CN110487777A - The method that acridinium ester chemiluminescent detects HLA-G prediction pre-eclampsia - Google Patents
The method that acridinium ester chemiluminescent detects HLA-G prediction pre-eclampsia Download PDFInfo
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- CN110487777A CN110487777A CN201910723495.3A CN201910723495A CN110487777A CN 110487777 A CN110487777 A CN 110487777A CN 201910723495 A CN201910723495 A CN 201910723495A CN 110487777 A CN110487777 A CN 110487777A
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- acridinium ester
- eclampsia
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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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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
Abstract
The present invention is the method that acridinium ester chemiluminescent detects that HLA-G predicts pre-eclampsia, includes the following steps: 1) to be coupled supperparamagnetic particles as general solid phase carrier using " Avidin ";2) the differential responses group of different antigen/antibody molecules is coupled from different biotins and is marked;3) the differential responses group of different antigen/antibody molecules is coupled by different coupling agents using different acridinium esters or acridine sulfonamide;4) it is based on NaOH-H2O2Substrate system, using acid and surfactant as reinforcing agent.This method detection sensitivity is high, specificity is good, the range of linearity is wide, precision is high, stability is good, "dead" pollution, safe operation, and method is easy quickly.Gold-magnetic particles are a kind of novel magnetic inorganic composite particles, it combines in external magnetic field the separability of magnetic-particle and magnetic-particle surface is modified can quickly fix to biomolecule.
Description
Technical field
The invention belongs to biology and pharmaceutical technology fields, are related to a kind of acridinium ester chemiluminescent detection HLA-G prediction tendency
The method of eclampsia.
Background technique
Pre-eclampsia is the syndrome of hypertension, oedema and albuminuria, accounts for 7-15% in all gestation, and is its mother
The main reason for body falls ill and is lethal.200,000 maternal deaths are at least caused every year in the whole world.The symptom of pre-eclampsia is usually
Pregnancy the 20th week after occur and be usually by the blood pressure and urine of routine monitoring women detect come.However these are supervised
Survey method can not effectively diagnose early stage syndrome, if it is possible to which early monitoring to pre-eclampsia takes effective treatment, just
The danger of fetus in subject or development can be reduced.
The prior art mainly has the following deficiencies: in terms of detection method selection
1 Enzyme Linked Immunoadsorbent Assay (ELISA): principle: enzyme linked immunosorbent assay (ELISA) (ELISA) is most popular at present exempts from
Epidemic disease measurement.Basic principle is the effective catalytic action of the specific binding and enzyme of antigen and antibody to substrate.Conventional method
It is that the antibody for being directed to target protein is coated on microtest plate, the target protein of the enzyme label of the solution and known quantity of test substances,
And if analyte contains target protein, the coated antibody of target protein competitive binding related with enzyme.Washing flat board
Afterwards, enzyme reaction substrate colour developing is added, measures OD value with microplate reader.Disadvantage: repeatability is bad;Due to autoantibody and the thermophilic opposite sex
The interference of antibody, is easy to appear false positive;No matter instrument and manual operation, have many disturbing factors, most influential is temperature
Degree and time.
2 co-agglutination methods: principle: coagglutination is analogous to the determination of serology of indirect agglutination, is that height is special
It is anisotropic and sensibility.It is examined by using staphylococcal protein A (SPA) labelled protein specific antisera preparation SPS
Disconnected solution, and SPS diagnostic solutions are mixed with the white dilution to measure on glass slide to observe agglutination phenomenon.Reaction
As a result it is co-agglutination to have occurred, and be based on this with appearance " ++ ", calculates the minimum dfetectable quantity of testing protein.Disadvantage:
It is influenced by protein body size;It is influenced by solution viscosity;It is influenced by enzyme effect.
3 immunity test strip methods: principle: compared with ELISA, test-strips detect albumen and combine also based on antigen and antibody specific
Principle.One of difference is nitrocellulose substituted polystyrene reaction plate as solid phase carrier.With extraneous protein spy
The antibody that the opposite sex combines is fixed in test-strips in conjunction with color developing agent.When one end of test-strips is discharged into containing foreign protein
When in the plant tissue extract of matter, the capillarity of other end absorption pad is extracted.Liquid flows upwards work as specific antibody
When in conjunction with extraneous protein, chromogenic reaction is presented in it.Test-strips include 2 " capture " regions: one " capture " and external egg
The antibody protein compound that white matter combines, another " capture " color developing agent.When the interlayer composite material of formation and unreacted
When color developing agent is tested corresponding " capture " the area capture in strip, these trapping regions show red color area.When only display in test-strips
When one region (quality control line), it is negative findings, shows that it does not include the transgenic protein detected, when display two
When a band, it is positive findings, shows that it includes to detect transgene protein.Disadvantage: test strips method is a kind of fast and convenient
Qualitative checking method.Test-strips are placed in sample to be tested extract, as a result can be obtained in 5 to 10 minutes.Test process
Special instrument and skilled technical ability are not needed, and be economical and conveniently.Its detection especially suitable for field sample, and answered
Detection for various genetically modified crops.However, test-strips can only detect a kind of protein and can only detect extraneous protein
Presence or absence, and cannot distinguish between specific transformed variety.
4 immunoblotting assay methods: principle: immunoblotting, also referred to as Western blotting are anti-by specific antibody identification
Former effective ways.This method is a kind of novel immune developed on the basis of gel electrophoresis and solid-phase immunoassay technology
Technology.Contained first with SDS- polyacrylamide gel electrophoresis (SDS-PAGE) or non denatured electrophoresis (Native-PAGE) separation
There is the sample of target protein (antigen), is then transferred on nitrocellulose filter by electrophoretic blotting or surface.Then pass through film
Anti- antigen-antibody reaction on surface specifically detects other films.For example, in the protein bars that will be separated by SDS-PAGE
It after band is transferred on film, with lock solution process film, is then reacted with first antibody, and with coupled horseradish peroxidase
(HRP) or the secondary antibody of alkaline phosphatase (ester) enzyme (AP) is reacted.After chromogenic substrate reaction is added, reaction display target protein
Position.Disadvantage: Western blotting is expressed in the form of characteristic bands, this is more convincing.It is reported that Western blotting is height
Sensitive, if purifying, can detecte to 1 to 5ng.However, its is cumbersome and expensive, and be not suitable for carrying out at port fast
Speed and large sample test.
5 immuno-PCR technologies: principle: Immuno polymerase chain reaction (IM-PCR) is to detect to be immunoreacted by PCR.
PCR is a kind of amplification in vitro method of simulation n DNA duplication.In template, primer in the presence of substrate and archaeal dna polymerase, leads to
It crosses denaturation-and anneals-extend in and generate the specific part of very low amount DNA in several circulations.One hour 1,000,000 times.
ImmunoPCR is that the DNA fragmentation of known array is tagged on anti-antigen-antibody complex, then by pcr amplified DNA, so
Detect the process of PCR product by conventional method afterwards.The presence of specific PCR products shows that there are DNA fragmentation labelled antibody institutes
For specific antigen.Therefore, immuno-PCR is the immunoassay of DNA marker.Immuno-PCR does not need special signal
Measuring device, detection sensitivity are high.Currently, the immuno-PCR sensibility reported both at home and abroad is generally higher than current ELISA method
102-108 times.Disadvantage: immune PCR technique is still in conceptual phase, and there is presently no the supplies of commercially available reagent box.
ImmunoPCR is antigen detection method most sensitive so far.It theoretically can detecte single antigen molecule, but actually
Its sensitivity is affected by many factors, such as linkers, the selection of display system, the concentration of DNA reporter molecule, PCR
Cycle-index etc..
Summary of the invention
In order to solve the above problem, it is an object of that present invention to provide a kind of acridinium ester chemiluminescent detection HLA-G to predict tendency
The method of eclampsia.
To achieve the above object, the present invention is by the following technical programs are as follows: acridinium ester chemiluminescent detects HLA-G prediction first
The method of million eclampsias, includes the following steps:
1) supperparamagnetic particles are coupled as general solid phase carrier using " Avidin ";
2) the differential responses group of different antigen/antibody molecules is coupled from different biotins and is marked;
3) difference of different antigen/antibody molecules is coupled by different coupling agents using different acridinium esters or acridine sulfonamide
Reactive group;
4) it is based on NaOH-H2O2Substrate system, using acid and surfactant as reinforcing agent.
Preferably, " Avidin " is selected from Avidin, streptavidin, neutral Avidin or can be in the step (1)
In conjunction with the class avidin molecule of biotin molecule.
Further, the partial size of the supperparamagnetic particles in the step (1) is 50nm~5 μm, and particle is coated with four oxygen
Change three-iron and on the surface thereof with the core of various active groups
Further, the various active groups are amino, carboxyl, hydroxyl or sulfydryl.
Further, the biotin in the step (1) refer toThe coupling done by core
Biotin derivative.
Further, the coupling agent in the step (3) is dimethylimino, glutaraldehyde, N, and N '-dicyclohexyl carbon two is sub-
Amine, SATA or SMCC.
Further, step (4) substrate system is with NaOH-H2O2Based on, using acid and surfactant as
Reinforcing agent carries out the luminous substrate system for being suitable for acridinium ester or acridine sulphonyl amine system of different ratio combination.
Further, the acid in the step (4) is nitric acid, sulfuric acid or hydrochloric acid.
Further, the surfactant in the step (4) is SDS, Tween, Triton-X, Brij or CTAC.
Advantages of the present invention are as follows: 1. this method detection sensitivities are high, specificity is good, the range of linearity is wide, precision is high, stablize
Property good, "dead" pollution, safe operation, method is easy quickly.Especially in detection sensitivity, specificity, accuracy and precision
The immune detection that degree aspect is participated in than enzyme is significantly increased.2, the sensitivity of chemiluminescent enzyme immunoassay method and accuracy want general
All over enzyme-linked immunization is higher than, while each requirement is set to detecting instrument and is far below Electrochemiluminescince again.3, gold-magnetic particles are one
The novel magnetic inorganic composite particles of kind, it combines the separability of magnetic-particle and magnetic-particle surface in external magnetic field and changes
Biomolecule can be fixed quickly after property.
Specific embodiment:
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, if being commercially available without specified otherwise.
Embodiment 1: the preparation of gold-magnetic particles
The preparation of washing buffer: being 4.8-5.0 with distilled water and MES preparation pH value, and the MES that concentration is 45mmol/ml is slow
Fliud flushing, the ultimate density that Tween-20 to final concentration of 0.15%, Proclin-300 is added is that 0.1,0.22 μ um microporous barrier exists
It sterilizes at 2-8 DEG C after filtering, validity period is three months.
Preparation work buffer: preparation pH7.0-7.4, final concentration of 45mM phosphate buffer, with distilled water, di(2-ethylhexyl)phosphate
Hydrogen sodium and disodium hydrogen phosphate, be added PVP, BSA, Tween-20, sucrose, Proclin-300, ultimate density be 1 %, 1%,
0.1%, 3%, 0.2%, 0.22 μ μ micro-filtrate membrane filtration, 2-8 DEG C of sterilizing, validity period is 1 year.
Golden magnetic-particle (0.5-5 μm) is washed with washing buffer, EDC and NHS, which is added, makes the ultimate density of the two
45mmol reacts 60 minutes at room temperature, and after washing 3 times, streptavidin is added.Prepare streptavidin
Albumen is allowed to be 5:1 with the molar ratio of magnetic-particle, and reaction carries out 3 hours at room temperature.By mixture and contain 0.5%BSA
45mM PBS be incubated at room temperature 30 minutes, wash 5 times, and with working buffer solution (1mg magnetic-particle and 20ml working buffer
Liquid) rebuild magnetic-particle, be configured to again, be assigned to 5ml/, 2-8 °C storage.
Example 2: the acridinium ester chemiluminescent detection reagent box preparation method of HLA-G in quantitative detection serum
1. preparing biotinylated antibody and acridinium ester label antibody hybrid working solution
By anti-HLA-G detection antibody at 2-8 DEG C to 0.05M MES dialysed overnight concentration is adjusted to 2mg/ml, and will
Preactivated acridinium ester is dissolved in pure water to final concentration of 10mmol/ml.With the molecular proportion of 10-60:1 by the desired amount of acridine
Ester (or acridine sulfonamide) solution is added in antibody-solutions, is reacted 60 minutes, is then transferred into bag filter at room temperature, point
Son retention diameter is 4000-8000 to 0.05M MES 2-8 °C.Overnight dialysis is added isometric glycerol, is protected from light at -20 DEG C
It saves.
Biotinylated capture antibody is mixed with the ratio of 1/3000-6000, the detection antibody of acridinium ester label is with 1
The ratio of/1000-2000 mixes, and dilutes in working buffer solution, and add HAMA antibody inhibition, final concentration of 2-5%,
It is packed into 10ml/2-8 °C with brown bottle, is kept in dark place in case using in the future.
2. the preparation of HLA-G calibrator
After setting, HLA-G net product is taken, is diluted to 5 ng/ml and 400 ng with cow's serum (containing 0.1%Proclin-300)
/ ml, distribution to 1ml/piece, and it is stored in -20 °C for future use.
3. the preparation of concentrated cleaning solution
With distilled water, sodium dihydrogen phosphate and disodium hydrogen phosphate preparation pH value are 7.0-7.4, the phosphate-buffered of final concentration of 1M
Liquid.NaCL to final concentration of 300mM, addition Tween-20 to final concentration of 5% is added, and distributes to 50ml/2-8 DEG C of support,
In addition to this.
4. the preparation of substrate liquid
Solution A: preparing 0.01-0.03M dilute nitric acid solution with distilled water and nitric acid, and hydrogen peroxide is added to final concentration 0.1-
0.5%, then it is loaded into spare use in 20ml/2-8 DEG C of brown bottle.
Solution B: preparing 0.02-0.06M dilute alkaline soln with distilled water and NaOH, and surfactant SDS is added to final concentration
For 0.5-2%, distributes to 20ml/2-8 DEG C and stored.
5. assembling external member
By magnetic-particle working solution, the antibody hybrid working solution of biotinylated antibody and acridinium ester label, HLA-G calibrates molten
Liquid, washing buffer and substrate liquid are fitted into kit, packaging, and by test be transmitted to 2-8 °C it is stored refrigerated.
6. kit test experience result:
Linear: sample linear regression is 0.95 or more with expected concentration coefficient R value.
Repeatability: variation within batch coefficient and interassay coefficient of variation should be respectively smaller than 10% and 15%.
Detection range: 2U/mL-64U/mL.
Sensitivity: minimal detectable concentration is less than 0.1U/mL.
The contents of the present invention are not limited in the above embodiments, recorded in one of them or more technical solution
Technical characteristic can be combined with arbitrary one or more technical solutions, technical solution obtained from these are combined also exists
In the protection scope of the application.
Claims (9)
1. the method that acridinium ester chemiluminescent detects HLA-G prediction pre-eclampsia, which comprises the steps of:
1) supperparamagnetic particles are coupled as general solid phase carrier using " Avidin ";
2) the differential responses group of different antigen/antibody molecules is coupled from different biotins and is marked;
3) difference of different antigen/antibody molecules is coupled by different coupling agents using different acridinium esters or acridine sulfonamide
Reactive group;
4) it is based on NaOH-H2O2Substrate system, using acid and surfactant as reinforcing agent.
2. the method for acridinium ester chemiluminescent detection HLA-G prediction pre-eclampsia according to claim 1, feature exist
In " Avidin " is selected from Avidin, streptavidin, neutral Avidin or can combine biotin molecule in the step (1)
Class avidin molecule.
3. the method for acridinium ester chemiluminescent detection HLA-G prediction pre-eclampsia according to claim 1, feature exist
In the partial size of the supperparamagnetic particles in the step (1) is 50nm~5 μm, and particle is coated with ferroso-ferric oxide and at it
With the core of various active groups on surface.
4. the method for acridinium ester chemiluminescent detection HLA-G prediction pre-eclampsia according to claim 3, feature exist
In the various active groups are amino, carboxyl, hydroxyl or sulfydryl.
5. the method for acridinium ester chemiluminescent detection HLA-G prediction pre-eclampsia according to claim 1, feature exist
In, the biotin in the step (1) refer toBy the biotin derivative for the coupling that core is done.
6. the method for acridinium ester chemiluminescent detection HLA-G prediction pre-eclampsia according to claim 1, feature exist
In, coupling agent in the step (3) be dimethylimino, glutaraldehyde, N, N '-dicyclohexylcarbodiimide, SATA or
SMCC。
7. the method for acridinium ester chemiluminescent detection HLA-G prediction pre-eclampsia according to claim 1, feature exist
In step (4) substrate system is with NaOH-H2O2Based on, using acid and surfactant as reinforcing agent carry out difference
The luminous substrate system for being suitable for acridinium ester or acridine sulphonyl amine system of proportion combination.
8. the method for acridinium ester chemiluminescent detection HLA-G prediction pre-eclampsia according to claim 1, feature exist
In the acid in the step (4) is nitric acid, sulfuric acid or hydrochloric acid.
9. the method for acridinium ester chemiluminescent detection HLA-G prediction pre-eclampsia according to claim 1, feature exist
In the surfactant in the step (4) is SDS, Tween, Triton-X, Brij or CTAC.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101493462A (en) * | 2008-01-25 | 2009-07-29 | 广州天美生物技术有限公司 | HLA-G hypersensitization detecting method by chemiluminescence immune analysis |
CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101493462A (en) * | 2008-01-25 | 2009-07-29 | 广州天美生物技术有限公司 | HLA-G hypersensitization detecting method by chemiluminescence immune analysis |
CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
Non-Patent Citations (1)
Title |
---|
中国科协组织人事部编: "《第五届博士生学术年会论文集 (下)》", 30 June 2008, 中国科学技术出版社 * |
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