CN101393214A - Multichannel ingestion type allergen rapid detection kit and method for making same - Google Patents
Multichannel ingestion type allergen rapid detection kit and method for making same Download PDFInfo
- Publication number
- CN101393214A CN101393214A CNA2008101222901A CN200810122290A CN101393214A CN 101393214 A CN101393214 A CN 101393214A CN A2008101222901 A CNA2008101222901 A CN A2008101222901A CN 200810122290 A CN200810122290 A CN 200810122290A CN 101393214 A CN101393214 A CN 101393214A
- Authority
- CN
- China
- Prior art keywords
- nitrocellulose membrane
- allergen
- membrane bar
- protein
- ige antibody
- 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.)
- Pending
Links
Images
Abstract
The invention discloses a multi-channel reagent kit for rapid detection of edible allergens and a preparation method thereof. The reagent kit comprises solidified milk, egg, meat, grain, nut, fruit, vegetable, aquatic product, bean food allergen proteins, a nitrocellulose membrane strip of a human IgE antibody, a colloidal gold labeled anti-human IgE antibody, a buffer liquid, and a classifying colorimetric card. The adaptation of the reagent kit can quantitatively judge results by visual measurement, can perform semi-quantitative classification to detection results by using the colorimetric card, can add samples at one time during clinical detection, ensure that each channel can quickly detect 10 to 20 kinds of allergens and a plurality of channels can synchronously detect 30 to 60 kinds of different allergens, has the advantages of simplicity, convenience, quickness, sensitivity, and stability, and provides an effective means for determining the allergens which cause patients to be sensitized and the degree of allergic reactions through in vitro assistant diagnosis.
Description
Technical field
The hyperchannel that the present invention relates to allergen specificity antibody IgE content in a kind of qualitative and half-quantitative detection human serum is eaten type allergen rapid detection kit and preparation method thereof.
Background technology
Anaphylactia is modal a kind of disease in the modern society, it is to be caused by the anaphylactogen of extensive existence, the about 5-10% of China, US and European have the people of 5-25% invaded and harassed by anaphylactia approximately, and wherein child and teen-age illness are particularly evident, threaten more serious.Allergic reaction (allergic reaction) claim allergic reaction again; Anaphylactogen (allergen) claim allergen again.Irritated and (the European Academy of Allergy and Clinical Immunology of clinical immunology association according to Europe, EAACI) viewpoint of delivering, immune-mediated allergic reaction has two types: allergic reaction (the Johansson SGO.et al of the allergic reaction of IgE mediation and non-IgE mediation, 2001, Allergy, 56:813-824).The allergic reaction of IgE mediation is that the monokaryon of organs such as the anaphylactogen lymph node that stimulates antibody, liver, spleen is engulfed system, triggers the thick liquid cell reaction and produces specific IgE (sIgE) antibody.On the IgE acceptor of Fc end attached to mast cell or basophilic granulocyte of IgE molecule, make body be in the sensitization state.When body is stimulated by anaphylactogen of the same type again, anaphylactogen makes IgE molecule crosslinked, cause that mast cell or basophilic granulocyte take off granulating, discharge histamine, chemical substance such as leukotriene and cell factor produces allergic reaction, cause the telangiectasis of body, oedema, smooth muscle spasm, the endocrine hyperactivity, cause the clinical symptoms of anaphylactia, as: pollinosis, allergic rhinitis, allergic asthma, anaphylactic shock, nettle rash, eczema, angioedema, arthritis, headache, (period-luminosity inflammation etc. is translated for functions of intestines and stomach imbalance and other chronic sympton, 2002, immunology, PP323-369, the People's Health Publisher; Sampson HA, 1999, Food allergy, part1, J Allergy Clin Immunol, 103:717-728).
Anaphylactogen sensitization transfer mode divides three classes: the food type, by oral cavity dietary intake anaphylactogen sensitization; Induction type is by enviromental allergen sensitization such as suction pollen, dust such as nasal cavities; Contact-type by contact skin, causes allergy through the pore transmission, as nickel, chromium goods, rubber etc.The anaphylactogen that this kit relates to is the big molecule allergen protein or the polypeptide of food type and induction type.
Food allergen divides two big classes: vegetable protein and animal protein are generally the glycoprotein of molecular weight 10kD-70kD.What be divided into the vegetable protein that contains anaphylactogen by their 26S Proteasome Structure and Function characteristic is the superfamily of the alcohol soluble protein in legume, tree nut, cereal, fruit, the vegetables etc., protein family (the Aalberse RC of plant defense systems such as the cup-shaped superfamily protein of the seed storage protein of peanut, tree nut, soybean etc. and the AMS of cereal, protease inhibitors, 2000, Structural biology of allergens, J AllergyClin Immunol, 106:228-238; Breiteneder H er al, 2004, A classification of plantfood allergens, J Allergy Clin Immunol, 113:821-830); The source of animal protein that contains anaphylactogen is for being meat, the lactoprotein of the mammal and the birds of representative with ox IgE albumin, with the little pure albumen fish meat albumen that is representative, with former myogen is meat albumen (the Chapman JA et al of crustacean, mollusc, amphibian and the Reptilia etc. of representative, 2006, Food allergy:a practice parameter, Ann Allergy Asthma Immunol, 96:S1-S68); With they be raw material also produced unnumbered " nutritious and healthy food " (WalJM, 1999, Nahrung, 43:s168-174).
The anaphylactoid sensitization anaphylactogen of IgE mediation detects clinically can be by Skin-test and various external detection method, former absorption detects (RAST), Western blotting (IS) and EIA enzyme immunoassay (EIA) etc. as radioanaphylaxis, determines the anaphylactogen of sensitization in conjunction with the interrogation of medical history, illness.Vitro detection is IgE, particularly allergenic specific IgE (sIgE) antibody concentration of measuring in the blood sample.The method that multiple detection desmoenzyme is arranged in the EIA enzyme immunoassay, it is fluorescence method, chemoluminescence method and immunochromatographic method etc., and product is all arranged by drugs approved by FDA (Chapman JA et al, 2006, Food allergy:a practice parameter, Ann Allergy Asthma Immunol, 96:S1-S68).The standardization council of U.S. clinical labororatory (CLSI) has formulated the outline (I/LA20-A, NCCLS, 1997,17 (20)) that people IgE antibody immunoassay method is estimated specially.Use the hyperchannel immunochromatographic method, only just can detect several easy, apace simultaneously to tens of kinds of anaphylactogens with small amount of sample, range estimation can qualitatively judge the positive or negative result, but use colorimetric card sxemiquantitative classification, reagent is easy to operate, both can be used for hospital's bedside quick diagnosis, also can be used for community clinic or rural hospital even family.Especially be fit to the current national conditions of China.
The hyperchannel immunochromatographic method, the antigen-antibody reaction of employing high degree of specificity and immunochromatographiassays assays technology are come the specific IgE antibody in qualitative and the half-quantitative detection serum.When solidify after the sensitizing antigen of nitrocellulose membrane carrier surface and patients serum react together, specific IgE will be attached on the antigen specifically in the serum, the anti human IgE antibody that the antibody of this specific bond is labeled is discerned, and specific IgE content is proportional in the intensity of color and the serum; According to the size scale colour atla that the concentration of specific IgE in the human serum is set up, the sensitization degree of the anaphylactogen of judge.
The multiple technologies that immunochromatographic method is used, method for extraction and purification as allergen protein, the preparation of people IgE antibody and anti human IgE antibody, purifying, colloid gold label anti human IgE antibody to produce purifying etc. all ripe, eat the commercialization production of type anaphylactogen quick detection reagent for hyperchannel and established technical foundation.
Summary of the invention
The object of the present invention is to provide hyperchannel to eat type anaphylactogen quick detection reagent kit and preparation method thereof, but 10-20 kinds of anaphylactogens of each passage fast detecting, a plurality of passages can detect 60-100 kinds of different anaphylactogens simultaneously, hyperchannel is eaten type anaphylactogen quick detection reagent accuracy height, high specificity, favorable reproducibility, easy to use, quick, but content that both can qualitative also half-quantitative detection human serum allergen specificity antibody IgE is in order to examination with make a definite diagnosis the degree that two-stage reagent detects the anaphylactogen of patient's sensitization and determines patient's sensitivity response.
Hyperchannel of the present invention is eaten the type allergen rapid detection kit, its composition comprises the nitrocellulose membrane bar of the milk, eggs, meat, cereals, nut fruits, fruits, greengrocery, aquatic product, bean food allergen protein and the people IgE antibody that have solidified, the antibody of colloid gold label, damping fluid, the size scale colour atla;
Wherein:
The nitrocellulose membrane bar of the milk food allergen albumen that has solidified comprises: milk, goat milk;
The nitrocellulose membrane bar of the eggs food allergen albumen that has solidified comprises: poultry albumen, poultry yolk;
The nitrocellulose membrane bar of the meat food allergen protein that has solidified comprises: pork, beef, mutton, duck, chicken, goose, turkey meat, donkey meat;
The nitrocellulose membrane bar of the cereals food allergen albumen that has solidified comprises: corn, wheat, oat, barley, buckwheat, rice, millet;
The nitrocellulose membrane bar of the nut fruits food allergen albumen that has solidified comprises: almond, peanut, fibert, cashew nut, walnut, American pistachios, Brazilian nut, sesame;
The nitrocellulose membrane bar of the fruits food allergen albumen that has solidified comprises: watermelon, pineapple, orange, mango, banana, citrus, Hu shaddock, lemon, apple, grape, pears, strawberry, peach;
The nitrocellulose membrane bar of the greengrocery food allergen albumen that has solidified comprises: celery, spinach, onion, eggplant, garlic, capsicum, potato, tomato;
The nitrocellulose membrane bar of the aquatic product food allergen albumen that has solidified comprises: hairtail, yellow croaker, flatfish, salmon; Carp, crucian, grass carp, silver carp, shrimp, crab, freshwater mussel, mussel, oyster, swamp eel, eel;
The nitrocellulose membrane bar of the bean food allergen protein that has solidified comprises: French beans, broad bean, soya bean, pea;
Colloid gold label antibody: the anti human IgE antibody of colloid gold label;
Multi-channel loading groove: by the multi-channel loading groove of polystyrene or pvc material manufacturing;
Damping fluid: contain 2% hyclone among Tris-HCl of 0.05mol/L pH8.0,0.15mol/L Nacl, 0.5%Tween-20 and 20mg/L gentamicin;
The size scale colour atla: according to the general in the world specific IgE antibody concentration and the relation of grade scale, the size scale colour atla of making (specific IgE content is proportional in the intensity of color and the serum, and with the rising of specific IgE concentration, color deepens gradually).
The nitrocellulose membrane bar of the food allergen albumen of above-mentioned curing and people IgE antibody places the multi-channel loading groove.
Hyperchannel of the present invention is eaten the preparation method of type allergen rapid detection kit, comprises the steps:
1) preparation of allergen protein: the material that will contain allergen protein is through pulverizing, degreasing, extraction, SDS-PAGE electrophoresis conclusive evidence, wash-out, drying;
2) preparation of colloid gold label antibody: with the anti human IgE antibody of colloid gold label;
3) solidify the nitrocellulose membrane bar with allergen protein, and sealing;
4) allergen protein is solidified the nitrocellulose membrane bar and put into the multi-channel loading groove;
5) preparation damping fluid: contain 2% hyclone among Tris-HCl of 0.05mol/L pH8.0,0.15mol/LNacl, 0.5%Tween-20 and 20mg/L gentamicin;
6), make the size scale colour atla according to the general in the world specific IgE antibody concentration and the relation of grade scale.(specific IgE content is proportional in the intensity of color and the serum, and with the rising of specific IgE concentration, color deepens gradually).
Above-mentioned multi-channel loading groove is by polystyrene or pvc material manufacturing.
The using method of kit of the present invention comprises following operation steps:
(1) adds sample liquid in well, allergenic specific IgE antibody in the sample liquid combines with corresponding solid phase allergen protein separately, allergenic specific IgE antibody again with the anti human IgE antibodies of colloid gold label, the anti human IgE antibody of colloid gold label also combines with people IgE in the Quality Control district simultaneously.
(2) add in the damping fluid well, accelerate the serum specimen migration velocity, the colour developing of combined zone;
After (3) 20-30 minutes, with size scale colour atla observations; Specific IgE content is proportional in the intensity of color and the serum.
Be solidificated in the specific IgE antibody in the lip-deep antigen capture sample of the nitrocellulose membrane bar serum, colloid gold label anti human IgE antibody recognition specific IgE and combination with it, form the compound of antigen-specific IgE-colloid gold label anti human IgE antibody, specific IgE content is proportional in the intensity of color and the serum, range estimation can qualitatively judge the result, uses colorimetric card to carry out the sxemiquantitative classification to testing result.
Beneficial effect of the present invention is:
The present invention has set up the allergenic specific IgE antibody content in the hyperchannel immunochromatographic method working sample liquid, qualitative and sxemiquantitative examination sensitization anaphylactogen, detection sensitivity uses colorimetric card to carry out the sxemiquantitative classification to testing result less than 0.3IU/ml, and range estimation can qualitatively judge the result.Can an application of sample in the clinical detection, but 10-20 kinds of anaphylactogens of each passage fast detecting, a plurality of passages can detect 60-100 kinds of different anaphylactogens simultaneously, have easy, quick, sensitive, stable advantage, determining to make the anaphylactogen of patient's sensitization and allergic reaction degree for external auxiliary diagnosis provides effective means.Both can be used for hospital's bedside quick diagnosis, also can be used for community clinic or rural hospital even family.
Description of drawings
Fig. 1 is the vertical view that a hyperchannel is eaten type anaphylactogen quick detection reagent plate;
Fig. 2 is the amino acid composition sequence (background color with same amino acid residue is a grey) of antibodies epitope regions of the storage protein of cereal such as barley;
Fig. 3 is the amino acid composition sequence (background color with same amino acid residue is a grey) of antibodies epitope regions of the fat transfer protein of fruit such as apple;
Specific implementation method
Further specify the present invention below in conjunction with embodiment.
Hyperchannel of the present invention is eaten the type allergen rapid detection kit, its composition comprises the nitrocellulose membrane bar of the milk, eggs, meat, cereals, nut fruits, fruits, greengrocery, aquatic product, bean food allergen protein and the people IgE antibody that have solidified, the multi-channel loading groove, colloid gold label anti human IgE antibody, damping fluid, size scale colour atla.
Hyperchannel of the present invention is eaten the preparation method of type allergen rapid detection kit:
1. the preparation of allergen protein
1.1 the thick leaching liquor of allergen protein
1.1.1 milk extract
The milk immersion liquid be manufactured with two kinds of methods:
(1) uses centrifugal 15 minutes of centrifuge method (2400 rev/mins) earlier, remove upper strata fat with little spoon, get the 400ml skimmed milk, add 5ml1% renin or junket sheet, do not stir, put into 37 ℃ of waters bath with thermostatic control or incubator 30 minutes, separate with casein (casein) as lactalbumin, use clean filtered through gauze, use common filter paper filtering again.In the ratio of 1:2 (W/V), be the lactalbumin extract with buffer saline extract dilution lactalbumin.
(2) filtrate that will contain whey adds 3 times of acetone, puts into refrigerator 24h, makes the whey precipitation; Remove supernatant with centrifuge method, embathe several times with acetone, dry back porphyrize becomes powder to store.Press 1:50 (W/V) during extraction and extracted in refrigerator 48 hours with the buffer saline extract, stir or vibrated 2 hours every day.
Casein is with acetone and ether degreasing repeatedly, grind at last, sifts out with No. 3 or No. 4, extracts in 1:50 (W/V) ratio.
1.1.2 eggs extract
Albumen and yolk all dilute with the buffer saline extract in the ratio of 1:20 (W/V), and fully direct filtration and degerming behind the mixing need do not extracted, and also need not to do toxicity test.
Yolk also can be made into dry powder, and method is to add acetone degreasing 2-3 hour, the acetone layer that inclines, treat volatile dry after, smash with beating crusher, use ether defatting 4 hours standby again.Yolk dry powder extracts with the buffer saline extract in 1:50 ratio (W/V).
1.1.3 meat extract
Get fresh lean meat, remove fat and connective tissue, cut into broken end, or rub with meat grinder.Alternately, dry under the room temperature with different solvents degreasings repeatedly such as toluene, dimethylbenzene, acetone and ether, sieve, be stored in the airtight vial standby.
Extracted 48 hours with the buffer saline extract in the ratio of 1:25 (W/V), stir or vibrated 2 hours every day in the leaching process.
After the coarse filtration as the liquid muddiness, the degreasing again that adds diethyl ether of available separating funnel.
1.1.4 cereal, nut, peas protein extract
(1) dry, shell, decortication, abrasive dust store; In apparatus,Soxhlet's in powder: the ratio degreasing of normal hexane=1:10 (W/V) 6 hours, the drying defatted powder is levigate again store in the airtight container under-20 ℃ standby.
(2) (contain 20mmol NaH2PO4,1mol/L NaCl pH7.0) extracts albumen to skimmed milk with extracting damping fluid; Skimmed milk: the ratio of extracting damping fluid is 1g:10ml (W/V), and room temperature was extracted 1.5 hours, and 4 ℃ of following 20000g centrifugal 30 minutes then, preserve supernatant.
1.1.5 fruit, greengrocery protein extract
100g fruit (comprising pericarp, stoning), vegetables are cleaned, added 150ml 20mmol/lPBS (pH7.4) solution behind the airing, chopping, wherein contain 2% crospovidone (PVPP) suspended particle, the EDTA of 2mmol/l, diethyl disulfide group carbamate (DIECA) sodium of 10mmol/l and the NaN3 of 3mmol/l, homogenate is extracted after 4 hours for 4 ℃, 12000g4 ℃ was descended centrifugal 30 minutes, supernatant-20 ℃ preservation down.
1.1.6 aquatic product protein extract
Get fresh aquatic products meat, degrease is pulverized.With dry under acetone or ether degreasing repeatedly, the room temperature, sieve, be stored in the airtight vial standby.
Extracted 48 hours with the buffer saline extract in the ratio of 1:25 (W/V), stir or vibrated 2 hours every day in the leaching process.
After the coarse filtration as the liquid muddiness, the degreasing again that adds diethyl ether of available separating funnel.
1.2 the extraction of allergen protein
The crude extract of allergen protein needs the purer allergen protein component through dialysis, the affirmation of SDS-PAGE electrophoresis Western blotting, rubber tapping, centrifugal extraction.
(1) with leaching liquor in bag filter (MWCO 10000) to 10mmol/L PBS (pH7.2) dialysis 48 hours, during slowly stir PBS solution, and change PBS liquid 4 times;
(2) the allergen protein solution after the dialysis is crossed 0.45 μ m filter membrane, and is diluted to the protein solution of 4mg/ml;
(3) allergen protein solution pours into the glue post of SDS-PAGE, electrophoretic separation; Zone at molecular weight 10-100kD is carried out Western blotting with patient's positive serum at a running gel post, colour developing;
(4) the following respective strap of all the other glue post allergen proteins location of cutting, add an amount of PBS solution after, centrifugal 10 minutes of room temperature 2000g;
(5) measure the concentration of allergen protein solution, add an amount of antiseptic ,-20 ℃ of following refrigerated storages.
2. the preparation of colloid gold label antibody
2.1 Preparation of Colloidal Gold
Reducing process is adopted in the preparation of collaurum more.Gold chloride (HAuCl
4) be mainly to be reduced material, reductive agent commonly used has sodium citrate, tannic acid, ascorbic acid, white phosphorus, sodium borohydride etc.According to the power of reductive agent type and reducing action, can prepare the collaurum that 0.8nm～150nm does not wait.The most frequently used preparation method is the citrate reducing process.Concrete operation method is as follows: a. is with HAuCl
4Be mixed with 0.01% aqueous solution earlier, get 100ml and be heated to and boil; B. stir and accurately add a certain amount of 1% trisodium citrate (Na3C6H5O72H2O) aqueous solution down; C. continue heated and boiled 15min.Can be observed flaxen aqueous solution of chloraurate and gray very soon after sodium citrate adds this moment, continuous and change into black, stablizes gradually subsequently to become red.About 2～the 3min of overall process; Return to original volume with distilled water after being cooled to room temperature.The collaurum that can prepare 16～147nm particle diameter with this method.The amount of the trisodium citrate that adds when the size of gold grain depends on preparation.
2.2 colloid gold label anti human IgE antibody
Using colloid gold label protein, is exactly that finger protein matter is adsorbed onto the colloid gold particle surface.The collaurum surface has negative charge, can form firm combining with the positive charge electrostatic attraction of protein.Because this combination mainly is a physical action, so can not cause the change of protein active.Before the mark, the isoelectric point or slightly high (pH8.2) that need to determine the ratio of collaurum and protein to be marked and the pH value is adjusted to labelled protein IgG.During mark, anti human IgE antibody 1mg to be marked is added 15ml colloidal gold solution (1OD), mix, stirring at room 15 minutes, adding 5% bovine serum albumin(BSA) (BSA) under magnetic stirs, to make its final concentration be 1%, mixed 5 minutes, centrifugal, the anti human IgE antibody by centrifuge method or gel chromatography purifying mark again.Being placed on 4 ℃ reaches and still kept superperformance in 2 years half.
3. solidify the nitrocellulose membrane bar with allergen protein
3.1 material and instrument
(1) nitrocellulose filter (Millipore, MDI, the S﹠amp that cross of activation processing; S, the nitrocellulose filter of offshore companies such as whatman), can form stable covalent bond with the amino group reaction in the protein molecule.
(2) allergen protein;
(3) 0.05mol/L Tris-HCl damping fluid (pH8.0) or activation reinforcing agent;
(4) 0.01mol/L PBS solution;
(5) 3%BSA confining liquid;
(6) nano chips specking system;
(7) metallic foil bag and vacuum sealer etc.
3.2 step
(1) the nitrocellulose filter bar of allergen protein preparation
Adopt Millipore, MDI, S﹠amp; S, the nitrocellulose filter of whatman medium flow rate.Reagent pad, sample pad, absorption pad adopt Millipore company product, compare with slide, and the advantage of film is strong with protein affinity, and the detection technique maturation need not other modification usually.Nitrocellulose filter, nylon membrane, PvDF film etc. all are the filter membranes that has micropore, and its aperture about 0.45 μ m, is exactly the network of fibers of a solid at this film of microscopically generally, can make detection sensitive more in conjunction with more protein probe.Simultaneously, this porous structure can make liquid carry out therein freely spreading, and when when an end of film is given the liquid diffusion pressure, liquid will spread in a certain direction, be the good material of chromatography, is the very desirable carrier of making the chromatography allergen protein.
(2) point sample
The concrete 0.05mol/L carbonic acid buffer that adopts, pH9.6 or the damping fluid of selecting according to the characteristic of allergen protein suitably dilute the concentration that allergen protein solution becomes 0.05 μ g/ml-10 μ g/ml.After the dilution allergen protein (100 μ l) is added in the spray sample liquid storage bottle, use micro-continuous type point film machine (as the product of BIO-DOT company), allergen solution is sprayed line in the contactless continuous point sample in the corresponding site of film, i.e. specking, each point sample 1 μ l, continuous 3 times; Reaction was spent the night under nitrocellulose filter behind the specking was placed 2-8 ℃.Use the 3%BSA confining liquid, 37 ℃ are incubated 2 hours down.General 25-the 30mm of chromatographic film bar is long, can spray 2-10 lines of line or specking 2-30 points,
(3) the Quality Control district of the curing people IgE positive;
A. material
People IgE antibody: derive from the human plasma through affinity chromatography purifying, purity〉95% people IgE antibody (available from U.S. Biodesign company).
B. step
With sample diluting liquid people IgE antibody being mixed with concentration is 50IU/ml, solidifies people IgE antibody in allergen protein nitrocellulose filter bar upper end and is the positive quality control district.
4. allergen protein is solidified the nitrocellulose membrane bar and put into the multi-channel loading groove
Adopt cutting machine that the allergen protein nitrocellulose filter bar that is cured is cut into required specification and shape, put into corresponding multi-channel loading groove, add the drying agent sealing in the metallic foil bag of packing into again, store down at 2-8 ℃.
Hyperchannel is eaten the type allergen rapid detection kit and is had 20 hyperchannels and eat type anaphylactogen quick detection reagent plate, every agent plate as shown in Figure 1,1 is the sample pipetting volume window among the figure, the 2 nitrocellulose membrane bars that solidify for allergen protein, 3 is the loading slot microchannel, and 4 are adsorptive pads outlet window.
5. preparation damping fluid: contain 2% hyclone among Tris-HCl of 0.05mol/L pH8.0,0.15mol/LNacl, 0.5%Tween-20 and 20mg/L gentamicin;
6. the size scale colour atla is made
According to the relation of specific IgE antibody concentration and grade scale in the world, print the size scale colour atla.
Hyperchannel of the present invention is eaten the using method of type allergen rapid detection kit
1. detection step
(1) with all reagent balances to room temperature (20-25 ℃).
On check-out console, add 2～3 in the specimen hole with the application of sample suction pipe when (2) measuring and bleed clearly (if use quantitative sample injector, every hole adds 100～150 μ l serum), add 1 of damping fluid when serum specimen migrates to Quality Control district index line.
(3) 20-30 minutes observationss.
2. the result judges
A. range estimation:
(1) positive findings: the Quality Control district red reaction zone occurs with the detection line at the corresponding parallel place of anaphylactogen on check-out console.
(2) feminine gender: only a red stripes appears in the Quality Control district
(3) invalid: red stripes does not appear in the Quality Control district
But b. use size scale colour atla sxemiquantitative classification to judge positive findings:
The index that is positive should be answered basically identical with size scale colour atla rank, and its classification results only allows to change between adjacent two ranks.
The stage division of allergenic specific IgE concentration in the world:
Embodiment 2 examination type hyperchannels are eaten the common pathergy of inferring protein on the molecular level by cell in conjunction with the identification of the IgE conjugated antigen determinant of the reactive protein of IgE that is chosen in of solidifying allergen protein in the type allergen rapid detection kit.U.S.'s allergy, (the theAmerican Academy of Allergy of asthma and immunology association, Asthma and Immunology (AAAAI)) and U.S.'s allergy, (the American college of Allergy of asthma and immunology institute, Asthma and Immunology (ACAAI)) unites the file of acceptance: think in " food hypersenstivity: practical parameter (Food allergy:a practiceparameter) ", it is because the function amino acid sequence that protein exists that protein inspires allergic reaction, the structure that comprises on the linear order or can combine with specific receptor protein on the B cell on the conformation, i.e. antigenic determinant.If the identity property of the amino acid sequence of one or several main allergen protein that two kinds of protein or polypeptide all contain reaches 70% or two protein or polypeptide is shared amino acid sequence consecutive identical more than 8 or 8, just can think that these two protein or polypeptide exist the antigenic determinant of identical allergic symptom (Chapman JA et al, 2006, Food allergy:a practice parameter, Ann Allergy AsthmaImmunol, 96:S1-S68).According to this principle, the amino acid sequence of the main allergen protein of retrieval comparison sensitization material of the same type large database from the internet: NCBI, PubMed, EBI or the document, the allergen protein mixed in equal amounts that will meet the material of this principle is solidificated in the same site surface of nitrocellulose filter bar jointly.For example the identity property of the amino acid sequence in the antigenic determinant zone of the main allergen protein (seed storage protein) of five kinds of cereal such as barley is about 40% in the comparison diagram 2, and the identity property of three kinds of cereal such as corn, rice, millet is about 70%, so just the allergen protein of these three kinds of cereal is solidificated in the same site surface of nitrocellulose filter bar.Main allergen protein (the fat transfer protein of six kinds of fruit such as apple, the identity property of the amino acid sequence in antigenic determinant zone LTP) reaches 72%, and the zone (Fig. 3) that has two 8 consecutive identical amino acid sequences also is solidificated in their allergen protein the same site surface of nitrocellulose filter bar jointly.
To 73 routine autopaths (2-18 years old 30 people, 18-70 years old 43 people), extract blood and prepare serum qualitatively screening (milk; Albumen/yolk; Fresh-water fishes; Shrimp/crab; Marine fish; Cockroach) anaphylactogen.
Eat the using method of type allergen rapid detection kit by above-mentioned hyperchannel and measure the patients serum, be judged as positive findings according to the size scale colour atla in different allergenic specific IgE concentration.Testing result such as following table:
| Anaphylactogen | Milk | Albumen yolk | The shrimp crab | Marine fish | Fresh-water fishes | Nut fruits |
| The positive detects number | 6 | 7 | 9 | 5 | 3 | 5 |
| Positive rate (%) | 8.2 | 9.5 | 10.8 | 12.3 | 4.0 | 6.8 |
Testing result shows, adopts kit of the present invention to detect the sensitization anaphylactogen easy, quickly and accurately, and sensitivity reaches 92.0%, and specificity reaches 85.0%, accuracy 86%.
Claims (3)
1, hyperchannel is eaten the type allergen rapid detection kit, it is characterized in that this kit composition comprises: the nitrocellulose membrane bar of the milk that has solidified, eggs, meat, cereals, nut fruits, fruits, greengrocery, aquatic product, bean food allergen protein and people IgE antibody, the antibody of colloid gold label, damping fluid, the size scale colour atla;
Wherein:
The nitrocellulose membrane bar of the milk food allergen albumen that has solidified comprises: milk, goat milk;
The nitrocellulose membrane bar that has solidified the eggs food allergen albumen of quilt comprises: poultry albumen, poultry yolk;
The nitrocellulose membrane bar of the meat food allergen protein that has solidified comprises: pork, beef, mutton, duck, chicken, goose, turkey meat, donkey meat;
The nitrocellulose membrane bar of the cereals food allergen albumen that has solidified comprises: corn, wheat, oat, barley, buckwheat, rice, millet;
The nitrocellulose membrane bar of the nut fruits food allergen albumen that has solidified comprises: almond, peanut, fibert, cashew nut, walnut, American pistachios, Brazilian nut, sesame;
The nitrocellulose membrane bar of the fruits food allergen albumen that has solidified comprises: watermelon, pineapple, orange, mango, banana, citrus, Hu shaddock, lemon, apple, grape, pears, strawberry, peach;
The nitrocellulose membrane bar of the greengrocery food allergen albumen that has solidified comprises: celery, spinach, onion, eggplant, garlic, capsicum, potato, tomato;
The nitrocellulose membrane bar of the aquatic product food allergen albumen that has solidified comprises: hairtail, yellow croaker, flatfish, salmon; Carp, crucian, grass carp, silver carp, shrimp, crab, freshwater mussel, mussel, oyster, swamp eel, eel;
The nitrocellulose membrane bar of the bean food allergen protein that has solidified comprises: French beans, broad bean, soya bean, pea;
The antibody of colloid gold label: the anti human IgE antibody of colloid gold label;
Damping fluid: contain 2% hyclone among Tris-HCl of 0.05mol/L pH8.0,0.15mol/L Nacl, 0.5%Tween-20 and 20mg/L gentamicin;
Colorimetric card: according to the general in the world specific IgE antibody concentration and the relation of grade scale, the size scale colour atla of making.
2. hyperchannel according to claim 1 is eaten the type allergen rapid detection kit, it is characterized in that the food allergen albumen that solidifies and the nitrocellulose membrane bar of people IgE antibody place the multi-channel loading groove.
3. hyperchannel according to claim 1 is eaten the preparation method of type allergen rapid detection kit, it is characterized in that comprising the steps:
1) preparation of allergen protein: the material that will contain allergen protein is through pulverizing, degreasing, extraction, SDS-PAGE electrophoresis conclusive evidence, wash-out, drying;
2) preparation of colloid gold label antibody: with the anti human IgE antibody of colloid gold label;
3) solidify the nitrocellulose membrane bar with allergen protein and people IgE antibody, constitute p-wire and Quality Control district, and sealing;
4) will solidify the nitrocellulose membrane bar of handling and put into the multi-channel loading groove;
5) preparation damping fluid: contain 2% hyclone among Tris-HCl of 0.05mol/L pH8.0,0.15mol/LNacl, 0.5%Tween-20 and 20mg/L gentamicin;
6), make the size scale colour atla according to the general in the world specific IgE antibody concentration and the relation of grade scale.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101222901A CN101393214A (en) | 2008-11-17 | 2008-11-17 | Multichannel ingestion type allergen rapid detection kit and method for making same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101222901A CN101393214A (en) | 2008-11-17 | 2008-11-17 | Multichannel ingestion type allergen rapid detection kit and method for making same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101393214A true CN101393214A (en) | 2009-03-25 |
Family
ID=40493609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008101222901A Pending CN101393214A (en) | 2008-11-17 | 2008-11-17 | Multichannel ingestion type allergen rapid detection kit and method for making same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101393214A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103439502A (en) * | 2013-06-28 | 2013-12-11 | 北京新华联协和药业有限责任公司 | Rapid specific antibody IgE detection kit and preparation method thereof |
| CN103995109A (en) * | 2014-05-28 | 2014-08-20 | 中生北控生物科技股份有限公司 | Test strip for screening specific fruits allergens IgE (Immunoglobulin E) and preparation method thereof |
| CN103995111A (en) * | 2014-05-28 | 2014-08-20 | 中生北控生物科技股份有限公司 | Test strip for screening vegetables food allergens IgE (Immunoglobulin E) and preparation method thereof |
| CN103995110A (en) * | 2014-05-28 | 2014-08-20 | 中生北控生物科技股份有限公司 | Test strip for screening specific aquatic products allergens IgE (Immunoglobulin E) and preparation method thereof |
| CN104280555A (en) * | 2014-10-24 | 2015-01-14 | 江南大学 | Colloidal gold test strip for detecting peanut allergen Arah2 rapidly and preparation method thereof |
| CN107727864A (en) * | 2016-07-01 | 2018-02-23 | 首都医科大学附属北京佑安医院 | The protein chip of abnormal decarboxyprothrombin, kit and preparation method thereof in a kind of detection serum |
| CN116087500A (en) * | 2022-12-26 | 2023-05-09 | 科赫生物科技（北京）有限公司 | Multi-joint detection device and application method thereof |
-
2008
- 2008-11-17 CN CNA2008101222901A patent/CN101393214A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103439502A (en) * | 2013-06-28 | 2013-12-11 | 北京新华联协和药业有限责任公司 | Rapid specific antibody IgE detection kit and preparation method thereof |
| CN103439502B (en) * | 2013-06-28 | 2015-12-23 | 北京新华联协和药业有限责任公司 | A kind of rapid specific antibody IgE detection kit and preparation method thereof |
| CN103995109A (en) * | 2014-05-28 | 2014-08-20 | 中生北控生物科技股份有限公司 | Test strip for screening specific fruits allergens IgE (Immunoglobulin E) and preparation method thereof |
| CN103995111A (en) * | 2014-05-28 | 2014-08-20 | 中生北控生物科技股份有限公司 | Test strip for screening vegetables food allergens IgE (Immunoglobulin E) and preparation method thereof |
| CN103995110A (en) * | 2014-05-28 | 2014-08-20 | 中生北控生物科技股份有限公司 | Test strip for screening specific aquatic products allergens IgE (Immunoglobulin E) and preparation method thereof |
| CN103995110B (en) * | 2014-05-28 | 2016-03-02 | 中生北控生物科技股份有限公司 | For the test strips and preparation method thereof of specificity aquatic product anaphylactogen IgE examination |
| CN103995111B (en) * | 2014-05-28 | 2016-03-23 | 中生北控生物科技股份有限公司 | For the test strips and preparation method thereof of greengrocery food allergen IgE examination |
| CN104280555A (en) * | 2014-10-24 | 2015-01-14 | 江南大学 | Colloidal gold test strip for detecting peanut allergen Arah2 rapidly and preparation method thereof |
| CN107727864A (en) * | 2016-07-01 | 2018-02-23 | 首都医科大学附属北京佑安医院 | The protein chip of abnormal decarboxyprothrombin, kit and preparation method thereof in a kind of detection serum |
| CN116087500A (en) * | 2022-12-26 | 2023-05-09 | 科赫生物科技（北京）有限公司 | Multi-joint detection device and application method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101387643A (en) | Multichannel allergen rapid detection kit and method for making same | |
| CN101178406A (en) | Allergen specificity antibody IgE ELISA detection reagent box and method of producing the same | |
| CN101393214A (en) | Multichannel ingestion type allergen rapid detection kit and method for making same | |
| CN101109750A (en) | Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof | |
| CN101393215A (en) | Multichannel attractive type allergen rapid detection kit and method for making same | |
| Vieths et al. | Apple allergy: the IgE‐binding potency of apple strains is related to the occurrence of the 18‐kDa allergen | |
| CN102993299B (en) | Food allergens, method of detecting food allergens and method of detecting food allergy-inducing foods | |
| CN101256186A (en) | Food anaphylactogen specificity IgG4 antibody ELISA detection kit and preparation method thereof | |
| Lemanske et al. | Standardized extracts, foods | |
| CN101696973A (en) | Allergenic specific IgE antibody immunoassay detection kit and preparation method thereof | |
| Achilova et al. | Clinical-immunological and medico-Social aspects of allergic diseases in children, development of criteria for early diagnosis and prognosis of the course of the disease (literature review) | |
| KR20170054522A (en) | Compositions, devices, and methods of ibs sensitivity testing | |
| Steinhoff et al. | Comparison of extraction conditions for milk and hen's egg allergens | |
| CN103454412A (en) | Liquid phase chip for detecting allergen specific antibody and preparation method of liquid phase chip | |
| CN102798721A (en) | Food allergen detection kit and food allergen detection method | |
| KR101510376B1 (en) | Shrimp allergen, anti-shrimp allergen antibody, and use thereof | |
| Yman | Standardization of in vitro methods | |
| CN102341705A (en) | Diagnostic reagent for crohn's disease | |
| JP4958369B2 (en) | Food allergen, method for detecting food allergen and method for detecting food allergy-inducing food | |
| Kharrazian et al. | Detection of islet cell immune reactivity with low glycemic index foods: Is this a concern for type 1 diabetes? | |
| Ding et al. | Reducing the allergenicity of pea protein based on the enzyme action of alcalase | |
| Boye et al. | Food allergens | |
| US8377696B2 (en) | Highly sensitive method for detecting protein in food | |
| Panase et al. | Visceral and serum lysozyme activities in some freshwater fish (three catfish and two carps) | |
| Alessandri et al. | Molecular allergology approach to allergic diseases in the paediatric age |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20090325 |