CN106771130A - A kind of enzyme-linked immune analytic method - Google Patents
A kind of enzyme-linked immune analytic method Download PDFInfo
- Publication number
- CN106771130A CN106771130A CN201611013283.9A CN201611013283A CN106771130A CN 106771130 A CN106771130 A CN 106771130A CN 201611013283 A CN201611013283 A CN 201611013283A CN 106771130 A CN106771130 A CN 106771130A
- Authority
- CN
- China
- Prior art keywords
- antigen
- enzyme
- solid phase
- antibody
- analytic method
- 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.)
- Granted
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of enzyme-linked immune analytic method, the method bonds together to form hrp-antibody complex by glucose oxidase and monoclonal antibody, after being detected antigen and hrp-antibody complex specific binding, add enzyme reaction substrate glucose, nascent state active oxygen is produced with glucose oxidase effect, then under the horseradish peroxidase enzyme catalytic effect of catalytic amount, nascent state active oxygen amplifies detection of the signal realization to determined antigen with developer through chromogenic reaction.The immunoassay method that the present invention sets up is sandwich method enzyme-linked immunosorbent assay, it is immunoassay method that is a kind of easy, quick and being capable of high flux detection, and testing result is accurate, reliable, significant meaning is respectively provided with several aspects such as production cost, shipping storage characteristic, safety in utilization and the feature of environmental protection, with application value very high.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of new enzyme-linked immune analytic method.
Background technology
Enzyme-linked immunosorbent assay (Enzyme-linked immunoassay, abbreviation ELISA), is a kind of anti-using antigen
The method that the characteristic of selectivity bond is detected to a corpse or other object for laboratory examination and chemical testing between body.Elisa assay method is special due to its quick, easy analysis
Property, it is widely used in various biochemical indicators, the clinical examination of disease marker in the samples such as serum, blood plasma, urine, feces, tissue fluid.Its
In, double antibody sandwich method is the elisa assay method for detecting that antigen is the most frequently used, and its basic functional principle is：It is solid using being connected to
Antibody and enzyme labelled antibody on phase carrier are combined with two antigenic determinants being detected on antigen molecule in sample respectively, are formed
Solid matrix antibody-antigen-enzyme labelled antibody immune complex.Amount due to insolubilized antibody and enzyme labelled antibody in reaction system is relative to treating
It is excessive to survey antigen, therefore the forming amount of compound is directly proportional to the content of determined antigen in detection range.Determine compound
Enzyme effect in thing is in the colored substance quality generated after the substrate for adding, you can determine determined antigen content.
Existing enzyme-linked immunoassay kit, the more principle based on antigen and antibody specificity identification build.Wherein, it is peppery
Root peroxidase (Horseradish Peroxidase, HRP) is because its specific activity is high, stable, molecular weight is small and pure enzyme holds
The characteristic for easily preparing, is used frequently as signal mediation material to realize the signal reading to destination object.The catalytic reaction of HRP
Need substrate hydrogen peroxide (H2O2) and hydrogen donor (DH2).Hydrogen donor is generally colourless reduction type dye, can be generated by reaction
Coloured oxidation type dye (D).The process of HRP catalytic reactions is as follows：
DH2+H2O2→D+2H2O
Using HRP catalyzing hydrogen peroxides (H2O2) oxidation dye produces chromogenic reaction, and determined using photometric detection instrument
Property or quantitative analysis results realize the detection to test substance.Therefore, all immunoreagents mediated as signal using HRP
Box, is required to exogenous H2O2Supported the use as chromogenic reaction reagent.
H2O2Due to the presence of the low-symmetry and peroxide bridge of molecular structure, cause its chemical stability poor, easily occur certainly
There is redox reaction with Coexisting component in decomposition reaction.In addition, H2O2High temperature, illumination or with some incompatible chemicals
Under matter (such as transiting state metal ion, combustable organic thing) effect, can rapidly decompose generation water and oxygen and release substantial amounts of heat,
Its thermal hazard is excited, and then triggers thermal runaway reaction, ultimately result in the generation of explosion accident.Therefore, H2O2Category is inflammable and explosive
Product, no matter kit the link such as production, transport, storage, be required to more harsh storage environment and packaging material；Due to
Must not be coexisted with other reagents, usually require that and use individually packaging；H2O2Selfdecomposition, can cause different batches kit examine
The comparativity for surveying result is deteriorated.
The content of the invention
The technical problems to be solved by the invention are to overcome the above-mentioned use HRP to carry out enzyme linked immunological as antibody labeling thing
Analyze the shortcoming for existing, there is provided enzyme-linked immune analytic method that is a kind of easy, quick and being capable of high flux detection.
The technical scheme that solution above-mentioned technical problem is used is made up of following step：
1st, in being 7.4 PBS by determined antigen standard items addition pH value, 1~10000pg/L antigen standards are prepared
Product solution, antigen standard solution is added to the surface of solid phase carriers of coated antibody, makes antigen and antibody linked, then with washing
Wash buffer solution washing solid phase carrier and pat dry.
2nd, after the monoclonal antibody that glucose oxidase is marked being dissolved in into the PBS that pH value is 7.4, it is added to step
The surface of solid phase carriers of antigen is bonded in rapid 1, with the antigen-reactive for being bonded in surface of solid phase carriers, is then washed with lavation buffer solution
Wash solid phase carrier and pat dry.
The 3rd, D/W is added to the surface of solid phase carriers obtained in step 2,30~37 DEG C are reacted 10~20 points
Clock, adds glucose oxidase developer and the horseradish peroxidase of catalytic amount to carry out chromogenic reaction, and determines optical density, paints
Optical density processed with the logarithm value changes of antigen standard concentration standard curve.
4th, according to the corresponding optical density of method test determined antigen sample of above-mentioned steps 1~3, the line of combined standard curve
Property equation is the content that can determine that antigen in determined antigen sample.
In above-mentioned steps 3, the described preferred 4-AA of glucose oxidase developer and N- ethyl-N- (2-
Hydroxyl -3- sulfopropyls) -3- methylaniline sodium salts color development system.
Above-mentioned lavation buffer solution is made up of PBS that pH value is 7.4 and Tween-20, and wherein Tween-20 is accounted for and washed
Wash the 0.1% of buffer solution quality.
Above-mentioned solid phase carrier is polystyrene ELISA Plate.
The monoclonal antibody glutaraldehyde two step method of above-mentioned glucose oxidase mark is prepared, and specific preparation method is such as
Under：
1st, 25mg glucose oxidases are dissolved in the glutaraldehyde water solution that 1mL mass fractions are 1.25%, are stored at room temperature
12 hours, gained reaction solution was eluted through Sephadex G-25 chromatographic columns with physiological saline, and flow velocity is 1mL/min, collects brown
Efflux.If the brown effluent volume that collection is obtained is concentrated into 5mL more than 5mL with polyethylene glycol, it is placed in 25mL beakers.
2nd, by monoclonal antibody normal saline dilution 12.5mg to be marked to 5mL, step 1 is added dropwise under stirring
In the enzyme solutions for obtaining, and the carbonic acid buffer of 0.25mL 1mol/L pH=9.5 is added, continue to stir 3 hours, added
0.25mL 0.2mol/L lysine solutions, after mixing, place 2 hours at room temperature, are then added dropwise under agitation and reaction
The isometric saturated ammonium sulfate of mixed liquor, 4 DEG C stand 1 hour, and 3000rpm is centrifuged 0.5 hour, takes lower sediment thing semi-saturation
Ammonium sulfate is washed twice, and is fitted into bag filter after sediment is dissolved in into the PBS of 5mL0.15mol/L pH=7.4, is used
The PBS dialysis of 0.15mol/L pH=7.4, (detects) that 10000rpm is centrifuged with Nai Shi reagents after removal ammonium ion
30min removes sediment, and supernatant is the monoclonal antibody of glucose oxidase mark.
Immunoassay method of the invention bonds together to form enzyme labelled antibody by glucose oxidase (GOD) and monoclonal antibody
Compound, after being detected antigen and the specific binding of enzyme mark compound, adds enzyme reaction substrate glucose, is acted on GOD and produced
Nascent state active oxygen, then under the HRP catalytic action of catalytic amount, nascent state active oxygen amplifies with developer through chromogenic reaction to be believed
Number realize detection to determined antigen.The present invention acts on the nascent oxygen for producing and replaces by being labeled GOD and substrate glucose
Exogenous H2O2, successfully avoid H2O2Easily decompose unstable so as to cause the shortcoming of testing result stability difference, and detect
Result is accurate, reliable.
The immunoassay method that the present invention sets up is sandwich method enzyme-linked immunosorbent assay, is a kind of easy, quick and can
The immunoassay method of high flux detection, and in several sides such as production cost, shipping storage characteristic, safety in utilization and the feature of environmental protection
Face is respectively provided with significant meaning.The inventive method can both be fabricated to kit commercialization and widely use, it is also possible to make company or
Laboratory uses from test sample, with application value very high.
Brief description of the drawings
Fig. 1 is that optical density is bent with the standard of the logarithm value changes of VEGF standard concentration in embodiment 1
Line.
Specific embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to
These embodiments.
Embodiment 1
The content of VEGF (VEGF) is determined, is comprised the following steps that：
1st, in being 7.4 PBS by VEGF standard items addition 0.1mol/L pH value, 1pg/L, 10pg/ are prepared respectively
The VEGF standard solutions of L, 100pg/L, 1000pg/L and 10000pg/L；The polystyrene enzyme of Anti-VEGF antibody will be coated with
Each hole of target respectively with washing dilution (be made up of PBS and Tween-20 that 0.05mol/L pH value is 7.4, its
Middle Tween-20 account for lavation buffer solution quality 0.05%) wash after pat dry (1 time), blank well is set respectively, and (blank well is not added with to be measured
Sample and enzyme marking reagent, remaining each step operation are identical), gauge orifice, testing sample hole, 50 μ L concentration are separately added into gauge orifice
It is the VEGF standard solutions of 1pg/L, 10pg/L, 100pg/L, 1000pg/L and 10000pg/L, testing sample first Jia 40 in hole
μ L 0.1mol/L pH are 7.4 PBS, and 10 μ L serum samples (the final dilution factor of testing sample is 5 times) are then added again,
37 DEG C incubate 1 hour, discard liquid in ELISA Plate, pat dry, then with lavation buffer solution (by PBS that pH value is 7.4 and
Tween-20 is constituted, and wherein Tween-20 accounts for 0.1%) the washing ELISA Plate of lavation buffer solution quality and pats dry (3 times).
2nd, the monoclonal antibody (Recombinant people VEGF 165A protein) for marking glucose oxidase is added
During 0.1mol/L pH value is 7.4 PBS, the solution of 0.038U/mL is configured to, in each gauge orifice and testing sample
Each in hole to add the 50 μ L solution, except blank well, 37 DEG C incubate 1 hour, discard liquid in ELISA Plate, pat dry, then with washing
Wash buffer solution washing ELISA Plate (identical with step 1) and pat dry (3 times).
3rd, the D/W of 50 μ L 1mol/L, 30 are added in each gauge orifice, testing sample hole, blank well
DEG C reaction 10 minutes, add the 50 μ L 0.005mol/L 4-AAs aqueous solution and 50 μ L0.004mol/L N- second
Base-N- TOOSs the aqueous solution, the 10 μ L 40U/mL HRP aqueous solution, gently shake mixed
Even, 37 DEG C of lucifuges develop the color 15 minutes；Using ELIASA, returned to zero with blank well, measure the light in each hole respectively under 555nm wavelength
Density, with the logarithm value (lgC) of VEGF standard concentrations as abscissa, optical density is ordinate, draws optical density and is marked with VEGF
The standard curve (result is shown in Fig. 1) of the logarithm value changes of quasi- product concentration, its detection is limited to 3.24 × 10-4ng/L。
4th, the corresponding optical density in testing sample hole obtained according to above-mentioned steps 1~3, the linear side of combined standard curve
Journey, calculates the content of VEGF in serum sample for 5.30ng/L, contains multiplied by the reality with extension rate, as testing sample
Amount.
Claims (4)
1. a kind of enzyme-linked immune analytic method, it is characterised in that it is made up of following step：
(1) in being 7.4 PBS by determined antigen standard items addition pH value, 1~10000pg/L antigen standards are prepared
Solution, antigen standard solution is added to the surface of solid phase carriers of coated antibody, makes antigen and antibody linked, then with washing
Buffer solution washs solid phase carrier and pats dry；
(2) after the monoclonal antibody that glucose oxidase is marked being dissolved in into the PBS that pH value is 7.4, it is added to step
(1) surface of solid phase carriers of bonding antigen, with the antigen-reactive for being bonded in surface of solid phase carriers, is then washed with lavation buffer solution in
Wash solid phase carrier and pat dry；
(3) D/W is added to the surface of solid phase carriers obtained in step (2), 30~37 DEG C are reacted 10~20 points
Clock, adds glucose oxidase developer and the horseradish peroxidase of catalytic amount to carry out chromogenic reaction, and determines optical density, paints
Optical density processed with the logarithm value changes of antigen standard concentration standard curve；
(4) according to above-mentioned steps (1)~corresponding optical density of (3) method test determined antigen sample, the line of combined standard curve
Property equation is the content that can determine that antigen in determined antigen sample.
2. enzyme-linked immune analytic method according to claim 1, it is characterised in that：In step (3), described glucose
Oxidizing ferment developer is the colour developing of 4-AA and N- ethyl-N- TOOSs
System.
3. enzyme-linked immune analytic method according to claim 1, it is characterised in that：Described lavation buffer solution is by pH value
PBS and Tween-20 for 7.4 are constituted, and wherein Tween-20 accounts for the 0.1% of lavation buffer solution quality.
4. enzyme-linked immune analytic method according to claim 1, it is characterised in that：Described solid phase carrier is polystyrene
ELISA Plate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611013283.9A CN106771130B (en) | 2016-11-17 | 2016-11-17 | A kind of enzyme-linked immune analytic method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611013283.9A CN106771130B (en) | 2016-11-17 | 2016-11-17 | A kind of enzyme-linked immune analytic method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106771130A true CN106771130A (en) | 2017-05-31 |
| CN106771130B CN106771130B (en) | 2018-12-28 |
Family
ID=58968609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611013283.9A Active CN106771130B (en) | 2016-11-17 | 2016-11-17 | A kind of enzyme-linked immune analytic method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106771130B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503732A (en) * | 2009-03-13 | 2009-08-12 | 温州东瓯津玛生物科技有限公司 | Glucose oxidase single liquid detection reagent |
| CN103266166A (en) * | 2013-05-24 | 2013-08-28 | 宁波美康生物科技股份有限公司 | Glucose detecting reagent |
| CN104245953A (en) * | 2012-04-27 | 2014-12-24 | 协和梅迪克斯株式会社 | Method for assaying component to be assayed in specimen |
| CN105295441A (en) * | 2015-11-20 | 2016-02-03 | 三诺生物传感股份有限公司 | Stabilizer of chromogenic reagent and applications thereof |
| CN106104259A (en) * | 2014-03-14 | 2016-11-09 | 泰尔茂株式会社 | component measuring device, method and program |
-
2016
- 2016-11-17 CN CN201611013283.9A patent/CN106771130B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503732A (en) * | 2009-03-13 | 2009-08-12 | 温州东瓯津玛生物科技有限公司 | Glucose oxidase single liquid detection reagent |
| CN104245953A (en) * | 2012-04-27 | 2014-12-24 | 协和梅迪克斯株式会社 | Method for assaying component to be assayed in specimen |
| CN103266166A (en) * | 2013-05-24 | 2013-08-28 | 宁波美康生物科技股份有限公司 | Glucose detecting reagent |
| CN106104259A (en) * | 2014-03-14 | 2016-11-09 | 泰尔茂株式会社 | component measuring device, method and program |
| CN105295441A (en) * | 2015-11-20 | 2016-02-03 | 三诺生物传感股份有限公司 | Stabilizer of chromogenic reagent and applications thereof |
Non-Patent Citations (6)
| Title |
|---|
| DINGBIN LIU, ET.AL.: "Glucose Oxidase-Catalyzed Growth of Gold Nanoparticles Enables Quantitative Detection of Attomolar Cancer Biomarkers.", 《ANAL CHEM》 * |
| JI YOUNG EUM, ET.AL.: "A highly sensitive immunoassay using antibodyconjugated spherical mesoporous silica with immobilized enzymes.", 《CHEM COMMUN》 * |
| JULIO RABA, HORACIO A. MOTTOLA: "Glucose Oxidase as an Analytical Reagent.", 《CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY》 * |
| MITSUNE YAMAGUCHI, ET.AL.: "A Rapid Enzyme Immunoassay for Cocaine and Benzoylecgonine Using Glucose Oxidase.", 《JOURNAL OF HEALTH SCIENCE》 * |
| ROY B. JOHNSON JR., ET.AL.: "Comparison of Glucose Oxidase and Peroxidase as Labels for Antibody in Enzyme-linked Immunosorbent Assay.", 《J IMMUNOASSAY》 * |
| 焦奎、张书圣主编: "《酶联免疫分析技术及应用》", 31 August 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106771130B (en) | 2018-12-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Recent developments and applications of chemiluminescence sensors | |
| JPH0670625B2 (en) | Quantitative analysis device and method | |
| EP2574926B1 (en) | Chromatographic kit and chromatography method | |
| CN109781976A (en) | Fluorescence immune analysis method based on carbon quantum dot | |
| CN102959397B (en) | Method for electrochemically detecting binding reactions | |
| KR101990301B1 (en) | Optical biosensor | |
| JPS6071957A (en) | Method of promoting immune reaction by ultrasonic treatment | |
| Zuo et al. | Rapid detection of severe fever with thrombocytopenia syndrome virus via colloidal gold immunochromatography assay | |
| Cho et al. | An enzyme immunoanalytical system based on sequential cross-flow chromatography | |
| JP3532669B2 (en) | Chemiluminescent composition, test kit, test device, and method for detecting analyte | |
| JPS61107158A (en) | Testing method | |
| Sun et al. | A colorimetric method for determination of the prostate specific antigen based on enzyme-free cascaded signal amplification via peptide-copper (II) nanoparticles | |
| CA1271419A (en) | Signal enhancement in immunoassay by modulation of chemical catalysis | |
| CN111693721A (en) | Preparation method and application of enzyme-linked immunosorbent assay based on prussian blue nano enzyme label | |
| JPS59178362A (en) | Method of detecting body to be tested | |
| JPH11153600A (en) | Test piece for immunoassay and measuring method using the same | |
| JPS61500152A (en) | Device for rapid quantitative analysis of fluids | |
| Cao et al. | An integrated electrochemical immunochromatographic test strip based on the amplification of gold nanoparticles for quantitative detection of alpha-fetoprotein | |
| CN106771130B (en) | A kind of enzyme-linked immune analytic method | |
| US4433060A (en) | Chemiluminescent immunoassays with triphenylmethane dyes activated by H.sub. O2 and a chloramine | |
| CN105785019A (en) | Method for detecting prostate specific antigens | |
| CN110333348A (en) | The nano particle and preparation method and application that polypeptide and copper ion are formed | |
| JPS61501047A (en) | Method for measuring immobilized reporter groups | |
| JPS58206966A (en) | Simultaneous calibration heterogeneous immunoassay | |
| CN118549365A (en) | Method for detecting prostate specific antigen based on EP tube and nano-enzyme hydrogel film |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |