CN112834751B - 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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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
- 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
- G01N33/577—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
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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 following components: test strip: the detection line on the NC film 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 paper strip is composed of water absorbing paper, NC film, sample pad, and is fixed on PVC rubber plate from top to bottom; wherein, the overlapping part of the water absorbing paper, the sample pad and the NC film is 2mm; gold-labeled microwells: adding the mixed solution of the complex solution of the rabbit anti-sesame polyclonal antibody and the colloidal gold and the freeze-drying diluent into micropores of the low-adsorption ELISA plate, and freeze-drying to obtain the anti-sesame polyclonal antibody; murine anti-sesame monoclonal antibodies and antibody protection dilutions. 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 detecting sesame allergen at home.
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 allergens, a detection method and application thereof.
Background
Food allergy is a public health problem of global concern, and it is counted 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 to 200 people die from allergic reactions every year, while in our country there are up to two hundred million people with allergic diseases, of which about 90% are caused by food allergies. Sesame as a common vegetable belongs to one of eight major types of sensitizers, and can cause serious allergic reaction, thereby seriously threatening the life health of sesame allergic people. At present, no thorough and effective radical treatment method for sesame allergic diseases exists at home and abroad, and the only effective means for preventing sesame allergy is to avoid eating food containing sesame.
Currently, the sesame allergen detection methods mainly comprise electrophoresis, chromatography, enzyme-linked immunosorbent assay (ELISA), a biological immunosensor, mass spectrum-based proteomics (LC-MS/MS), polymerase Chain Reaction (PCR) and the like. The electrophoresis method, the chromatography method and the ELISA method are traditional sesame detection methods based on proteins, and can only analyze single proteins, and the requirements of the development of the method are not met although the development of the method is mature; the mass spectrum-based proteomics can simultaneously and rapidly detect various 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 forms a cycle through three links of high-temperature denaturation, low-temperature annealing, medium-temperature extension and the like, so as to achieve the purpose of specific amplification of target genes. PCR technology is divided into two types, ordinary PCR and real-time fluorescent quantitative PCR. The common PCR technology is mainly used for qualitative detection, and the detection result needs to be verified by electrophoresis and is easy to generate false positive result due to cross contamination. The real-time fluorescence PCR realizes quantitative analysis of the initial template by detecting the fluorescence signal of each cycle product in the amplification reaction in real time, and has the advantages of strong specificity, high accuracy, less PCR pollution, high automation degree and the like compared with the traditional PCR, but has the advantages of complex operation, high cost, long time consumption, high skill requirement on experimenters and the like.
The existing sesame allergen detection methods have high requirements on the technical level of operators and require complex and precise instruments and equipment for cooperation, some methods consume long time, some methods have relatively high cost, and various severe requirements make the methods impossible to walk into 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 requirement on operators and difficult running into families of the existing sesame allergen detection method, the invention provides a high-sensitivity kit for detecting sesame allergens in a household mode, a detection method and application thereof.
In order to solve the problems, the invention provides the following technical scheme:
in a first aspect, the present invention provides a high sensitivity kit for home-use detection of sesame allergens, comprising:
test strip: the detection line on the NC film 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 paper strip is composed of water absorbing paper, NC film, sample pad, and is fixed on PVC rubber plate from top to bottom; wherein, the overlap of the absorbent paper and sample pad with NC film is 2mm.
Gold-labeled microwells: adding the mixed solution of the complex solution of the rabbit anti-sesame polyclonal antibody and the colloidal gold and the freeze-drying diluent into micropores of the low-adsorption ELISA plate, and freeze-drying to obtain the anti-sesame polyclonal antibody;
murine anti-sesame monoclonal antibodies and antibody protection dilutions.
Preferably, the kit further comprises: the kit also comprises an extraction liquid for extracting sesame antigens in the sample, wherein the extraction liquid mainly comprises the following components in concentration: tris-HCl with molar concentration of 0.05-0.1M, triton X-100 with mass volume concentration of 0.5-1.5%, naCl with mass volume concentration of 0.5-3%, ethanol with volume concentration of 5-30%, naN with mass volume concentration of 0.01% -0.1% 3 The solvent was water and the pH was 9.0.
In a second aspect, the present invention also provides a method for home-type detection of sesame allergen, the method 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 sesame murine monoclonal antibody into the gold-labeled micropores, blowing for multiple times to completely dissolve pink powder in the micropores, incubating at room temperature, inserting the MAX end of the test strip into the micropores downwards, and judging whether the sample contains sesame allergen according to the color development result of the test strip after 5 min;
the judgment basis is as follows: when only the quality control line develops color, judging that the detection result is negative; when the detection line and the quality control line develop color at the same time, judging that the detection result is positive; when the quality control line does not develop color, whether the detection result develops color or not is judged to be invalid.
In a third aspect, the present invention also provides a method for preparing the above-mentioned high-sensitivity kit for detecting sesame allergen, which 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 re-suspending with 10ml of complex solution to obtain a sesame gold-labeled antibody solution;
the complex solution contains the following components in concentration: PB at pH7.4 at a molar concentration of 0.01M, bovine serum albumin at a mass volume concentration of 1-5% and sucrose at a mass volume concentration of 1-5%.
(2) Preparing gold-labeled micropores: mixing sesame gold-labeled antibody solution and freeze-dried diluent in a volume ratio of 1 (10-15), subpackaging the mixed solution into a low-adsorption ELISA plate, freezing overnight in a refrigerator, and buckling the ELISA plate cap after vacuum freeze-drying for at least 20 hours to obtain gold-labeled micropores;
(3) Preparation of monoclonal antibody solution: diluting the monoclonal antibody by 8-20 ten thousand times with antibody protective solution for standby;
(4) Preparation of a test strip: the NC film is coated with the goat anti-mouse with the concentration of 0.1-0.5 mg/ml, the quality control line is coated with the goat anti-mouse with the concentration of 2-8 mg/ml, and the mixture is dried for 16h at 37 ℃; fixing the water absorbing paper, the NC film, the sample pad and the MAX line on a PVC adhesive plate from top to bottom to prepare a test strip;
(5) Sample testing: extracting an antigen in a sample to be detected to obtain an extracting solution; adding 3-4 drops of extracting solution and sesame murine monoclonal antibody into the gold-labeled microwells, blowing for multiple times to completely dissolve pink powder in the microwells, incubating at room temperature, inserting the MAX end of the test strip into the microwells downwards, and judging whether the sample contains sesame allergen according to the color development result of the test strip after 5 min.
Preferably, in the method, the method for extracting the sesame antigen in the sample to be detected comprises the following steps:
adding 2-4 ml of the extract 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 sucking the supernatant to obtain an extracting solution;
wherein the extract mainly comprises the following components in concentration: tris-HCl with molar concentration of 0.05-0.1M, triton X-100 with mass volume concentration of 0.5-1.5%, naCl with mass volume concentration of 0.5-3%, ethanol with volume concentration of 5-30%, naN with mass volume concentration of 0.01% -0.1% 3 The solvent was water and the pH was 9.0.
Preferably, in the method, the antibody protection solution contains the following components in the following concentrations: 0.01M pH7.4PB, fresh bovine serum at a volume concentration of 5% and sodium chloride at a mass volume concentration of 0.85%.
Preferably, in the method, the colloidal gold is prepared by adopting 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 allergen in foods, air, water sources and surfaces of articles.
The invention has the following beneficial effects:
1. the invention provides a household detection method of sesame allergen components, which improves the preparation method of gold-labeled micropores on the basis of a colloidal gold immunological method.
The traditional method is that monoclonal antibodies are coated on a detection line part of an NC membrane, and when a rabbit multi-antibody gold-labeled complex and an antigen are chromatographed on the NC membrane to the detection line, the rabbit multi-antibody gold-labeled complex and the antigen are combined with the monoclonal antibodies on the detection line to develop color. The detection method of the invention is that the mouse anti-sesame monoclonal antibody is prepared into a solution, the solution and the solution to be detected (containing antigen) are sequentially added into a gold-labeled micropore to react for 5 minutes at room temperature, so that the mouse anti-sesame monoclonal antibody, the antigen and the rabbit multi-antibody gold-labeled substance are fully combined into a compound, and then are combined with goat anti-mouse IgG on a detection line for color development. Based on the principle, the sensitivity of the method is improved by at least 100 times compared with that of the traditional method.
In addition, the method is simple and convenient to operate, and common people can perform screening detection without barriers, so that life health of sesame allergen groups is protected.
2. The kit comprises the test paper strip, the gold-labeled microwell, the sesame murine 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 quick, other equipment is not needed, the reagent is not needed to be configured in another time, and the convenience of detecting the sesame allergen at home 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 sub-packaging the extract into small bottles and adding special magnetic beads, greatly shortens the extraction time, simplifies pretreatment parts, is ready to use and ready to take, and allows allergic people to screen whether the suspicious food contains sesame ingredients or not without going out, thereby avoiding anaphylactic reaction caused by taking the food containing sesame.
Drawings
FIG. 1 is a schematic illustration of a test strip and method of use according to the present invention;
FIG. 2 shows the results of detecting sample antigens at different concentrations using the test strip of the present invention; the sample antigen concentration is 0ppb,1ppb, 10ppb, 100ppb, 1000ppb and 10000ppb in order from left to right;
wherein, the left graph A shows the test result of the kit of the invention, the sensitivity is 1ppb, the right graph B shows the test result of the traditional kit, the sensitivity is 100ppb;
FIG. 3 shows the results of a 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 gliadin and 10ppm of peanut extracted egg respectively.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Experimental material sources:
1. trisodium citrate, tris, potassium carbonate, sucrose, chloroauric acid, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, purchased from the national drug group; bovine serum albumin, purchased from beijing solebone technologies limited; the low-adsorption ELISA plate and the hole cover are purchased from Xiamen Yijiame experiment equipment limited company; sample pad, absorbent paper, MAX wire, colored paper, PVC backing, purchased from Shanghai Jiening biotechnology limited; nitrocellulose membranes were purchased from Shanghai gold standard biotechnology Co.
2. Various antibodies employed in the present invention
(1) Rabbit anti-sesame polyclonal antibody:
the laboratory was used to feed New Zealand white rabbits, which were temporarily fed for 7 days and then were subjected to an immunization injection. Diluting sesame allergen protein extract with sterile deionized water to a concentration of 1mg/mL, collecting blood of the auricular vein before immunization, collecting negative control serum, taking 500 mu L of antigen during immunization, fully emulsifying with equal volume of Freund's complete adjuvant, respectively immunizing 2 rabbits, performing subcutaneous multipoint injection on the back, and injecting 1mL of antigen emulsion each time. 300 mu L of antigen is fully emulsified with an equal volume of Freund's incomplete adjuvant after 14 days, the immunization is enhanced by the same method, then the immunization is enhanced every 10 days for six times, the ear margin intravenous injection without the adjuvant is carried out for the last time, the heart is used for blood sampling after three days, serum is centrifugally collected, and the serum is stored at the temperature of minus 20 ℃ after split charging.
(2) Murine anti-sesame monoclonal antibody:
the sesame allergen protein (gene expression method) prepared in the laboratory is used for immunizing a mouse to generate sensitized B lymphocytes, spleen cells and syngeneic myeloma cells are taken and fused under the action of polyethylene glycol to form hybridoma cells, positive cell strain 2-5G1 capable of stably secreting sesame monoclonal antibodies is obtained through screening, and then the cells are injected into the abdominal cavity of the mouse after expanded culture, and the ascites is purified to obtain the required mouse anti-sesame monoclonal antibodies.
(3) Sheep anti-mouse IgG: purchased from beijing solebao technologies limited.
Example 1 preparation of a high sensitivity kit for household detection of sesame allergen
1. Preparation of colloidal gold
The colloidal gold is prepared by adopting a trisodium citrate reduction method, and the preparation method mainly comprises the following steps:
accurately measuring 800mL of ultrapure water (UP grade water), adding into a 1000mL conical flask, adding 8mL of 1% chloroauric acid solution, heating to boiling state while stirring, rapidly adding 1mL of 12% trisodium citrate aqueous solution, changing the solution from gray to black within 2min, changing the solution into red finally, continuing heating and stirring for 10min, cooling to room temperature, adding ultrapure water to 800mL, obtaining colloidal gold, and preserving in a dark place for standby.
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 stirring, reacting for 2 hours at room temperature, adding 1000ul of 10% bovine serum albumin dropwise, reacting for 1 hour at room temperature, 13000r/min, centrifuging for 15min, discarding supernatant, and re-suspending the precipitate by 10ml of complex solution.
Preferably, the complex solution is composed of the following concentrations of the ingredients: 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 the 1L 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 are weighed, dissolved in 500ml of water, and then the volume is fixed to 1000ml by water.
3. Gold mark micropore preparation
Mixing 4ml of the prepared sesame gold-labeled antibody with 56ml of freeze-dried diluent, respectively loading into a low-adsorption ELISA plate according to the amount of 60ul per hole, and pre-cooling in a refrigerator at-80 ℃ for overnight; the next day, the ELISA plate is placed into a freeze dryer for vacuumizing for at least 20 hours, and then an ELISA plate cap is buckled to obtain gold-labeled micropores, and the gold-labeled micropores are dried and stored for later use.
4. Murine anti-sesame monoclonal antibody solution configuration:
after 10 ten thousand times dilution of the mouse anti-sesame monoclonal antibody with the antibody protective solution, the mouse anti-sesame monoclonal antibody dilution was stored in a glass bottle subjected to high-temperature autoclaving treatment.
The antibody protection liquid comprises the following components: 0.01M pH7.4PB, fresh bovine serum at a volume concentration of 5% and sodium chloride at a mass volume concentration of 0.85%.
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 are weighed and dissolved in 900ml of water, 50ml of new born calf serum is added into the solution, and the volume is fixed to 1000ml.
5. Test strip preparation
The NC film detection line is coated with goat anti-mouse IgG with the concentration of 0.1-0.5 mg/ml, the quality control line is coated with goat anti-rabbit IgG with the concentration of 2-8 mg/ml, and the NC film detection line is dried for 16h at 37 ℃. The absorbent paper, NC film, sample pad and MAX line were fixed on PVC glue plate in the order from top to bottom, wherein the overlapping part of absorbent paper and sample pad and NC film was 2mm each.
6. Preparation of extract
The extract mainly comprises the following components in concentration: tris-HCl with molar concentration of 0.05-0.1M, triton X-100 with mass volume concentration of 0.5-1.5%, naCl with mass volume concentration of 0.5-3%, ethanol with volume concentration of 5-30%, naN with mass volume concentration of 0.01% -0.1% 3 The solvent was water and the pH was 9.0.
PreferablyThe extract consists of the following components in concentration: tris-HCl with molar concentration of 0.05M, triton X-100 with mass volume concentration of 1%, naCl with mass volume concentration of 0.85%, ethanol with volume concentration of 15%, naN with mass volume concentration of 0.01% 3 The solvent was water and the pH was 9.0.
The preparation method of 1L of the extract liquid comprises weighing 6.05g of Tris,10g of Triton X-100,8.5g of sodium chloride, 150ml of absolute ethyl alcohol and 0.1g of NaN 3 Dissolved in 950ml of water, adjusted to pH 9.0, and then fixed to volume of 1L.
7. Detection of allergens in a sample
(1) Extraction of sesame allergen in sample
3ml of extract and one magnetic bead were added to a 5ml vial. In the sample test, 10 drops of liquid sample are directly dripped into a 5ml small bottle by a 0.5ml straw, in the solid sample, two flat spoons of sample 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, and the liquid sample is kept still for 1min, and the supernatant is collected.
(2) Detection of samples
3-4 drops of clear liquid are added into the gold-labeled microwells by using a 0.5ml straw, 2 drops of diluted monoclonal antibody are added, the solution is blown for 5-6 times to completely dissolve pink powder in the microwells, after incubation for 5min at room temperature, the MAX end of the test strip is inserted into the microwells downwards, and the result is judged when 5 min.
(3) Analysis of detection results:
the judgment basis is as follows: when only the quality control line (C line) develops color, judging that the detection result is negative; when the detection line (T line) and the quality control line (C line) develop color at the same time, judging that the detection result is positive; when the quality control line does not develop color, whether the detection result develops color or not is judged to be invalid.
Example 2 test of sensitivity and specificity of the kit
1. The experimental method comprises the following steps:
(1) Sensitivity experiment: sesame allergen proteins were prepared in different concentrations of 10000ppb,1000ppb,100ppb,10ppb,1ppb,0ppb respectively with a diluent, and then tested according to the above-described test procedure. Meanwhile, the prior reagent strip is used as a control for experiments, 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 marks gold, a mouse anti-sesame monoclonal antibody coats a detection line, and a goat anti-mouse IgG coats a quality control line.
(2) Specificity experiments: milk casein, cod extract protein, almond extract protein, egg extract protein, peanut extract protein, wheat bran protein and soybean extract protein are respectively taken, diluted solution is used for preparing the concentration of 10ppm, and the test is carried out according to the detection step.
2. Experimental results and analysis:
(1) FIG. 2 shows the sensitivity test results, which show 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 a factor of 100.
(2) FIG. 3 shows the cross-reaction results of the test strip of the present invention with milk casein, cod extract protein, almond extract protein, egg extract protein, peanut extract protein, wheat bran protein, and soybean extract protein, respectively. The results show that: the test strip has no cross reaction with the allergen proteins, and the specificity of the test strip is good.
Example 3 quality inspection of kits
1. Minimum detection limit test
Negative corn flour is taken for marking experiments, and marking levels are respectively 0ppb, 5ppb, 10ppb, 100ppb and 1000ppb.
The results show that: 0ppb and 5ppb were negative, 10ppb, 100ppb and 1000ppb were positive, and the results indicate that: the minimum detection limit of the test strip is 10ppb.
2. Negative sample recombination rate
50 samples of known sesame negative 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 with positive sesame are purchased from the market, and tested by the test strip, and the results are positive, and the recombination rate is 100%.
4. Parallelism detection
Taking negative corn flour, adding 10ppb, repeating the test 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 investigation of the kit
After the test strip prepared in example 1 of the present invention was left at 37℃and 4℃for 24 days, respectively, a comparative test was performed using a standard substance, and the test result showed no significant difference, and the test strip was stably stored at 2 to 8℃for 2 years according to a conventional conversion method in the art (a period of 37℃being equivalent to a period of 4℃being left for one month).
It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.
Claims (8)
1. A high-sensitivity kit for detecting sesame allergens in a household type, comprising:
test strip: the detection line on the NC film 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 paper strip is composed of water absorbing paper, NC film, sample pad, and is fixed on PVC rubber plate from top to bottom; wherein, the overlapping part of the water absorbing paper, the sample pad and the NC film is 2mm;
gold-labeled microwells: adding the mixed solution of the complex solution of the rabbit anti-sesame polyclonal antibody and the colloidal gold and the freeze-drying diluent into micropores of the low-adsorption ELISA plate, and freeze-drying to obtain the anti-sesame polyclonal antibody;
the sesame murine monoclonal antibody and antibody protection diluent are added to the gold-labeled microwell when in use.
2. The high sensitivity kit of claim 1, further comprising: an extract for extracting sesame antigens in a sample, the extract mainly comprising the following components in concentration: tris-HCl with molar concentration of 0.05-0.1M and massTriton 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%, naN with a mass volume concentration of 0.01-0.1% 3 The solvent was water and the pH was 9.0.
3. A household detection method of sesame allergen is characterized in that: the method for detection 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 sesame murine monoclonal antibody into the gold-labeled micropores, blowing for multiple times to completely dissolve pink powder in the micropores, incubating at room temperature, inserting the MAX end of the test strip into the micropores downwards, and judging whether the sample contains sesame allergen according to the color development result of the test strip after 5 min;
the judgment basis is as follows: when only the quality control line develops color, judging that the detection result is negative; when the detection line and the quality control line develop color at the same time, judging that the detection result is positive; when the quality control line does not develop color, whether the detection result develops color or not is judged to be invalid.
4. The method for preparing the household high-sensitivity kit for detecting sesame allergen according to claim 1, comprising 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 re-suspending with 10ml of complex solution to obtain a sesame gold-labeled antibody solution;
the complex solution contains the following components in concentration: PB with a molar concentration of 0.01M and pH7.4, bovine serum albumin with a mass and volume concentration of 1-5% and sucrose with a mass and volume concentration of 1-5%;
(2) Preparing gold-labeled micropores: mixing sesame gold-labeled antibody solution and freeze-dried diluent in a volume ratio of 1 (10-15), subpackaging the mixed solution into a low-adsorption ELISA plate, freezing overnight in a refrigerator, and buckling the ELISA plate cap after vacuum freeze-drying for at least 20 hours to obtain gold-labeled micropores;
(3) Preparation of monoclonal antibody solution: diluting the monoclonal antibody by 8-20 ten thousand times with antibody protection liquid for standby;
(4) Preparation of a test strip: the NC film is coated with the goat anti-mouse with the concentration of 0.1-0.5 mg/ml, the quality control line is coated with the goat anti-mouse with the concentration of 2-8 mg/ml, and the mixture is dried for 16 hours at 37 ℃; fixing the water absorbing paper, the NC film, the sample pad and the MAX line on a PVC adhesive plate from top to bottom to prepare a test strip;
(5) Sample testing: extracting an antigen in a sample to be detected to obtain an extracting solution; 3-4 drops of extracting solution and sesame murine monoclonal antibody are added into the gold-labeled micropores, the mixture is blown for multiple times to completely dissolve pink powder in the micropores, after incubation at room temperature, the MAX end of the test strip is downwards inserted into the micropores, and after 5min, whether sesame allergen is contained in the sample is judged according to the color development result of the test strip.
5. The method according to claim 4, wherein the method for extracting sesame antigen in the sample to be tested comprises the steps of:
adding 2-4 ml of the extract and one magnetic bead into a 5ml small bottle; 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 sucking 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%, naN with a mass volume concentration of 0.01-0.1% 3 The solvent was water and the pH was 9.0.
6. The method of claim 4, wherein the antibody-protecting solution contains the following components in the following concentrations: PB at pH7.4 at 0.01M, neonatal bovine serum at 5% by volume and sodium chloride at 0.85% by mass.
7. The method of claim 4, wherein the colloidal gold is prepared by a method of reducing trisodium citrate.
8. Use of the high sensitivity kit for detecting sesame allergens in domestic use according to claim 1 for detecting sesame allergens in food, air, water sources and surfaces of objects.
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