CN112834750B - Household high-sensitivity kit for detecting celery allergen, detection method and application thereof - Google Patents
Household high-sensitivity kit for detecting celery 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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention discloses a household high-sensitivity kit for detecting celery allergen, a detection method and application thereof, wherein the high-sensitivity kit comprises the following components: test strip: the detection line on the NC film is coated with a mouse anti-celery monoclonal antibody 1 with the concentration of 0.1-0.5 mg/ml, and the quality control line is coated with a sheep anti-mouse 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 celery gold-labeled antibody and the freeze-dried diluent into micropores of the low-adsorption ELISA plate, and freeze-drying to obtain the celery gold-labeled antibody; the celery gold-labeled antibody is prepared by labeling the biotinylated celery antibody 2 by a reticular structure formed by a streptavidin gold-labeled compound and a biotin-labeled bovine serum albumin gold-labeled compound. 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 home detection of celery allergen.
Description
Technical Field
The invention relates to the technical field of allergen detection methods, in particular to a household high-sensitivity kit for detecting celery allergens, a detection method and application thereof.
Background
Food allergy, also called food allergy or digestive system allergy, allergic gastroenteritis, is an allergy in the digestive system or systemic due to IgE-mediated and non-IgE-mediated immune reactions caused by certain foods or food additives, etc. People suffering from allergic diseases in China are up to two hundred million, and about 90% of them are caused by food allergy. Celery is a common vegetable, belongs to one of eight major types of sensitizers, can cause serious allergic reaction, and seriously threatens the life health of celery allergic people. At present, no thorough and effective radical treatment method for the allergic diseases of celery is available at home and abroad, and the most effective means for preventing the allergy of celery is to avoid eating food containing celery.
The current celery allergen detection methods mainly comprise electrophoresis, chromatography, enzyme-linked immunosorbent assay (ELISA), 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 celery detection methods based on proteins, and can only analyze single proteins, and the development is mature but can not meet the requirements; 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 celery 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 method impossible to walk into families.
Therefore, the existing celery 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 celery allergen detection method, the invention provides a household high-sensitivity kit for detecting celery allergens, 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 application discloses a high sensitivity kit for home-use detection of celery allergen comprising:
test strip: the detection line on the NC film is coated with a mouse anti-celery monoclonal antibody 1 with the concentration of 0.1-0.5 mg/ml, and the quality control line is coated with a sheep anti-mouse 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 celery gold-labeled antibody and the freeze-dried diluent into micropores of the low-adsorption ELISA plate, and freeze-drying to obtain the celery gold-labeled antibody;
the celery gold-labeled antibody is prepared by labeling the biotinylated celery antibody 2 by a reticular structure formed by a streptavidin gold-labeled compound and a biotin-labeled bovine serum albumin gold-labeled compound.
Preferably, the high sensitivity kit further comprises: the method also comprises an extraction liquid for extracting the celery 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 invention provides a home-type detection method of celery allergen, which adopts the kit for detection, and comprises the following steps:
(1) Extracting an antigen in a sample to be detected to obtain an extracting solution;
(2) Adding 3-4 drops of extracting solution 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 celery allergen 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 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.
Preferably, the method for preparing the extracting solution from the sample 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.
In a third aspect, the invention also provides a preparation method of the household high-sensitivity kit for detecting celery allergen, which comprises the following steps:
(1) Preparation of celery gold-labeled antibody:
A. adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding streptavidin while stirring, reacting for 1-4 hours at room temperature, then dropwise adding bovine serum albumin, reacting for 1-4 hours at room temperature, centrifugally collecting precipitate, and re-suspending by using a complex solution to obtain a streptavidin gold-labeled compound solution;
B. adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding biotin-labeled bovine serum albumin while stirring, reacting for 1-4 hours at room temperature, then dropwise adding the bovine serum albumin, centrifuging after the reaction, collecting precipitate, and re-suspending the precipitate with a complex solution to obtain the biotin-labeled bovine serum albumin gold-labeled compound;
C. the streptavidin gold-labeled compound and the biotin-labeled bovine serum albumin gold-labeled compound obtained in the previous two steps are mixed according to the following ratio of 1: uniformly mixing the components according to the ratio of 3-1:30, adding the biotinylated mouse anti-celery monoclonal antibody 2 according to the ratio of 8-24 ug/ml while stirring, and reacting for 1-4 h or overnight at the room temperature to obtain the celery gold-labeled antibody.
(2) Preparing gold-labeled micropores: mixing the solution of the celery gold-labeled antibody with the freeze-dried diluent according to the 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 a test strip: 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; the NC film detection line is coated with mouse anti-celery monoclonal antibody 1, the concentration is 0.5-2 mg/ml, the quality control line is coated with sheep anti-mouse IgG, and the concentration is 1-5 mg/ml.
Preferably, the method of step (1) is specifically:
A. adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding streptavidin while stirring, reacting at room temperature for 2 hours, then dropwise adding 10% bovine serum albumin, reacting at room temperature for 2 hours, centrifuging, collecting precipitate, and re-suspending with 10ml of complex solution to obtain a streptavidin gold-labeled complex solution;
B. adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding biotin-labeled bovine serum albumin while stirring, reacting for 2 hours at room temperature, dropwise adding 1000ul of 10% bovine serum albumin, reacting for 2 hours at room temperature, dropwise adding 10% bovine serum albumin, reacting for 1 hour, centrifuging, collecting precipitate, and re-suspending the precipitate with 10ml of complex solution to obtain biotin-labeled bovine serum albumin gold-labeled complex;
C. uniformly mixing the streptavidin gold-labeled compound and the biotin-labeled bovine serum albumin gold-labeled compound obtained in the previous two steps in a ratio of 1:5, adding the biotinylated mouse anti-celery monoclonal antibody 2 while stirring, and reacting for 2 hours or overnight at the room temperature to obtain the celery gold-labeled antibody.
The prepared kit can be directly used for sample testing according to the following method:
extracting an antigen in a sample to be detected to obtain an extracting solution; 3-4 drops of extracting solution are added into the gold-labeled microwells, the solution is blown for a plurality of times to completely dissolve pink powder in the microwells, after incubation at room temperature, the MAX end of the test strip is downwards inserted into the microwells, and after 5 minutes, whether the sample contains celery allergen is judged according to the color development result of the test strip.
Preferably, the colloidal gold is prepared by adopting a method of reducing trisodium citrate.
Preferably, the complex solution contains the following concentrations of each component: phosphate buffer solution 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%.
In a fourth aspect, the invention also provides an application of the household high-sensitivity kit for detecting celery allergens in food, air, water source and article surfaces.
The invention has the following beneficial effects:
1. the invention provides a household detection method for celery allergen components, which is characterized in that a mesh structure formed by the reaction of a streptavidin gold-labeled compound and a biotin-labeled bovine serum albumin gold-labeled compound is used for labeling a biotinylated celery antibody 2, so that the labeling efficiency of the celery antibody is greatly improved, the effect of cascade amplification is achieved, and the detection sensitivity of the prepared celery gold-labeled antibody to antigens is improved by at least 100 times. In addition, the method is simple and convenient to operate, common people can perform screening detection without barriers, and life health of celery allergen groups is protected.
In the gold-labeled micropores, the biotinylated celery antibody 2 marked by the reticular structure is combined with an antigen in a sample to be detected and a mouse anti-celery monoclonal antibody 1 coated on a detection line to form a sandwich structure, and a positive sample is detected through a chromogenic reaction.
2. The kit provided by the invention comprises the test paper strip and the gold-labeled microwells, and the kit is provided with main kit antibodies and the like required by celery 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 home detection of celery allergens 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 suspicious food contains celery components or not without going out, thereby avoiding anaphylactic reaction caused by taking food containing celery.
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 1-8 are milk casein, shrimp extract protein, almond extract protein, soybean extract protein, egg extract protein, wheat gliadin, peanut extract protein and cod extract protein 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. various antibodies employed in the present invention
(1) Preparation of murine anti-celery monoclonal antibody 1 and murine anti-celery monoclonal antibody 2: the method comprises the steps of immunizing a mouse with celery allergen protein (gene expression method) prepared in a laboratory to generate sensitized B lymphocytes, then taking spleen cells and syngeneic myeloma cells, fusing under the action of polyethylene glycol to form hybridoma cells, screening to obtain 10 positive cell strains capable of stably secreting the monoclonal antibodies of the celery, performing expanded culture on the 10 positive cell strains, injecting the 10 positive cell strains into the abdominal cavity of the mouse, purifying ascites to obtain the corresponding mouse anti-sesame monoclonal antibodies, and screening and pairing to obtain the available mouse anti-celery monoclonal antibodies 1 and 2, wherein the clone numbers of the corresponding cell strains are 10-14G1 and 2-2G2 respectively.
(2) Biotinylated murine anti-celery monoclonal antibody 2: the activated lipid with biotin-N-hydroxysuccinimide was reacted with the mouse anti-celery monoclonal antibody 2 in PBS (0.01M) at pH8.0 for a certain period of time. biotin-N-hydroxysuccinimide activated lipid was purchased from Beijing Soy Bao technology Co.
(3) Sheep anti-mouse IgG: purchased from beijing solebao technologies limited.
Example 1 preparation of a high sensitivity kit for household detection of celery 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 celery gold-labeled antibody
(1) Taking 100ml of prepared colloidal gold, adding 0.4ml of 0.2M potassium carbonate, uniformly mixing, adding 500ug of streptavidin under stirring, reacting for 2 hours at room temperature, dropwise adding 1000ul of 10% bovine serum albumin, reacting for 1 hour at room temperature, 13000r/min, centrifuging for 15 minutes, discarding the supernatant, and re-suspending the precipitate by using 10ml of complex solution.
(2) Taking 100ml of prepared colloidal gold, adding 0.8ml of 0.2M potassium carbonate, uniformly mixing, adding 1000ug of biotin-labeled bovine serum albumin 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.
(3) Streptavidin gold-labeled antibody and biotin-labeled bovine serum albumin gold-labeled antibody were mixed at a ratio of 1: mixing evenly according to the proportion of 5, dropwise adding the biotinylated celery antibody 2 while stirring according to the proportion of 8ug/ml, and reacting for 2 hours at room temperature or overnight at 4 ℃ to obtain the celery gold-labeled antibody.
In this example, the preparation method of 1L of the complex solution comprises: 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 celery gold-labeled antibody with 56ml of freeze-dried diluent, respectively loading the mixture into a low-adsorption ELISA plate according to the amount of 60ul per hole, and pre-cooling the mixture 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. Test strip preparation
The detection line on the NC film is coated with a mouse anti-celery monoclonal antibody 1 with the concentration of 0.1-0.5 mg/ml, and the quality control line is coated with a sheep anti-mouse 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.
5. 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.
Preferably, the extract consists of the following concentrations of the components: 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 method for preparing 1L of the preferable extract is as follows: 6.05g of Tris,10g of Triton X-100,8.5g of sodium chloride, 150ml of absolute ethanol, 0.1g of NaN are weighed 3 Dissolved in 950ml of water, adjusted to pH 9.0, and then fixed to volume of 1L.
6. Detection of allergens in a sample
(1) Extraction of celery 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, the solution is blown for 5-6 times to completely dissolve pink powder in the microwells, the MAX end of the test strip is inserted into the microwells downwards, and the result is judged when 10 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: the celery allergen proteins were formulated with dilutions to different concentrations of 0ppm,0.1ppm,1ppm,10ppm,100ppm respectively, and then tested according to the previous test procedure. Meanwhile, a reagent strip (namely, a mouse anti-celery monoclonal antibody 1 is marked with a gold-labeled antibody, a mouse anti-celery monoclonal antibody 2 is coated with a detection line, and a goat anti-mouse IgG is coated with a quality control line) prepared by a traditional method is used as a control for experiments, and the results are shown in figure 2.
(2) Specificity experiments: milk casein, shrimp extract protein, almond extract protein, soybean extract protein, egg extract protein, wheat gliadin, peanut extract protein and cod extract protein were each prepared to a concentration of 10ppm with a diluent, and tested according to the test procedure, the results of which are shown in FIG. 3.
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 0.1ppm and the sensitivity of the test strip of the control group is 10ppm, which indicates that the sensitivity of the test strip of the present invention is improved by 100 times.
(2) FIG. 3 shows the cross-reaction results of the test strip of the present invention with milk casein, shrimp extract protein, almond extract protein, soybean extract protein, egg extract protein, wheat gliadin, peanut extract protein and cod extract protein, respectively (from left to right).
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
The negative corn flour was used as a standard test at standard levels of 0ppm, 0.5ppm, 1ppm,10ppm and 100ppm, respectively.
The results show that: the test results marked with 0ppm and 0.5ppm are negative, and the test results of other marked groups are positive, which indicates that the lowest detection limit of the test strip is 1ppm.
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 strips prepared in example 1 of the present invention were left at 37℃and 4℃for 18 days, respectively, a comparative test was performed using a standard. The test results show no obvious difference, and the test strip can be stably stored for 1.5 years at 2-8 ℃ according to the conventional conversion method (the placement at 37 ℃ for one day is equivalent to the placement at 4 ℃ for one month) in the field.
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 home-use detection of celery allergen, comprising:
test strip: the detection line on the NC film is coated with a mouse anti-celery monoclonal antibody 1 with the concentration of 0.1-0.5 mg/ml, and the quality control line is coated with a sheep anti-mouse 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 celery gold-labeled antibody and the freeze-dried diluent into micropores of the low-adsorption ELISA plate, and freeze-drying to obtain the celery gold-labeled antibody;
the celery gold-labeled antibody is prepared by labeling the biotinylated celery antibody 2 by a network structure formed by connecting a streptavidin gold-labeled compound and a biotin-labeled bovine serum albumin gold-labeled compound.
2. The high sensitivity kit according to claim 1, further comprising an extract for extracting apigenin in the sample, the extract consisting essentially of the following concentrations of the components: 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.
3. A household detection method of celery 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) 3-4 drops of extracting solution are added into the gold-labeled micropores, the solution is blown for multiple times to completely dissolve pink powder in the micropores, the MAX end of the test strip is downwards inserted into the micropores, and after 10min, whether the sample contains celery allergen is judged according to the color development result of the test strip;
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. A method according to claim 3, wherein the method of preparing the extract from the sample is:
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.
5. The method for preparing the household high-sensitivity kit for detecting celery allergen according to claim 1, comprising the following steps:
(1) Preparation of celery gold-labeled antibody:
A. adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding streptavidin while stirring, reacting for 1-4 hours at room temperature, then dropwise adding bovine serum albumin, reacting for 1-4 hours at room temperature, centrifugally collecting precipitate, and re-suspending by using a complex solution to obtain a streptavidin gold-labeled compound solution;
B. adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding biotin-labeled bovine serum albumin while stirring, reacting for 1-4 hours at room temperature, then dropwise adding the bovine serum albumin, centrifuging after the reaction, collecting precipitate, and re-suspending the precipitate with a complex solution to obtain the biotin-labeled bovine serum albumin gold-labeled compound;
C. the streptavidin gold-labeled compound and the biotin-labeled bovine serum albumin gold-labeled compound obtained in the previous two steps are mixed according to the following ratio of 1: uniformly mixing the materials according to the ratio of 3-1:30, adding the biotinylated mouse anti-celery monoclonal antibody 2 according to the ratio of 8-24 ug/ml while stirring, and reacting for 1-4 hours or overnight at room temperature to obtain the celery gold-labeled antibody;
(2) Preparing gold-labeled micropores: mixing the celery gold-labeled antibody solution with freeze-dried diluent according to the 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 a test strip: 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; the NC film upper detection line is coated with a mouse anti-celery monoclonal antibody 1, the concentration is 0.5-2 mg/ml, the quality control line is coated with goat anti-mouse IgG, and the concentration is 1-5 mg/ml.
6. The method of claim 5, wherein the colloidal gold is prepared by a method of reducing trisodium citrate.
7. The method of claim 5, wherein the multiple solution contains the following concentrations of each component: phosphate buffer solution with a molar concentration of 0.01M and a pH of 7.4, bovine serum albumin with a volume concentration of 1-5% and sucrose with a mass volume concentration of 1-5%.
8. Use of the home-use kit for detecting celery allergen according to claim 1 for detecting celery allergen in food, air, water source and surface of articles.
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