CN107561271A - Colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen and preparation method thereof in food - Google Patents
Colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen and preparation method thereof in food Download PDFInfo
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- CN107561271A CN107561271A CN201710716638.9A CN201710716638A CN107561271A CN 107561271 A CN107561271 A CN 107561271A CN 201710716638 A CN201710716638 A CN 201710716638A CN 107561271 A CN107561271 A CN 107561271A
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- fibert
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Abstract
The invention discloses colloidal gold immuno-chromatography test paper strip of fibert 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-fibert protein monoclonal antibody colloid gold label thing is fixed in gold standard pad, the monoclonal antibody of another capture fibert albumen is coated with the detection line of nitrocellulose filter, antigen line is coated with fibert albumen, 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 fibert protein allergies original detection technique, 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 fibert anaphylactogen in food
Golden immuno-chromatographic test paper strip and preparation method thereof.
Background technology
Fibert, also known as Zhen Li, mountain Chinese chestnut, sharp chestnut etc., it is rich in protein, grease（It is mostly unrighted acid）, recklessly
Radish element, vitamin, mineral matter etc., there is the laudatory title of " king of nut ".Fibert was both direct-edible, can also be used as food ingredient to use
In the various cakes of making, candy.It is also one of most common food allergen simultaneously though fibert is nutritious.International food
The code committee exists《Prepackaged food universal tag standard》Middle clear stipulaties, nut and its product including fibert are being eaten
It should be illustrated all the time in product label.Fibert is classified as by the countries and regions such as the U.S., European Union, Japan, New Zealand, Australia
Force the food allergen of mark.Fibert allergen detection methods have ELISA, immunosensor detection technique, real-time fluorescence at present
Quantitative PCR technique, SPR, mass-spectrometric technique etc., these detection methods have very high to instrument and equipment, surface environment, operating personnel etc.
It is required that and detection time it is longer, costly, thus it is very necessary to establish a kind of ELISA test strip of new fast and stable.
Colloidal gold immunochromatographimethod technology (colloidal gold immunochromatography assay, GICA)
A kind of solid phase for organically combining a variety of methods such as colloidal gold-labeled method, immunoassay technology and Chromatographic techniques
Labeled immunoassay technology.Colloidal gold-labeled method is introduced immunohistochemistry, glue by Faulk and Taylor first within 1971
Body gold labelling technique is developed rapidly.At present, the colloidal gold test gone out by this technological development oneself be widely used in
The fields such as clinical diagnosis, drug test, environmental pollution.
The content of the invention
It is an object of the invention to develop the fibert 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 the monoclonal pairing antibody based on a pair of mutual coordinations that can detect fibert albumen, 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 fibert anaphylactogen in food, including assemble successively adsorptive pads, nitrocellulose filter,
Gold standard pad, sample pad, a kind of anti-fibert protein monoclonal antibody-colloid gold label thing is fixed in described gold standard pad, it is described
Another anti-fibert with the coordination of the anti-fibert 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 fibert 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 4-16 μ in a kind of described anti-fibert protein monoclonal antibody-colloid gold label thing
g/mL。
The concentration of another anti-fibert protein monoclonal antibody is 0.5-2.5mg/mL in described detection line.
It by pH is 7.0-8.5 that described antigen line, which is, and concentration is the 0.01 mol/L PBS solution containing fibert albumen
Film is drawn as coating buffer to obtain, wherein the concentration of fibert albumen is 500-10000ppm.
The concentration of sheep anti-mouse igg is 0.5-3mg/mL in described nature controlling line.
The preparation method of the colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen, comprises the following steps in described food：
（1）Using one kind in anti-fibert protein monoclonal antibody as labelled antibody, with collaurum according to（0.04-0.16）：1
Volume ratio is mixed, and a kind of anti-fibert 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, 60-65 DEG C of dry 1-2h is used after being disposed, is put into moisture-resistant cabinet and stores for future use；
（3）Sample pad processing：
From contain 0.2%Tween20, concentration be 0.01mol/L-0.05mol/L BBS solution as sample pad dilution pair
Sample pad carries out immersion treatment, processing terminate after with standby after 60-65 DEG C of baking oven forced air drying 1-2h；
（4）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 fibert anaphylactogen colloidal gold immunochromatographimethod paper slip in food.
The present invention Cleaning Principle be：By a kind of anti-fibert protein monoclonal antibody be tagged on Au colloidal nanoparticles into
For the gold labeling antibody of the present invention, fibert allergen protein can be captured.It is anti-that another is fixed on nitrocellulose filter
Fibert 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 fibert
When detecting material, fibert 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 fibert 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 fibert protein allergies original detection technique 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 fibert test strips sensitivity determination result figure described in the embodiment of the present invention 2；
Fig. 2 is the fibert test strips specific assay result figure described in the embodiment of the present invention 3；
Fig. 3 is the fibert test strips repeatability measurement result figure described in the embodiment of the present invention 4；
Fig. 4 is the testing result figure of the embodiment of the present invention 5.
Reference：T, detection line；H, antigen line；C, nature controlling line；SAM1, Bertholletia excelsa sample；SAM2, peanut sample；
SAM3, fibert sample；Lot1, first batch test strips；Lot2, second lot test strips；SAM4, negative control；SAM5、4ppm
Fibert chocolate samples；SAM6,40ppm fibert chocolate samples.
Embodiment
The preparation of the colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen in embodiment 1, food
The preparation method of the colloidal gold immuno-chromatography test paper strip of fibert 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-fibert protein monoclonal antibody-colloid gold label thing：
2ml colloidal gold solutions are taken, add 4 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 fibert albumen 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-fibert albumen
Monoclonal antibody-colloid gold label thing；
Obtained anti-fibert 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- fibert protein monoclonal antibody-colloid gold label thing is fixed in gold standard pad, with 65 DEG C of dryings after being disposed
1h, it is put into moisture-resistant cabinet and stores for future use；
（5）Sample pad processing：
The 0.01mol/L BBS solution for containing 0.2%Tween2 is chosen to carry out at immersion sample pad as sample pad dilution
Reason, processing terminate after with standby after 65 DEG C of baking oven forced air drying 1h；
（6）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-fibert protein monoclonal antibody, H lines draw the antigen liquid that concentration is 2500ppm, the solvent of the antigen liquid for pH=
7.0th, 0.01 mol/L PBS solutions, C lines draw 1.5mg/mL sheep anti-mouse igg.Nitrocellulose filter after coating is sent into drying
Case, 65 DEG C of dry 1h, store for future use in moisture-resistant cabinet.
（7）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 fibert anaphylactogen colloidal gold immunochromatographimethod paper slip in food.
In use, sample is added in sample pad, when containing fibert material to be detected in sample, fibert 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 fibert anaphylactogen in embodiment 2, food
Standard items are diluted to various concentrations with sample diluting liquid：100000ppm、1000ppm、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；100-1000ppm is
Higher concentration is positive, the colour developing of TC lines；2-10ppm 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 fibert anaphylactogen in embodiment 3, food
Bertholletia excelsa, peanut, almond is taken to be diluted to 100ppm as test sample after grinding is broken, prepared with embodiment 1 respectively
Test strips detected, result is observed after 5min, testing result is as shown in Figure 2.As a result show, Bertholletia excelsa, peanut, almond
C lines are shown at 100ppm, the test paper and Bertholletia excelsa, peanut, almond no cross reaction, it is good to show that this test strips has
Good specificity.
The repeatability measure of the colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen in embodiment 4, food
The test strips of 2 batches are completed according to the method for embodiment 1 in the different time respectively, between the test strips of 2 batches
It is 4 days every the time, contrasts differences between batches.Standard items are diluted to various concentrations with sample diluting liquid：1000ppm、100ppm、
10ppm, 5ppm, 2ppm, 0ppm, detected respectively with the test strips of 2 batches, result is observed after 5min, testing result is such as
Shown in Fig. 3.As a result show, the test strips of 2 batches contrast no notable differences between batches.
The feasibility of fibert allergen measurement in embodiment 5, food
It is pure skilful to provide the chocolate food containing 40ppm fiberts, the chocolate food of 4ppm fiberts, negative control by FAPAS
Gram force food（Without fibert composition）, detected respectively using the two batches test strips of the preparation of embodiment 4, testing result such as Fig. 4
It is shown.As a result show, be capable of detecting when chocolate food, the chocolate food of 4ppm fiberts of the 40ppm fiberts that FAPAS is provided
Product, negative control are shown correctly.Chocolate-like food belongs to the sample of more difficult detection, can smoothly and repeatedly detect,
Illustrate that test paper has good feasibility.
Claims (8)
1. the colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen in food, it is characterised in that including assemble successively adsorptive pads,
Nitrocellulose filter, gold standard pad, sample pad, a kind of anti-fibert protein monoclonal antibody-collaurum is fixed in described gold standard pad
Label, is coated with described nitrocellulose filter and the anti-fibert protein monoclonal antibody of colloid gold label coordination successively
Another anti-fibert 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 fibert 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 fibert 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 fibert anaphylactogen in food as claimed in claim 1 or 2,
Characterized in that, in a kind of described anti-fibert protein monoclonal antibody-colloid gold label thing monoclonal antibody concentration
For 4-16 μ g/mL.
5. the colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen in the food as described in claim 1 or 2, it is characterised in that
The concentration of another anti-fibert protein monoclonal antibody is 0.5-2.5mg/mL in described detection line.
6. the colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen in food as claimed in claim 1 or 2, it is characterised in that
It by pH is 7.0-8.5 that described antigen line, which is, and the PBS solution for containing fibert albumen that concentration is 0.01 mol/L is as coating
Liquid is drawn film and obtained, and wherein the concentration of fibert albumen is 500-10000ppm.
7. the colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen in food as claimed in claim 1 or 2, it is characterised in that
The concentration of sheep anti-mouse igg is 0.5-3mg/mL in described nature controlling line.
8. the preparation side of the colloidal gold immuno-chromatography test paper strip of fibert 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-fibert protein monoclonal antibody as labelled antibody, with collaurum according to（0.04-0.16）：1
Volume ratio is mixed, and a kind of anti-fibert 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, 60-65 DEG C of dry 1-2h is used after being disposed, is put into moisture-resistant cabinet and stores for future use；
（3）Sample pad processing：
From contain 0.2%Tween20, concentration be 0.01mol/L-0.05mol/L BBS solution as sample pad dilution pair
Sample pad carries out immersion treatment, processing terminate after with standby after 60-65 DEG C of baking oven forced air drying 1-2h；
（4）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 fibert anaphylactogen colloidal gold immunochromatographimethod paper slip in food.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710716638.9A CN107561271A (en) | 2017-08-21 | 2017-08-21 | Colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen and preparation method thereof in food |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710716638.9A CN107561271A (en) | 2017-08-21 | 2017-08-21 | Colloidal gold immuno-chromatography test paper strip of fibert anaphylactogen and preparation method thereof in food |
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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 |
-
2017
- 2017-08-21 CN CN201710716638.9A patent/CN107561271A/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 |
|---|
| 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》 * |
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Application publication date: 20180109 |