CN106771130B - A kind of enzyme-linked immune analytic method - Google Patents
A kind of enzyme-linked immune analytic method Download PDFInfo
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- CN106771130B CN106771130B CN201611013283.9A CN201611013283A CN106771130B CN 106771130 B CN106771130 B CN 106771130B CN 201611013283 A CN201611013283 A CN 201611013283A CN 106771130 B CN106771130 B CN 106771130B
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a kind of enzyme-linked immune analytic methods, this method bonds together to form hrp-antibody complex by glucose oxidase and monoclonal antibody, after being detected antigen and hrp-antibody complex specific binding, enzyme reaction substrate glucose is added, it is acted on glucose oxidase and generates nascent state active oxygen, then under the horseradish peroxidase enzyme catalytic effect of catalytic amount, nascent state active oxygen and color developing agent realize the detection to determined antigen through chromogenic reaction amplified signal.The immunoassay method that the present invention establishes is sandwich method enzyme-linked immunosorbent assay, it is a kind of immunoassay method that is easy, quick and being capable of high-throughput detection, and testing result is accurate, reliable, significant meaning is all had at several aspects such as production cost, transport storage characteristic, safety in utilization and the feature of environmental protection, there is very high application value.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of novel enzyme-linked immune analytic method.
Background technique
Enzyme-linked immunosorbent assay (Enzyme-linked immunoassay, abbreviation ELISA) is a kind of anti-using antigen
The method that the characteristic that specificity is bonded between body detects a specimen.Analysis spy of the elisa assay method due to it quickly, easy
Property, it is widely used in the clinical examination of various biochemical indicators, disease marker in the samples such as serum, blood plasma, urine, feces, tissue fluid.Its
In, double antibody sandwich method is the detection most common elisa assay method of antigen, and basic functional principle is: solid using being connected to
Antibody and enzyme labelled antibody on phase carrier in conjunction with two antigenic determinants being detected on antigen molecule in sample, are formed respectively
Solid matrix antibody-antigen-enzyme labelled antibody immune complex.Due in reaction system the amount of solid phase antibody and enzyme labelled antibody relative to
It is excessive for surveying antigen, therefore the forming amount of compound and the content of determined antigen are directly proportional in detection range.It measures compound
The colored substance quality that enzyme effect in object generates after the substrate of addition, that is, can determine determined antigen content.
Existing enzyme-linked immunoassay kit, the principle mostly based on antigen and antibody specificity identification construct.Wherein, peppery
Root peroxidase (Horseradish Peroxidase, HRP) is since its specific activity is high, stablize, molecular weight is small and pure enzyme holds
The characteristic easily prepared is used frequently as signal and mediates substance, to realize the signal reading to target object.The catalysis of HRP is reacted
Need substrate hydrogen peroxide (H2O2) and hydrogen donor (DH2).Hydrogen donor is mostly colourless reduction type dye, is produced by reacting
Coloured oxidation type dye (D).The process of HRP catalysis reaction is as follows:
DH2+H2O2→D+2H2O
Utilize HRP catalyzing hydrogen peroxide (H2O2) oxidation dye generates chromogenic reaction, and is determined using photometric detection instrument
Property or quantitative analysis results, to realize the detection to test substance.Therefore, all immunoreagents mediated using HRP as signal
Box is required to exogenous H2O2It is matched as chromogenic reaction reagent.
H2O2Due to the presence of the low-symmetry and peroxide bridge of molecular structure, cause its chemical stability poor, easily occurs certainly
With Coexisting component redox reaction occurs for decomposition reaction.In addition, H2O2High temperature, illumination or with some incompatible chemicals
Under matter (such as transiting state metal ion, combustable organic object) effect, it can decompose rapidly and generate water and oxygen and a large amount of heat of releasing,
Its thermal hazard is excited, and then causes thermal runaway reaction, eventually leads to the generation of explosion accident.Therefore, H2O2Belong to inflammable and explosive
No matter product are required to more harsh storage environment and packaging material in links such as the productions, transport, storage of kit；Due to
It must not coexist, be usually required that using individually packaging with other reagents；H2O2Selfdecomposition, will lead to different batches kit inspection
The comparativity for surveying result is deteriorated.
Summary of the invention
Technical problem to be solved by the present invention lies in overcome above-mentioned use HRP to carry out enzyme linked immunological as antibody marker
Analysis there are the shortcomings that, provide it is a kind of it is easy, quickly and be capable of the enzyme-linked immune analytic method of high-throughput detection.
Technical solution used by above-mentioned technical problem is solved to be made of following step:
1, determined antigen standard items are added in the PBS buffer solution that pH value is 7.4, prepare 1~10000pg/L antigen standard
Antigen standard solution is added to the surface of solid phase carriers of coated antibody, makes antigen and antibody linked by product solution, then with washing
It washs buffer washing solid phase carrier and pats dry.
2, after the monoclonal antibody that glucose oxidase marks being dissolved in the PBS buffer solution that pH value is 7.4, it is added to step
The surface of solid phase carriers for being bonded antigen in rapid 1 is then washed with washing buffer with the antigen-reactive for being bonded in surface of solid phase carriers
It washs solid phase carrier and pats dry.
3, glucose solution is added to the surface of solid phase carriers obtained in step 2,30~37 DEG C are reacted 10~20 points
Clock, the horseradish peroxidase that glucose oxidase color developing agent and catalytic amount is added carries out chromogenic reaction, and measures optical density, draws
The standard curve that optical density processed changes with the logarithm of antigen standard concentration.
4, 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 be that can determine the content of antigen in determined antigen sample.
In above-mentioned steps 3, the preferred 4-AA of glucose oxidase color developing agent and N- ethyl-N- (2-
Hydroxyl -3- sulfopropyl) -3- methylaniline sodium salt color development system.
The PBS buffer solution and Tween-20 that above-mentioned washing buffer is 7.4 by pH value form, and wherein Tween-20, which accounts for, washes
Wash the 0.1% of buffer quality.
Above-mentioned solid phase carrier is polystyrene ELISA Plate.
The monoclonal antibody of above-mentioned glucose oxidase label is prepared with glutaraldehyde two step method, and specific preparation method is such as
Under:
1,25mg glucose oxidase is dissolved in the glutaraldehyde water solution that 1mL mass fraction is 1.25%, is stored at room temperature
12 hours, gained reaction solution was eluted through Sephadex G-25 chromatographic column with physiological saline, flow velocity 1mL/min, collected brown
Efflux.If collecting obtained brown effluent volume is greater than 5mL, it is concentrated into 5mL with polyethylene glycol, is placed in 25mL beaker.
2, by monoclonal antibody normal saline dilution 12.5mg to be marked to 5mL, step 1 is added dropwise under stirring
In obtained enzyme solutions, and the carbonic acid buffer of 0.25mL 1mol/L pH=9.5 is added, continues stirring 3 hours, add
0.25mL 0.2mol/L lysine solution after mixing, is placed 2 hours at room temperature, is then added dropwise and reacts under stiring
The isometric saturated ammonium sulfate of mixed liquor, 4 DEG C stand 1 hour, and 3000rpm is centrifuged 0.5 hour, take lower sediment thing semi-saturation
Ammonium sulfate washes twice, and is fitted into bag filter after sediment to be dissolved in the PBS buffer solution of 5mL0.15mol/L pH=7.4, uses
The PBS buffer solution of 0.15mol/L pH=7.4 is dialysed, and 10000rpm centrifugation (is detected) with Nai Shi reagent after removing ammonium ion
30min removes sediment, and supernatant is the monoclonal antibody of glucose oxidase label.
Immunoassay method of the invention bonds together to form enzyme labelled antibody by glucose oxidase (GOD) and monoclonal antibody
Compound is added enzyme reaction substrate glucose, acts on and generating with GOD after being detected antigen and the specific binding of enzyme mark compound
Nascent state active oxygen, then under the HRP catalytic action of catalytic amount, nascent state active oxygen and color developing agent are believed through chromogenic reaction amplification
Number realize detection to determined antigen.The present invention is replaced by being labeled the nascent oxygen of GOD and substrate glucose effect generation
Exogenous H2O2, successfully avoid H2O2The unstable disadvantage so as to cause testing result stability difference is easily decomposed, and is detected
As a result accurate, reliable.
The immunoassay method that the present invention establishes is sandwich method enzyme-linked immunosorbent assay, is a kind of easy, quickly and can
The immunoassay method of high throughput detection, and in several sides such as production cost, transport storage characteristic, safety in utilization and the feature of environmental protection
Face all has significant meaning.The method of the present invention can both be fabricated to kit commercialization be widely used, it is also possible to make company or
Laboratory is used from test sample, has very high application value.
Detailed description of the invention
Fig. 1 is that the standard that optical density changes with the logarithm of vascular endothelial growth factor standard concentration in embodiment 1 is bent
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
Measure the content of vascular endothelial growth factor (VEGF), the specific steps are as follows:
1, VEGF standard items are added in the PBS buffer solution that 0.1mol/L pH value is 7.4, prepare 1pg/L, 10pg/ respectively
L, the VEGF standard solution of 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 (by 0.05mol/L pH value be 7.4 PBS buffer solution and Tween-20 form,
Middle Tween-20 accounts for the 0.05% of washing buffer quality) washing after pat dry (1 time), setting blank well respectively, (blank well is not added to be measured
Sample and enzyme marking reagent, remaining each step operation are identical), gauge orifice, sample to be tested hole, 50 μ L concentration are separately added into gauge orifice
For the VEGF standard solution of 1pg/L, 10pg/L, 100pg/L, 1000pg/L and 10000pg/L, in sample to be tested hole first plus 40
Then the PBS buffer solution that μ L 0.1mol/L pH is 7.4 adds 10 μ L serum samples (the final dilution of sample to be tested is 5 times) again,
37 DEG C incubate 1 hour, discard liquid in ELISA Plate, pat dry, then with washing buffer (by pH value be 7.4 PBS buffer solution and
Tween-20 composition, wherein Tween-20 accounts for the 0.1% of washing buffer quality) it washs ELISA Plate and pats dry (3 times).
2, the monoclonal antibody (Recombinant people VEGF 165A protein) of glucose oxidase label is added
In the PBS buffer solution that 0.1mol/L pH value is 7.4, it is configured to the solution of 0.038U/mL, in each gauge orifice and sample to be tested
Respectively it is added the 50 μ L solution in hole, except blank well, 37 DEG C are incubated 1 hour, are discarded liquid in ELISA Plate, are patted dry, then with washing
It washs buffer washing ELISA Plate (identical as step 1) and pats dry (3 times).
3, it is added the glucose solution of 50 μ L 1mol/L in each gauge orifice, sample to be tested hole, blank well, 30
DEG C reaction 10 minutes, add 50 μ L 0.005mol/L 4-AA aqueous solutions and 50 μ L0.004mol/L N- second
Base-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt aqueous solution, 10 μ L 40U/mL HRP aqueous solutions, gently concussion is mixed
Even, 37 DEG C are protected from light colour developing 15 minutes；It using microplate reader, is returned to zero with blank well, measures the light in each hole respectively under 555nm wavelength
Density, with the logarithm (lgC) of VEGF standard concentration for abscissa, optical density is ordinate, draws optical density and marks with VEGF
The standard curve (the result is shown in Figure 1) of the logarithm variation of quasi- product concentration, detection are limited to 3.24 × 10-4ng/L。
4, the corresponding optical density in sample to be tested hole obtained according to above-mentioned steps 1~3, the linear side of combined standard curve
Journey, the content for calculating VEGF in serum sample is 5.30ng/L, and the reality multiplied by extension rate, as sample to be tested contains
Amount.
Claims (2)
1. a kind of enzyme-linked immune analytic method, it is characterised in that it is made of following step:
(1) vascular endothelial growth factor standard items to be measured are added in the PBS buffer solution that pH value is 7.4, prepare 1~10000
Vascular endothelial growth factor standard solution is added to coating Anti- by pg/L vascular endothelial growth factor standard solution
The polystyrene ELISA Plate surface of VEGF antibody, makes vascular endothelial growth factor and Anti-VEGF antibody linked, then with washing
Buffer washing polystyrene ELISA Plate simultaneously pats dry；
(2) after the monoclonal antibody that glucose oxidase marks being dissolved in the PBS buffer solution that pH value is 7.4, it is added to step
(1) the polystyrene ELISA Plate surface of bonding vascular endothelial growth factor in, with the blood for being bonded in polystyrene ELISA Plate surface
The reaction of endothelial tube growth factor, then washs polystyrene ELISA Plate with washing buffer and pats dry；
(3) glucose solution is added to the polystyrene ELISA Plate surface obtained in step (2), 30~37 DEG C of reactions 10
~20 minutes, the colour developing of 4-AA and N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt is added
The horseradish peroxidase of system and catalytic amount carries out chromogenic reaction, and measures optical density, draws optical density with blood vessel endothelium
The standard curve of the logarithm variation of growth factor standard concentration；
(4) the corresponding optical density of vascular endothelial growth factor sample to be measured is tested according to above-mentioned steps (1)~(3) method, in conjunction with
The linear equation of standard curve is the content that can determine vascular endothelial growth factor sample medium vascular endothelial growth factor to be measured.
2. enzyme-linked immune analytic method according to claim 1, it is characterised in that: the washing buffer is by pH value
It is formed for 7.4 PBS buffer solution and Tween-20, wherein Tween-20 accounts for the 0.1% of washing buffer quality.
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Citations (5)
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| 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 (5)
| Title |
|---|
| A highly sensitive immunoassay using antibodyconjugated spherical mesoporous silica with immobilized enzymes.;Ji Young Eum, et.al.;《Chem Commun》;20131204;第50卷;附加材料第1页、第5页 * |
| A Rapid Enzyme Immunoassay for Cocaine and Benzoylecgonine Using Glucose Oxidase.;Mitsune Yamaguchi, et.al.;《Journal of Health Science》;20011231;第47卷(第4期);第420页 * |
| Comparison of Glucose Oxidase and Peroxidase as Labels for Antibody in Enzyme-linked Immunosorbent Assay.;Roy B. Johnson Jr., et.al.;《J Immunoassay》;19801231;第1卷(第1期);第33页 * |
| Glucose Oxidase as an Analytical Reagent.;Julio Raba, Horacio A. Mottola;《Critical Reviews in Analytical Chemistry》;19951231;第25卷(第1期);第1-42页 * |
| Glucose Oxidase-Catalyzed Growth of Gold Nanoparticles Enables Quantitative Detection of Attomolar Cancer Biomarkers.;Dingbin Liu, et.al.;《Anal Chem》;20140526;第86卷;第5802页 * |
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