentration in the height and sample at peak is in positive
It closes.It therefore, is vertical sit with the precipitating peak formed after different dilution standard antigen swimmings when antibody concentration is fixed in agar
Mark, antigen concentration is abscissa, draws standard curve.Containing for determined antigen can be calculated according to the precipitating peak length of sample
Amount；Conversely, the content (i.e. reversed rocket immunoelectrophoresis) of test antibodies can be measured when antigen concentration is fixed in agar.On
State two methods have the defects that it is certain: accuracy is poor, and the operating time is long, the degree of automation is low, is unable to satisfy clinically big
Amount sample quickly detects demand.The testing principle of radioimmunology are as follows: utilize radioisotope labeling antigen or antibody, then
With tested antibody or antigen binding, the principle of antigen antibody complex is formed to be analyzed.This method detection process influences
Factor is more, but main is there are radiocontamination, and this problem is not easy to solve, to limit its development and application.
Therefore, for the deficiency of existing detection method, it is necessary to a kind of quality stabilization is provided, rapid and convenient is detected, it is sensitive
Degree is high, the kit for being used to detect glucagon in blood of high specificity.
Summary of the invention
It is an object of the invention to provide a kind of complete homogeneous determination pancreas hyperglycemia in place of overcome the deficiencies in the prior art
The detection kit and its method of element, the detection kit realize completely homogeneous detection pancreas using spatial neighbor chemiluminescence
Glucagons has the characteristics of stability is high, detection rapid and convenient, high sensitivity, high specificity.
To achieve the above object, the technical solution adopted by the present invention is as follows:
A kind of detection kit of complete homogeneous determination glucagon, including reagent R1, reagent R2 and reagent R3, it is described
Reagent R1 includes that 9,10- acridan (Acridan) marks anti-human glucagon antibody and horseradish peroxidase-labeled anti-
Human glucagon antibody, the reagent R2 includes antioxidant, and the reagent R3 includes triggering agent.
The testing principle of kit of the present invention are as follows: be separately added into Acridan mark anti-human glucagon antibody, antigen,
The anti-human glucagon antibody of horseradish peroxidase-labeled forms antibody-antigen-antibody compound, is not necessarily to washing process, adds
Enter after the reagent R2 short time is incubated for, the reagent R3 continuous strong light in detection a period of time (usually 1~3s) immediately is added
Degree, and its peak area is calculated, it is compared with standard curve, the content of glucagon in sample, glucagon can be extrapolated
Content be positively correlated with its peak area.
The key point that detection kit of the invention can obtain said effect is, the oxidable examination of horseradish peroxidase
Triggering agent in agent R3 generates free radicals, and free radical aoxidizes the Acridan on anti-human glucagon antibody immediately, due to certainly
It is very short by the half-life period of base, it can only spread in the molecule, therefore hair could be reacted by only forming antibody-antigen-antibody compound
Light.
Compared with previous chemoluminescence method, most important difference is for it, and detection method is spatial neighbor
Learn luminescence method, belong to completely homogeneous detection, be closest to the reaction of internal nature, have simple and quick, examination scope is wide,
The high feature of high sensitivity, accuracy.
Kit of the invention is not necessarily to washing process, process flow is greatly optimized, to reagent without carrying out coating processing
Each performance of box has biggish guarantee.Therefore, kit of the present invention has detection range wide, detects rapid and convenient, sensitivity
The features such as high, specific and reproducible, and the discharge of a large amount of wastes is reduced, it is more suitable for clinically using.In reagent R2
Antioxidant can effectively remove free radical or other oxidizing substances in sample and reagent, effectively reduce background, thus
Improve the accuracy and sensitivity of detection kit.
Further, the antioxidant be at least one of ascorbic acid, tea polyphenols, glutathione, further
Ground, the antioxidant are ascorbic acid.
Ascorbic acid, tea polyphenols, glutathione can be used as background remover, remove sample and free radical in reagent or
Other oxidizing substances reduce the background signal in detection process, effectively improve the accuracy and sensitivity of detection kit.
Further, the triggering agent be at least one of p-Coumaric Acid, hydrogen peroxide, urea peroxide, more into
One step, the triggering agent is hydrogen peroxide.
Further, the reagent R1 includes the component of following concentration:
9,10- acridans mark anti-human glucagon antibody 1-5mg/L
The anti-human glucagon antibody 0.1-0.3mg/L of horseradish peroxidase-labeled
The reagent R2 includes the component of following concentration:
Antioxidant 0.05-10w/v%
The reagent R3 includes the component of following concentration:
Trigger agent 0.01-10w/v%.
Further, the kit further includes reinforcing agent and stabilizer, and the reagent R1 includes the component of following concentration:
The reagent R2 includes the component of following concentration:
Antioxidant 0.05-10w/v%
The reagent R3 includes the component of following concentration:
Trigger agent 0.01-10w/v%.
Further, the reagent R1 includes the component of following concentration:
The reagent R2 includes the component of following concentration:
Antioxidant 0.05w/v%
The reagent R3 includes the component of following concentration:
Trigger agent 0.01-10w/v%.
Further, the kit further includes buffer, reinforcing agent, stabilizer and preservative, and the reagent R1 includes
The component of following concentration:
The reagent R2 includes the component of following concentration:
Antioxidant 0.05-10w/v%
Preservative 0.01-10w/v%
The reagent R3 includes the component of following concentration:
Trigger agent 0.01-10w/v%.
Further, the pH value of the buffer is 6.5-8.5, and the buffer is phosphate buffer, Tris buffering
At least one of liquid, MES buffer and HEPES buffer solution, further, the pH value of the buffer are 7.4, described slow
Fliud flushing is HEPES buffer solution.
Further, the stabilizer is sorbierite, polyvinyl pyrrolidone, glycerol, mannitol, ethylenediamine tetra-acetic acid
At least one of disodium, further, the stabilizer are sorbierite and polyvinyl pyrrolidone.Stabilization of the invention
Agent marks anti-human glucagon antibody and the anti-human glucagon antibody of horseradish peroxidase-labeled to 9,10- acridan
With protective effect, antibody activity is avoided to reduce, improves the stability of testing result.
Further, the reinforcing agent is chlorination trimethylcetylammonium, in 4- phenylboric acid, TritonX-100
At least one, further, the reinforcing agent are chlorination trimethylcetylammonium.Reinforcing agent and antioxidant there are energy
Kit engaged background of the present invention is enough effectively reduced, significantly increases luminous signal intensity, to improve kit
Sensitivity, accuracy and repeatability.
Further, the preservative be at least one of Sodium Mercurothiolate, proclin 300, KY100 biological preservative,
Further, the preservative is Sodium Mercurothiolate.
The present invention also provides the detection method of the detection kit of above-mentioned complete homogeneous determination glucagon, to
Reagent R1 is added in test sample sheet, in 37 DEG C of 5~30min of incubation, reagent R2 is added and mixes, reagent R3 is added, immediately detection reaction
The luminous intensity values of object calculate the content of glucagon in sample to be tested, wherein sample to be tested, reagent R1, reagent R2 and examination
The volume ratio of agent R3 is 1:16:1:15.Detection method is spatial neighbor chemoluminescence method, can homogeneously detect pancreas completely
Glucagons, it is simple and quick, examination scope is wide, high sensitivity, accuracy are high.
Compared with prior art, the invention has the benefit that
(1) kit of the invention realizes completely homogeneous detection glucagon using spatial neighbor chemoluminescence method, makes
Antigen-antibody reaction is more abundant, to improve the utilization rate of antibody, reduces the cost of kit.
(2) compared with existing chemoluminescence method, antigen-antibody of the present invention does not change anti-without being fixed on solid phase carrier
The space structure of body exposes antibody binding epitope sufficiently, retains strongest specificity, and kit is made to have higher sensitivity
And accuracy.
(3) compared with existing chemoluminescence method, kit of the invention is without coating and the process of sessile antibody, significantly
Shorten the production time of kit, to guarantee that antigen-antibody activity is unaffected, ensure that testing result has preferably weight
Renaturation.
(3) detection kit of the present invention does not need washing process in the detection process, does not need a large amount of cleaning solution, reduces
The discharge of waste, more conducively clinically uses and its processing to waste.
(4) present invention passes through reinforcing agent and reagent R2 in each component, especially reagent R1 in preferred reagent R1 and reagent R2
The signal background in detection process is effectively reduced in the presence of middle antioxidant, enhances luminous signal intensity, to significantly improve examination
Sensitivity, accuracy and the repeatability of agent box.
Detailed description of the invention
Fig. 1 is the canonical plotting obtained using the kit measurement of embodiment 6, and wherein X-axis indicates that glucagon is dense
Degree, Y-axis indicate peak area；
Fig. 2 is the kit and the correlation of the glucagon detection kit of Mercodia AB of the embodiment of the present invention 6
Comparison diagram, wherein X-axis indicates the serum of the kit measurement of embodiment 6 as a result, Y-axis expression is Mercodia AB kit
The serum result of measurement.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiment to the present invention
It further illustrates.It will be appreciated by those skilled in the art that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.
Acridan:9,10- acridan.
HEPES: ethoxy croak piperazine second thiosulfonic acid, nonionic both sexes buffer can control constant pH range the long period.
MES:2- (N- morpholine) ethanesulfonic acid.
EDTA K2: EDTAP dipotassium ethylene diamine tetraacetate.
In embodiment, used experimental method is conventional method unless otherwise specified, material used, reagent etc.,
It is commercially available unless otherwise specified.
The anti-glucagon antibody of following embodiment is purchased from BioPorto Diagnostics A/S
(ANTIBODYSHOP)。
The preparation of embodiment 1, glucagon calibration object
The matrix liquid of calibration object of the present invention includes the component of following concentration:
Calibration object matrix liquid is prepared according to aforementioned proportion, with this matrix liquid by glucagon antigen diluent at respective concentration
Calibration object, concentration is respectively 1pmol/L, 4pmol/L, 16pmol/L, 32pmol/L, 128pmol/L, separately plus matrix liquid
0pmol/L, is saved totally in 2~8 DEG C by 6 bottles, every bottle of 1mL.
Embodiment 2, Acridan mark anti-glucagon Antibody preparation
The anti-glucagon antibody of 250 μ g (being first diluted to 1mg/mL with 20mM PBS) is taken, the Acridan of 40 μ L is added,
The 20mM PBS for adding 710 μ L is incubated at room temperature 1h on gyroscope；With marker buffer, (30mM pH 7.6PBS is slow again
Fliud flushing, 0.1w/v% sodium thiosulfate, 0.5w/v%BSA, 0.4w/v% mannitol and 0.05w/v%ProClin300) dilution
To working concentration.
Method according to this, being prepared for concentration is respectively 1.5mg/L, 3mg/L, 6mg/L, 12mg/L, 24mg/L, 48mg/L
Acridan marks anti-glucagon antibody.
The anti-glucagon Antibody preparation of embodiment 3, horseradish peroxidase-labeled
Anti- glucagon antibody is marked using commercialization HRP labelling kit, then with marker buffer (30mM pH
7.6PBS buffer, 0.05w/v%VC or 0.05w/v% reduced glutathione, 0.03 w/v%EC Oxyrase, 0.5w/
Mannitol, the 0.5w/v% PEG 20000 of v%, the enzyme stabilizers of 0.1w/v%, 0.05w/v%ProClin 300.) dilute
Release working concentration.
Method according to this, be prepared for concentration be respectively 0.025mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L, 0.4mg/L,
The HRP of 0.8mg/L marks anti-glucagon antibody.
Embodiment 4, Acridan mark anti-human glucagon antibody and the anti-human pancreas hyperglycemia of horseradish peroxidase-labeled
The concentration of plain antibody selectes standard
Mark anti-human glucagon antibody and the anti-human pancreas of horseradish peroxidase-labeled high Acridan using square matrix method
Blood glucose element antibody is matched with various concentration, is greater than 5 with lowest signal-to-noise, highest signal to noise ratio is greater than on the basis of 200, preferentially
The proportion relation for selecting cost minimum.
The Acridan that concentration is 1.5mg/L, 3mg/L, 6mg/L, 12mg/L, 24mg/L, 48mg/L will be marked respectively
Anti-human glucagon antibody is 0.025mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L, 0.4mg/L, 0.8mg/ with concentration
The anti-human glucagon antibody of the horseradish peroxidase-labeled of L carries out the investigation of square matrix method.Two groups of ratios intersect proportion, and discovery is most
Excellent ratio is that 3mg/L Acridan marks anti-human glucagon antibody and 0.2mg/L horseradish peroxidase-labeled
Anti-human glucagon antibody, therefore select this suitable ratio.
Embodiment 5, reinforcing agent and background remover influence reagent performance
1. the reinforcing agent chlorination trimethylcetylammonium in reagent R1 is to the reactivity of reagent R1 and the influence of signal-to-noise ratio
Chlorination trimethylcetylammonium is not added when reagent preparation R1, other components unchangeds investigate each concentration calibration product
Peak area and signal-to-noise ratio.As a result such as table 1.
Influence of 1 reinforcing agent of table to reagent R1 reactivity and signal-to-noise ratio
Reinforcing agent chlorination trimethylcetylammonium has stronger raising effect, school to calibration object response value in reagent R1
The progressive ratio of quasi- product concentration and peak area is more preferable, while also improving the signal-to-noise ratio of reagent R1.
2. reinforcing agent chlorination trimethylcetylammonium concentration influences reactivity
Change chlorination trimethylcetylammonium concentration, other components unchangeds detect background such as table 2.
2 various concentration reinforcing agent of table is on reactive influence
From table 2 it can be seen that calibration object reactivity is not when chlorination trimethylcetylammonium concentration is greater than 0.01w/v%
It significantly alters again, therefore chlorination trimethylcetylammonium optium concentration is 0.01w/v%.
3. ascorbic acid (VC) influences background
VC is not added when reagent preparation R2, other components unchangeds detect the peak area of background S0, as a result such as table 3.
3 VC of table influences background
Addition antioxidant VC can prevent free radical from reacting with free Acridan, it is ensured that free radical can only be in antibody-
The intramolecular of antigen-antibody complex is transmitted, to reduce background, improves the accuracy of testing result.
4.VC concentration influences background
Change background remover VC concentration, other components unchangeds detect background such as table 4.
4 various concentration VC of table influences background
When VC concentration is greater than 0.05w/v%, background is held essentially constant as can be seen from Table 4, therefore VC optium concentration is
0.05w/v%.
Embodiment 6, a kind of detection kit of complete homogeneous determination glucagon
The detection kit of the complete homogeneous determination glucagon of the present embodiment includes reagent R1, reagent R2 and reagent
R3,
Reagent R1 includes the component of following concentration:
Reagent R2 includes the component of following concentration:
Ascorbic acid 0.05w/v%
300 0.045w/v% of proclin
Reagent R3 includes the component of following concentration:
Hydrogen peroxidase 10 .68w/v%
300 0.045w/v% of proclin.
Kit semi-finished product are prepared according to above-mentioned formula, glucagon inspection can be just assembled into after verifying is qualified
Test agent box.
Embodiment 7, detection method
Using glucagon detection kit described in embodiment 6, for multi-function microplate reader LB942S, parameter is such as
Table 5.
Analysis method: 5 μ L samples to be tested and 80 μ L reagent R1,37 DEG C of incubation 30min are added in one-step method, and 5 μ L examination is added
Agent R2 is mixed, and 75 μ L reagent R3 are added, and the luminous intensity values RLU in continuous detection a period of time (1~3s), passes through instrument immediately
Peak area is calculated in device.Using the Glucagon concentrations of calibration object as X-coordinate after measurement, using being sat as Y for peak area
Mark, makes standard curve as shown in Figure 1, equation y=999.92x+10.558, R2=1, sample is calculated according to the standard curve
The concentration of middle glucagon.
Table 5
When being detected using kit of the invention, the incubation time after reagent R1 is added can be according to actually detected needs
It is adjusted to 5~30min.
Embodiment 8, correlation test
Reagent is measured using the glucagon of kit of the present invention (specific formula is with embodiment 6) and Mercodia AB
Multi-function microplate reader LB942S is respectively adopted in box, Coase steps SMART1000 full-automatic illumination instrument to 40 parts of fresh human serums by each
From parameter be measured simultaneously, correlation regression analysis is carried out to measured value, measurement result is shown in Fig. 2.
Found out by the result of Fig. 2, the coefficient R of two kinds of reagents2=0.9941, regression equation y=0.9923x-
0.0121.The result shows that this reagent with listed contrast agent measurement patients serum's correlation it is good, have well specificity
And accuracy.
Embodiment 9, accuracy and precision test
Reagent: kit (specific formula is with embodiment 6) of the present invention, calibration object, quality-control product.
Instrument: multi-function microplate reader LB942S.
Operating procedure: being calibrated using calibration object, measures each Quality Control 10 times, calculates test mean value, SD, CV and relative deviation.
Table 6 the result shows that, the relative deviation that kit of the present invention detects each Quality Control is respectively less than 3%, and accuracy is good.Measurement
10 CV values with sample are respectively less than 5%, and precision is preferable.
6 measurement result of table (pmol/L)
Embodiment 10, linear test
Using kit of the present invention (specific formula is with embodiment 6), calibration object, high concentration glucagon sample, Quality Control
Product.
Instrument: multi-function microplate reader LB942S.
Operating procedure: taking high concentration glucagon sample to be configured to 256pmol/L, using blank solution as dilution,
It is diluted by 0:1,1:1,1:3,1:7,1:63,1:255, each sample replication 3 times.
Table 7 the result shows that, kit of the present invention is linear good within the scope of 1~128pmol/L, and the range of linearity is wider.
7 measurement result of table (pmol/L)
Embodiment 11, specific test
Take enteroglucagon, oxyntomodulin and glucagon-like-peptide-1 be diluted to respectively with dilution 10 μ g/L,
50 μ g/L, 2 μ g/L are measured with kit of the present invention (specific formula is with embodiment 6), and measurement result is as shown in table 8.
Table 8 the result shows that, measurement result of the enteroglucagon of 10 μ g/L on this kit be 0.47pmol/L, 50 μ
Measurement result of the oxyntomodulin of g/L on this kit is 0.73pmol/L, the glucagon-like-peptide-1 of 2 μ g/L
Measurement result on this kit is 0.68pmol/L, is not higher than 1pmol/L, illustrates the specificity of kit of the present invention
Preferably.
8 measurement result of table
Embodiment 12, anti-interference test
Fresh mix serum is taken, 5 equal portions are divided into, corresponding interfering substance is added according to the following table 9, takes kit of the present invention
(specific formula is with embodiment 6) measures the content of glucagon in serum, and measurement result is as shown in table 10.
Relative deviation (%)=(measurement mean value-noiseless object sample measurement mean value of interference sample)/noiseless object sample
This measurement mean value × 100%.
10 result of table indicates that kit of the present invention is not by jaundice (bilirubin < 20mg/dL), piarhemia (triglycerides <
2000mg/dL), the interference of haemolysis (hemoglobin < 200mg/dL), illustrates that the anti-interference ability of kit of the present invention is stronger.
9 interfering substance concentration of table
| Chaff interferent | Bilirubin (mg/dL) | Triglycerides (mg/dL) | Hemoglobin (mg/dL) |
| Concentration | 20 | 2000 | 200 |
10 measurement result of table
In conclusion the present invention is spatial neighbor chemoluminescence method, belong to completely homogeneous detection, detection is very quick, greatly
The reaction time is shortened greatly, and the utilization rate of antibody can be improved, reduces the cost of kit；Detection kit of the present invention is without packet
The process of quilt and sessile antibody, greatly shortens the production time of kit, so that antigen-antibody activity is unaffected, ensure that inspection
Surveying result has preferably repeatability；Washing process is not needed in the detection process, reduces the discharge of waste, is more conducive to face
It is used on bed and its processing to waste；Reinforcing agent and antioxidant in kit can be effectively reduced in detection process
Signal background enhances luminous signal intensity, to significantly improve the sensitivity of kit, accuracy and repeatability.
Inventor has found that the detection kit concentration of component that the present invention uses can be according to actual needs through overtesting
Appropriate adjustment is carried out, the reagent R1 includes the component of following concentration: buffer 5-100mmol/L, 9,10- acridan mark
Remember anti-human glucagon antibody 1-5mg/L, the anti-human glucagon antibody 0.1-0.3mg/L of horseradish peroxidase-labeled,
Stabilizer 0.01-10w/v%, reinforcing agent 0.01-10w/v%, preservative 0.01-10w/v%；The reagent R2 includes following dense
The component of degree: antioxidant 0.05-10w/v%, preservative 0.01-10w/v%；The reagent R3 includes the group of following concentration
Point: triggering agent 0.01-10w/v%.According to the high sensitivity of the kit of above-mentioned formula preparation, high specificity is reproducible.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of detection kit of complete homogeneous determination glucagon, which is characterized in that including reagent R1, reagent R2 and examination
Agent R3, the reagent R1 include that 9,10- acridan marks anti-human glucagon antibody and horseradish peroxidase-labeled anti-
Human glucagon antibody, the reagent R2 includes antioxidant, and the reagent R3 includes triggering agent.
2. the detection kit of complete homogeneous determination glucagon according to claim 1, which is characterized in that described anti-
Oxidant is at least one of ascorbic acid, tea polyphenols, glutathione, it is preferable that the antioxidant is ascorbic acid.
3. the detection kit of complete homogeneous determination glucagon according to claim 1, which is characterized in that the touching
Hair agent is at least one of p-Coumaric Acid, hydrogen peroxide, urea peroxide, it is preferable that the triggering agent is hydrogen peroxide.
4. the detection kit of described in any item complete homogeneous determination glucagons, feature exist according to claim 1~3
In the reagent R1 includes the component of following concentration:
9,10- acridans mark anti-human glucagon antibody 1-5mg/L
The anti-human glucagon antibody 0.1-0.3mg/L of horseradish peroxidase-labeled
The reagent R2 includes the component of following concentration:
Antioxidant 0.05-10w/v%
The reagent R3 includes the component of following concentration:
Trigger agent 0.01-10w/v%.
5. the detection kit of complete homogeneous determination glucagon according to claim 4, which is characterized in that further include
Buffer, reinforcing agent, stabilizer and preservative.
6. the detection kit of complete homogeneous determination glucagon according to claim 5, which is characterized in that the examination
Agent R1 includes the component of following concentration:
The reagent R2 includes the component of following concentration:
Antioxidant 0.05-10w/v%
Preservative 0.01-10w/v%
The reagent R3 includes the component of following concentration:
Trigger agent 0.01-10w/v%.
7. the detection kit of complete homogeneous determination glucagon according to claim 5 or 6, which is characterized in that institute
The pH value for stating buffer is 6.5-8.5, and the buffer is phosphate buffer, Tris buffer, MES buffer and HEPES
At least one of buffer.
8. the detection kit of complete homogeneous determination glucagon according to claim 5 or 6, which is characterized in that institute
Stating stabilizer is at least one of sorbierite, polyvinyl pyrrolidone, glycerol, mannitol, disodium ethylene diamine tetraacetate.
9. the detection kit of complete homogeneous determination glucagon according to claim 5 or 6, which is characterized in that institute
Stating reinforcing agent is at least one of chlorination trimethylcetylammonium, 4- phenylboric acid, TritonX-100, the preservative
For at least one of Sodium Mercurothiolate, proclin 300, KY100 biological preservative.
10. the detection side of the detection kit of described in any item complete homogeneous determination glucagons according to claim 1~9
Method, which is characterized in that reagent R1 is added in sample to be tested, in 37 DEG C of 5~30min of incubation, reagent R2 is added and mixes, examination is added
Agent R3 detects the luminous intensity values of reactant immediately, calculates the content of glucagon in sample to be tested, wherein sample to be tested,
The volume ratio of reagent R1, reagent R2 and reagent R3 are 1:16:1:15.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811579133.3A CN109655626A (en) | 2018-12-21 | 2018-12-21 | A kind of detection kit and its method of complete homogeneous determination glucagon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811579133.3A CN109655626A (en) | 2018-12-21 | 2018-12-21 | A kind of detection kit and its method of complete homogeneous determination glucagon |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109655626A true CN109655626A (en) | 2019-04-19 |
Family
ID=66115494
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811579133.3A Pending CN109655626A (en) | 2018-12-21 | 2018-12-21 | A kind of detection kit and its method of complete homogeneous determination glucagon |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109655626A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110057641A (en) * | 2019-04-30 | 2019-07-26 | 山东博科生物产业有限公司 | The compound calibration object and preparation method thereof containing 6 indexs of strong antijamming capability |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060121626A1 (en) * | 2004-12-03 | 2006-06-08 | Genzyme Corporation | Diagnostic assay device |
| CN102333886A (en) * | 2009-02-27 | 2012-01-25 | 贝克曼考尔特公司 | Solution phase homogeneous assays |
| CN102680456A (en) * | 2011-03-16 | 2012-09-19 | 北京联众泰克科技有限公司 | ECLI (Electro ChemiLuminescence Immunoassay) determining method |
| CN103344764A (en) * | 2013-06-19 | 2013-10-09 | 天津美德太平洋科技有限公司 | Reagent, method and kit for detection of biological activity of glucagon-like peptide-1 (GLP-1) |
| CN105116140A (en) * | 2015-09-21 | 2015-12-02 | 广州市进德生物科技有限公司 | Glucagon detection kit and detection method thereof |
-
2018
- 2018-12-21 CN CN201811579133.3A patent/CN109655626A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060121626A1 (en) * | 2004-12-03 | 2006-06-08 | Genzyme Corporation | Diagnostic assay device |
| CN102333886A (en) * | 2009-02-27 | 2012-01-25 | 贝克曼考尔特公司 | Solution phase homogeneous assays |
| CN102680456A (en) * | 2011-03-16 | 2012-09-19 | 北京联众泰克科技有限公司 | ECLI (Electro ChemiLuminescence Immunoassay) determining method |
| CN103344764A (en) * | 2013-06-19 | 2013-10-09 | 天津美德太平洋科技有限公司 | Reagent, method and kit for detection of biological activity of glucagon-like peptide-1 (GLP-1) |
| CN105116140A (en) * | 2015-09-21 | 2015-12-02 | 广州市进德生物科技有限公司 | Glucagon detection kit and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 荆春艳等: "初诊2型糖尿病患者胰高血糖素水平的变化及临床意义", 《安徽医药》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110057641A (en) * | 2019-04-30 | 2019-07-26 | 山东博科生物产业有限公司 | The compound calibration object and preparation method thereof containing 6 indexs of strong antijamming capability |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108362688B (en) | Detection kit for chemiluminescence of 25-hydroxy vitamin D magnetic particles | |
| US20190219569A1 (en) | Fluorescence immunochromatographic detection card and a preparation method therefor and use thereof | |
| CN102735833B (en) | Thyroperoxidase antibody homogeneous-phase luminescent immunoassay kit and detection method thereof | |
| CN102072957B (en) | Hepatitis C virus antibody diagnostic kit and preparation method thereof | |
| CN106582848B (en) | A kind of preparation method and application for the double catalysis analogue enztmes mediating Gold Mineralization approach based on ferroheme | |
| CN106918708A (en) | A kind of competition law turbid kit of latex enhancing immune transmittance for detecting insulin | |
| CN108169497A (en) | Human prolactin detection kit and preparation method and application | |
| CN109239367A (en) | Measure the method and kit of small molecule compound | |
| CN102998466A (en) | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for growth hormone (GH), and preparation method of kit | |
| CN109725162A (en) | A kind of detection kit and its method of complete homogeneous determination insulin | |
| CN108931652A (en) | A kind of kit with Magnetism particulate immuno chemistry luminescence method detection myoglobin content | |
| Jin et al. | Magnetic particle-based chemiluminescence enzyme immunoassay for free thyroxine in human serum | |
| US20210247392A1 (en) | Direct immunoassay measurement of autoantibodies | |
| TW201802472A (en) | Anti-human hemoglobin monoclonal antibody or antibody kit, insoluble carrier particle to which anti-human hemoglobin monoclonal antibody is immobilized, and measurement reagent and measurement method using same | |
| CN110082537A (en) | Vascular endothelial growth factor chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN109655626A (en) | A kind of detection kit and its method of complete homogeneous determination glucagon | |
| CN105758846A (en) | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol | |
| US20220260558A1 (en) | Antigen measuring method and measuring apparatus | |
| CN105891501A (en) | Colloidal gold immunocolorimetry kit for detecting retinol conjugated protein (RBP) and preparation method of kit | |
| CN114112957B (en) | Adiponectin determination kit and application thereof | |
| CN116338163A (en) | Method for quantitatively detecting CD3/GPRC5D bispecific antibody by one-step method | |
| CN108918888A (en) | It is a kind of detect Endostatin latex enhancing immune than turbid kit and its application | |
| CN113109325A (en) | Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof | |
| CN113552372A (en) | Magnetic particle chemiluminescence immunoassay kit for SAA detection and preparation method thereof | |
| CN103163302B (en) | Oligopeptide antibody kit prepared by directional cross-coupling technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190419 |