CN109030829A - A kind of quantification kit and its application method of homogeneous chemistry luminescence method detection dog IL-6 - Google Patents
A kind of quantification kit and its application method of homogeneous chemistry luminescence method detection dog IL-6 Download PDFInfo
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- CN109030829A CN109030829A CN201810694904.7A CN201810694904A CN109030829A CN 109030829 A CN109030829 A CN 109030829A CN 201810694904 A CN201810694904 A CN 201810694904A CN 109030829 A CN109030829 A CN 109030829A
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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
- G01N33/68—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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The present invention relates to chemiluminescence immune assay, the quantification kit and its application method of specifically a kind of homogeneous chemistry luminescence method detection dog IL-6；The detection solution that the kit uses includes 1 conjugate of DNA1-IL-6 antibody, 2 conjugate of DNA2-IL-6 antibody, DNA3, restriction enzyme and the graphene oxide for marking acridinium ester (AE), the DNA1 and DNA2 have 6 base complementrities, the DNA3 contains 2 restriction enzyme sites, and there are 9 bases and DNA1, DNA2 complementary pairing respectively, DNA3 is adsorbed on surface of graphene oxide by pi-pi accumulation effect, and the chemiluminescence of the AE of end mark is quenched because CRET occurs；This method is homogeneous immunoassay method, it is easy to operate, analysis time is substantially reduced simultaneously, the measurement of single sample can be completed in 5-10 minutes, in conjunction with ortho position striking effect and chemiluminescent molecule beacon, the switch of graphene quenching mechanism is induced by immune response, so that chemiluminescence signal is discharged, without washing and purification procedures.
Description
Technical field
The present invention relates to chemiluminescence immune assay, the quantitative examination of specifically a kind of homogeneous chemistry luminescence method detection dog IL-6
Agent box and its application method.
Background technique
Chemiluminescence immune assay (chemiluminescence immunoassay, CLIA), is will have high sensitivity
Chemical luminescent detecting technology combined with the immune response of high specific, for various antigens, haptens, antibody, hormone,
The detection and analysis technology of enzyme, fatty acid, vitamin and drug etc..By whether there is separation cleaning step, it is divided into out-phase chemistry hair
Light method and homogeneous chemistry luminescence method.Currently, diagnosing detection field in vitro, domestic and international testing product is substantially all using out-phase chemistry
Luminescence method.External producer includes Roche, Abbott Laboratories, Beckman, Siemens, the Meikang Suo Lin and Xi Si etc., and domestic manufacturer includes new produces
Industry, Antu, mikey, Mai Rui, pool are at, long brilliance doctor etc..Out-phase chemoluminescence method depends on physical separation, separates typical require
Be that crucial reactant is optionally immobilized on some solid substrates so that some physical processes for example filter, deposit, coalescing or
Person's magnetic separation can be used；And washing step is required, also to remove free ingredient.Therefore out-phase chemoluminescence method is whole
A analytic process step is more, time-consuming, complicated for operation, at high cost, and professional technician is needed to operate Special instrument in most cases
Device.And homogeneous chemistry luminescence immunoassay directly carries out chemiluminescence inspection without separation and cleaning step under pure liquid-phase condition
It surveys, it is easy to operate quick, it is suitble to on-site test.
It is external at this stage to only have one homogeneous chemistry luminescence method (light-induced chemiluminescent) mediated using singlet oxygen of Siemens
Launch, detection need special LOCI module.LOCI technology is single step chemiluminescence sandwich immune detecting method, reagent
In containing there are two types of synthesis pearl reagent and a kind of biotin chemistry monoclonal antibody.The first pearl reagent (sensitive pearl) is coated with chain
Mould Avidin, and contain photaesthesia dyestuff；Second of pearl reagent (chemical pearl) is coated with another antibody, and contaminates containing chemiluminescence
Material；Sample and chemical pearl and biotinylated antibody form sandwich complex after being incubated for；Then sensitive pearl is added, with biotin
In conjunction with the rear immune complex for forming aggregation；Compound sensitive pearl therein under the irradiation of 680nm light can generate singlet oxygen, single
Line state oxygen can cause chemiluminescence reaction after permeating into chemical pearl, and generated chemiluminescence is reacted in measurement under 612nm wavelength
Signal.This method the high requirements on the equipment, marker material is special, is not easy to obtain.
Striking effect in ortho position makes distance between a pair of and antibody coupling DNA further by immune response, and then causes DNA group
Dress, triggering concatenated dna assemble and generate detection signal, make to be converted into the detection of protein the detection to DNA.By with it is various
DNA signal amplification technique combines the Sensitive Detection, it can be achieved that protein.It can homogeneously carry out in this way, simple, quick,
It is sensitive.
Graphite oxide dilute (GO) is a kind of two-dimentional carbon material of monoatomic thickness, and the energy transfer property of wide scope makes it
Performance is quenched with good, is a kind of good energy acceptor.We have found that acridinium ester can be effectively quenched in graphene oxide
Chemiluminescence between hydrogen peroxide.Mechanism is quenched between the illuminator and graphene oxide in the chemical luminous system
Photoelectron transfer or long-range Resonance energy transfer.
Interleukin-6 (Interleukin-6, IL-6) is a kind of glycoprotein, and body is after by inflammatory stimulus by T cell, B
The secretion such as cell, mononuclear macrophage and endothelial cell.IL-6 is a kind of manifold effect cell factor, and there are many biology to live for it
Property, including reaction and cytoprotection function are infected before mediating.IL-6 participates in the occurrence and development of many diseases, blood plasma level with
Inflammation, virus infection, autoimmune disease are closely related, its variation ratio CRP is earlier.It is presently considered to be inflammation, pyemic
The quantization marker of early stage sensitivity label object and chronic inflammation, effectively instructs the use of antibiotic.The IL-6 of different animals
The similitude of exon is very low, and showing IL-6, there are different species specificities, and perhaps this with living environment and be immunized
The Different Evolutionary that system progress is different and generates plays better biological effect to adapt to the environment present in itself.
Summary of the invention
The object of the present invention is to provide a kind of simple, quick, sensitive single step homogeneous chemistry luminescence immunoassay dogs
The quantification kit and its application method of IL-6.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
A kind of quantification kit of homogeneous chemistry luminescence method detection dog IL-6, the detection solution that the kit uses include
1 conjugate of DNA1- IL-6 antibody, 2 conjugate of DNA2- IL-6 antibody, mark the DNA3 of acridinium ester, restriction enzyme and
Graphene oxide, the DNA1 and DNA2 have 6 base complementrities, and the DNA3 contains 2 restriction enzyme sites, and has 9 bases respectively
With DNA1, DNA2 complementary pairing, DNA3 is adsorbed on surface of graphene oxide by pi-pi accumulation effect, the AE's of end mark
Chemiluminescence is quenched because CRET occurs.
Further, the DNA1 contains 53 bases, and 3 ' terminal modified sulfydryls pass through coupling agent succinimide -4- ring
Hexane -1- carbonic ester forms 1 conjugate of DNA1-IL-6 antibody in conjunction with the amino covalence on IL-6 antibody 1.
Further, the DNA2 contains 51 bases, and 5 ' terminal modified sulfydryls pass through coupling agent SMCC and IL-6 antibody 2
On amino covalence combine, formed 2 conjugate of DNA2- IL-6 antibody；
Further, the DNA3 contains 22 bases, 5 ' terminal modified acridinium esters (AE), 3-9 and 12-18 since 5 ' ends
The base sequence of base position is all CCTCAGC, can be identified by restriction enzyme Nt.BbvCI, to shear its formation
Double-strand；
The base complete complementary of 1-9 and DNA2 1-9 base position since 3 ' ends of the DNA3 since 5 ' ends；
The base complete complementary of 5-13 and DNA1 1-9 base position since 5 ' ends of the DNA3 since 3 ' ends.
A kind of application method of the quantification kit of homogeneous chemistry luminescence method detection dog IL-6, the application method include with
Lower step:
(1) sample solution and detection solution are mixed, and is incubated 5-10 minutes under the conditions of 37 DEG C；
(2) chemiluminescent substrate is added, light signal collection is carried out by chemiluminescence detector；
(3) concentration of IL-6 in sample is found out from standard curve.
Further, the luminous substrate is hydrogen peroxide and sodium hydroxide.
It is using the beneficial effect of technical solution of the present invention:
Chemiluminescence is quenched by graphene oxide, simultaneously in present invention combination ortho position striking effect and chemiluminescence detection technology
Restriction endonuclease circulation amplification strategy is introduced, proposes a kind of simple, quick, sensitive single step homogeneous chemistry luminescence immunoassay side
Method.Compared to existing immunoassay method, have the following characteristics that
(1) this method is homogeneous immunoassay method, can complete the one of protein with directly mixing for detection solution by sample
The detection of step formula, it is easy to operate, while analysis time is substantially reduced, the measurement of single sample can be completed in 5-10 minutes.
(2) ortho position striking effect and chemiluminescent molecule beacon are combined, graphene quenching mechanism is induced by immune response
Switch, to discharge chemiluminescence signal, without washing and purification procedures.
(3) restriction enzyme cyclic policy is combined, it is in place to carry out signal amplification, detection sensitivity is improved, keeps it more suitable
In the detection of low abundance proteins.
(4) using double identifications or more recognition mechanisms, cross reaction probability can be greatly reduced, while Solid Free carrier
Non-specific adsorption has very high specificity.
(5) graphene oxide is introduced as energy acceptor, and it is flat to construct a kind of versatile, simply at low cost analysis
Platform.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is the detection schematic diagram for the quantification kit that homogeneous chemistry luminescence method of the invention detects dog IL-6.
Fig. 2 is that ortho position striking effect induction immune complex forms schematic diagram.
Specific embodiment
In conjunction with the accompanying drawings, the present invention is further explained in detail.These attached drawings are simplified schematic diagram, only with
Illustration illustrates basic structure of the invention, therefore it only shows the composition relevant to the invention.
As shown in Fig. 1, the principle of the invention lies in: the detection solution of this method is coupled comprising DNA1- IL-6 antibody 1
Object, 2 conjugate of DNA2- IL-6 antibody, the DNA3 for marking acridinium ester (AE), restriction enzyme (Nt.BbvCI) and oxidation stone
Black alkene (GO).The DNA3 of design contains 2 restriction enzyme sites, and has 9 bases and DNA1, DNA2 complementary pairing respectively.When there is no mesh
In the presence of marking protein, DNA3 is adsorbed on the surface GO by pi-pi accumulation effect, and the chemiluminescence of the AE of end mark is because occurring
CRET and be quenched, show as " signal-off ".In the presence of having target protein, the antibody on two Ab-DNA compounds is simultaneously
It is identified, keep DNA1 and DNA2 close enough and form ortho position compound, and can hybridize with DNA3.Double-strand once being formed,
Nt.BbvCI can be cut from specific site, release ortho position compound, the ortho position compound released again with another
DNA3 hybridization causes the second repeating query ring cutting and cuts.In this way, each molecule ortho position compound can generate the short of a large amount of AE label
Chain DNA, these short chain DNAs hardly adsorb by GO, and chemiluminescent substrate is added in detection solution, can get and be exaggerated
Chemiluminescence (CL) signal.Regulate and control CRET based on ortho position hybridization reaction, the method that the present invention constructs a kind of " signal opening " is come
Realize high sensitivity, the high specific detection of protein.
DNA1 contains 53 bases, and 3 ' terminal modified sulfydryls pass through coupling agent succinimide -4- thiacyclohexane -1- carbonic ester
(SMCC), in conjunction with the amino covalence on antibody 1,1 conjugate of DNA1- antibody is formed.DNA2 contains 51 bases, and 5 ' ends are repaired
Sulfydryl is adornd, through coupling agent SMCC in conjunction with the amino covalence on antibody 2, forms 2 conjugate of DNA2- antibody.DNA1 and DNA2
There are 6 base complementrities.
DNA3 contains 22 bases, 5 ' terminal modified acridinium esters (AE), 3-9 the and 12-18 base position since 5 ' ends
Base sequence be all CCTCAGC, can be identified by restriction enzyme Nt.BbvCI, thus shear its formation double-strand.DNA3
The base complete complementary of the 1-9 base position of 1-9 and DNA2 since 3 ' ends since 5 ' ends；5- of the DNA3 since 3 ' ends
The base complete complementary of 1-9 base position of the 13 and DNA1 since 5 ' ends, as shown in Figure 2.
DNA3 is adsorbed on the surface GO by pi-pi accumulation effect, and the chemiluminescence of the AE of end mark is due to occurring CRET
It is quenched.
Above-mentioned detection solution is mixed with the sample solution containing object to be measured protein, is carried out under 37 DEG C of constant temperatures anti-
The short chain DNA that a large amount of AE modifications should be generated afterwards, is added chemiluminescent substrate hydrogen peroxide and sodium hydroxide, generates chemiluminescence letter
Number.
Fig. 1 is homogeneous chemistry chemiluminescence immunoassay method schematic diagram of the invention.
In conjunction with attached drawing 1, illustrate that homogeneously exempting from for acridinium ester chemiluminescent is quenched based on ortho position striking effect and graphene oxide
Detection of the epidemic disease analysis method to target protein (IL-6)
1, configuration detection solution: by 1 conjugate of DNA1-IL-6 antibody, 2 conjugate of DNA2-IL-6 antibody, modify AE DNA3,
Restriction enzyme and GO mixing, making their ultimate density is respectively 10nM, 10nM, 0.1 μM, 80U/mL and 20 μ g/ml.
2, by the calibration solution of 50 μ L various concentrations or contain target protein (IL-6) solution to be measured and 100 μ L
Solution mixing is detected, is incubated 5-10 minutes under the conditions of 37 DEG C.
3,200 μ L chemiluminescent substrates are added in solution after incubation, and detect the chemiluminescence letter of the solution immediately
Number, detection time 3s.According to the values of chemiluminescence of record, mesh in the calibration curve and solution to be measured of target protein quality detection is obtained
Mark the concentration of protein.
Taking the above-mentioned ideal embodiment according to the present invention as inspiration, through the above description, relevant staff is complete
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.It is all in essence of the invention
Within mind and principle, any modification, equivalent substitution, improvement and etc. done be should all be included in the protection scope of the present invention.This
The technical scope of item invention is not limited to the contents of the specification, it is necessary to its technology is determined according to scope of the claims
Property range.
Claims (6)
1. a kind of quantification kit of homogeneous chemistry luminescence method detection dog IL-6, it is characterised in that: the inspection that the kit uses
Surveying solution includes 1 conjugate of DNA1- IL-6 antibody, 2 conjugate of DNA2- IL-6 antibody, the DNA3 for marking acridinium ester, limitation
Property restriction endonuclease and graphene oxide, the DNA1 and DNA2 have 6 base complementrities, and the DNA3 contains 2 restriction enzyme sites, and respectively
There are 9 bases and DNA1, DNA2 complementary pairing, DNA3 is adsorbed on surface of graphene oxide, end mark by pi-pi accumulation effect
The chemiluminescence of the AE of note is quenched because CRET occurs.
2. the quantification kit of homogeneous chemistry luminescence method detection dog IL-6 according to claim 1 a kind of, it is characterised in that:
The DNA1 contains 53 bases, 3 ' terminal modified sulfydryls, by coupling agent succinimide -4- thiacyclohexane -1- carbonic ester, with
Amino covalence on IL-6 antibody 1 combines, and forms 1 conjugate of DNA1-IL-6 antibody.
3. the quantification kit of homogeneous chemistry luminescence method detection dog IL-6 according to claim 1 a kind of, it is characterised in that:
The DNA2 contains 51 bases, 5 ' terminal modified sulfydryls, through coupling agent SMCC in conjunction with the amino covalence on IL-6 antibody 2,
Form 2 conjugate of DNA2- IL-6 antibody.
4. the quantification kit of homogeneous chemistry luminescence method detection dog IL-6 according to claim 1 a kind of, it is characterised in that:
The DNA3 contains 22 bases, 5 ' terminal modified acridinium esters (AE), the alkali of the 3-9 and 12-18 base position since 5 ' ends
Basic sequence is all CCTCAGC, can be identified by restriction enzyme Nt.BbvCI, to shear the double-strand of its formation；
The base complete complementary of 1-9 and DNA2 1-9 base position since 3 ' ends of the DNA3 since 5 ' ends；
The base complete complementary of 5-13 and DNA1 1-9 base position since 5 ' ends of the DNA3 since 3 ' ends.
5. such as a kind of making for quantification kit of homogeneous chemistry luminescence method detection dog IL-6 of any of claims 1-4
With method, it is characterised in that: the application method the following steps are included:
(1) sample solution and detection solution are mixed, and is incubated 5-10 minutes under the conditions of 37 DEG C；
(2) chemiluminescent substrate is added, light signal collection is carried out by chemiluminescence detector；
(3) concentration of IL-6 in sample is found out from standard curve.
6. a kind of application method of the quantification kit of homogeneous chemistry luminescence method detection dog IL-6 according to claim 5,
It is characterized by: the luminous substrate is hydrogen peroxide and sodium hydroxide.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110836967A (en) * | 2019-12-12 | 2020-02-25 | 南京浦光生物科技有限公司 | Kit for detecting canine pancreatic lipase based on homogeneous chemiluminescence immunoassay method |
| CN110836976A (en) * | 2019-12-12 | 2020-02-25 | 南京浦光生物科技有限公司 | Kit for detecting C-reactive protein of dog based on homogeneous chemiluminescence immune competition method |
| CN110850103A (en) * | 2019-12-12 | 2020-02-28 | 南京浦光生物科技有限公司 | Kit for detecting cat serum amyloid A based on homogeneous chemiluminescence immune competition method |
| CN111007239A (en) * | 2019-10-31 | 2020-04-14 | 南京浦光生物科技有限公司 | Homogeneous immunoassay method based on ortho-position touch effect and acridine ester chemiluminescence quenched by graphene oxide and using equipment |
| CN113125730A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Homogeneous detection kit for interleukin 6 and application thereof |
| CN113125732A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Homogeneous detection kit for interleukin 6 and application thereof |
| CN114544967A (en) * | 2020-11-11 | 2022-05-27 | 艾克发(北京)生物技术有限公司 | Multiple signal amplification system and application thereof in immunoadsorption direct method detection |
| WO2023245853A1 (en) * | 2022-06-23 | 2023-12-28 | 南京浦光生物科技有限公司 | Reagent combination, kit, system, and method for detecting target protein |
| WO2023245854A1 (en) * | 2022-06-23 | 2023-12-28 | 南京浦光生物科技有限公司 | Reagent combination, kit, detection system and detection method for detecting small molecular substance |
| CN118091110A (en) * | 2024-04-03 | 2024-05-28 | 中国科学院过程工程研究所 | Biological probe for single-molecule immunodetection, detection method and application |
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| CN111007239A (en) * | 2019-10-31 | 2020-04-14 | 南京浦光生物科技有限公司 | Homogeneous immunoassay method based on ortho-position touch effect and acridine ester chemiluminescence quenched by graphene oxide and using equipment |
| CN110836967B (en) * | 2019-12-12 | 2021-07-20 | 南京浦光生物科技有限公司 | Kit for detecting canine pancreatic lipase based on homogeneous chemiluminescence immunoassay method |
| CN110836976A (en) * | 2019-12-12 | 2020-02-25 | 南京浦光生物科技有限公司 | Kit for detecting C-reactive protein of dog based on homogeneous chemiluminescence immune competition method |
| CN110850103A (en) * | 2019-12-12 | 2020-02-28 | 南京浦光生物科技有限公司 | Kit for detecting cat serum amyloid A based on homogeneous chemiluminescence immune competition method |
| CN110850103B (en) * | 2019-12-12 | 2021-06-01 | 南京浦光生物科技有限公司 | Kit for detecting cat serum amyloid A based on homogeneous chemiluminescence immune competition method |
| CN110836976B (en) * | 2019-12-12 | 2021-08-13 | 南京浦光生物科技有限公司 | Kit for detecting C-reactive protein of dog based on homogeneous chemiluminescence immune competition method |
| CN110836967A (en) * | 2019-12-12 | 2020-02-25 | 南京浦光生物科技有限公司 | Kit for detecting canine pancreatic lipase based on homogeneous chemiluminescence immunoassay method |
| CN113125732A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Homogeneous detection kit for interleukin 6 and application thereof |
| CN113125730A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Homogeneous detection kit for interleukin 6 and application thereof |
| CN113125730B (en) * | 2019-12-31 | 2023-07-07 | 科美博阳诊断技术(上海)有限公司 | Homogeneous detection kit for interleukin 6 and application thereof |
| CN113125732B (en) * | 2019-12-31 | 2023-08-08 | 科美博阳诊断技术(上海)有限公司 | Homogeneous detection kit for interleukin 6 and application thereof |
| CN114544967A (en) * | 2020-11-11 | 2022-05-27 | 艾克发(北京)生物技术有限公司 | Multiple signal amplification system and application thereof in immunoadsorption direct method detection |
| WO2023245853A1 (en) * | 2022-06-23 | 2023-12-28 | 南京浦光生物科技有限公司 | Reagent combination, kit, system, and method for detecting target protein |
| WO2023245854A1 (en) * | 2022-06-23 | 2023-12-28 | 南京浦光生物科技有限公司 | Reagent combination, kit, detection system and detection method for detecting small molecular substance |
| CN118091110A (en) * | 2024-04-03 | 2024-05-28 | 中国科学院过程工程研究所 | Biological probe for single-molecule immunodetection, detection method and application |
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