CN112834751A - Household high-sensitivity kit for detecting sesame allergen, detection method and application thereof - Google Patents
Household high-sensitivity kit for detecting sesame allergen, detection method and application thereof Download PDFInfo
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Images
Classifications
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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
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
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- 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
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- 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
Abstract
The invention discloses a household high-sensitivity kit for detecting sesame allergen, a detection method and application thereof, wherein the kit comprises: the test paper strip: the detection line on the NC membrane is coated with goat anti-mouse IgG with the concentration of 0.1-0.5 mg/ml, and the quality control line is coated with goat anti-rabbit IgG with the concentration of 2-8 mg/ml; the test strip is composed of absorbent paper, an NC membrane and a sample pad from top to bottom and is fixed on a PVC rubber plate; wherein the overlapped part of the absorbent paper, the sample pad and the NC membrane is 2 mm; gold-labeled micropores: adding a mixed solution of a compound solution of a rabbit anti-sesame polyclonal antibody and colloidal gold and a freeze-drying diluent into micropores of a low-adsorption enzyme label plate, and freeze-drying to obtain the low-adsorption enzyme label plate; and a mouse anti-sesame monoclonal antibody and an antibody protection diluent. The detection kit disclosed by the invention is convenient to use, high in detection sensitivity and strong in specificity, and greatly improves the convenience of household detection of sesame allergens.
Description
Technical Field
The invention relates to the technical field of allergen detection methods, in particular to a household high-sensitivity kit for detecting sesame allergen, a detection method and application thereof.
Background
Food allergy is a public health problem of global concern, and it is statistically estimated that about 1% to 2% of adults and 5% to 8% of children worldwide have allergic reactions to certain foods. In the United states alone, 150-200 people die annually of allergic reactions, and up to two hundred million people with allergic diseases in China, about 90% of which are caused by food allergy. Sesame, as a common vegetable, belongs to one of eight major allergens, and can cause severe anaphylactic reaction and seriously threaten the life health of sesame allergic people. At present, no thorough and effective method for radically treating sesame allergic diseases exists at home and abroad, and the only effective means for preventing sesame allergy is to avoid eating sesame-containing food.
At present, sesame allergen detection methods mainly comprise electrophoresis, chromatography, enzyme-linked immunosorbent assay (ELISA), a biological immunosensor, proteomics (LC-MS/MS) based on mass spectrum, Polymerase Chain Reaction (PCR) technology and the like. Among them, electrophoresis, chromatography and enzyme-linked immunoassay are traditional sesame detection methods based on protein, and often only can analyze single protein, and although the development is mature, the requirement cannot be met; the proteomics based on the mass spectrum can simultaneously detect a plurality of food allergens with high flux and high sensitivity. The Polymerase Chain Reaction (PCR) technology is a detection technology developed on the basis of DNA/RNA, and a cycle period is formed by three steps of high-temperature denaturation, low-temperature annealing, medium-temperature extension and the like, so that the purpose of specific amplification of a target gene is achieved. The PCR technology is divided into two types, namely common PCR and real-time fluorescent quantitative PCR. The common PCR technology is mainly used for qualitative detection, and the detection result needs electrophoresis verification and is easy to generate false positive result by cross contamination. The real-time fluorescence PCR realizes the quantitative analysis of the initial template by detecting the fluorescence signal of each cycle product in the amplification reaction in real time, and although compared with the traditional PCR, the real-time fluorescence PCR has the advantages of strong specificity, high accuracy, less PCR pollution, high automation degree and the like, the operation is more complex, the cost is high, the consumed time is long, the requirement on the skill of an experimenter is high, and the like.
The existing sesame allergen detection methods have high requirements on the technical level of operators, complicated and precise instruments and equipment are needed for matching, some methods are long in time consumption, some methods are relatively high in cost, and various strict requirements make the methods impossible to move to families.
Therefore, the existing sesame allergen detection method needs to be further improved.
Disclosure of Invention
Aiming at the problems of high operation difficulty, long time consumption, high technical requirements on operators and difficulty in entering families of the existing sesame allergen detection method, the invention provides a household high-sensitivity kit for detecting sesame allergen, a detection method and application thereof.
In order to solve the above problems, the present invention provides the following technical solutions:
in a first aspect, the present invention provides a high-sensitivity kit for detecting sesame allergen in a home-use type, comprising:
the test paper strip: the detection line on the NC membrane is coated with goat anti-mouse IgG with the concentration of 0.1-0.5 mg/ml, and the quality control line is coated with goat anti-rabbit IgG with the concentration of 2-8 mg/ml; the test strip is composed of absorbent paper, an NC membrane and a sample pad from top to bottom and is fixed on a PVC rubber plate; wherein the overlapping part of the absorbent paper and the sample pad with the NC membrane is 2 mm.
Gold-labeled micropores: adding a mixed solution of a compound solution of a rabbit anti-sesame polyclonal antibody and colloidal gold and a freeze-drying diluent into micropores of a low-adsorption enzyme label plate, and freeze-drying to obtain the low-adsorption enzyme label plate;
and a mouse anti-sesame monoclonal antibody and an antibody protection diluent.
Preferably, the kit further comprises: the sesame antigen extraction method further comprises an extraction liquid for extracting sesame antigen in a sample, wherein the extraction liquid mainly comprises the following components in concentration: Tris-HCl with a molar concentration of 0.05-0.1M, Triton X-100 with a mass volume concentration of 0.5-1.5%, NaCl with a mass volume concentration of 0.5-3%, and a volume5 to 30 percent of ethanol and 0.01 to 0.1 percent of NaN3The solvent was water, pH 9.0.
In a second aspect, the present invention also provides a method for home-use detection of sesame allergen using the kit according to claim 1, comprising the steps of:
(1) extracting an antigen in a sample to be detected to obtain an extracting solution;
(2) adding 3-4 drops of extracting solution and the sesame mouse-derived monoclonal antibody into the gold-labeled micropores, blowing and beating for multiple times to completely dissolve pink powder in the micropores, after incubation at room temperature, downwards inserting the MAX end of the test strip into the micropores, and judging whether the sample contains sesame allergens or not according to the color development result of the reagent strip after 5 min;
the judgment basis is as follows: when only the quality control line is developed, judging that the detection result is negative; when the detection line and the quality control line are simultaneously developed, judging that the detection result is positive; when the quality control line is not colored, whether the detection result is colored or not is judged to be invalid.
In a third aspect, the invention also provides a preparation method of the household high-sensitivity kit for detecting sesame allergen, wherein the preparation method comprises the following steps:
(1) preparation of gold-labeled antibody: adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding sesame rabbit polyclonal antibody while stirring, reacting at room temperature for 2 hours, then dropwise adding 10% bovine serum albumin, reacting at room temperature for 1 hour, centrifuging, collecting precipitate, and resuspending with 10ml of complex solution to obtain a sesame gold-labeled antibody solution;
the redissolution contains the following components in concentration: PB with the molar concentration of 0.01M and the pH value of 7.4, bovine serum albumin with the mass volume concentration of 1-5% and cane sugar with the mass volume concentration of 1-5%.
(2) Preparing gold-labeled micropores: mixing the sesame gold-labeled antibody solution with the freeze-dried diluent in a volume ratio of 1 (10-15), subpackaging the mixed solution into a low-adsorption enzyme label plate, freezing in a refrigerator overnight, performing vacuum freeze-drying for at least 20h, and then fastening an enzyme label plate cap to obtain gold-labeled micropores;
(3) preparation of monoclonal antibody solution: diluting the monoclonal antibody by 8-20 ten thousand times by using an antibody protective solution for later use;
(4) preparing a test strip: coating goat-anti-mouse on the NC membrane detection line with the concentration of 0.1-0.5 mg/ml, coating goat-anti-mouse on the quality control line with the concentration of 2-8 mg/ml, and drying for 16h at 37 ℃; fixing the absorbent paper, the NC membrane, the sample pad and the MAX line on a PVC rubber plate from top to bottom to prepare a test strip;
(5) and (3) sample testing: extracting an antigen in a sample to be detected to obtain an extracting solution; adding 3-4 drops of the extracting solution and the sesame mouse-derived monoclonal antibody into the gold-labeled micropores, blowing and beating for multiple times to completely dissolve pink powder in the micropores, after incubation at room temperature, downwards inserting the MAX end of the test strip into the micropores, and judging whether the sample contains sesame allergens or not according to the color development result of the reagent strip after 5 min.
Preferably, in the method, the method for extracting the sesame antigen in the sample to be detected comprises:
adding 2-4 ml of extraction liquid and one magnetic bead into a 5ml vial; directly adding a liquid sample or a solid sample into the small bottle, shaking the small bottle up and down for 30s, standing for at least 1min, and absorbing the supernatant to obtain an extracting solution;
wherein the extract mainly comprises the following components in concentration: Tris-HCl with a molar concentration of 0.05-0.1M, Triton X-100 with a mass volume concentration of 0.5-1.5%, NaCl with a mass volume concentration of 0.5-3%, ethanol with a volume concentration of 5-30%, and NaN with a mass volume concentration of 0.01-0.1%3The solvent was water, pH 9.0.
Preferably, in the method, the antibody protection solution contains the following components in concentration: 0.01M pH7.4PB, 5% volume concentration of newborn bovine serum and 0.85% mass volume concentration of sodium chloride.
Preferably, in the method, the colloidal gold is prepared by a method of reducing trisodium citrate.
In a fourth aspect, the invention also provides the application of the household high-sensitivity kit for detecting sesame allergens in food, air, water sources and the surfaces of articles.
The invention has the following beneficial effects:
1. the invention provides a household detection method of sesame allergen components, which improves a preparation method of gold-labeled micropores on the basis of a colloidal gold immunological method.
The existing traditional method is to coat a monoclonal antibody on a detection line part of an NC membrane, and when a compound of a rabbit multi-antibody gold-labeled compound and an antigen is chromatographed on the NC membrane to the detection line, the compound is combined with the monoclonal antibody on the detection line for color development. The detection method of the invention is that the mouse anti-sesame monoclonal antibody is prepared into solution, and the solution and the liquid to be detected (containing antigen) are added into the gold-labeled micropores in sequence, and react for 5min at room temperature, so that the mouse anti-sesame monoclonal antibody, the antigen and the rabbit polyclonal antibody are fully combined into a compound, and then are combined with the goat anti-mouse IgG on the detection line for color development. Based on the principle, the sensitivity of the method is improved by at least 100 times compared with the traditional method.
In addition, the method is simple and convenient to operate, and ordinary people can perform screening detection without obstacles, so that the method is a life health protection and protection navigation method for sesame allergen people.
2. The kit corresponding to the method provided by the invention comprises the test strip, the gold-labeled micropores, the sesame mouse-derived monoclonal antibody and the antibody protection diluent, and the kit provides main kit antibodies and the like required by sesame allergen detection, so that the detection of a sample to be detected is simple and rapid, other equipment is not required, the reagent is not required to be prepared in additional time, and the convenience of household sesame allergen detection is greatly improved.
3. The sample pretreatment of the invention adopts the self-prepared high-efficiency extract, improves the extraction efficiency of sesame allergen in food by subpackaging the extract into small bottles and adding special magnetic beads, greatly shortens the extraction time and simplifies the pretreatment part, namely, the extract is taken immediately, so that allergic people can screen whether the suspicious food contains sesame components or not without leaving home, thereby avoiding the allergic reaction caused by the intake of the food containing sesame.
Drawings
FIG. 1 shows the structure and method of use of the test strip of the present invention;
FIG. 2 shows the test results of the test strip of the present invention for different concentrations of sample antigens; the concentration of the sample antigen is 0ppb, 1ppb, 10ppb, 100ppb, 1000ppb and 10000ppb from left to right;
wherein, the left graph A is the test result of the kit of the invention, and the display sensitivity is 1ppb, and the right graph B is the test result of the traditional kit, and the display sensitivity is 100 ppb;
FIG. 3 shows the result of the specificity test of the kit of the present invention;
wherein, 1-7 are 10ppm of milk casein, 10ppm of cod extracted protein, 10ppm of almond extracted protein, 10ppm of soybean extracted protein, 10ppm of egg extracted protein, 10ppm of wheat alcohol soluble protein and 10ppm of peanut extracted protein respectively.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.
Sources of experimental materials:
1. trisodium citrate, Tris, potassium carbonate, sucrose, chloroauric acid, disodium hydrogen phosphate dodecahydrate, and sodium dihydrogen phosphate dihydrate, purchased from the national pharmaceutical group; bovine serum albumin, purchased from beijing solibao technologies ltd; the low adsorption enzyme label plate and the hole cover are purchased from Xiamen Yijiamei experimental equipment Co.Ltd; sample pad, absorbent paper, MAX thread, colored leather paper, PVC back plate, purchased from shanghai jiening biotechnology limited; nitrocellulose membrane, purchased from shanghai gold-labeled biotechnology limited.
2. Multiple antibodies employed in the invention
(1) Rabbit anti-sesame polyclonal antibody:
the experimental rabbit is a new zealand white rabbit, which is raised in the laboratory, and after the rabbit is raised temporarily for 7 days, the rabbit starts to carry out immune injection. Diluting the sesame allergen protein extract to the concentration of 1mg/mL by using sterile deionized water, collecting blood from the marginal vein before immunization, collecting negative control serum, taking 500 mu L of antigen during immunization, fully emulsifying with equal volume of Freund complete adjuvant, respectively immunizing 2 rabbits, injecting subcutaneous multiple injection at the back, and injecting 1mL of antigen emulsion into each injection. Fully emulsifying 300 mu L of antigen with equivalent volume of Freund incomplete adjuvant after 14 days, strengthening the immunity by the same method, strengthening the antigen once every 10 days for six times, carrying out intravenous injection on the margin of ear without adjuvant for the last time, collecting blood from the heart after three days, centrifuging, collecting serum, subpackaging and storing at-20 ℃.
(2) Mouse anti-sesame monoclonal antibody:
the sesame allergen protein (gene expression method) prepared in the laboratory is used for immunizing a white mouse to generate sensitized B lymphocytes, spleen cells and homologous myeloma cells of the white mouse are taken to be fused under the action of polyethylene glycol to form hybridoma cells, the hybridoma cells are screened to obtain a positive cell strain 2-5G1 capable of stably secreting sesame monoclonal antibodies, the cells are expanded and cultured and then injected into the abdominal cavity of the mouse, and ascites is taken to be purified to obtain the required mouse anti-sesame monoclonal antibodies.
(3) Goat anti-mouse IgG: purchased from Beijing Solaibao Tech technologies, Inc.
EXAMPLE 1 preparation of a Home-use high sensitivity kit for detecting sesame allergen
1. Preparation of colloidal gold
The colloidal gold is prepared by a trisodium citrate reduction method, and the preparation method mainly comprises the following steps:
accurately measuring 800mL of ultrapure water (UP grade water), adding the ultrapure water into a 1000mL conical flask, adding 8mL of 1% chloroauric acid solution, heating to a boiling state while stirring, quickly adding 1mL of trisodium citrate aqueous solution with the mass fraction of 12%, changing the solution from gray to black within 2min, finally changing the solution into red, continuously heating and stirring for 10min, cooling to room temperature, adding the ultrapure water to a constant volume of 800mL to obtain colloidal gold, and storing in the dark for later use.
2. Preparation of colloidal gold-labeled antibody (i.e., sesame gold-labeled antibody)
Taking 100ml of prepared colloidal gold, adding 0.6ml of 0.2M potassium carbonate, uniformly mixing, adding 600ug of rabbit anti-sesame polyclonal antibody under the condition of stirring, reacting for 2h at room temperature, then dropwise adding 1000ul of 10% bovine serum albumin, reacting for 1h at room temperature, 13000r/min, centrifuging for 15min, removing supernatant, and re-suspending the precipitate with 10ml of re-solution.
Preferably, the double solution consists of the following components in concentration: PB pH7.4 with molar concentration of 0.01M, bovine serum albumin with mass volume concentration of 1%, sucrose with mass volume concentration of 2%; the solvent is water.
The preparation method of 1L of complex solution comprises the following steps: 2.88g of disodium hydrogen phosphate dodecahydrate, 0.296g of sodium dihydrogen phosphate dihydrate, 10g of bovine serum albumin and 20g of sucrose were weighed and dissolved in 500ml of water, and then the volume was adjusted to 1000ml with water.
3. Preparation of gold-labeled micropores
Mixing 4ml of the prepared gold-labeled sesame antibody with 56ml of freeze-dried diluent, packaging the mixture in a low-adsorption enzyme label plate according to the amount of 60ul per hole, and precooling the plate in a refrigerator at minus 80 ℃ for a night; and (4) placing the ELISA plate into a freeze dryer, vacuumizing for at least 20h, then covering an ELISA plate cap to obtain gold-labeled micropores, and drying and storing for later use.
4. Preparing a mouse anti-sesame monoclonal antibody solution:
after diluting the mouse anti-sesame monoclonal antibody by 10 ten thousand times with the antibody protective solution, the mouse anti-sesame monoclonal antibody diluted solution is stored in a glass bottle which is sterilized at high temperature and high pressure.
The antibody protection solution contains the following components: 0.01M pH7.4PB, 5% by volume of newborn bovine serum, and 0.85% by mass of sodium chloride.
The preparation method of the 1L antibody protection solution comprises the following steps: 2.88g of disodium hydrogen phosphate dodecahydrate, 0.296g of sodium dihydrogen phosphate dihydrate, and 8.5g of sodium chloride were weighed and dissolved in 900ml of water, and 50ml of newborn bovine serum was added thereto, followed by volume adjustment to 1000 ml.
5. Test strip preparation
And (3) coating goat anti-mouse IgG on the detection line on the NC membrane at the concentration of 0.1-0.5 mg/ml, coating goat anti-rabbit IgG on the quality control line at the concentration of 2-8 mg/ml, and drying at 37 ℃ for 16 h. And fixing the absorbent paper, the NC membrane, the sample pad and the MAX line on the PVC rubber plate in the sequence from top to bottom, wherein the overlapped parts of the absorbent paper, the sample pad and the NC membrane are 2mm in length.
6. Preparation of extraction liquid
The extract mainly comprises the following components in concentration: Tris-HCl with a molar concentration of 0.05-0.1M, Triton X-100 with a mass volume concentration of 0.5-1.5%, NaCl with a mass volume concentration of 0.5-3%, ethanol with a volume concentration of 5-30%, and NaN with a mass volume concentration of 0.01-0.1%3The solvent was water, pH 9.0.
Preferably, the extract consists of the following components in the following concentrations: Tris-HCl with a molar concentration of 0.05M, Triton X-100 with a mass volume concentration of 1%, NaCl with a mass volume concentration of 0.85%, ethanol with a volume concentration of 15%, and NaN with a mass volume concentration of 0.01%3The solvent was water, pH 9.0.
1L of the above-mentioned extract was prepared by weighing 6.05g of Tris, 10g of Triton X-100, 8.5g of sodium chloride, 150ml of absolute ethanol, and 0.1g of NaN3Dissolving in 950ml of water, adjusting the pH value to 9.0, and then making the volume constant to 1L.
7. Detection of allergens in a sample
(1) Extraction of sesame allergen from sample
3ml of extract and one magnetic bead were added to a 5ml vial. When the sample is tested, a liquid sample is directly dripped into a 5ml small bottle by 10 drops through a 0.5ml sucker, when the solid sample is tested, two flat spoon samples are taken by a disposable small spoon and added into the 5ml small bottle, then the small bottle is shaken up and down by hands for 30s, the stand is carried out for 1min, and the supernatant is collected.
(2) Detection of samples
And (3-4) drops of supernatant is dripped into the gold-labeled micropores by using a 0.5ml suction pipe, 2 drops of diluted monoclonal antibody are added, the pink powder in the micropores is completely dissolved by blowing and beating for 5-6 times, the mixture is incubated at room temperature for 5min, the MAX end of the test strip is downwards inserted into the micropores, and the result is judged at 5 min.
(3) Analysis of detection results:
the judgment basis is as follows: when only the quality control line (C line) is developed, the detection result is judged to be negative; when the detection line (T line) and the quality control line (C line) are simultaneously developed, judging that the detection result is positive; when the quality control line is not colored, whether the detection result is colored or not is judged to be invalid.
EXAMPLE 2 testing of sensitivity and specificity of the kit
1. The experimental method comprises the following steps:
(1) sensitivity test: sesame allergen proteins were prepared in different concentrations of 10000ppb, 1000ppb, 100ppb, 10ppb, 1ppb, and 0ppb, respectively, using a diluent, and then tested according to the aforementioned detection procedure. Meanwhile, the test is carried out by using the existing reagent strip as a control, and the result is shown in figure 2. The existing reagent strip is a test strip prepared by a traditional method, namely a rabbit anti-sesame polyclonal antibody gold label, a mouse anti-sesame monoclonal antibody coating detection line and a goat anti-mouse IgG coating quality control line.
(2) Specific experiments: milk casein, cod extracted protein, almond extracted protein, egg extracted protein, peanut extracted protein, wheat bran protein and soybean extracted protein are respectively taken, diluted solution is used for preparing the concentration of 10ppm, and the concentration is tested according to the detection steps.
2. Experimental results and analysis:
(1) fig. 2 is a sensitivity test result, and the result shows that the sensitivity of the test strip of the present invention is 1ppb, and the sensitivity of the test strip of the control group is 100ppb, which indicates that the sensitivity of the test strip of the present invention is improved by 100 times.
(2) FIG. 3 shows the results of cross-reactions between the test strip of the present invention and casein milk, cod extracted protein, almond extracted protein, egg extracted protein, peanut extracted protein, wheat bran protein and soybean extracted protein, respectively. The results show that: the test strip has no cross reaction with the allergen protein, and the specificity of the test strip is good.
EXAMPLE 3 quality testing of the kit
1. Minimum detection limit test
The negative corn flour is taken as a marking and is used for an experiment, and the marking levels are 0ppb, 5ppb, 10ppb, 100ppb and 1000ppb respectively.
The results show that: 0ppb and 5ppb were negative, 10ppb, 100ppb and 1000ppb were positive, which indicates that: the test strip had a minimum detection limit of 10 ppb.
2. Negative sample recombination rate
50 samples of known sesame negative samples on the market are purchased and tested by the test strip, and the result shows that the recombination rate is 100%.
3. Positive sample recombination rate
50 samples of known sesame positive on the market are purchased, and the test paper strip is used for testing, so that the results are all positive, and the recombination rate is 100%.
4. Parallelism detection
And taking negative corn flour, adding 10ppb of standard, repeatedly testing for 8 times, and obtaining positive results with good repeatability.
The detection result shows that the detection result of the kit is accurate and reliable.
Example 4 stability study of the kit
After the test strip prepared in the embodiment 1 of the invention is respectively placed at 37 ℃ and 4 ℃ for 24 days, a standard substance is used for a comparison test, the detection result shows no obvious difference, and the test strip can be stably stored at 2-8 ℃ for 2 years according to a conventional conversion method in the field (the condition that the test strip is placed at 37 ℃ for one day is equal to the condition that the test strip is placed at 4 ℃ for one month).
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Claims (8)
1. A high sensitivity kit for detecting sesame allergen in a household type, comprising:
the test paper strip: the detection line on the NC membrane is coated with goat anti-mouse IgG with the concentration of 0.1-0.5 mg/ml, and the quality control line is coated with goat anti-rabbit IgG with the concentration of 2-8 mg/ml; the test strip is composed of absorbent paper, an NC membrane and a sample pad from top to bottom and is fixed on a PVC rubber plate; wherein the overlapping part of the absorbent paper and the sample pad with the NC membrane is 2 mm.
Gold-labeled micropores: adding a mixed solution of a compound solution of a rabbit anti-sesame polyclonal antibody and colloidal gold and a freeze-drying diluent into micropores of a low-adsorption enzyme label plate, and freeze-drying to obtain the low-adsorption enzyme label plate;
and a mouse anti-sesame monoclonal antibody and an antibody protection diluent.
2. The high sensitivity kit according to claim 1, further comprising: the sesame antigen extraction method further comprises an extraction liquid for extracting sesame antigen in a sample, wherein the extraction liquid mainly comprises the following components in concentration: Tris-HCl with a molar concentration of 0.05-0.1M, Triton X-100 with a mass volume concentration of 0.5-1.5%, NaCl with a mass volume concentration of 0.5-3%, ethanol with a mass volume concentration of 5-30%, and NaN with a mass volume concentration of 0.01-0.1%3The solvent was water, pH 9.0.
3. A household detection method of sesame allergen is characterized in that: the method of detecting using the kit of claim 1, comprising the steps of:
(1) extracting an antigen in a sample to be detected to obtain an extracting solution;
(2) adding 3-4 drops of extracting solution and the sesame mouse-derived monoclonal antibody into the gold-labeled micropores, blowing and beating for multiple times to completely dissolve pink powder in the micropores, after incubation at room temperature, downwards inserting the MAX end of the test strip into the micropores, and judging whether the sample contains sesame allergens or not according to the color development result of the reagent strip after 5 min;
the judgment basis is as follows: when only the quality control line is developed, judging that the detection result is negative; when the detection line and the quality control line are simultaneously developed, judging that the detection result is positive; when the quality control line is not colored, whether the detection result is colored or not is judged to be invalid.
4. The method for preparing a home-use high-sensitivity kit for detecting sesame allergens according to claim 1, comprising the steps of:
(1) preparation of gold-labeled antibody: adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding sesame rabbit polyclonal antibody while stirring, reacting at room temperature for 2 hours, then dropwise adding 10% bovine serum albumin, reacting at room temperature for 1 hour, centrifuging, collecting precipitate, and resuspending with 10ml of complex solution to obtain a sesame gold-labeled antibody solution;
the redissolution contains the following components in concentration: PB with the molar concentration of 0.01M and the pH value of 7.4, bovine serum albumin with the mass volume concentration of 1-5% and cane sugar with the mass volume concentration of 1-5%.
(2) Preparing gold-labeled micropores: mixing the sesame gold-labeled antibody solution with the freeze-dried diluent in a volume ratio of 1 (10-15), subpackaging the mixed solution into a low-adsorption enzyme label plate, freezing in a refrigerator overnight, performing vacuum freeze-drying for at least 20h, and then fastening an enzyme label plate cap to obtain gold-labeled micropores;
(3) preparation of monoclonal antibody solution: diluting the monoclonal antibody by 8-20 ten thousand times by using an antibody protective solution for later use;
(4) preparing a test strip: coating goat-anti-mouse on the NC membrane detection line with the concentration of 0.1-0.5 mg/ml, coating goat-anti-mouse on the quality control line with the concentration of 2-8 mg/ml, and drying for 16h at 37 ℃; fixing the absorbent paper, the NC membrane, the sample pad and the MAX line on a PVC rubber plate from top to bottom to prepare a test strip;
(5) and (3) sample testing: extracting an antigen in a sample to be detected to obtain an extracting solution; adding 3-4 drops of the extracting solution and the sesame mouse-derived monoclonal antibody into the gold-labeled micropores, blowing and beating for multiple times to completely dissolve pink powder in the micropores, after incubation at room temperature, downwards inserting the MAX end of the test strip into the micropores, and judging whether the sample contains sesame allergens or not according to the color development result of the reagent strip after 5 min.
5. The method according to claim 4, wherein the method for extracting the sesame antigen in the sample to be tested comprises:
adding 2-4 ml of extraction liquid and one magnetic bead into a 5ml vial; directly adding a liquid sample or a solid sample into the small bottle, shaking the small bottle up and down for 30s, standing for at least 1min, and absorbing the supernatant to obtain an extracting solution;
wherein the extract mainly comprises the following components in concentration: Tris-HCl with a molar concentration of 0.05-0.1M, Triton X-100 with a mass volume concentration of 0.5-1.5%, NaCl with a mass volume concentration of 0.5-3%, ethanol with a volume concentration of 5-30%, and NaN with a mass volume concentration of 0.01-0.1%3The solvent was water, pH 9.0.
6. The method of claim 4, wherein the antibody protecting solution comprises the following concentrations of each component: 0.01M PB, pH7.4, 5% by volume neonatal bovine serum and 0.85% by mass by volume sodium chloride.
7. The method of claim 4, wherein the gold colloid is prepared by reducing trisodium citrate.
8. The use of the high-sensitivity kit for the household-type detection of sesame allergen according to claim 1 for the detection of sesame allergen in food, air, water sources and surfaces of articles.
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