CN112834750A - 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
- Publication number
- CN112834750A CN112834750A CN202011619365.4A CN202011619365A CN112834750A CN 112834750 A CN112834750 A CN 112834750A CN 202011619365 A CN202011619365 A CN 202011619365A CN 112834750 A CN112834750 A CN 112834750A
- Authority
- CN
- China
- Prior art keywords
- celery
- gold
- labeled
- concentration
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 title claims abstract description 66
- 235000010591 Appio Nutrition 0.000 title claims abstract description 66
- 238000001514 detection method Methods 0.000 title claims abstract description 57
- 239000013566 allergen Substances 0.000 title claims abstract description 35
- 244000101724 Apium graveolens Dulce Group Species 0.000 title 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 71
- 240000007087 Apium graveolens Species 0.000 claims abstract description 65
- 238000012360 testing method Methods 0.000 claims abstract description 48
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 28
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 27
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 27
- 239000012528 membrane Substances 0.000 claims abstract description 16
- 238000003908 quality control method Methods 0.000 claims abstract description 16
- 229960002685 biotin Drugs 0.000 claims abstract description 14
- 235000020958 biotin Nutrition 0.000 claims abstract description 14
- 239000011616 biotin Substances 0.000 claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 14
- 239000002250 absorbent Substances 0.000 claims abstract description 10
- 230000002745 absorbent Effects 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 239000003085 diluting agent Substances 0.000 claims abstract description 8
- 238000001179 sorption measurement Methods 0.000 claims abstract description 6
- 241000283707 Capra Species 0.000 claims abstract description 5
- 239000011259 mixed solution Substances 0.000 claims abstract description 5
- 230000006287 biotinylation Effects 0.000 claims abstract description 4
- 238000007413 biotinylation Methods 0.000 claims abstract description 4
- 238000004108 freeze drying Methods 0.000 claims abstract description 3
- 238000002372 labelling Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 22
- 238000002156 mixing Methods 0.000 claims description 18
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- 108010090804 Streptavidin Proteins 0.000 claims description 13
- 239000002244 precipitate Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000000427 antigen Substances 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 8
- 229920004890 Triton X-100 Polymers 0.000 claims description 7
- 239000013504 Triton X-100 Substances 0.000 claims description 7
- 235000013305 food Nutrition 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 4
- 238000010009 beating Methods 0.000 claims description 4
- 238000007664 blowing Methods 0.000 claims description 4
- 238000011161 development Methods 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 4
- 229940038773 trisodium citrate Drugs 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 2
- 229960004793 sucrose Drugs 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 11
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 abstract description 4
- 108090001008 Avidin Proteins 0.000 abstract description 2
- 229960005322 streptomycin Drugs 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 19
- 235000018102 proteins Nutrition 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 18
- 238000003752 polymerase chain reaction Methods 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 5
- 244000000231 Sesamum indicum Species 0.000 description 4
- 235000003434 Sesamum indicum Nutrition 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 244000144725 Amygdalus communis Species 0.000 description 3
- 235000011437 Amygdalus communis Nutrition 0.000 description 3
- 235000017060 Arachis glabrata Nutrition 0.000 description 3
- 244000105624 Arachis hypogaea Species 0.000 description 3
- 235000010777 Arachis hypogaea Nutrition 0.000 description 3
- 235000018262 Arachis monticola Nutrition 0.000 description 3
- 241000238557 Decapoda Species 0.000 description 3
- 108010000912 Egg Proteins Proteins 0.000 description 3
- 102000002322 Egg Proteins Human genes 0.000 description 3
- 208000004262 Food Hypersensitivity Diseases 0.000 description 3
- 206010016946 Food allergy Diseases 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 108010073771 Soybean Proteins Proteins 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 208000026935 allergic disease Diseases 0.000 description 3
- 235000020224 almond Nutrition 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 235000020932 food allergy Nutrition 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 235000020232 peanut Nutrition 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 2
- 108010061711 Gliadin Proteins 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 230000000172 allergic effect Effects 0.000 description 2
- 208000030961 allergic reaction Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 208000031058 celery allergy Diseases 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 230000037029 cross reaction Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000013568 food allergen Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- 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
-
- 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
-
- 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
- G01N33/558—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 test paper strip: the detection line on the NC membrane 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 goat anti-mouse 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 celery gold-labeled antibody and a freeze-dried diluent into micropores of a low-adsorption enzyme label plate, and freeze-drying to obtain the celery gold-labeled antibody; the celery gold-labeled antibody is prepared by labeling a biotinylation celery antibody 2 by using a network structure consisting of a streptomycin avidin 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 household detection of celery 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 celery allergen, a detection method and application thereof.
Background
Food allergy is also called food allergy or digestive allergy, allergic gastroenteritis, and is caused by an allergic reaction in the digestive system or systemically due to IgE-mediated and non-IgE-mediated immune reactions caused by certain foods or food additives. People suffering from allergic diseases in China are as many as two hundred million, and about 90 percent of people are caused by food allergy. Celery is a common vegetable, belongs to one of eight major allergens, can cause serious anaphylactic reaction, and seriously threatens the life health of people with celery allergy. At present, no thorough and effective method for radically treating celery allergic diseases exists at home and abroad, and the most effective means for preventing celery allergy is to avoid eating food containing celery.
At present, celery 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) technology and the like. Among them, electrophoresis, chromatography and enzyme-linked immunoassay are traditional celery detection methods based on proteins, and can only analyze a 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 celery 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 detection method for celery allergen is in need of further improvement.
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 celery allergen detection method, the invention provides a household high-sensitivity kit for detecting celery 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 application discloses a household high-sensitivity kit for detecting celery allergen, which comprises:
the test paper strip: the detection line on the NC membrane 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 goat anti-mouse 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 celery gold-labeled antibody and a freeze-dried diluent into micropores of a low-adsorption enzyme label plate, and freeze-drying to obtain the celery gold-labeled antibody;
the celery gold-labeled antibody is prepared by labeling a biotinylation celery antibody 2 by using a network structure consisting of a streptomycin avidin gold-labeled compound and a biotin-labeled bovine serum albumin gold-labeled compound.
Preferably, the high-sensitivity kit further comprises: bag for returningThe method comprises the following steps of extracting a celery antigen from a sample, wherein the extracting solution 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.
In a second aspect, the invention provides a household detection method for celery allergens, which adopts the kit for detection, and the method 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 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 celery 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.
Preferably, the household-type detection method comprises the following steps of:
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.
In a third aspect, the invention further provides a preparation method of the household high-sensitivity kit for detecting celery allergens, which comprises the following steps:
(1) preparing a celery gold-labeled antibody:
A. adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding streptavidin while stirring, reacting at room temperature for 1-4 h, then dropwise adding bovine serum albumin, reacting at room temperature for 1-4 h, centrifuging, collecting precipitate, and re-suspending with a 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 at room temperature for 1-4 h, then dropwise adding bovine serum albumin, centrifugally collecting precipitate after reaction, and re-suspending the precipitate with a re-solution to obtain a biotin-labeled bovine serum albumin gold-labeled compound;
C. and (3) performing the following steps of mixing the streptavidin gold-labeled complex obtained in the first two steps and the biotin-labeled bovine serum albumin gold-labeled complex with the weight ratio of 1: and (3) uniformly mixing the components in a ratio of 3-1: 30, adding a biotinylated mouse anti-celery monoclonal antibody 2 according to a ratio of 8-24 ug/ml while stirring, and reacting at room temperature for 1-4 h or overnight at 4 ℃ to obtain the celery gold-labeled antibody.
(2) Preparing gold-labeled micropores: mixing the celery 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) preparing a test strip: 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; the detection line on the NC membrane is coated with a mouse anti-celery monoclonal antibody 1 with the concentration of 0.5-2 mg/ml, and the quality control line is coated with a sheep anti-mouse IgG with the concentration of 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, centrifugally collecting precipitate, and resuspending the precipitate 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 at room temperature for 2 hours, then dropwise adding 1000ul of 10% bovine serum albumin, reacting at room temperature for 2 hours, then dropwise adding 10% bovine serum albumin, reacting for 1 hour, centrifugally collecting precipitates, and re-suspending the precipitates with 10ml of a complex solution to obtain a biotin-labeled bovine serum albumin gold-labeled compound;
C. and uniformly mixing the streptavidin gold-labeled compound and the biotin-labeled bovine serum albumin gold-labeled compound obtained in the first two steps in a ratio of 1:5, adding a biotinylated mouse anti-celery monoclonal antibody 2 while stirring, and reacting at room temperature for 2h or overnight at 4 ℃ to obtain the celery gold-labeled antibody.
The prepared kit can be directly used for sample test according to the following method:
extracting an antigen in a sample to be detected to obtain an extracting solution; adding 3-4 drops of extracting solution 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 celery allergens or not according to the color development result of the reagent strip after 5 min.
Preferably, the colloidal gold is prepared by a method of reducing trisodium citrate.
Preferably, the redissolution contains the following concentrations of ingredients: a phosphate buffer solution with a molar concentration of 0.01M and a pH value of 7.4, bovine serum albumin with a mass volume concentration of 1-5%, and sucrose with a mass 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 sources 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 biotinylated celery antibody 2 is marked by a net structure formed by the reaction of a streptavidin gold-labeled compound and a biotin-marked bovine serum albumin gold-labeled compound, so that the marking 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 an antigen is improved by at least 100 times. In addition, the method is simple and convenient to operate, ordinary people can carry out screening detection without obstacles, and life health protection and navigation protection are provided for celery allergen people.
In the gold-labeled micropores, the biotinylated celery antibody 2 marked by the network structure is combined with the antigen in the sample to be detected and the mouse anti-celery monoclonal antibody 1 coated on the detection line to form a sandwich structure, and the positive sample is detected through a color reaction.
2. The kit corresponding to the method provided by the invention comprises the test strip and the gold-labeled micropores, 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 rapid, other equipment is not required, the reagent is not required to be prepared in additional time, and the convenience of household detection of celery allergen is greatly improved.
3. The sample pretreatment of the invention adopts the self-prepared high-efficiency extract, improves the extraction efficiency of the sesame allergen in the 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 obtained immediately, so that allergic people can screen whether the suspicious food contains celery component without going out of home, thereby avoiding the allergic reaction caused by ingesting the food containing celery.
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 1-8 are milk casein, shrimp extracted protein, almond extracted protein, soybean extracted protein, egg extracted protein, wheat gliadin, peanut extracted protein and cod 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. multiple antibodies employed in the invention
(1) Preparing a mouse anti-celery monoclonal antibody 1 and a mouse anti-celery monoclonal antibody 2: a celery allergen protein (gene expression method) prepared in a 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 and fused under the action of polyethylene glycol to form hybridoma cells, 10 strains of positive cell strains capable of stably secreting the sesame celery monoclonal antibody are obtained through screening, 10 strains of cells are subjected to expanded culture and then injected into the abdominal cavity of the mouse, ascites is taken and purified to obtain the corresponding mouse anti-sesame monoclonal antibody, and then, available mouse anti-celery monoclonal antibody 1 and mouse anti-celery monoclonal antibody 2 are obtained through screening and pairing, and the clone numbers of the corresponding cell strains are respectively 10-14G1 and 2-2G 2.
(2) Biotinylation mouse anti-celery monoclonal antibody 2: reacting biotin-N-hydroxysuccinimide activated ester with mouse anti-celery monoclonal antibody 2 in 0.01M PBS (with the pH value of 8.0) for a certain time to obtain the compound. Biotin-N-hydroxysuccinimide activated esters were purchased from Beijing Sorleibao technologies, Inc.
(3) Goat anti-mouse IgG: purchased from Beijing Solaibao Tech technologies, Inc.
Example 1 preparation of a household high-sensitivity kit for detecting celery 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 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 the condition of stirring, reacting at room temperature for 2h, then dropwise adding 1000ul of 10% bovine serum albumin, reacting at room temperature for 1h, 13000r/min, centrifuging for 15min, removing supernatant, and re-suspending the precipitate with 10ml of re-solution.
(2) Taking 100ml of prepared colloidal gold, adding 0.8ml of 0.2M potassium carbonate, uniformly mixing, adding 1000ug of biotin standard bovine serum albumin 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.
(3) And (2) performing the steps of mixing a streptavidin gold-labeled antibody and a biotin-labeled bovine serum albumin gold-labeled antibody in a ratio of 1: and 5, uniformly mixing the mixture according to the proportion of 8ug/ml, dropwise adding a biotinylated celery antibody 2 while stirring, and reacting at room temperature for 2 hours or overnight at 4 ℃ to obtain the celery gold-labeled antibody.
In this example, the preparation method of 1L of the complex solution is: 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 celery gold-labeled 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 mixture 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. Test strip preparation
The detection line on the NC membrane 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 goat anti-mouse 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.
5. 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.
The preparation method of 1L of the preferable extraction liquid 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 were weighed3Dissolving in 950ml of water, adjusting the pH value to 9.0, and then making the volume constant to 1L.
6. Detection of allergens in a sample
(1) Extraction of celery allergen from a 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) dropwise adding 3-4 drops of the supernatant into the gold-labeled micropores by using a 0.5ml suction pipe, blowing and beating for 5-6 times to completely dissolve pink powder in the micropores, downwards inserting the MAX end of the test strip into the micropores, and judging the result after 10 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: celery allergen proteins were prepared in different concentrations of 0ppm, 0.1ppm, 1ppm, 10ppm, 100ppm using diluents and then tested according to the aforementioned test procedure. Meanwhile, the test was performed using a reagent strip prepared by the conventional method (i.e., a gold-labeled antibody labeled with a mouse-anti-celery monoclonal antibody 1, a detection line coated with a mouse-anti-celery monoclonal antibody 2, and a quality control line coated with goat-anti-mouse IgG) as a control, and the results are shown in FIG. 2.
(2) Specific experiments: milk casein, shrimp extracted protein, almond extracted protein, soybean extracted protein, egg extracted protein, wheat alcohol soluble protein, peanut extracted protein and cod extracted protein are respectively taken, the concentration of the diluent is 10ppm, the test is carried out according to the detection steps, and the result is shown in figure 3.
2. Experimental results and analysis:
(1) FIG. 2 shows the results of the sensitivity test, which shows 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 results of cross-reactions of the test strip of the present invention with milk casein, shrimp-derived protein, almond-derived protein, soy-derived protein, egg-derived protein, wheat gliadin, peanut-derived protein, and cod-derived protein (left to right), 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 for standard addition experiment, and the standard addition levels are 0ppm, 0.5ppm, 1ppm, 10ppm and 100ppm respectively.
The results show that: the test results of the labeled 0ppm and 0.5ppm are negative, and the test results of other labeled groups are positive, which indicates that the test strip has the lowest detection limit of 1 ppm.
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
The test strips prepared in example 1 of the present invention were left at 37 ℃ and 4 ℃ for 18 days, respectively, and then subjected to a comparative test using a standard. The detection result shows that no obvious difference exists, and the result shows that the test strip can be stably stored for 1.5 years at the temperature of 2-8 ℃ according to the conventional conversion method in the field (the condition that the test strip is placed at 37 ℃ for one day is equivalent 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 household high-sensitivity kit for detecting celery allergens is characterized by comprising:
the test paper strip: the detection line on the NC membrane 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 goat anti-mouse 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 celery gold-labeled antibody and a freeze-dried diluent into micropores of a low-adsorption enzyme label plate, and freeze-drying to obtain the celery gold-labeled antibody;
the celery gold-labeled antibody is prepared by labeling a biotinylation celery antibody 2 by using 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 of claim 1, further comprising an extraction liquid for extracting celery antigen from the 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 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 celery allergen is characterized by comprising the following steps: 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 into the gold-labeled micropores, blowing and beating for multiple times to completely dissolve pink powder in the micropores, inserting the MAX end of the test strip downwards into the micropores, and judging whether the sample contains celery allergen or not according to the color development result of the reagent strip after 10 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 of claim 3, wherein the method of preparing the extract from the sample 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.
5. The preparation method of the household high-sensitivity kit for detecting celery allergens as claimed in claim 1, comprising the following steps:
(1) preparing a celery gold-labeled antibody:
A. adding potassium carbonate into the prepared colloidal gold, uniformly mixing, adding streptavidin while stirring, reacting at room temperature for 1-4 h, then dropwise adding bovine serum albumin, reacting at room temperature for 1-4 h, centrifuging, collecting precipitate, and re-suspending with a 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 at room temperature for 1-4 h, then dropwise adding bovine serum albumin, centrifugally collecting precipitate after reaction, and re-suspending the precipitate with a re-solution to obtain a biotin-labeled bovine serum albumin gold-labeled compound;
C. and (3) performing the following steps of mixing the streptavidin gold-labeled complex obtained in the first two steps and the biotin-labeled bovine serum albumin gold-labeled complex with the weight ratio of 1: and (3) uniformly mixing the components in a ratio of 3-1: 30, adding a biotinylated mouse anti-celery monoclonal antibody 2 according to a ratio of 8-24 ug/ml while stirring, and reacting at room temperature for 1-4 h or overnight at 4 ℃ to obtain the celery gold-labeled antibody.
(2) Preparing gold-labeled micropores: mixing the celery 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) preparing a test strip: 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; the detection line on the NC membrane is coated with a mouse anti-celery monoclonal antibody 1 with the concentration of 0.5-2 mg/ml, and the quality control line is coated with a sheep anti-mouse IgG with the concentration of 1-5 mg/ml.
6. The method of claim 5, wherein the colloidal gold is prepared by a method of trisodium citrate reduction.
7. The method according to claim 5, wherein the double solution comprises the following concentrations of the respective components: phosphate buffer solution with the molar concentration of 0.01M and the pH value of 7.4, bovine serum albumin with the volume concentration of 1-5 percent and cane sugar with the mass volume concentration of 1-5 percent.
8. The use of the household-type high-sensitivity kit for detecting celery allergen according to claim 1 in detecting celery allergen in food, air, water sources and surfaces of articles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011619365.4A CN112834750B (en) | 2020-12-30 | 2020-12-30 | Household high-sensitivity kit for detecting celery allergen, detection method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011619365.4A CN112834750B (en) | 2020-12-30 | 2020-12-30 | Household high-sensitivity kit for detecting celery allergen, detection method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112834750A true CN112834750A (en) | 2021-05-25 |
| CN112834750B CN112834750B (en) | 2024-01-26 |
Family
ID=75923987
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011619365.4A Active CN112834750B (en) | 2020-12-30 | 2020-12-30 | Household high-sensitivity kit for detecting celery allergen, detection method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112834750B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005069997A (en) * | 2003-08-27 | 2005-03-17 | Sharp Corp | Method for detecting allergen protein, detecting chip and detecting apparatus |
| CN101387643A (en) * | 2008-10-20 | 2009-03-18 | 杭州浙大生物基因工程有限公司 | Multichannel allergen rapid detection kit and method for making same |
| CN103667205A (en) * | 2013-12-10 | 2014-03-26 | 潍坊医学院 | Biologically labeled bimolecular site-directed biotinylated recombinant alkaline phosphatase (AP) |
| CN108459159A (en) * | 2018-02-11 | 2018-08-28 | 莎穆(上海)生物科技有限公司 | Supramolecular self assembly mediates the new method of mesh nano gold enhancing immuno-chromatographic test paper strip sensitivity |
| CN109374887A (en) * | 2018-10-12 | 2019-02-22 | 北京纳百生物科技有限公司 | Bovine viral diarrhea virus antigen colloidal gold detection kit and its application |
-
2020
- 2020-12-30 CN CN202011619365.4A patent/CN112834750B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005069997A (en) * | 2003-08-27 | 2005-03-17 | Sharp Corp | Method for detecting allergen protein, detecting chip and detecting apparatus |
| CN101387643A (en) * | 2008-10-20 | 2009-03-18 | 杭州浙大生物基因工程有限公司 | Multichannel allergen rapid detection kit and method for making same |
| CN103667205A (en) * | 2013-12-10 | 2014-03-26 | 潍坊医学院 | Biologically labeled bimolecular site-directed biotinylated recombinant alkaline phosphatase (AP) |
| CN108459159A (en) * | 2018-02-11 | 2018-08-28 | 莎穆(上海)生物科技有限公司 | Supramolecular self assembly mediates the new method of mesh nano gold enhancing immuno-chromatographic test paper strip sensitivity |
| CN109374887A (en) * | 2018-10-12 | 2019-02-22 | 北京纳百生物科技有限公司 | Bovine viral diarrhea virus antigen colloidal gold detection kit and its application |
Non-Patent Citations (2)
| Title |
|---|
| NAM HYUN KIM 等: "Highly sensitive biomolecule detection on a quartz crystal microbalance using gold nanoparticles as signal amplification probes", ANALYTICAL SCIENCES, vol. 23, pages 177 - 181 * |
| 陈峥嵘: "犬瘟热病毒AS-PCR核酸试纸条的研究", 中国优秀硕士学位论文全文数据库基础科学辑, no. 02, pages 006 - 619 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112834750B (en) | 2024-01-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103033619B (en) | A kind of protein chip kit of comprehensive detection lung cancer marker and method | |
| CN112679605B (en) | Antibodies or antigen binding fragments thereof against novel coronavirus nucleocapsid proteins and uses thereof | |
| CN110441512B (en) | Colloidal gold immunochromatography detection device for ethyl maltol hapten and ethyl maltol | |
| CN108445230B (en) | Procalcitonin chemiluminescence detection reagent based on nano antibody and detection method | |
| CN106645729A (en) | Chemiluminescent kit for quantitatively detecting mycobacterium tuberculosis r interferon as well as preparation method, detection method and evaluation method of chemiluminescent kit | |
| CN112213491B (en) | Kit for rapidly detecting drugs in hair and application of kit | |
| CN109180519B (en) | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method | |
| CN116925218B (en) | Antibody of small heat shock protein HSPB1, antibody composition, hybridoma cell strain and application thereof | |
| CN108359642B (en) | Hybridoma cell of shrimp tropomyosin monoclonal antibody and application thereof | |
| CN112834750A (en) | Household high-sensitivity kit for detecting celery allergen, detection method and application thereof | |
| CN112834751B (en) | Household high-sensitivity kit for detecting sesame allergen, detection method and application thereof | |
| EP1347301A1 (en) | Method for ultra-rapid and ultra-sensitive measurement | |
| CN114058594B (en) | Hybridoma cell strain secreting vitamin A monoclonal antibody and application thereof | |
| CN108132348B (en) | Sudan red III hapten, coupling antigen, antibody and colloidal gold rapid detection device and application thereof | |
| CN110988325A (en) | Blocking agent and kit containing same | |
| CN108277206A (en) | A kind of wheat gliadin monoclonal antibody hybridoma cell strain and its application | |
| CN104987380A (en) | Composition, kit and method for detection of blood plasma inflammatory cytokine autoantibodies | |
| CN110386979A (en) | A kind of method preparing mulberry Ralstonia solanacearum antibody and its application in DAS-ELISA detection method | |
| CN109799352A (en) | It chemical illuminating reagent and its is applied in immune detection | |
| KR102653196B1 (en) | VHH single domain antibody with high affinity against nonstructural protein 3B of foot-and-mouth disease virus | |
| CN117003882B (en) | Hybridoma cell strain combination and antibody combination for detecting golimumab and application of hybridoma cell strain combination and antibody combination | |
| CN113185530B (en) | Hybridoma cell strain, 2-type euglena brevibacterium toxin monoclonal antibody secreted by hybridoma cell strain and application of monoclonal antibody | |
| CN115627264B (en) | anti-Malneffei mannan protein hybridoma cell strain and application thereof | |
| CN117003870B (en) | Hybridoma cell strain combination and antibody combination for detecting adalimumab and application of hybridoma cell strain combination and antibody combination | |
| CN113912719B (en) | Monoclonal antibody for detecting mouse interleukin 6 and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |