CN101109750A - Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof - Google Patents
Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof Download PDFInfo
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- CN101109750A CN101109750A CNA2007100701980A CN200710070198A CN101109750A CN 101109750 A CN101109750 A CN 101109750A CN A2007100701980 A CNA2007100701980 A CN A2007100701980A CN 200710070198 A CN200710070198 A CN 200710070198A CN 101109750 A CN101109750 A CN 101109750A
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Abstract
The invention discloses a food allergen specificity IgG ELISA inspection reagent box, which comprises a standard enzyme board for such food allergen protein as milk, egg, meat, grain, nut, fruit, vegetable, aquatic products and beans, etc., a standard liquid of human IgG, an enzyme-marking antibody, a TMB background color-developing liquid, a sample diluting liquid, a concentrated cleaning mixture and a terminating reaction liquid. The invention is for detecting the concentration of food allergen specificity IgG antibody in the serum of human being, the sensitizing food allergen is screed by an allergen sieve of the sample serum with absorbancy higher than the cut-off value; the specificity IgG concentration in the sample serum is calculated from a standard curve, the anaphylactic reaction degree is determined in a quantitative grading way. The invention is flexible, rapid, simple and steady, can be used for out-body clinic auxiliary diagnosis and scientific research of anaphylaxis diseases.
Description
Technical field
The present invention relates to Enzyme Linked Immunoadsorbent Assay (ELISA) detection kit and the production method thereof of food allergen specificity IgG in a kind of detection by quantitative human serum, sensitization food allergen and allergic reaction degree are determined in examination.
Background technology
Anaphylactia is modal a kind of disease in the modern society, and it is to be caused by the anaphylactogen of extensive existence, and the people of the nearly 5-10% of China is invaded and harassed by anaphylactogen.Allergic reaction (allergic reaction) claim allergic reaction again, and anaphylactogen (allergen) claim allergen again.Allergic reaction has two main types: by the type of IgE mediation, take place immediately in the contact allergy former back short time usually, illness is obvious; Delayed hypersensitivity by IgG and subclass IgG4 mediation thereof just reaction symptom can occur in contact allergy after former several hours or several days usually, and chronic fatigue, arthritis, nettle rash, eczema, headache, functions of intestines and stomach imbalance and other chronic sympton are arranged.The allergic reaction of IgG mediation, normally cause by food allergen, and patient's skin test is negative more, press for that examination goes out to cause anaphylactoid anaphylactogen from numerous foods, and then prevention and treatment to the ill well (period-luminosity inflammation etc. is translated, and 2002, immunology, PP323-369, the People's Health Publisher; Sampson HA, 1999, Food allergy, part1, J Allergy ClinImmunol, 103:717-728).
Behind autopath's dietary intake anaphylactogen, IgG antibody that produces in the body and subclass thereof with take off granular basophilic granulocyte and loose giant cell combines, graded cell and tissue that activation is complementary, initiation allergic reaction; In autopath's blood, can detect IgG and subclass concentration thereof rising (BrightonWD, 1980, Frequency of occurrence of IgG (S-TS), Clin Allergy, 10:97-100).The autopath is once more behind the dietary intake anaphylactogen, improve specific IgG concentration in the blood circulation, carried out the detection of immunization therapy patient's serum IgG concentration and repeatedly confirmed (Kemeny DM etal for alleviating the allergic reaction illness, 1986, Subclass of IgG in allergic disease, Clim Allergy, 16:571-574).
According to the big quantity research over nearly 40 years, food allergen divides two big classes: vegetable protein and animal protein.By their 26S Proteasome Structure and Function characteristic, contain anaphylactogen vegetable protein be the superfamily of the alcohol soluble protein in legume, tree nut, cereal, fruit, the vegetables etc., protein family (the Breiteneder H er al 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, 2004, A classification of plant food allergens, J AllergyClim Immunol, 113:821-830); The source that contains the animal protein of anaphylactogen is mammiferous meat and lactoprotein, the meat albumen of birds, fish, crustacean, mollusc, amphibian and Reptilia etc., and the source is extensively sent out, is enriched; With they be raw material also produced unnumbered " nutritious and healthy food " (WalJM, 1999, Nahrung 43:s168-174), presses for the vitro detection food allergen.
Allergic reaction clinically can be by Skin-test and various external detection method, as radioimmunoassay (RIA) method, Western blotting (IS), immunoprecipitation (IP) and Enzyme Linked Immunoadsorbent Assay (ELISA) method etc., wait to determine anaphylactogen (the Sampson HA of sensitization in conjunction with disease symptom, 1999, Foodallergy, part2, J Allergy Clin Immunol, 103:981-989).The ELISA method is than method safety such as RIA, IS, IP, simple, fast economic, be current in the world with fastest developing speed and the most frequently used vitro detection sensitization anaphylactogen and irritated degree methods thereof (Yunginger JW er al, 2002, J Allergy ClinImmunol, 105:1077-1084).
The ELISA kit detects the required technology of allergen protein in the food, method of purification as allergen protein, human IgG antibody's preparation, purifying, anti-human IgG antibody produces purification, all ripe (Porstmann T such as the preparation purifying of enzymic-labelled antibody, kiessig ST, 1992, Enzyme immunoassay techniques, Anoverview, J Immunol Methods 150:5-21) has set up technical foundation for the commercialization production of food allergen specificity IgG ELISA detection kit, also produced commercialization vitro detection kit abroad, as the immuno CAP of Pharmacia company (www.purduepharma.com)
TM100 specificIgG kit, the Allerquant IgG food allergyacreening ELISA kit of Biomerica company (www.biomerica.com) etc.
Summary of the invention
The object of the present invention is to provide a kind of highly sensitive, high specificity, easy to use, the food allergen specificity IgG ELISA detection kit in the detection by quantitative human serum apace, in order to the food allergen of examination patient sensitization and determine the degree of patient's sensitivity response.
Food allergen specificity IgG ELISA detection kit of the present invention, its composition comprise ELISA Plate, human IgG titer, enzymic-labelled antibody, tmb substrate colour developing liquid, sample diluting liquid, concentrated cleaning solution and the cessation reaction liquid of the milk that wraps quilt, eggs, meat, cereals, nut fruits, fruits, greengrocery, aquatic product, bean food allergen protein;
Wherein:
The ELISA Plate of having wrapped the milk food allergen albumen of quilt comprises: milk, goat milk;
The ELISA Plate of having wrapped the eggs food allergen albumen of quilt comprises: poultry albumen, poultry yolk;
The ELISA Plate of having wrapped the meat food allergen protein of quilt comprises: pork, beef, mutton, duck, chicken, goose, turkey meat, donkey meat;
The ELISA Plate of having wrapped the cereals food allergen albumen of quilt comprises: corn, wheat, oat, barley, buckwheat, rice, millet;
The ELISA Plate of having wrapped the nut fruits food allergen albumen of quilt comprises: almond, peanut, fibert, cashew nut, walnut, American pistachios, Brazilian nut, sesame;
The ELISA Plate of having wrapped the fruits food allergen albumen of quilt comprises: watermelon, pineapple, orange, mango, banana, citrus, Hu shaddock, lemon, apple, grape, pears, strawberry, peach;
The ELISA Plate of having wrapped the greengrocery food allergen albumen of quilt comprises: celery, spinach, onion, eggplant, garlic, capsicum, potato, tomato;
The ELISA Plate of having wrapped the aquatic product food allergen albumen of quilt comprises: hairtail, yellow croaker, flatfish, salmon; Carp, crucian, grass carp, silver carp, shrimp, crab, freshwater mussel, mussel, oyster, swamp eel, eel;
The ELISA Plate of having wrapped the bean food allergen protein of quilt comprises: French beans, broad bean, soya bean, pea;
Enzymic-labelled antibody: the anti-human IgG monoclonal antibody of horseradish peroxidase-labeled;
Tmb substrate colour developing liquid: 2.08mmol/L 3,3 ', 5,5 '-tetramethyl benzidine, 12mmol/L hydrogen peroxide, 10mmol/L beta-schardinger dextrin-, the acetate buffer solution of 0.1mol/L pH5.0.
Sample diluting liquid: contain 1%BSA in the phosphate buffer of 10mmol/L pH7.4-7.6,0.05%Tween-20 and 1mg/L gentamicin;
Concentrated cleaning solution: contain 1% hyclone in the phosphate buffer of 0.1mol/L pH7.4-7.6,0.5%Tween-20 and 20mg/L gentamicin;
Cessation reaction liquid: 1.0mol/L H
2SO
4
The preparation method of food allergen specificity IgG ELISA detection kit of the present invention comprises the steps:
(1) preparation of food allergen albumen: the food that will contain allergen protein is through pulverizing, degreasing, extraction, SDS-PAGE electrophoresis conclusive evidence, wash-out, drying;
(2) preparation of enzymic-labelled antibody: with horseradish peroxidase (Horseradish peraxidase, HRP) the anti-human IgG antibody of mark purifying;
(3) preparation human IgG titer, tmb substrate colour developing liquid, concentrated cleaning solution and cessation reaction liquid;
(4) ELISA Plate bag quilt: with anti-human IgG antibody and food allergen albumen coated elisa plate, and sealing.
The using method of kit of the present invention comprises following operation steps:
(1) adds sample liquid and titer in corresponding ELISA Plate micropore, food allergen specificity IgG antibody in the sample liquid is combined with corresponding solid phase allergen protein separately or titer in human IgG combine the unconjugated foreign protein of flush away and other materials with anti-human IgG antibody on the solid phase carrier;
(2) make the human IgG antibody of combination combine the enzymic-labelled antibody that flush away is unnecessary with the anti-human IgG antibody of HRP-;
(3) add tmb substrate colour developing liquid, incubated at room reaction, the intensity of detection chromogenic reaction after the termination enzyme reaction.
Beneficial effect of the present invention is:
The present invention has set up the letter of sandwich ELISA method and has connect food allergen specificity IgG antibody content and qualitatively screening sensitization food allergen in the working sample liquid, detection sensitivity is less than 0.7U/ml, the linearly dependent coefficient of dose-response curve 〉=0.99, have easy, quick, sensitive, stable advantage, determining to make the food allergen of patient's sensitization and allergic reaction degree for external auxiliary diagnosis provides effective means.
Specific implementation method
Further specify the present invention below in conjunction with embodiment.
Embodiment 1
Food allergen specificity IgG ELISA detection kit of the present invention, its composition comprise ELISA Plate, human IgG titer, enzymic-labelled antibody, tmb substrate colour developing liquid, sample diluting liquid, concentrated cleaning solution and the cessation reaction liquid of the milk that wraps quilt, eggs, meat, cereals, nut fruits, fruits, greengrocery, aquatic product, bean food allergen protein.
Above-mentioned ELISA Plate is 96 (12 8 holes or 8 12 holes).
The preparation method of food allergen specificity IgG ELISA detection kit:
1. the preparation of food allergen albumen
1.1 the thick leaching liquor of food allergen albumen
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 5ml 1% 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.Extracted in refrigerator 48 hours with the buffer saline extract by 1: 50 (W/V) during extraction, 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, in (W/V) ratio extraction in 1: 50.
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 in 1: 50 ratio (W/V) usefulness buffer saline extract.
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) skimmed milk (contains 20mmol NaH with extracting damping fluid
2PO
4, 1mol/L NaCl pH7.0) extracts albumen; 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
Clean, airing, blend, extract; Get part and measure moisture.Extract damping fluid by 1g (dry weight): 10ml and (contain 20mmol NaH
2PO
4, 1mol/L NaCl pH7.0) extracts albumen; Room temperature was extracted 1.5 hours, and 4 ℃ of following 20000g centrifugal 30 minutes then, preserve supernatant.
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 food allergen albumen
The crude extract of food allergen albumen 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 the 0.45um 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 human IgG titer
2.1 material
Human IgG antibody: derive from the human plasma through the affinity chromatography purifying human IgG antibody of purity＞95% (available from U.S. Biodesign company).
2.2 step
To resist the human IgG antibody to be mixed with concentration with sample diluting liquid is 200ug/ml human IgG titer.
3. coated elisa plate
3.1 material
(1) ELISA Plate crossed of activation processing (Nunc, Nunc-Immuno platesTM, USA or Greiner, Greiner labortechnik, Germany);
(2) food allergen albumen;
(3) 0.05mol/L carbonic acid buffer (pH9.6) or activation coating buffer;
(4) 0.01mol/L PBS solution;
(5) 3%BSA confining liquid;
(6) metallic foil bag and vacuum sealer etc.
3.2 step
(1) with coating buffer (0.05mol/L carbonic acid buffer, pH9.6 or the activation coating buffer of selecting according to the characteristic of allergen protein) suitably dilute allergen protein solution and become the bag of 0.05ug/ml-10ug/ml by concentration, mixing waits to wrap that every hole adds 100ul in the ELISA Plate micropore that the activation processing of quilt crosses;
(2) 4 ℃ of following reactions are spent the night behind the ELISA Plate envelope film;
(3) outwell antibody and the antigenic solution that wraps quilt, wash plate, every hole adds 250ul 3%BSA confining liquid, and 4 ℃ of following sealings are spent the night;
(4) the turned letter confining liquid is washed plate 4 times with cleansing solution, pats dry;
(5) with after the ELISA Plate vacuum drying, be encapsulated in the metallic foil bag of band drying agent, store down at 4 ℃.
4. the preparation of enzymic-labelled antibody (the anti-human IgG antibody of horseradish peroxidase (HRP) mark)
4.1 material
(1) horseradish peroxidase (HRP, RZ＞3.2, activity＞200U/ml; Available from U.S. SIGMA company);
(2) 0.1mol/L NaIO
4Solution;
(3) 0.2mol/L carbonic acid buffer, pH9.5;
(4) anti-human IgG antibody (available from U.S. KPL company);
(5) NaBH of 4.0mg/ml
4Solution;
(6) PBS of 0.15mol/L, pH7.4;
(7) Protein A-Sepharose 4 Fast Flow affinity chromatography column material and chromatographic columns;
(8) citric acid, phosphoric acid and the Tris damping fluid used of affinity chromatography purifying;
(9) Sephadex G25 gel chromatographic columns;
(10) spectrophotometer etc.
4.2 step
(1) takes by weighing 5mg HRP and be dissolved in the 1ml distilled water, add the 0.1mol/LNaIO that 0.2ml newly joins then
4Solution, lucifuge stirred 20 minutes under the room temperature;
(2) above-mentioned solution is packed in the bag filter, to the sodium-acetate buffer dialysis of 1mol/L pH4.4,4 ℃ are spent the night;
(3) add 20 μ l 0.2mol/L pH9.5 carbonate buffer solutions, make the pH of the HRP solution of above hydroformylation be elevated to 9.0～9.5, add the anti-human IgG antibody of 10mg then immediately in 1ml 0.01mol/L carbonate buffer solution, the room temperature lucifuge stirred 2 hours gently;
(4) add the 4mg/ml NaBH that 0.1ml newly joins
4Liquid, mixing left standstill 2 hours under 4 ℃ again;
(5) above-mentioned solution is packed in the bag filter, to 0.15mol/L pH7.4PBS dialysis, 4 ℃ are spent the night.Small amount of precipitate is answered centrifugal discarding;
(6) the 2ml Protein A-Sepharose 4 Fast Flow that pack in the chromatographic column; Clean post with the 5mlpH3.0 sodium citrate buffer solution, then the phosphate buffer with 10mlpH8.0 cleans;
(7) regulate the pH value to 8.0 of anti-human IgG antibody's solution of the HRP mark of above-mentioned preparation with Tris, with the HRP-anti human IgE antibody-solutions upper prop of about 5ml/ hour speed with the mark preparation;
(8) phosphate buffer with pH8.0 cleans chromatographic column, and the purge flow fluid contains foreign proteins such as NIg;
(9) the anti-human IgG antibody of sodium citrate buffer solution wash-out HRP-of usefulness 10ml 0.1mol/L.Enzymic-labelled antibody wash-out rear pillar cleans with the 10ml phosphate buffer;
(10) in the absorbance of 280nm place monitoring eluent, collect fraction greater than baseline.The fraction of collecting is rapidly with the neutralization of 2mol/L Tris solution.
(11) fraction of Shou Jiing is crossed PD10 (Sephadex G25) post and with the medium solution of PBS exchange wash-out fraction;
(12) after the sterilization, add the carrier protein freeze drying, the enzymic-labelled antibody of collection is stored in the dark place below 4 ℃.
4. the using method of food allergen specificity IgG antibody ELISA detection kit
(1) ELIAS strip is installed
The ELIAS strip balance of having wrapped quilt is opened packaging bag to room temperature, take out the ELIAS strip combination of required examination food allergen albumen bag quilt, is fixedly mounted on the ELISA Plate framework.Do not need with ELISA Plate place packaging bag, the purging good seal is placed on 2-8 ℃ and stores down.
(2) sample is hatched
Sample liquid after getting 100ul human IgG titer series and diluting joins in the corresponding coated elisa plate hole; Only add the 100ul sample diluting liquid in the blank well; Seal the ELISA Plate hole with film, under 37 ℃, hatched 45 minutes.
(3) wash plate
Siphon away or evacuation apertures in liquid, pat dry after washing plate 4 times.
(4) enzymic-labelled antibody is hatched
Add the 100ul enzyme mark antibody solution with the every hole of pipettor; Seal ELISA Plate, 37 ℃ of following incubation reaction with 45 minutes; Wash plate 4 times as step (3).
(5) substrate reactions colour developing
Every hole adds 100ulTMB substrate colour developing liquid, rocks for 30 seconds gently, and room temperature leaves standstill reaction 15 minutes.
(6) cessation reaction detects
Every hole adds 100ul cessation reaction liquid, measures the absorbance in every hole in 20 minutes under the 450nm of microplate reader wavelength.
(7) result calculates
Calculate the mean light absorbency value of repeating hole; Logarithm value with the mean light absorbency value of standard solution series is carried out linear regression to the logarithm value of respective concentration, makes up working curve; Calculate the specific IgG antibodies content of patient this food allergen greater than the absorbance of that irritated foramen primum of cut-off value from working curve by the mean light absorbency value of sample repeating hole, and by given classification with this anaphylactogen sensitivity response deciding grade and level.
The stage division of food allergen specificity IgG concentration
The absorbance classification
＜0.20 0 (feminine gender)
0.21-0.60 1 (the weak positive)
0.61-1.20 2 (positives)
＞1.20 3 (strong positives)
Embodiment 2: with the test of ELISA method examination food allergen
To 32 routine food allergy patients (21 people in 2-18 year, 11 people in 18-56 year), extract blood and prepare serum qualitatively screening food (milk; Albumen/yolk; Corn, wheat, oat, barley; Shrimp, crab, freshwater mussel; Hairtail, yellow croaker, flatfish, salmon; Watermelon, pineapple, orange, mango, banana; Potato, tomato; French beans, broad bean, soya bean, pea; Pork, beef, mutton) anaphylactogen.
Measure the absorbance of patients serum in different food hypersenstivity primordial coverings hole by the using method of above-mentioned food allergen specificity IgG antibody ELISA detection kit, its intermediate value is greater than the positive that is judged as of cut-off value (0.20).Testing result such as following table:
| Food allergen | Milk | Albumen yolk | Corn wheat oat barley | Shrimp crab freshwater mussel | Hairtail yellow croaker flatfish salmon | Watermelon pineapple orange mango banana | Pomato | French beans broad bean soya bean pea | Pork beef mutton |
| The positive detects number | 2 | 6 | 1 | 7 | 10 | 1 | 0 | 1 | 1 |
| Positive rate (%) | 7 | 19 | 4 | 22 | 32 | 4 | 0 | 4 | 4 |
Testing result shows, adopts kit of the present invention to detect the sensitization food allergen easy, quickly and accurately, and sensitivity reaches 91.2%, and specificity reaches 86.5%.
Claims (3)
1. food allergen specificity IgG ELISA detection kit is characterized in that this kit is formed to comprise: wrapped milk, eggs, meat, cereals, nut fruits, fruits, greengrocery, the aquatic product of quilt, ELISA Plate, human IgG titer, enzymic-labelled antibody, tmb substrate colour developing liquid, sample diluting liquid, concentrated cleaning solution and the cessation reaction liquid of bean food allergen protein;
Wherein:
The ELISA Plate of having wrapped the milk food allergen albumen of quilt comprises: milk, goat milk;
The ELISA Plate of having wrapped the eggs food allergen albumen of quilt comprises: poultry albumen, poultry yolk;
The ELISA Plate of having wrapped the meat food allergen protein of quilt comprises: pork, beef, mutton, duck, chicken, goose, turkey meat, donkey meat;
The ELISA Plate of having wrapped the cereals food allergen albumen of quilt comprises: corn, wheat, oat, barley, buckwheat, rice, millet;
The ELISA Plate of having wrapped the nut fruits food allergen albumen of quilt comprises: almond, peanut, fibert, cashew nut, walnut, American pistachios, Brazilian nut, sesame;
The ELISA Plate of having wrapped the fruits food allergen albumen of quilt comprises: watermelon, pineapple, orange, mango, banana, citrus, Hu shaddock, lemon, apple, grape, pears, strawberry, peach;
The ELISA Plate of having wrapped the greengrocery food allergen albumen of quilt comprises: celery, spinach, onion, eggplant, garlic, capsicum, potato, tomato;
The ELISA Plate of having wrapped the aquatic product food allergen albumen of quilt comprises: hairtail, yellow croaker, flatfish, salmon; Carp, crucian, grass carp, silver carp, shrimp, crab, freshwater mussel, mussel, oyster, swamp eel, eel;
The ELISA Plate of having wrapped the bean food allergen protein of quilt comprises: French beans, broad bean, soya bean, pea;
Enzymic-labelled antibody: the anti-human IgG monoclonal antibody of horseradish peroxidase-labeled;
Tmb substrate colour developing liquid: 2.08mmol/L 3,3 ', 5,5 '-tetramethyl benzidine, 12mmol/L hydrogen peroxide, 10mmol/L beta-schardinger dextrin-, the acetate buffer solution of 0.1mol/L pH5.0.
Sample diluting liquid: contain 1%BSA in the phosphate buffer of 10mmol/L pH7.4-7.6,0.05%Tween-20 and 1mg/L gentamicin;
Concentrated cleaning solution: contain 1% hyclone in the phosphate buffer of 0.1mol/L pH7.4-7.6,0.5%Tween-20 and 20mg/L gentamicin;
Cessation reaction liquid: 1.0mol/L H
2SO
4
2. food allergen specificity IgG ELISA detection kit according to claim 1 is characterized in that said ELISA Plate is 96 holes.
3. the preparation method of food allergen specificity IgG ELISA detection kit according to claim 1 is characterized in that comprising the steps:
(1) preparation of food allergen albumen: the food that will contain allergen protein is through pulverizing, degreasing, extraction, SDS-PAGE electrophoresis conclusive evidence, wash-out, drying;
(2) preparation of enzymic-labelled antibody: with the anti-human IgG antibody of horseradish peroxidase-labeled purifying;
(3) preparation human IgG titer, tmb substrate colour developing liquid, concentrated cleaning solution and cessation reaction liquid;
(4) ELISA Plate bag quilt: with anti-human IgG antibody and food allergen albumen coated elisa plate, and sealing.
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| CNA2007100701980A CN101109750A (en) | 2007-07-27 | 2007-07-27 | Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof |
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