CN107860916A - Colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen and preparation method thereof in food - Google Patents
Colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen and preparation method thereof in food Download PDFInfo
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- CN107860916A CN107860916A CN201711064925.2A CN201711064925A CN107860916A CN 107860916 A CN107860916 A CN 107860916A CN 201711064925 A CN201711064925 A CN 201711064925A CN 107860916 A CN107860916 A CN 107860916A
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
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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
- G01N33/54306—Solid-phase reaction mechanisms
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- Chemical Kinetics & Catalysis (AREA)
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- Coloring Foods And Improving Nutritive Qualities (AREA)
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Abstract
The invention discloses colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen and preparation method thereof in food, the chromatograph test strip includes adsorptive pads, nitrocellulose filter, gold standard pad and the sample pad being sequentially connected, and can also include weight tray；Nitrocellulose filter is provided with detection line, antigen line and nature controlling line, a kind of anti-walnut protein monoclonal antibody colloid gold label thing is fixed in gold standard pad, the monoclonal antibody of another capture walnut protein is coated with the detection line of nitrocellulose filter, antigen line is coated with walnut protein, nature controlling line coating sheep anti-mouse igg；It is negative when a nature controlling line, an antigen line occur in test strips, detection line occurs（No matter either with or without antigen line）All it is positive, compared with existing walnut protein Allergic skin test technology, high sensitivity, high specificity, the concentration of anaphylactogen can also be made and intuitively evaluate, can be directly used for food inspection.
Description
Technical field
The invention belongs to technical field of food safety detection, and in particular to a kind of colloid for detecting walnut anaphylactogen in food
Golden immuno-chromatographic test paper strip and preparation method thereof.
Background technology
Walnut allergy is a kind of serious nut allergies reaction.Serious clinical symptoms are frequently accompanied by, are even resulted in dead
Die.Walnut all be present in the food processing of many, this may cause potential sensitization dangerous.Walnut allergy is mainly Ig E
The type Ⅰ hypersensitivity reaction of mediation.Jug r 1 are considered as the major allergen protein in walnut.Walnut allergy detection side at present
Method has ELISA, immunosensor detection technique, Real-Time Fluorescent Quantitative PCR Technique, SPR, mass-spectrometric technique etc., these detection methods
There is very high requirement to instrument and equipment, surface environment, operating personnel etc. and detection time is longer, costly, thus establish one
The ELISA test strip of the new fast and stable of kind is very necessary.
The content of the invention
It is an object of the invention to develop the walnut anaphylactogen test strips in a kind of detection food that can be quick and harmless, no
It is only capable of verifying allergen protein micro in food, also can operate with the quality management and control of food processing process, such as charging, production
Process and finished product detection, prevent unnecessary allergy puzzlement in advance.
The present invention is that the monoclonal being mutually paired that walnut protein can be detected based on a pair matches antibody, and this is to monoclonal
Match antibody to be provided by BioFront companies of the U.S., by preparing 40nm collaurums, mark antibody after purification, optimize every layer
Analysis condition etc. completes test strips invention.
The present invention is achieved by the following technical solutions：
The colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in food, including assemble successively adsorptive pads, nitrocellulose filter,
Gold standard pad, sample pad, a kind of anti-walnut protein monoclonal antibody-colloid gold label thing is fixed in described gold standard pad, it is described
Another anti-walnut with the pairing of the anti-walnut protein monoclonal antibody of colloid gold label is coated with nitrocellulose filter successively
Protein monoclonal antibody detection line, antigen line and sheep anti-mouse igg nature controlling line.
The colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen also includes carrying adsorptive pads, cellulose nitrate in described food
Plain film, gold standard pad, the bottom plate of sample pad.
Mutually overlapping connection between described adsorptive pads, nitrocellulose filter, gold standard pad, sample pad.
The concentration of monoclonal antibody is 4-20 μ in a kind of described anti-walnut protein monoclonal antibody-colloid gold label thing
g/mL。
The concentration of another anti-walnut protein monoclonal antibody is 0.2-2.5mg/mL in described detection line.
It by pH is 5.0-7.4 that described antigen line, which is, and concentration is that the 0.01-0.2 mol/L PB containing walnut protein is molten
Liquid is drawn film as coating buffer and obtained, and wherein the concentration of walnut protein is 15000-60000ppm.
The concentration of sheep anti-mouse igg is 0.5-2.5mg/mL in described nature controlling line.
The preparation method of the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen, comprises the following steps in described food：
（1）Using one kind in anti-walnut protein monoclonal antibody as labelled antibody, with collaurum according to（0.04-0.20）：1
Volume proportion is mixed, and a kind of anti-walnut protein monoclonal antibody-colloid gold label thing is obtained after purified, concentration；
（2）By step（1）The colloid gold label thing spraying of preparation is fixed in gold standard pad, and detection is coated with nitrocellulose filter
Line, antigen line, nature controlling line, 37-65 DEG C of dry 2-24h is used after being disposed, is put into moisture-resistant cabinet and stores for future use；
（3）The assembling of test strips：
Adsorptive pads, nitrocellulose filter, gold standard pad, sample pad are sequentially connected assembling or are carried on bottom plate, is produced described
For detecting walnut anaphylactogen colloidal gold immunochromatographimethod paper slip in food.
The present invention Cleaning Principle be：By on a kind of anti-walnut protein labeling of monoclonal antibody to Au colloidal nanoparticles into
For the gold labeling antibody of the present invention, walnut allergen protein can be captured.It is anti-that another is fixed on nitrocellulose filter
Walnut protein monoclonal antibody is as detection line, while fixed suitable Quality Control antibody is as nature controlling line.When sample is added dropwise to sample
When product pad, sample can utilize the capillarity of micropore on film to do horizontal swimming on chromatography strip.When being treated in sample containing walnut
When detecting material, walnut determinand can combine to form compound with gold labeling antibody, and with the capture protein binding in detection line and
It is trapped, the colloidal gold aggregation in compound forms macroscopic red lines（T lines）, therefore TC two lines occur；If
Gold labeling antibody can close in antigen knot without being captured completely, form H lines, therefore tri- lines of THC occur；If determinand is dense
Du Taigao, gold labeling antibody is captured all combined with determinand without unnecessary gold labeling antibody by T lines, therefore C lines one occur
Bar line.When being free of material to be detected in sample, the gold labeling antibody not combined with determinand can be captured by antigen line, and collaurum gathers
Collection forms obvious red lines（H lines）, therefore HC two lines occur.Therefore, can be according to detection line in test strips and Quality Control
Whether whether failed containing determinand and test strips in the colour developing situation judgement sample of line.
The detection method for preparing walnut Allergic skin test test strips used in the present invention belongs to sandwich method, and result judgement is when examination
There is a nature controlling line in paper slip, an antigen line is negative, detection line occurs（No matter either with or without antigen line）All it is positive, with showing
There is walnut protein Allergic skin test technology to compare, high sensitivity, high specificity, the concentration of anaphylactogen can also be made intuitively
Evaluation, can be directly used for food inspection.
It is further elaborated with reference to specific embodiment.
Brief description of the drawings
Fig. 1 is the walnut test strips sensitivity determination result figure described in the embodiment of the present invention 2；
Fig. 2 is the walnut test strips specific assay result figure described in the embodiment of the present invention 3；
Fig. 3 is the walnut test strips repeatability measurement result figure described in the embodiment of the present invention 4.
Embodiment
The preparation of the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in the food of embodiment 1
The preparation method of the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen, comprises the following steps in food：
（1）Solution is prepared：
The preparation of trisodium citrate：0.1g trisodium citrates accurately are weighed, are dissolved with ultra-pure water, and are settled to 10mL, 0.22
μm membrane filtration, it is now with the current；
Chlorauric acid solution is prepared：1.0g trisodium citrates accurately are weighed, are dissolved with ultra-pure water, and are settled to 100mL, 0.22 μ
M membrane filtrations, brown reagent bottle be stored in 4 DEG C it is standby；
The preparation of solution of potassium carbonate：1.38g potassium carbonate accurately is weighed, is dissolved with ultra-pure water, and is settled to 50mL, 0.22 μm of filter
Membrane filtration, 4 DEG C store for future use；
The preparation of 5% bovine serum albumin(BSA) (BSA) solution：0.5g potassium carbonate accurately is weighed, is dissolved with ultra-pure water, and be settled to
10mL, 0.22 μm of membrane filtration, 4 DEG C store for future use；
（2）The preparation of collaurum：
250mL conical flasks are taken, 99mL ultra-pure waters is added, adds 1mL1% chlorauric acid solutions, place it on magnetic stirring apparatus,
Heating stirring is rapidly added 1% citric acid three sodium solution that 1mL is now prepared to seething with excitement, and continues to be heated to claret appearance, color
Continue heating stirring 10min after stable, saved backup after cooling with ultra-pure water constant volume to 100mL, 4 DEG C of brown bottles；
（3）A kind of preparation and purifying of anti-walnut protein monoclonal antibody-colloid gold label thing：
2ml colloidal gold solutions are taken, add 8 μ L 0.2mol/L solution of potassium carbonate, low speed mixes on horizontal shaker；
Using one kind in the monoclonal antibody of a pair of detection walnut proteins as labelled antibody, 16 μ g are taken into colloidal gold solution,
30min is mixed, it is 1% then to add 5% bovine serum albumin(BSA) (BSA) to concentration, mixes 30min, obtains a kind of anti-walnut protein
Monoclonal antibody-colloid gold label thing；
Obtained anti-walnut protein monoclonal antibody-colloid gold label thing is dispensed into 2ml centrifuge tubes, in 4 DEG C, 10000rpm
Under the conditions of centrifuge 30min, be subsequently added into isometric Tris-HCl, at 4 DEG C, centrifuge 30min under the conditions of 10000rpm, in removing
Clearly, add and redissolve liquid, be concentrated into 1/10 times of original volume, 4 DEG C save backup；
（4）Anti- walnut protein monoclonal antibody-colloid gold label thing is fixed in gold standard pad, with 37 DEG C of dryings after being disposed
2h, it is put into moisture-resistant cabinet and stores for future use；
（5）The preparation of nitrocellulose filter：
Nitrocellulose filter is selected from Millipore Corp., after lamination instrument assembles, draws THC lines respectively；It is 1mg/ that wherein T lines, which draw concentration,
ML another anti-walnut protein monoclonal antibody, H lines draw the antigen liquid that concentration is 25000ppm, and the solvent of the antigen liquid is pH
=6.0,0.05 mol/L PB solution, C lines draw 1.0mg/mL sheep anti-mouse igg.Nitrocellulose filter after coating is sent into drying
Case, 37 DEG C of dry 3h, store for future use in moisture-resistant cabinet.
（6）The assembling of test strips：
Adsorptive pads, nitrocellulose filter, gold standard pad, sample pad are pasted onto PVC bottoms in the way of part end to end is overlapping successively
On plate, obtain being used to detect walnut anaphylactogen colloidal gold immunochromatographimethod paper slip in food.
In use, sample is added in sample pad, when containing walnut material to be detected in sample, walnut determinand can be with
Gold labeling antibody combines to form compound, and is trapped with the capture protein binding in detection line, and the collaurum in compound gathers
Collection forms macroscopic red detection lines（T lines）, therefore TC two lines occur；If gold labeling antibody is not caught completely
Obtain, can be closed in antigen knot, form H lines, therefore tri- lines of THC occur；If testing concentration is too high, gold labeling antibody is all
Combined with determinand, therefore captured without unnecessary gold labeling antibody by T lines, therefore one line of C lines occurs.It is to be checked when being free of in sample
When surveying material, the gold labeling antibody not combined with determinand can be captured by antigen line, and colloidal gold aggregation forms obvious red lines
（H lines）, therefore HC two lines occur.Therefore, can be according to detection line in test strips and the colour developing situation judgement sample of nature controlling line
In whether failed containing determinand and test strips.C lines develop the color, and interpretation is that high concentration is positive（+++）；TC lines develop the color, and sentence
Read as the higher concentration positive（++）；THC develops the color, and interpretation is the positive（+）；HC develops the color, and interpretation is feminine gender（-）；C lines do not develop the color,
Illustrate that test strips fail.
The sensitivity determination of the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in the food of embodiment 2
Standard items are diluted to various concentrations with sample diluting liquid：100000ppm、10000ppm、100ppm、10ppm、5ppm、
2ppm, 0ppm, the test paper prepared with embodiment 1 are detected, and negative control is used as by the use of sample diluting liquid（0ppm）, see after 5min
Result is examined, testing result is as shown in Figure 1.As a result show, 100000ppm is that high concentration is positive, the colour developing of C lines；10000ppm be compared with
High concentration is positive, the colour developing of TC lines；2-100ppm is the positive, and THC develops the color；0ppm is negative control, and HC lines develop the color.The test strips
Sensitivity be 2ppm.
The specific assay of the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in the food of embodiment 3
Green fruit, Bertholletia excelsa, peanut, almond, fibert is taken to be diluted to 100ppm after crushing as test sample, grinding respectively,
The test strips prepared with embodiment 1 are detected, and result are observed after 5min, testing result is as shown in Figure 2.As a result show, green
Fruit, Bertholletia excelsa, peanut, almond, fibert show C lines at 100ppm, the test paper and green fruit, Bertholletia excelsa, peanut, apricot
Benevolence, fibert no cross reaction, show that this test strips has good specificity.
Walnut in the food of embodiment 4（Walnut）The repeatability measure of the colloidal gold immuno-chromatography test paper strip of anaphylactogen
Respectively the test strips of 2 batches are completed in the different time according to the method for embodiment 1, respectively Lot1, Lot2,2
The test strips interval time of individual batch is 1 day, contrasts differences between batches.Standard items are diluted to various concentrations with sample diluting liquid：
100ppm, 10ppm, 5ppm, 2ppm, 0ppm, detected respectively with the test strips of 2 batches, result is observed after 5min, detected
As a result it is as shown in Figure 3.As a result show, the test strips of 2 batches contrast no notable differences between batches.
Claims (8)
1. the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in food, it is characterised in that including assemble successively adsorptive pads,
Nitrocellulose filter, gold standard pad, sample pad, a kind of anti-walnut protein monoclonal antibody-collaurum is fixed in described gold standard pad
Label, is coated with described nitrocellulose filter and the anti-walnut protein monoclonal antibody of colloid gold label pairing successively
Another anti-walnut protein monoclonal antibody detection line, antigen line and sheep anti-mouse igg nature controlling line.
2. the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in food as claimed in claim 1, it is characterised in that also wrap
Include carrying adsorptive pads, nitrocellulose filter, gold standard pad, the bottom plate of sample pad.
3. the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in food as claimed in claim 1 or 2, it is characterised in that
Mutually overlapping connection between described adsorptive pads, nitrocellulose filter, gold standard pad, sample pad.
4. the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in food as claimed in claim 1 or 2,
Characterized in that, in a kind of described anti-walnut protein monoclonal antibody-colloid gold label thing monoclonal antibody concentration
For 4-20 μ g/mL.
5. the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in the food as described in claim 1 or 2, it is characterised in that
The concentration of another anti-walnut protein monoclonal antibody is 0.2-2.5mg/mL in described detection line.
6. the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in food as claimed in claim 1 or 2, it is characterised in that
It by pH is 5.0-7.4 that described antigen line, which is, and the PB solution for containing walnut protein that concentration is 0.01-0.2 mol/L is as bag
Film is drawn by liquid to obtain, wherein the concentration of walnut protein is 15000-60000ppm.
7. the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in food as claimed in claim 1 or 2, it is characterised in that
The concentration of sheep anti-mouse igg is 0.5-2.5mg/mL in described nature controlling line.
8. the preparation side of the colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen in the food as described in claim 1-7 is any
Method, it is characterised in that comprise the following steps：
（1）Using one kind in anti-walnut protein monoclonal antibody as labelled antibody, with collaurum according to（0.04-0.20）：1
Volume proportion is mixed, and a kind of anti-walnut protein monoclonal antibody-colloid gold label thing is obtained after purified, concentration；
（2）By step（1）The colloid gold label thing spraying of preparation is fixed in gold standard pad, and detection is coated with nitrocellulose filter
Line, antigen line, nature controlling line, 37-65 DEG C of dry 2-24h is used after being disposed, is put into moisture-resistant cabinet and stores for future use；
（3）The assembling of test strips：
Adsorptive pads, nitrocellulose filter, gold standard pad, sample pad are assembled or are carried on bottom plate successively, produces described be used for
Detect walnut anaphylactogen colloidal gold immunochromatographimethod paper slip in food.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711064925.2A CN107860916A (en) | 2017-11-02 | 2017-11-02 | Colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen and preparation method thereof in food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711064925.2A CN107860916A (en) | 2017-11-02 | 2017-11-02 | Colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen and preparation method thereof in food |
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| CN107860916A true CN107860916A (en) | 2018-03-30 |
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| CN201711064925.2A Pending CN107860916A (en) | 2017-11-02 | 2017-11-02 | Colloidal gold immuno-chromatography test paper strip of walnut anaphylactogen and preparation method thereof in food |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110161239A (en) * | 2019-05-08 | 2019-08-23 | 扬州大学 | A kind of double-antibody sandwich colloid gold test paper based on EFTu, staphylococcus detection method and application |
| CN110846410A (en) * | 2019-12-20 | 2020-02-28 | 西北民族大学 | Method for detecting walnut allergens in food by real-time fluorescent quantitative PCR |
| CN114544940A (en) * | 2022-02-21 | 2022-05-27 | 中国海洋大学 | Colloidal gold test strip for rapidly detecting hyaluronic acid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100317033A1 (en) * | 2009-06-10 | 2010-12-16 | Matthew Philip Abdel | Methods and materials for detecting food containing allergens |
| CN202814988U (en) * | 2012-08-23 | 2013-03-20 | 南京基蛋生物科技有限公司 | Full scale high-sensitivity C-reaction protein colloidal gold test paper strip |
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2017
- 2017-11-02 CN CN201711064925.2A patent/CN107860916A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100317033A1 (en) * | 2009-06-10 | 2010-12-16 | Matthew Philip Abdel | Methods and materials for detecting food containing allergens |
| CN202814988U (en) * | 2012-08-23 | 2013-03-20 | 南京基蛋生物科技有限公司 | Full scale high-sensitivity C-reaction protein colloidal gold test paper strip |
Non-Patent Citations (1)
| Title |
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| 王硕: "食物过敏原胶体金免疫层析检测试纸条的研制", 《中国优秀硕士论文数据库 工程科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110161239A (en) * | 2019-05-08 | 2019-08-23 | 扬州大学 | A kind of double-antibody sandwich colloid gold test paper based on EFTu, staphylococcus detection method and application |
| CN110846410A (en) * | 2019-12-20 | 2020-02-28 | 西北民族大学 | Method for detecting walnut allergens in food by real-time fluorescent quantitative PCR |
| CN110846410B (en) * | 2019-12-20 | 2023-08-08 | 西北民族大学 | Method for detecting walnut allergen in food by using real-time fluorescence quantitative PCR |
| CN114544940A (en) * | 2022-02-21 | 2022-05-27 | 中国海洋大学 | Colloidal gold test strip for rapidly detecting hyaluronic acid |
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Application publication date: 20180330 |