CN107860918A - Former colloidal gold immuno-chromatography test paper strip of gluten and preparation method thereof in food - Google Patents
Former colloidal gold immuno-chromatography test paper strip of gluten and preparation method thereof in food Download PDFInfo
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- CN107860918A CN107860918A CN201711064939.4A CN201711064939A CN107860918A CN 107860918 A CN107860918 A CN 107860918A CN 201711064939 A CN201711064939 A CN 201711064939A CN 107860918 A CN107860918 A CN 107860918A
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- 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
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
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Abstract
The invention discloses former colloidal gold immuno-chromatography test paper strip of gluten in food and preparation method thereof, 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-gluten protein monoclonal antibody colloid gold label thing is fixed in gold standard pad, the monoclonal antibody of another capture gluten protein is coated with the detection line of nitrocellulose filter, antigen line is coated with gluten 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 gluten 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 to detect the colloid that gluten is former in food
Golden immuno-chromatographic test paper strip and preparation method thereof.
Background technology
Wheat flour and gluten protein are because its heat resistance and such as quality, shrinkage and fragrant etc. characteristic are by extremely general
It is used as food material everywhere.Gluten protein is the mixture of prolamin and glutenin, is present in wheat, rye and big
Mai Zhong.
Chylous diarrhea is the normal hair property disease caused by the intolerance to gluten protein, if avoiding edible gluten protein,
The disease disappears certainly.The definition of " no seitan " food is to contain the gluten protein no more than 200 ppm in food in CODEX.
New food standard in preparation will then define natural " no seitan " food as the natural of the gluten protein containing no more than 20 ppm
Food, and " no seitan " processed food is processed food for the solidification of the gluten protein containing no more than 200 ppm.Many countries
Also more strict limit standard is had been carried out.
To avoid adverse reaction caused by food irritability, more healthy life is enjoyed, design Rapid detection test strip is
As public demand, apply in raw materials for production, production quality management and control, food production particular step quick sampling are examined, quickly
The detection process of test strip only needs 15 minutes, simple to operate, quickly, can confirm testing result immediately, can visually sentence
Determine result, realize the quick inspection to allergin of low cost, high efficiency, high selectivity, high sensitive.
The content of the invention
It is an object of the invention to develop the gluten original 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 gluten 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 former colloidal gold immuno-chromatography test paper strip of gluten in food, including assemble successively adsorptive pads, nitrocellulose filter,
Gold standard pad, sample pad, a kind of anti-gluten protein monoclonal antibody-colloid gold label thing is fixed in described gold standard pad, it is described
Another anti-seitan with the pairing of the anti-gluten 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 former colloidal gold immuno-chromatography test paper strip of gluten 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 2-30 μ in a kind of described anti-gluten protein monoclonal antibody-colloid gold label thing
g/mL。
The concentration of another anti-gluten protein monoclonal antibody is 0.5-3.0mg/mL in described detection line.
It by pH is 5.0-7.0 that described antigen line, which is, and concentration is the 0.01-0.2 mol/L CPBS containing gluten protein
Solution is drawn film as coating buffer and obtained, and wherein the concentration of gluten protein is 500-5000ppm.
The concentration of sheep anti-mouse igg is 0.5-3.0mg/mL in described nature controlling line.
The preparation method of the former colloidal gold immuno-chromatography test paper strip of gluten, comprises the following steps in described food：
（1）Using one kind in anti-gluten protein monoclonal antibody as labelled antibody, with collaurum according to（0.02-0.30）：1
Volume proportion is mixed, and a kind of anti-gluten 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 1-24h 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 gluten original colloidal gold immunochromatographimethod paper slip in food.
The present invention Cleaning Principle be：By a kind of anti-gluten protein monoclonal antibody be tagged on Au colloidal nanoparticles into
For the gold labeling antibody of the present invention, gluten original albumen can be captured.It is anti-that another is fixed on nitrocellulose filter
Gluten 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 seitan
When detecting material, seitan 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 gluten original test strip 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 gluten 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 seitan test strips sensitivity determination result figure prepared by embodiment 1；
Fig. 2 is seitan test strips specific assay result figure prepared by embodiment 1；
Fig. 3 is seitan test strips repeatability measurement result figure prepared by embodiment 1.
Embodiment
The preparation of the former colloidal gold immuno-chromatography test paper strip of gluten in the food of embodiment 1
The preparation method of the former colloidal gold immuno-chromatography test paper strip of gluten, 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-gluten 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 gluten proteins as labelled antibody, 12 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-gluten protein
Monoclonal antibody-colloid gold label thing；
Obtained anti-gluten protein monoclonal antibody-colloid gold label thing is dispensed into 2ml centrifuge tubes, in 4 DEG C, 9000rpm
Under the conditions of centrifuge 30min, be subsequently added into isometric Tris-HCl, at 4 DEG C, centrifuge 30min under the conditions of 9000rpm, in removing
Clearly, add and redissolve liquid, be concentrated into 1/10 times of original volume, 4 DEG C save backup；
（4）Anti- gluten 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；Wherein T lines draw concentration be
2.0mg/mL another anti-gluten protein monoclonal antibody, H lines draw the antigen liquid that concentration is 2000ppm, the antigen liquid it is molten
Agent is pH=6.2,0.1mol/L CPBS solution, and C lines draw 2.0mg/mL sheep anti-mouse igg.Nitrocellulose filter after coating is sent
Enter drying box, 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 gluten original colloidal gold immunochromatographimethod paper slip in food.
In use, sample is added in sample pad, when containing seitan material to be detected in sample, seitan 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 former colloidal gold immuno-chromatography test paper strip of gluten in the food of embodiment 2
Standard items are diluted to various concentrations with sample diluting liquid：1000000ppm、100000ppm、10000ppm、1000ppm、
100ppm, 10ppm, 5ppm, 2ppm, 1ppm, 0ppm, with embodiment 1 prepare test paper detected, by the use of sample diluting liquid as
Negative control（0ppm）, result is observed after 5min, and testing result is as shown in Figure 1.As a result show, 1000000ppm is high concentration sun
Property, the colour developing of C lines；100000-1000ppm is that higher concentration is positive, the colour developing of TC lines；1-100ppm is the positive, and THC develops the color；0ppm
It is negative control, HC lines develop the color.The sensitivity of the test strips is 1ppm.
The specific assay of the former colloidal gold immuno-chromatography test paper strip of gluten in the food of embodiment 3
Green fruit is taken respectively（Pecan）, Bertholletia excelsa (Brazil nut), peanut (Peanut), walnut (Walnut), almond
(Almond), fibert (Hazelnut), egg (Egg) are diluted to 100ppm after being used as test sample, grinding broken, use embodiment
1 test strips prepared are detected, and result are observed after 5min, testing result is as shown in Figure 2.As a result show, green fruit, Brazil are hard
Fruit, peanut, walnut, almond, fibert, egg show C lines at 100ppm, the test paper and green fruit, Bertholletia excelsa, peanut,
Walnut, almond, fibert, egg no cross reaction, show that this test strips has good specificity.
The repeatability measure of the former colloidal gold immuno-chromatography test paper strip of gluten in the food of embodiment 4
Test strips Lot1, Lot2 of 2 batches are completed according to the method for embodiment 1 in the different time respectively, 2 batches
Test strips interval time is 3 days, contrasts differences between batches.Standard items are diluted to various concentrations with sample diluting liquid：
100000ppm, 10000ppm, 1000ppm, 100ppm, 10ppm, 5ppm, 2ppm, 1ppm, 0ppm, respectively with the examination of 2 batches
Paper slip is detected, and result is observed after 5min, and testing result is as shown in Figure 3.As a result show, the test strips contrast of 2 batches does not have
There are notable differences between batches.
Claims (8)
1. the former colloidal gold immuno-chromatography test paper strip of gluten in food, it is characterised in that including assemble successively adsorptive pads,
Nitrocellulose filter, gold standard pad, sample pad, a kind of anti-gluten protein monoclonal antibody-collaurum is fixed in described gold standard pad
Label, is coated with described nitrocellulose filter and the anti-gluten protein monoclonal antibody of colloid gold label pairing successively
Another anti-gluten protein monoclonal antibody detection line, antigen line and sheep anti-mouse igg nature controlling line.
2. the former colloidal gold immuno-chromatography test paper strip of gluten 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 former colloidal gold immuno-chromatography test paper strip of gluten 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 former colloidal gold immuno-chromatography test paper strip of gluten in food as claimed in claim 1 or 2,
Characterized in that, in a kind of described anti-gluten protein monoclonal antibody-colloid gold label thing monoclonal antibody concentration
For 2-30 μ g/mL.
5. the former colloidal gold immuno-chromatography test paper strip of gluten in the food as described in claim 1 or 2, it is characterised in that
The concentration of another anti-gluten protein monoclonal antibody is 0.5-3.0mg/mL in described detection line.
6. the former colloidal gold immuno-chromatography test paper strip of gluten in food as claimed in claim 1 or 2, it is characterised in that
It by pH is 5.0-7.0 that described antigen line, which is, and concentration is the 0.01-0.2 mol/L CPBS solution conducts for containing gluten protein
Coating buffer is drawn film and obtained, and wherein the concentration of gluten protein is 500-5000ppm.
7. the former colloidal gold immuno-chromatography test paper strip of gluten in food as claimed in claim 1 or 2, it is characterised in that
The concentration of sheep anti-mouse igg is 0.5-3.0mg/mL in described nature controlling line.
8. the preparation side of the former colloidal gold immuno-chromatography test paper strip of gluten 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-gluten protein monoclonal antibody as labelled antibody, with collaurum according to（0.02-0.30）：1
Volume ratio is mixed, and a kind of anti-gluten 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 1-24h 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 gluten original colloidal gold immunochromatographimethod paper slip in food.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711064939.4A CN107860918A (en) | 2017-11-02 | 2017-11-02 | Former colloidal gold immuno-chromatography test paper strip of gluten and preparation method thereof in food |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711064939.4A CN107860918A (en) | 2017-11-02 | 2017-11-02 | Former colloidal gold immuno-chromatography test paper strip of gluten and preparation method thereof in food |
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| CN107860918A true CN107860918A (en) | 2018-03-30 |
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|---|---|---|---|
| CN201711064939.4A Pending CN107860918A (en) | 2017-11-02 | 2017-11-02 | Former colloidal gold immuno-chromatography test paper strip of gluten and preparation method thereof in food |
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Cited By (1)
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698832A (en) * | 2009-07-30 | 2010-04-28 | 山东出入境检验检疫局检验检疫技术中心 | Production of gluten protein monoclonal antibody and application of cell strain thereof |
| 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 |
-
2017
- 2017-11-02 CN CN201711064939.4A patent/CN107860918A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698832A (en) * | 2009-07-30 | 2010-04-28 | 山东出入境检验检疫局检验检疫技术中心 | Production of gluten protein monoclonal antibody and application of cell strain thereof |
| 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 |
|---|
| 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 (1)
| 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 |
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