CN105137087A - Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe - Google Patents
Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe Download PDFInfo
- Publication number
- CN105137087A CN105137087A CN201510501270.5A CN201510501270A CN105137087A CN 105137087 A CN105137087 A CN 105137087A CN 201510501270 A CN201510501270 A CN 201510501270A CN 105137087 A CN105137087 A CN 105137087A
- Authority
- CN
- China
- Prior art keywords
- igy
- chicken
- solution
- scattering
- rabbit anti
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—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
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention discloses a chicken IgY- standard curve producing method and detection method using a rabbit anti-chicken IgY- nanogold immune scattering probe. The method is as follows: preparing water-soluble well dispersed nanogold, the nanogold and a rabbit anti-chicken IgY antibody are conjugated into a specific immune probe used directly for IgY detection, and a new method for rapid and sensitive quantitative detection of IgY is established. The technology provides a new method for detecting IgY, has the advantages of high sensitivity, high accuracy, low detection limit, strong anti-interference ability, easy operation, and low equipment requirements, and is conducive to the promotion and application. The method can be applied to rapid and sensitive quantitative detection of IgY in egg products, health food, and other related products, and has broad application prospects.
Description
Technical field
The present invention relates to the method detecting chicken yolk immune globulin, refer to that one utilizes rabbit anti-chicken IgY-nm of gold immunity scattering probe to do to detect chicken IgY-typical curve method and detection method particularly.
Background technology
Chicken yolk immune globulin (IgY) is from oviparous animal, and mammal is played in most of antigen sources of present stage research, thus as oviparous animal compared with mammal, high to the degree of reaction of mammalian antigen, distinct antibodies formation rate is high, therefore IgY can be used as and produces better antibody sources, and immunology has advantage.In addition, the exploitation of IgY health food also had research in recent years.Antibody IgY can combine with the viral antigen in body, and then the chance suppressing germ to contact with intestines parietal cell, provides the immunocompetence that health is passive, resists antigen (bacterium, virus and the extraneous protein etc.) harm to health.Because IgY has certain acid-fast ability, can the non-inactivation by the gastric juice of the young smoothly.This provides certain foundation for developing IgY health food.In addition, because IgY has inhibiting effect (Wang Zhuling and Wang Qiang 2008) to oral cavity streptococcus mutans, be developed to anticarious IgY-toothpaste at present, home market has very ripe commodity.In view of IgY plays an important role, so the quantitative Clinical significance of detecting of IgY is remarkable in immunology, medical conditions prevention, food additives and commodity etc.
At present, the research about IgY mainly concentrates on the separation and purification of IgY, aspect (Huang Jing and Luo Jian 2002 such as immunizing potency research; Palomboetal1998), also few for the research quantitatively detecting IgY, that has reported has euzymelinked immunosorbent assay (ELISA), high performance liquid chromatography and electrochemical method etc.But euzymelinked immunosorbent assay (ELISA) is difficult to accomplish that accurate quantitative analysis detects (Song Hongxin etc. 2004); High performance liquid chromatography complicated operation, instrument is (Chen Weifeng etc. 2012) costly; Electrochemical method relates to electrode preparation and detects thing fixing at electrode surface, operates comparatively loaded down with trivial details (Bottarietal2014).
Therefore develop that a kind of sensitivity is high, reagent consumption is little, with low cost, it is significant to detect convenient, to be easy to penetration and promotion IgY quantitative detecting method.
Summary of the invention
Technical matters to be solved by this invention be just to provide a kind of utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe manufacturing to detect chicken yolk immune globulin typical curve method and utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe to detect chicken yolk immune globulin method fast.The method achieve the quantitative test to trace IgY, there is detection speed fast, highly sensitive, anti-interference strong feature, and experimental implementation is simple, and agents useful for same consumption is little, cost is low, achieve and IgY rapid sensitive is detected, meet the important development trend that modern food analysis detects.
For achieving the above object, a kind of method utilizing rabbit anti-chicken IgY-nm of gold immunity scattering probe manufacturing to detect chicken yolk immune globulin typical curve provided by the invention, comprises the following steps:
1) anti-for rabbit chicken IgY antibody (Anti-IgY) is joined in nano-Au solution (AuNPs), stir, add PEG 20000 again, continue stirring and obtain rabbit anti-chicken IgY-nm of gold immunity scattering probe solution, and save backup under being placed in 4 DEG C of conditions;
2) the immune scattering probe solution of anti-for rabbit chicken IgY-nm of gold (Anti-IgY-AuNPs), IgY and PBS damping fluid are mixed, be then settled to 5mL with distilled water; Namely obtain the mixed solution of the IgY containing variable concentrations, wherein, in mixed solution, the concentration of IgY is 0 ~ 200ng/mL;
3) the above-mentioned mixed solution obtaining IgY containing variable concentrations is placed in respectively fluorospectrophotometer to carry out synchronous scanning and measure its scattering spectrum, the scattering strength I of the mixed solution containing IgY
1, the mixed solution raw scattered intensity not containing IgY is I
0; Observe the change of scattered light spectrogram; Wherein, synchronous scanning is set as Δ λ=0, i.e. λ
em=λ
ex;
4) by contrast raw scattered intensity I
0with add IgY react after solution scattering strength I
1between scattering strength variation delta I=I
1-I
0, obtain the linear relationship between IgY concentration and Δ I, set up IgY and quantitatively detect equation.
Further, described step 1) in, the concentration of rabbit anti-chicken IgY antibody is 0.002mg/mL, and the concentration of nano-Au solution is 47.8 μ g/mL, pH=7.5, and the massfraction of PEG 20000 is 3%, wherein,
Described rabbit anti-chicken IgY-nm of gold immunity scattering probe solution is by joining in the nano-Au solution of 10mL by anti-for the rabbit of 1mL chicken IgY antibody, stirs 10 ~ 20min, then adds the PEG 20000 of 200 μ L; Continuation stirring 15 ~ 30min obtains.
Again further, described step 2) in, the consumption of rabbit anti-chicken IgY-nm of gold immunity scattering probe solution is 5mL; The pH=4.9 of PBS damping fluid, concentration is the consumption of 0.02mol/L, PBS damping fluid is 2.5mL; In the mixed solution of eight kinds, the concentration of IgY is respectively 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL and 200ng/mL.
Again further, described fluorospectrophotometer is the RF-5301 type fluorospectrophotometer of Japanese Shimadzu, its excitation wavelength is set as 225nm, emission wavelength is set as 225-800nm, excite and be 5nm with utilizing emitted light slit width, Sensitive is set to low, and Responsetime is set as 0.25s.
Again further, described step 4) in, it is Δ I=2.0764C-4.3059 that IgY quantitatively detects equation, and wherein, C is IgY concentration (ng/mL).
Present invention also offers one utilizes rabbit anti-chicken IgY-nm of gold immunity scattering probe to detect chicken yolk immune globulin method fast, comprises the following steps:
1) anti-for rabbit chicken IgY antibody (Anti-IgY) is joined in nano-Au solution (AuNPs), stir, add PEG 20000 again, continue stirring and obtain rabbit anti-chicken IgY-nm of gold immunity scattering probe solution, and save backup under being placed in 4 DEG C of conditions;
2) anti-for rabbit chicken IgY-nm of gold immunity scattering probe solution, IgY and PBS damping fluid are mixed, be then settled to 5mL with distilled water; Namely obtain the mixed solution of the IgY containing variable concentrations, wherein, in mixed solution, the concentration of IgY is 0 ~ 200ng/mL;
3) the above-mentioned mixed solution obtaining IgY containing variable concentrations is placed in respectively fluorospectrophotometer to carry out synchronous scanning and measure its scattering spectrum, the scattering strength I of the mixed solution containing IgY
1, the mixed solution raw scattered intensity not containing IgY is I
0; Observe the change of scattered light spectrogram; Wherein, synchronous scanning is set as Δ λ=0, i.e. λ
em=λ
ex;
4) by contrast raw scattered intensity I
0with add IgY react after solution scattering strength I
1between scattering strength variation delta I=I
1-I
0, obtain the linear relationship between IgY concentration and Δ I, set up IgY and quantitatively detect equation;
5) be dissolved in distilled water by determinand, ultrasonic vibration makes determinand fully dissolve, and centrifugal segregation high-abundance proteins, obtains supernatant;
6) by step 1) in the supernatant that obtains mix with the PBS damping fluid of the rabbit of 0.5mL anti-chicken IgY-nm of gold immunity scattering probe solution and 2.5mL, be settled to 5mL and obtain liquid to be measured;
7) utilize fluorospectrophotometer to detect the scattering strength of liquid to be measured, utilize the linear equation of typical curve obtained above to calculate IgY content in determinand.
Again further, described step 1) in, the concentration of rabbit anti-chicken IgY antibody is 0.002mg/mL, and the concentration of nano-Au solution is 47.8 μ g/mL, pH=7.5, and the massfraction of PEG 20000 is 3%, wherein,
Described rabbit anti-chicken IgY-nm of gold immunity scattering probe solution is by joining in the nano-Au solution of 10mL by anti-for the rabbit of 1mL chicken IgY antibody, stirs 10 ~ 20min, then adds the PEG 20000 of 200 μ L; Continuation stirring 15 ~ 30min obtains.
Again further, described step 2) in, the consumption of rabbit anti-chicken IgY-nm of gold immunity scattering probe solution is 5mL; The pH=4.9 of PBS damping fluid, concentration is the consumption of 0.02mol/L, PBS damping fluid is 2.5mL; In the mixed solution of eight kinds, the concentration of IgY is respectively 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL and 200ng/mL.
Again further, described fluorospectrophotometer is the RF-5301 type fluorospectrophotometer of Japanese Shimadzu, its excitation wavelength is set as 225nm, emission wavelength is set as 225-800nm, excite and be 5nm with utilizing emitted light slit width, Sensitive is set to low, and Responsetime is set as 0.25s.
Again further, described step 4) in, it is Δ I=2.0764C-4.3059 that IgY quantitatively detects equation, and wherein, C is IgY concentration (ng/mL).Beneficial effect of the present invention is:
1, the present invention is based on the Rayleigh scattering characteristic of AuNPs, it is become specific immunity probe with anti-IgY coupling, establish the new method of quick, sensitive quantitative detection IgY.This is the new detecting technique combined by the high degree of specificity that the high sensitivity of AuNPs labelling technique and anti-IgY and IgY react.
2, the range of linearity of this detection method is at 4ng/mL to 200ng/mL, and minimum detectability is 1.92ng/mL.Compare with electrochemical method with existing method such as euzymelinked immunosorbent assay (ELISA), the high performance liquid chromatography quantitatively detected about IgY both at home and abroad, the instrument and equipment that this method uses is simple, is conducive to promoting and application, and the quantitative detection for IgY opens a new way.
3, this method is mainly used in the detection of IgY in egg products, can detect rapidly and accurately for the content of IgY in different sample, and accuracy rate is high, this is not only significant for the detection in current fowls egg products, and the false distinguishing that can be IgY health products and Related product provides a kind of thinking and certain reference value.
Accompanying drawing explanation
Fig. 1: IgY (a), AuNPs (b), antiIgY-AuNPs (c) contrast with the scatter pattern of IgY-anti-IgY-AuNPs (d);
The ultra-violet absorption spectrum (a) of Fig. 2: IgY-antiIgY-AuNPs and scattering spectrum (b);
The scattered light variation diagram of Fig. 3: Anti-IgY-AuNPs detection IgY;
The change of Fig. 4: IgY-AntiIgY-AuNPs scattering strength and the linear relationship chart of IgY concentration.
Embodiment
In order to explain the present invention better, illustrate main contents of the present invention further below in conjunction with specific embodiment, but content of the present invention is not only confined to following examples.
The preparation of embodiment 1 nm of gold
Utilize trisodium citrate as reductive agent, prepare the nano colloid gold that particle is homogeneous, be evenly distributed.Glassware used all need at chloroazotic acid (HNO
3︰ HCl=1 ︰ 3, v/v) middle immersion 48h, take out, first use a large amount of tap water, more for subsequent use by distilled water cleaning post-drying.
The method that the synthesis of nm of gold has been delivered according to people such as Kim is carried out:
1) aqueous solution of chloraurate getting 100mL0.1mg/mL is heated to 95 DEG C, until boiling.Equally, the sodium citrate solution of 5mL10mg/mL is heated to 95 DEG C, until boiling;
2) under magnetic stirring apparatus 120rpm stirs, above-mentioned sodium citrate solution is dropwise joined in aqueous solution of chloraurate, and continue to keep boiling 10 minutes.Nm of gold will be formed in 2 ~ 3 minutes.The color of mixed solution becomes shiny red from mazarine; Keep in Dark Place in 4 DEG C of refrigerators after the nano-Au solution cooling of synthesis.
The concentration of above-mentioned nano-Au solution is 47.8 μ g/mL.
Embodiment 2 prepares AuNPs-Anti-IgY immunity scattering probe
Nm of gold combines with rabbit anti-chicken IgY antibody, forms IgY specific immunity scattering probe.Concrete steps are as follows:
The Anti-IgY of 1mL0.002mg/mL is added in the AuNPs solution of 10mL47.8 μ g/mL of suitable pH under the effect of magnetic agitation.Stir after 10 minutes, add 200 μ L massfractions be the PEG 20000 (PEG20000) of 3% as stabilizing agent, continue stirring after 15 minutes, stand-by at being kept at 4 DEG C.
Embodiment 3 sets up the typical curve of the immune scattering probe in detecting IgY of rabbit anti-chicken IgY-nm of gold (Anti-IgY-AuNPs)
The PBS damping fluid mixing that the 0.02mol/LpH of the Anti-IgY-AuNPs solution of 0.5mL, IgY and 2.5mL is 4.9, then 5mL is settled to distilled water, obtain eight kinds of mixed solutions, wherein, mixed solution, the concentration of IgY is respectively 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL and 200ng/mL.
Adopt its scattering spectrum of RF-5301 type fluorescent spectrophotometer assay of Japanese Shimadzu.Setting synchronous scanning △ λ=0 (λ
em=λ
ex), excitation wavelength is set as 225nm, and emission wavelength is set as 225-800nm, and excite and be 5nm with utilizing emitted light slit width, the response time is set as 0.25s.The product obtained is carried out scattering spectrum sign (Fig. 1).
Result shows that the maximum scattering wavelength of AuNPs is at 564nm place.The RRS intensity of IgY (a), AuNPs (b) and Anti-IgY-AuNPs (c) is very weak.
After AuNPs and Anti-IgY combines, its RRS intensity slightly rises (from b to c).After IgY adds, system significantly rises (d) in the RRS intensity at 564nm place, establishes the detection method of IgY based on this.
Anti-IgY-AuNPs solution is carried out to the sign (Fig. 2) of scattering spectrum and absorption spectrum, result shows that Anti-IgY-AuNPs immunity scattering probe (IgY-anti-IgY-AuNPs) combining IgY has wider ultraviolet absorption peak, and wherein the strongest absorption peak is positioned at 525nm (a).The RRS collection of illustrative plates of IgY-anti-IgY-AuNPs shows, after adding IgY, and the RRS intensity very high (b) of system.When quantitatively detecting, add the scattering strength I of solution after IgY
1, the Anti-IgY-AuNPs solution raw scattered intensity not adding IgY is I
0.
Observe the change of scattered light spectrogram.By contrast raw scattered intensity I
0with add IgY react after solution scattering strength I
1between scattering strength variation delta I (=I
1-I
0), obtain the linear relationship between IgY concentration and Δ I, set up IgY and quantitatively detect equation (Fig. 3, Fig. 4).Linear equation is Δ I=2.0764C-4.3095 (R
2=0.9946).
Embodiment 4 detects the antijamming capability of IgY method
In interfering material and synthetic sample, add IgY, obtain respective response signal by above-mentioned detection, calculate scattered light and change number percent, check antijamming capability of the present invention.Select 14 kinds of interfering materials and 5 kinds of synthetic samples as detection sample.
The Anti-IgY-AuNPs solution of 0.5mL, the 0.02mol/LpH of IgY and 2.5mL is the PBS damping fluid mixing of 4.9, add certain density interfering material or synthetic sample again, then 5mL is settled to distilled water, obtain eight kinds of mixed solutions, wherein, mixed solution, the concentration of IgY is respectively 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL and 200ng/mL.
Replace as interfering material or synthetic sample blank group with PBS.After reaction, at 37 DEG C, fluorospectrophotometer is adopted to detect its scattering strength.Wherein phosphate buffered solution proportioning is:
First prepare the Na of 0.2mol/L
2hPO
4: take Na
2hPO
412H
2o31.2g adds distilled water and dissolves to 1000mL.The NaH of 0.2mol/L
2pO
4: take NaH
2pO
412H
2o71.632g adds redistilled water and dissolves to 1000mL.Two kinds of solution therewith, regulate pH to be 4.9 with pH meter, then get 10mL and be diluted to 100mL, be PBS buffer solution used.
The scattering strength change number percent that each interfering material concentration and this concentration interfering material cause is as shown in table 1.
The scattering strength knots modification that table 1 interfering material causes
There is the materials such as salt, amino acid, protein, carbohydrate and vitamin in actual living things system and egg products.The present invention simulates a few class materials common in living things system, synthesizes 5 groups of samples, is often combined in sample all containing materials such as certain density salt, amino acid, protein, carbohydrate and vitamins.The composition of each synthetic sample and material concentration and the scattering strength change caused as shown in table 2.
Show to detect IgY in 2-in-1 one-tenth sample
The application of embodiment 5 in actual sample detects
The typical curve of above-mentioned preparation is applied to the quantitative detection of IgY in actual powdered egg.Concrete steps are as follows:
1) by yolk powder and dried whole-egg two kinds of sample dissolution in a certain amount of distilled water, ultrasonic vibration 10min makes it fully dissolve, and makes the abundant stripping of IgY.Centrifugal 15min in 5000rpm hydro-extractor, removes high-abundance proteins, gets supernatant to be measured;
2) the Anti-IgY-AuNPs solution of 0.5mL, the powdered egg sample solution comprising IgY of 25 μ L and the mixing of 2.5mLPBS damping fluid;
3), after each solution mixes, potpourri distilled water is settled to 5mL; In powdered egg sample solution, the concentration of IgY will be diluted within the scope of the concentration change of the linear equation that said method is set up; Detect the scattering strength of mixed liquor with fluorospectrophotometer, utilize linear equation to calculate IgY content in powdered egg.Testing result is as shown in table 3.
The testing result of IgY in table 3 actual sample
In actual sample detects, all samples all do not carry out complicated pre-service, overcome the difficult problem detecting food samples triviality to a great extent.From the interpretation of result of table 1, table 2 and table 3, it is also very wide that this method has very strong antijamming capability, the scope of application, established good theory and practice basis for detecting protein based on immune nanometer gold probe resonance Rayleigh scattering from now on
Other unspecified part is prior art.Although above-described embodiment is to invention has been detailed description; but it is only the present invention's part embodiment; instead of whole embodiment, people can also obtain other embodiments according to the present embodiment under without creative prerequisite, and these embodiments all belong to scope.
Claims (10)
1. utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe manufacturing to detect a method for chicken yolk immune globulin typical curve, it is characterized in that: comprise the following steps:
1) anti-for rabbit chicken IgY antibody is joined in nano-Au solution, stir, then add PEG 20000, continue stirring and obtain rabbit anti-chicken IgY-nm of gold immunity scattering probe solution, and save backup under being placed in 4 DEG C of conditions;
2) anti-for rabbit chicken IgY-nm of gold immunity scattering probe solution, IgY and PBS damping fluid are mixed, be then settled to 5mL with distilled water; Namely obtain the mixed solution of the IgY containing variable concentrations, wherein, in mixed solution, the concentration of IgY is 0 ~ 200ng/mL;
3) the above-mentioned mixed solution obtaining IgY containing variable concentrations is placed in respectively fluorospectrophotometer to carry out synchronous scanning and measure its scattering spectrum, the scattering strength I of the mixed solution containing IgY
1, the mixed solution raw scattered intensity not containing IgY is I
0; Observe the change of scattered light spectrogram; Wherein, synchronous scanning is set as Δ λ=0, i.e. λ
em=λ
ex;
4) by contrast raw scattered intensity I
0with add IgY react after solution scattering strength I
1between scattering strength variation delta I=I
1-I
0, obtain the linear relationship between IgY concentration and Δ I, set up IgY and quantitatively detect equation.
2. utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe manufacturing to detect the method for chicken yolk immune globulin typical curve according to claim 1, it is characterized in that: described step 1) in, the concentration of rabbit anti-chicken IgY antibody is 0.002mg/mL, the concentration of nano-Au solution is 47.8 μ g/mL, pH=7.5, the massfraction of PEG 20000 is 3%, wherein
Described rabbit anti-chicken IgY-nm of gold immunity scattering probe solution is by joining in the nano-Au solution of 10mL by anti-for the rabbit of 1mL chicken IgY antibody, stirs 10 ~ 20min, then adds the PEG 20000 of 200 μ L; Continuation stirring 15 ~ 30min obtains.
3. according to claim 1 or 2, utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe manufacturing to detect the method for chicken yolk immune globulin typical curve, it is characterized in that: described step 2) in, the consumption of rabbit anti-chicken IgY-nm of gold immunity scattering probe solution is 5mL; The pH=4.9 of PBS damping fluid, concentration is the consumption of 0.02mol/L, PBS damping fluid is 2.5mL; In the mixed solution of eight kinds, the concentration of IgY is respectively 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL and 200ng/mL.
4. according to claim 1 or 2, utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe manufacturing to detect the method for chicken yolk immune globulin typical curve, it is characterized in that: described fluorospectrophotometer is the RF-5301 type fluorospectrophotometer of Japanese Shimadzu, its excitation wavelength is set as 225nm, emission wavelength is set as 225-800nm, excite and be 5nm with utilizing emitted light slit width, Sensitive is set to low, and Responsetime is set as 0.25s.
5. utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe manufacturing to detect the method for chicken yolk immune globulin typical curve according to claim 4, it is characterized in that: described step 4) in, it is Δ I=2.0764C-4.3059 that IgY quantitatively detects equation, and wherein, C is IgY concentration.
6. utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe to detect a chicken yolk immune globulin method fast, it is characterized in that: comprise the following steps:
1) anti-for rabbit chicken IgY antibody is joined in nano-Au solution, stir, then add PEG 20000, continue stirring and obtain rabbit anti-chicken IgY-nm of gold immunity scattering probe solution, and save backup under being placed in 4 DEG C of conditions;
2) anti-for rabbit chicken IgY-nm of gold immunity scattering probe solution, IgY and PBS damping fluid are mixed, be then settled to 5mL with distilled water; Namely obtain the mixed solution of the IgY containing variable concentrations, wherein, in mixed solution, the concentration of IgY is 0 ~ 200ng/mL;
3) the above-mentioned mixed solution obtaining IgY containing variable concentrations is placed in respectively fluorospectrophotometer to carry out synchronous scanning and measure its scattering spectrum, the scattering strength I of the mixed solution containing IgY
1, the mixed solution raw scattered intensity not containing IgY is I
0; Observe the change of scattered light spectrogram; Wherein, synchronous scanning is set as Δ λ=0, i.e. λ
em=λ
ex;
4) by contrast raw scattered intensity I
0with add IgY react after solution scattering strength I
1between scattering strength variation delta I=I
1-I
0, obtain the linear relationship between IgY concentration and Δ I, set up IgY and quantitatively detect equation;
5) be dissolved in distilled water by determinand, ultrasonic vibration makes determinand fully dissolve, and centrifugal segregation high-abundance proteins, obtains supernatant;
6) by step 1) in the supernatant that obtains mix with the PBS damping fluid of the rabbit of 0.5mL anti-chicken IgY-nm of gold immunity scattering probe solution and 2.5mL, be settled to 5mL and obtain liquid to be measured;
7) utilize fluorospectrophotometer to detect the scattering strength of liquid to be measured, utilize the linear equation of typical curve obtained above to calculate IgY content in determinand.
7. utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe to detect chicken yolk immune globulin method fast according to claim 6, it is characterized in that: described step 1) in, the concentration of rabbit anti-chicken IgY antibody is 0.002mg/mL, the concentration of nano-Au solution is 47.8 μ g/mL, pH=7.5, the massfraction of PEG 20000 is 3%, wherein
Described rabbit anti-chicken IgY-nm of gold immunity scattering probe solution is by joining in the nano-Au solution of 10mL by anti-for the rabbit of 1mL chicken IgY antibody, stirs 10 ~ 20min, then adds the PEG 20000 of 200 μ L; Continuation stirring 15 ~ 30min obtains.
8. according to claim 6 or 7, utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe to detect chicken yolk immune globulin method fast, it is characterized in that: described step 2) in, the consumption of rabbit anti-chicken IgY-nm of gold immunity scattering probe solution is 5mL; The pH=4.9 of PBS damping fluid, concentration is the consumption of 0.02mol/L, PBS damping fluid is 2.5mL; In the mixed solution of eight kinds, the concentration of IgY is respectively 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL and 200ng/mL.
9. according to claim 1 or 2, utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe to detect chicken yolk immune globulin method fast, it is characterized in that: described fluorospectrophotometer is the RF-5301 type fluorospectrophotometer of Japanese Shimadzu, its excitation wavelength is set as 225nm, emission wavelength is set as 225-800nm, excite and be 5nm with utilizing emitted light slit width, Sensitive is set to low, and Responsetime is set as 0.25s.
10. utilize rabbit anti-chicken IgY-nm of gold immunity scattering probe to detect chicken yolk immune globulin method fast according to claim 4, it is characterized in that: described step 4) in, it is Δ I=2.0764C-4.3059 that IgY quantitatively detects equation, and wherein, C is IgY concentration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510501270.5A CN105137087A (en) | 2015-08-14 | 2015-08-14 | Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510501270.5A CN105137087A (en) | 2015-08-14 | 2015-08-14 | Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105137087A true CN105137087A (en) | 2015-12-09 |
Family
ID=54722510
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510501270.5A Pending CN105137087A (en) | 2015-08-14 | 2015-08-14 | Chicken IgY- standard curve producing method and detection method using rabbit anti-chicken IgY- nanogold immune scattering probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105137087A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050019842A1 (en) * | 2002-11-06 | 2005-01-27 | Prober James M. | Microparticle-based methods and systems and applications thereof |
| CN101320044A (en) * | 2008-07-03 | 2008-12-10 | 广西师范大学 | Resonance scattering spectrometry for detecting trace of albumin in human urine |
| CN101718779A (en) * | 2009-10-29 | 2010-06-02 | 广西师范大学 | Kit for detecting immuno-nanogold synchronous scattering spectrum of human serum ceruloplosmin and use method thereof |
-
2015
- 2015-08-14 CN CN201510501270.5A patent/CN105137087A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050019842A1 (en) * | 2002-11-06 | 2005-01-27 | Prober James M. | Microparticle-based methods and systems and applications thereof |
| CN101320044A (en) * | 2008-07-03 | 2008-12-10 | 广西师范大学 | Resonance scattering spectrometry for detecting trace of albumin in human urine |
| CN101718779A (en) * | 2009-10-29 | 2010-06-02 | 广西师范大学 | Kit for detecting immuno-nanogold synchronous scattering spectrum of human serum ceruloplosmin and use method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 宋宏新等: "抗风疹病毒卵黄免疫球蛋白的间接酶联免疫法检测的研究", 《中华检验医学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hu et al. | Advantages of time-resolved fluorescent nanobeads compared with fluorescent submicrospheres, quantum dots, and colloidal gold as label in lateral flow assays for detection of ractopamine | |
| CN108362688B (en) | Detection kit for chemiluminescence of 25-hydroxy vitamin D magnetic particles | |
| CN101949942B (en) | Kit for quantitatively testing free triiodothyronine | |
| CN102998445B (en) | Reagent and preparation method for determining glycocholic acid | |
| CN102967706B (en) | Preparation method and application of flow injection chemiluminiscence immuno sensor for detecting tumor marker | |
| CN109917134B (en) | Calibrator stabilizer, detection kit for determining C peptide and detection method | |
| CN101813699A (en) | Photo-induced chemiluminescence immunoassay quantitative kit and method for detecting content of milk protein | |
| CN105242047B (en) | AFB1 graphene oxide immuno-chromatographic test paper strip and its application | |
| CN105181680A (en) | Magnetic bead separation chemiluminescence immune detection method for melamine | |
| CN107543932A (en) | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of calcitonin | |
| CN106053826A (en) | III type procollagen N-terminal peptide quantitative measurement kit and preparation method thereof | |
| CN105277717A (en) | Magnetic particle separation and chemiluminescence immunoassay method of thyroglobulin | |
| CN105203748A (en) | Quantitative detection kit for full-measuring-range C reaction protein | |
| CN109307765A (en) | Type pepsinogen II detection kit | |
| CN103954776A (en) | NGAL (Neutrophil Gelatinase Associated Lipocalin) optical excitation chemiluminescence detection kit and preparation and use methods of kit | |
| Liao et al. | Lanthanide chelate-encapsulated polystyrene nanoparticles for rapid and quantitative immunochromatographic assay of procalcitonin | |
| CN108445214A (en) | A kind of kit and preparation method quantitatively detecting d-dimer | |
| CN103954766B (en) | Transferring receptor detection kit and preparation method thereof | |
| CN101865917B (en) | Triiodothyronine detection reagent kit and use method thereof | |
| CN113125696B (en) | Estradiol homogeneous chemiluminescence detection kit and application thereof | |
| CN102809654A (en) | Double-particle compounded C-reactive protein detection kit | |
| CN104330551B (en) | A kind of interleukin 6 quantitative determination reagent kit and preparation method thereof | |
| CN107843734A (en) | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of sex hormone binding globulin | |
| CN109459570A (en) | The colloid gold immune test paper preparation method of lead ion in a kind of quick detection blood of human body | |
| CN103048477A (en) | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151209 |
|
| RJ01 | Rejection of invention patent application after publication |