TWI342784B - A mixture of food allergens and a method for preparing the same - Google Patents

Description

1342784 IX. Description of the invention: [Technical field to which the invention belongs]

The present invention relates to a mixture comprising a plurality of natural and/or denatured food allergens identified by an Ig E antibody of a food allergy patient, an antibody prepared by immunizing an animal with the mixture, and a method for using the antibody And methods for detecting food allergens and foods that cause allergies. The present invention provides a mechanism for understanding the onset of food allergies, producing and confirming hypoallergenic foods, and detecting food allergens and food allergens in food manufacturing environments such as food manufacturing machines and processes, and providing food allergies Suffering from safety. [Prior Art] Food allergies are harmful immune reactions in sensitive individuals. Food allergies are caused by substances that are caused by allergies in food (hereinafter referred to as food allergens). Food allergies can cause dermatitis, asthma, digestive tract disorders, allergic shock, and so on. Allergies are classified as Types I to IV depending on the mechanism by which the disease begins. Food allergies are mainly caused by Type I allergies, in which IgE antibodies react with food allergens ingested. The number of food allergic patients has gradually increased in recent years. This phenomenon poses serious problems in the fields of medical science and food industry. To prevent this hazard, consumer information needs to be provided via the logo. The FA 0/Deleted Joint Food Standards Committee (Codex Alimentarius Commision) recognizes the need for foods containing any of the eight food ingredients known as food allergens and recommends that Member States implement labeling systems (1) June 9 9 9). In Sakamoto, take into account the severity and frequency of food allergies 326\total file\92\92 】01099VTF948034 5 © 1342784 and issue the label of 24 items (from 2 Ο Ο 2 years from April). It should be noted that these regulations do not require the labeling of allergic substances nor the food allergens themselves, but require the labeling of foods containing food allergens, ie foods that cause allergies.

Foods that cause allergies include eggs, milk, meat, fish, crustaceans, mollusks, grains, beans, nuts, fruits, vegetables, brewer's yeast, and gelatin. Similarly, egg protein, egg mucin, lysozyme, globoprotein, yS-lactoglobulin, α-lactalbumin, gluten protein, α-amylase inhibitor, and the like are known to cause allergic foods. In addition, (1) Regardless of the current determination, it is believed that there are still many other foods and ingredients that cause food allergies (food allergens); (2) foods and ingredients that cause allergies (food allergens) are quite diverse, and Food allergic patients have different responses to these allergens; and (3) as shown in the examples described later, there are many known and unknown allergies even in single allergenic foods. original. However, conventional methods cannot easily detect so many allergenic food or food allergens. Food is prepared by procedures such as heating, freezing, drying, salting, fermentation, enzyme treatment, etc. (hereinafter referred to as food processing) to improve its digestibility, storage life, taste, physical properties, etc., and to stabilize it. Chemical. Although food processes affect proteins and modify their molecular structure (for example, protein denaturation), few have discussed whether such food processes can lead to the production of food allergens. Development of the research work of the present invention clarifies the following: (1) some heated food ingredients may exhibit allergic; (2) cause allergic ingredients or individual 326V total file \92\92 ] 01099VTF948034 6 (S) 1342784 Allergies. In case of anti-allergic reactions, it will be collected or prepared b) and

Qing can not be antigen and antibiotics. Total food two now

White and 榖 and D e parts or a test for the detection of an allergy to detect an antigen with an inhibitory food, depending on whether the food of interest is heated or not, prepare a) unheated egg antigen, b) Heated egg antigen prepared by heating (a), c) via pooled serum from multiple food patients (referred to as pooled patient serum) and (a) serum containing no hot egg antigen Serum prepared by mixing, and d) serum mixed with patient serum and (b) serum containing no heated egg antigen. The results of the reaction between a) and c) or a) and d) and between b) and c) d) show that the blood containing no unheated egg antigen is immune to the unheated egg antigen, but It can be combined with the heated egg: in the same way, it shows that the serum without the heated egg antigen can not react with the hot egg antigen, but it can be countered by the unheated egg antigen, and the food allergy patient has the opposite Processing and unprocessed specific I g E antibodies, leading to food allergies. However, there are still no methods for the screening of food allergens in eggs, peanuts, casein, and stone-lactose egg gluten protein (food and development) for the detection of allergens in processed foods or their ingredients. Food velopment), Vol. 35, pp. 10-11). However, all of them have one or more of the following disadvantages: (1) The method is not always an allergen that is allergic to allergic patients. In other words, it is not the substance identified by the patient's I g E antibody; (2) the method is known only for allergens, and each method can only detect a single allergen (single); (3) using a single antigen test The method of the antibody is not applicable to the food of the manufactured substance; (4) The method of detecting the antibody using a single antigen 326V total file \92\92101099VTF948034 7 1342784 The allergy-producing food as shown in the examples described later cannot be accurately determined. Quantification; (5) The method of detecting an antibody using a single antigen cannot be applied to the analysis of an allergic food containing no antigen (for example, a method using a single antibody prepared by exposure to egg protein located in a protein cannot be used for analyzing egg yolk) , mayonnaise, etc.); (6) methods for detecting antibodies using a single antigen cannot be applied to the analysis of processed foods due to the inability to detect degenerative or molecularly modified allergens; and (7) against natural and denatured/5 - Monoclonal antibodies to lactoglobulin, egg protein and alpha-casein have been published (Allergy, No.

50 volumes, 3 0 9 pages). Since monoclonal antibodies detect only individual epitopes in a food allergen molecule, the effect is reduced if the epitope is removed or modified by a food process. Although it has been published for use (J apanese Patent Disclosure 2000-65820), eggs and milk (Japanese Meat Science, Book 39, 1 6 6 - 169 pages) The method of screening allergens for allergic patients' serum, but it is only used in hospitals, not in testing institutions and food manufacturing plants. Although food allergen detection methods (FFIJ., N 〇. 1 8 0, 7 7 - 8 2 pages) have been published as an allergic reaction, they cannot be used due to complicated methods and laboratory-animal husbandry. Most food manufacturing plants. Although methods for using mobile systems and enzyme-labeled antibodies and allergen sensors using microelectrodes have been developed (Japanese Food Industry, Vol. 42, pp. 53-56), such methods can be practically utilized. Before, there were still many problems to be solved. SUMMARY OF THE INVENTION The present invention is to solve the problems of the prior art described above. The object of the present invention is 326\总档\92\92 〗 01099VTF94S034 (δ) 1342784 % To provide (1) a variety of known and/or unknown and natural and recognized by I g E antibodies recognized by food allergic patients / or a mixture of denatured food allergens (referred to as the first invention of the present invention for convenience), (2) an antibody prepared by immunizing an animal with a mixture (referred to as the second aspect of the present invention) Inventions, and (3) A method for detecting food allergens and foods causing allergies (referred to as the third invention of the present invention). [Embodiment] The first invention of the present invention is completed by the following procedure:

(1 - i ) isolating the I g E antibody from a population of food allergic patients, and (1 - ii ) by using the aforementioned I g E antibody and an immunological method such as affinity chromatography and immunoprecipitation Foods that have been or are not processed by food processing or their ingredients (hereinafter collectively referred to as foods) are isolated from a variety of substances (in other words, a variety of food allergens that can be recognized by Ig E antibodies in food allergic patients). The first invention of the present invention can also be accomplished by the following procedure: (2 - i) using a conventional SDS-PAGE of a food ingredient, and then transferring the glue to the film, (2 - ii) using the film to collect the patient Serum, conjugated anti-human I g E antibody, and color reagents for western blotting (westernb 1 〇11 ing ) (in other words, the distribution of various food allergens identified by I g E antibodies from food allergic patients) (2 - iii ) Similarly, Western blotting, (2 - iv ) comparison using a film, animal serum prepared by immunizing food ingredients in a conventional manner, conjugated anti-animal I g G and coloring reagents The distribution of the two western ink points. The measurement did not appear in the former, but the 32 hatred file \92\92]01099VTF948034 9 8 1342784 % The molecular size of the band in the latter (in other words, the various substances that I g E cannot identify in food allergy patients), And (2 - v ) depending on the foregoing results, various food allergens identified by the I g E antibody of the food allergy patient are separated from the food component by a conventional method such as gel filtration chromatography.

The second invention of the present invention utilizes a variety of food allergens (including processed and/or processed) which are prepared by the first invention and which are identifiable by Ig E antibodies (referred to as antibodies recognizing multiple antigens) of food allergy patients. Or an animal antibody prepared against the food allergen prepared by immunizing the animal. The third invention of the present invention relates to a method for detecting food allergens and foods causing allergies using antibodies recognizing a plurality of antigens. The aforementioned heat treatment temperature range is equivalent to the range of the food processing, preferably between 40 and 250 ° C, and more preferably between 60 and 120 ° C. The antigen system is prepared by mixing the original antigen and heating it at two to six different temperatures. The food allergen portion identified by the I g E antibody of the food allergy patient is prepared from the mixture using the first invention of the present invention, and is used to immunize the animal. When the target of the test is limited to the heated food, the food allergen portion identified by the Ig E antibody of the food allergy patient is prepared from the heated food using the first invention of the present invention, and is utilized Animal immunity. Examples of animals are rabbits, goats, and mutton sheep 'rats, mice, guinea pigs, horses, pigs, chickens, and the like. During the immunization period, part of the blood collection and detection of anti-food allergen antibodies are preferred. The antibody of the present invention may be a single strain or a plurality of antibodies. As shown in the examples described later, even in the allergic foods of the single 10 326V total file \92\92101099\TF948034 1342784, there are many known and unknown 1 gE antibody identifications of food allergic patients. Food allergens. This is why multivalent antibodies that recognize multiple food allergens, i.e., antibodies that recognize multiple antigens, can be easily prepared by immunizing animals using such food allergens. Antisera of animals can be used as antibodies against food allergens identified by Ig E antibodies in food allergic patients. The antiserum can be used after absorption treatment of a portion which is not recognized by the I g E antibody of a food allergy patient. Antiserum can be used after the conventional I g G purification.

The method of the present invention for detecting food allergens and foods causing food allergies can be applied to foods containing allergens without any limitation. Examples of foods that cause allergies are eggs, milk, meat, fish, crustaceans, mollusks, yoke, beans, nuts, fruits, vegetables, brewer's yeast, and gelatin. More specifically, protein, egg yolk 'milk, cheese, pork, beef, chicken, lamb, squid, bamboo fish, sardines, fish, squid, fish, flounder, caviar, crab, shrimp, Mussels (m 〇u 1 e ), squid 'octopus, lobster, abalone, crustaceans, mollusks, wheat, rice, buckwheat, rye, barley, oats, corn, millet, foxtail millet, 稗, cereals, soybeans, peanuts, cocoa, peas, kidney beans, citrus, brazil, almonds, coconut, walnuts, beans, nuts, apples, bananas, oranges, peaches, kiwis, strawberries, melons, avocados, grapefruit , mango, pear, sesame, mustard, fruit, tomato, carrot, horse yam, spinach, onion, garlic, bamboo shoots, pumpkin, sweet potato, celery, parsley, mountain, sakamoto pine, vegetables, containing one or A variety of these items and their ingredients (for example, egg protein, egg mucin, lysozyme, protein, Lu-lactoglobulin, α-lactalbumin, 326\ total file\92\92101099\丁?948034 11 1342784 · Protein ' and α - powder killing Inhibitors) of food. Such foods can be prepared using procedures such as heating, cold, drying, salting, fermentation, enzyme treatment, and the like.

The allergen identified by the I g E antibody of a food allergy patient with a probability of 50% or more is referred to as a primary allergen, and less than 50% is referred to as a minor allergen. Food allergies are not only caused by major allergens, but also by minor allergens. Some patients are not allergic to major allergens but are allergic to secondary allergens. Therefore, methods for screening food allergens should be able to detect both primary and secondary allergens. The screening method of the present invention detects various natural and denatured allergens recognized by the Ig E antibody of food allergic patients. In order to prepare antibodies satisfying these needs, the inventors succeeded in discovering the following items. In other words, the food ingredients are subjected to conventional SDS-PAGE analysis and the gel is transferred to the film: (i) Western blotting using membranes, pooling patient serum, conjugated anti-human Ig E antibodies, and color reagents (ii) Similarly, using a film, a serum prepared by immunizing a food component with an animal in a conventional manner, a conjugated anti-animal I g G antibody, and a coloring reagent are subjected to a Western blot method, (iii) by two The comparison of the distribution of Western blotting methods reveals the following facts: (1) bands that are stained in both films, and (2) bands that are not dyed in the former but stained in the latter (ie, multiple non-allergic Substance), and (3) distributed in a higher and/or lower molecular weight position than in the former dyed zone only in the latter 12 326\total file\92\92101099\TF948034 1342784, dyed tape; and (iv From the foregoing results, a plurality of food allergens identified by the Ig E antibody of a food allergy patient are separated from the food component by gel filtration chromatography, and then the animal is immunized to prepare a multi-food allergen. antibody. Thermostated animals such as rabbits, goats, sheep, rats, mice, guinea pigs, horses, pigs, and chickens can be immunized. Any method of immunization known to those skilled in the art can be employed.

The antibody prepared as described above can be used in the food allergen detecting method of the present invention. The antibody can be immobilized on a microtiter plate, a PVDF film, a nitrocellulose film, a chromatographic strip (c h r 〇 m a t 〇 s t r i p ), a test tube 'bead, a nylon film, and the like. The antibody can be applied to immunological methods such as enzyme immunoassay, immunoblotting, dot blotting, immunochromatography, and antibody wafers (a n t i b 〇 d y c h i p ). As described above, the food allergen detecting method of the present invention aims to detect various food allergens in foods and food manufacturing environments such as food manufacturing machines and procedures. It is preferred to use liquid extracts from food for analysis. Although water is a preferred extractant, phosphate buffered saline, Tris-HCl buffer, alcohol, or the like can be used as long as the allergen can be extracted. In order to improve the efficiency of extracting allergens from food, a protein denaturant (e.g., S D S or urea), an antioxidant containing S ( (e.g., 2-thiol glycol), and the like may be added to the extractant if necessary. A variety of allergens can be determined by wiping (smearing) the food manufacturing environment or by trapping the air in the sachet bottle. As long as the principle of the food allergen detecting method of the present invention is immunology, the method is not limited to a specific method. The sandwich ELlSA (sandwich 13 326V total file \92\92101099\TF948034 1342784 ELISA), the competitive method, the direct method (direct m e t h o d ) and the like are examples of enzyme immunoassays. When the antibody is labeled, an enzyme (for example, a peroxidase, an alkali phosphatase, and a Θ-galactosidase), a fluorescent substance (for example, a fluorescent isothiocyanate), or a bioluminescent substance (for example, Luciferin-luciferase), chemiluminescent substances (for example, amino benzoquinone (1 umi η ο 1 ), oxime derivative and diamond powder derivative), biotin, avidin, Gold colloids, radioactive materials (eg, 3 2 P ), and the like.

Regarding the example of the detection method of the present invention, the sandwich E L I S A, the competition and the direct method are summarized below. To carry out the sandwich E L I S A, the labeled antibodies (identified antibodies recognizing multiple antigens) of the present invention which recognize various allergens are prepared. The unlabeled antibody recognizing a plurality of antibodies of the present invention is immobilized on an E L I S A plate via adsorption or chemical binding. Plates in areas where the antibody is not immobilized are blocked by proteins that do not interfere with the reaction system, such as gelatin and rabbit serum albumin. An extract (hereinafter referred to as a sample) or a standard antigen taken from food, food ingredients or a food manufacturing environment is added to the plate for the first antigen-antibody reaction. After the reaction, the plate was washed. For the second antigen-antibody reaction, the aforementioned labeled antibody is added to the plate where the allergen is captured by the immobilized antibody. After washing and removing the unreacted labeled antibody, a detection reagent is added (for example, 1,2-diamine and H2〇2 in the case of peroxidase: phosphoric acid in the case of alkali phosphatase) P-nitrophenyl ester). The allergen is detected or quantified by measuring the amount of reactant between the labeling and detection reagents. A calibration curve can be obtained by altering the amount of standard antigen on the E L I S A plate or the amount of food allergies (eg, eggs and milk). Food over 14 326V total file \92\92101099VTF948034 1342784 The amount of allergen or allergenic food can be quantified via a calibration curve. If the quantitation value is 1 ppm or more, the sample may be an allergenic food. The method of detecting an antibody using a single antigen causes a quantitative error because it is necessary to convert the value obtained by detecting the antibody from a single antigen to the content of the allergenic food.

For example, the concentrations of egg mucin and egg protein in whole eggs are about 4% and 2%, respectively. The concentrations in the protein are approximately 8% and 4%, respectively. However, egg yolk contains neither egg mucin nor egg protein. Therefore, (]) methods that only detect ovomucin or egg protein cannot be used to determine egg yolk protein; (2) when the calibration curve prepared from whole eggs is applied to the determination of protein, it will be quantified twice as much as the actual value. The above values; and (3) when the actual value exceeds the quantitative range, an extremely high quantitative value is generated. Similarly, since the concentration of casein and point-lactoglobulin in whole milk protein is about 80% and 10%, respectively, antibodies with a single antigen cannot accurately quantify the enhanced casein sodium and whey. The problem of processed milk such as protein. However, the antibodies of the present invention which recognize multiple antigens do not have or have such a problem. For competition methods, a certain amount of standard antigen is directly immobilized on a solid phase, which is blocked with a non-reactive protein. The antibodies and samples that recognize the allergen and the enzyme are then added to the plate. The plate is allowed to stand for a certain period of time and washed to remove unbound material. A colored substrate is added to the plate. The reaction between the immobilized allergen and the previously sample-treated antibody bound to the enzyme was stopped and measured. 32 Team General File\92\92101099\TF948034 15 1342784 For the direct method, the sample is directly adsorbed onto the solid phase, blocked with a non-reactive protein and reacted with an antibody that recognizes the allergen to the enzyme. The same procedure as previously described is then applied to detect allergens in the sample. For any of the foregoing methods, fluorescent, bioluminescent, and chemical luminescent substrates can be used for color rendering. Other conditions for the food allergen detection method of the present invention are modified by those skilled in the art, depending on the purpose, sample, measurement principle, and the like.

The food allergen detecting method of the present invention can detect 过敏.1 or 1. 〇 nanograms (n g ) or more of allergens in extracts from foods and their raw materials. (Industrial Applicability) A mixture of various natural and/or denatured food allergens recognized by an Ig E antibody of a food allergy patient and an antibody of the present invention have mechanisms for understanding the onset of food allergy and development to reduce food allergy The original allergic technology. Further, the food allergen detecting method of the present invention has utility for confirming the technique of reducing allergies, and detecting food allergens in foods, foods causing allergies, and food manufacturing environments such as machines and procedures. Accordingly, the present invention is useful for providing food safety in patients with food allergy and for solving medical and industrial problems arising from an increase in the number of recent food allergy patients. (Examples) The present invention is more specifically illustrated by the following examples, which should not be construed as limiting the scope of the invention. The abbreviations used in this specification are commonly used in the technical field. Example 1 (Preparation of standard antigens for various foods) 16 326V total file \92\92 ] 01099VTF948034

1342784 (1) Chicken, clams and duck eggs Standard egg antigens were prepared by shelling, dispersing, freeze-drying and finely grinding 1 kg of eggs. 10 g of the antigen was suspended in 10 times of p Η7.00 phosphate buffered saline (hereinafter abbreviated as PBS), and then distributed to 5 test tubes, which were not heated or respectively at 60. Heat at 1 0 0 and 1 2 0 °C for 30 minutes. Then, they were mixed together and prepared to prepare a sample. Samples of cockroaches and duck eggs were prepared in the same manner. (2) Milk Stir one liter of milk to solidify and precipitate the milk fat and pass it through absorbent cotton. After repeating this procedure three times, the filtrate was dried and then finely ground to prepare a standard milk antigen. The sample was also prepared as described in (1). (3) Wheat and rice 1 kg of wheat flour was stirred at room temperature for 2 hours, and the amount of 4 M urea in 0.1 M Tris-HCl buffer (pH 8. was taken, and then centrifuged again. The liquid wheat was dialyzed, freeze-dried and then micromilled to prepare a standard wheat antigen. Similarly, the sample was as described in (1). Samples were prepared from rice flour in the same manner. (4) Buckwheat passed 1 kg of naphtha powder through the chamber. Stir for 2 hours at a temperature, and extract the amount of 0.1 M Tris-HCl buffer (pH S, then centrifuge again), then dialyze the supernatant, freeze-dry and then grind to prepare standard buckwheat antigen. Similarly, as in (1), the sample 326\<the total file\92\92101099\TF948034 17 is prepared, and the volume is then 80' homogenized filtered frozen ground, 5 times 6). 4) Microfabrication 1342784 (5) Peanut One kilogram of peanut was ground and degreased in 5 times the amount of n-hexane by stirring at room temperature for 2 hours. After repeating this procedure 3 times and then removing n-hexane, it was extracted with 5 times the amount of 0.1 M Tris-HCl buffer (pH 8.4) added with 1 ° / 〇 NaCl, by stirring at room temperature for 2 hours. Formulation and then centrifuge again. The standard peanut antigen was prepared by dialysis, lyophilization and fine grinding. Similarly, samples were prepared as described in (1). (6) Soy

Standard soybean antigens were prepared as described in (5). Similarly, samples were prepared as described in (1). Example 2 (various purified food allergens) (1) Purified egg allergen The egg protein (the main allergen of the egg) was suspended in 10 volumes of P Η 8.0 PBS and distributed to In 5 test tubes, they were heated without heating or at 60, 80, 100 and 1 20 ° C for 30 minutes, respectively. The samples were then prepared by mixing them together and homogenizing them. A sample of ovomucin was prepared in the same manner. Egg proteins and egg mucins are proteins located in proteins. (2) Purified milk allergen A sample of casein, 9-lactoglobulin and α-lactalbumin was prepared as described in Example 2 (I). No-lactoglobulin and alpha-lactalbumin are proteins in the serum. Example 3 (Preparation of antibodies) (1) Preparation of rabbit antibodies against various standard food antigens Using F reund complete adjuvant (for first immunization) 18 326Y total file \92\92101099VTF94S034 1342784 Phosphoric acid The esterase was anti-rabbit I g G (4 Ο Ο 0-fold dilution) and allowed to react at room temperature for 1 hour. The membrane was washed with PBST as 4-methyl-4(3-phosphate phenyl)phosphonium [1,2-dipropylene oxide-3,2-adamantane] disodium salt-chemiluminescent reagent (Lumi-Phos) 530, Wako Pure Chemical) was treated at room temperature for 30 minutes and exposed to a photosensitive film to detect photons produced by the dephosphorylation of alkali phosphatase. These results are shown in Figure 1. From Figure 1, it is observed that the pooled patient's serum reacts with multiple bands (Figure 1,

4, and 7), and there are multiple bands identified by the patient I g E in each food. At the same time, rabbit antibodies detect food allergens identified by Ig E antibodies in food allergic patients (Figures 2, 5, and 8). The substances identified by the two sera are as follows: egg egg protein, egg mucin, lysozyme and egg transferrin; milk road protein, /3 - lactoglobulin and alpha-lactalbumin; wheat gliadin and alpha - amylase inhibitor; 132-, 84-, 27- and ΙΙ-kDa substances of buckwheat; and 107-, 72-, 35- and 28-kDa substances of peanut. In addition, rabbit antibodies did not detect non-food allergens that were not recognized by patients with I g E. (4) Concentration and fractionation of allergens The following antibodies were prepared for which any substance other than allergens could not be identified: Comparing the molecular weights of the stained and serum-stained bands of the pooled patients, it was noted that some bands of the latter were distributed. The former is high and / or low molecular weight position (Figure 1). The 326V total file of the food allergen identified by the I g E antibody recognized by the food allergic disease was collected from the standard antigen by gel filtration chromatography. The fraction of the molecular weight (hereinafter referred to as allergies) is 9.2V. Original part). An anti-allergen partial antibody was prepared by preparing a sample as described in Example 1 and immunizing the animal. From the Western blotting method as described above for the antibody, it was found that the antibody recognized the food allergen recognized by the pooled patient serum (Fig. 1, lines 3, 6, and 9).

Concentration and fractionation of such allergens can be accomplished by immunoprecipitation and affinity chromatography using patient Ig E antibodies and ion exchange chromatography. Example 5 (Detection of the main allergen and its heated preparation by the dot-ink method) After the PVDF film of the immersed PBS is installed in the spotting device, the purified main allergen (egg egg, egg sticky) Protein, egg transferrin, lysozyme, tropoelastin, cold-lactoglobulin, alpha-lactalbumin, ο:-housemic enzyme inhibitor and gliadin) and their heated preparations are absorbed into the film, Blocked with TBS with 3% RSA and washed with TBST. An antibody against the egg allergen fraction, the milk allergen fraction, and the wheat allergen fraction (diluted 2000 times) was added to the membrane and allowed to stand at room temperature for 1 hour. Next, the biotin-conjugated anti-rabbit Ig G sheep antibody and HR Ρ-conjugated avidin protein (400 times) are added to the film, which is washed and subjected to photons generated by reaction with a chemiluminescent reagent. It was measured and detected on a photosensitive film. The aforementioned antibodies detect the major allergens of egg protein, egg mucin 'egg transferrin, lysozyme, casein, 5-lactoglobulin, α-lactalbumin, α-amylase inhibitor and gliadin. In addition, the antibody is assayed for the aforementioned heated preparation of the purified allergen. Example 6 (Detection of egg, milk and wheat allergens using sandwich ELIS) 326V total file\92\92]01099\TF948034 21 8 1342784 (1) Antibody against the egg prepared in Example 4 (4) Antibodies to the allergen portion of milk and wheat and biotin-conjugated antibodies prepared according to conventional methods are used below. (2) Applying the antibody to the microtiter plate and blocking the above-mentioned antibody (10 μg/ml) in a volume of 100 μl was dispensed into the tank of the ELISA plate (N unc ) at 4 ° C Allow to stand overnight, wash (via

Add 150 mM-NaCl and (L05% Tween 20 in 20 mM Tris-HCl buffer, ρ Η 7.4) with 0. Γ /. R S A ( S i g m a ) is blocked at 25 ° C for 1 hour. (3) Detection of egg, milk and wheat allergens After removing the blocking solution in the tank, remove 9 5 μl of thinner.

(1%-RSA '150 mM-NaCl and 0. 05% Tween 20 of 20 11^1'1"丨5-1'101 buffer, mouth}]7.4) and 5 microliters The PBS extract from various foods was added to the tank and allowed to stand at 25 ° C for 2 hours. Similarly, a suspension of 5 μl of the egg, milk and wheat standard antigen mentioned in Example 1 was added to the tank, and allowed to stand at 25 ° C for 2 hours. After washing 5 times with 300 μl of washing liquid, add 100 μl of the bound biotin antibody (100-fold dilution) to the tank and let it at 25 ° C. After standing for 1 hour, 100 μl of a peroxidase-conjugated avidin protein (250-fold dilution) was added to the tank and allowed to stand at 25 ° C for 3 0. minute. After washing, a solution of 100 μl of a 3,3',5,5 'tetrabenzidine solution was added to the vessel and allowed to react under light-shielding at 25 ° C for 30 minutes. Then, 100 μl of 1 N sulfuric acid was added to stop the reaction. Lee 22 326\ Total file\92\92101099\TF948034 1342784 Use the microtiter plate reader to read the optical density of the bath (under the primary and secondary wavelengths of 450 and 630 nm, respectively). The results are shown in Table 1. Eggs, milk and wheat allergens in various food extracts can be measured as shown in Table 1. Table〗. A variety of food worries, milk and wheat test food against egg allergens against milk allergens against the antibody part of the wheat allergen part of the antibody A ---- sister partially cooked egg tart XX milk X 〇X Bread (prepared in the laboratory XX )) Pudding (prepared in the laboratory 〇〇X: containing eggs and milk) French toast (prepared in the experiment 〇〇〇 φ room: containing eggs, milk and wheat) 〇; detection To. X; not detected. (The same in the following table) Food ELISA method Example 7 (Evaluation using the basic properties of Sanming) (1) Dilution test of standard antigen (1) Standard antigen quantification according to the standard 6 ' will be in Example 1 Prepared eggs, milk and wheat. These results are plotted on the drawing paper, which confirms that the 326V total file \92\92101099\TF94S034 23 1342784 almost extends the calibration curve through the origin. (2) Evaluation of reproducibility between simultaneous measurements According to the aforementioned method, samples A to E of five different concentrations of egg standard antigen in a detectable range were prepared, and five simultaneous determinations were performed. As shown in Table 2, a C V value of less than 5% was obtained. Therefore, excellent simultaneous reproducibility was confirmed. Table 2. Evaluation of reproducibility between simultaneous assays of eggs using the sandwich ELISA method

1 2 3 4 5 Average SD CV(%) A 1. 165 1. 079 1.068 1. 075 1. 064 1. 090 0. 042 3. 87 B 0. 756 0. 711 0. 699 0. 689 0. 693 0. 710 0. 027 3. 84 C 0. 544 0. 505 0. 500 0. 497 0. 507 0. 511 0. 019 3. 74 D 0. 233 0. 222 0. 236 0. 222 0. 227 0. 228 0. 006 2. 79 E 0. 175 0. 165 0. 179 0. 173 0. 170 0. 172 0. 005 3. 06 (3) Evaluation of the reproducibility between each E 丨 measurement In the foregoing method, samples A to E of five different concentrations of milk standard antigens in the detectable range were prepared and subjected to continuous 5 day measurement, as shown in Table 3, to obtain CV values of less than 5%. Therefore, *9 is excellent in reproducibility between each measurement. 326V total file\92\92101099\TF948034 24 1342784 Table 3. Evaluation of reproducibility between daily measurements of milk standard antigen assays using the sandwich ELISA method

1 2 3 4 5 Average SD CV (%) A 2. 356 2. 387 2. 346 2. 321 2. 241 2. 330 0. 055 2. 37 B 1. 353 1. 306 1. 274 1 .246 1 254 1. 287 0. 044 3.40 C 0. 956 0. 949 0. 928 0 • 921 0. 899 0. 931 0. 023 2. 45 D 0. 295 0. 292 0. 275 0. 281 0. 278 0. 284 0. 009 3. 10 E 0. 195 0. 183 0. 186 0. 184 0. 182 0. 186 0. 005 2. 82 These results show that the method can detect food and food distributed quickly and steadily. Multi-day Pk and denatured food allergens in its composition have been shown to detect food allergens or food allergens containing 0.5 ng/ml or more. Example 8 (Food allergies) Taking IgE antibodies with anti-day Pk*»», and heated food allergens) As an example of egg allergy, it has been confirmed that food allergy patients not only have IgE antibodies against natural food allergens but also Confrontation Heat the food allergen I gE antibodies. In other words, the whole egg is dissolved in PBS (1.0%, W/V), half of the solution is not heated (unheated egg antigen), and the other half of the solution is heated at 120 °C. : 30 minutes (heated egg-antigen). The network P 1 0 0 microliters of each diluted 10 times solution was dispensed into EL] [SA plate tank, washed it) blocked with PBS added with 1% SHAS and washed 0 thus 5 prepared Plate coated with natural egg antigen and plate coated with heated egg antigen. Next 5 natural egg antigen or heated egg antigen (1 ' 5 ' 50, 326V total file \92\92 H) 1099\TF948034 25 1342784 1 Ο Ο, 5 Ο 0 and 1 Ο Ο 0 ng/ml) added to the pooled patient serum (1000 dilutions) described in Example 4 (1), reacted at 37 °C 2 hours, and centrifuge. Two serums were thus prepared. The former and the latter are referred to as serum without natural egg antigen and serum without heated egg antigen.

Each of the 100 microliter portions of the serum containing no natural egg antigen and the serum containing no heated egg antigen were then dispensed onto each plate coated with natural egg antigen and coated with heated egg antigen. It was allowed to stand at 3 7 ° C for 2 hours and washed with P B S T . 100 μl of the bound biotin-conjugated anti-human I g E - ε goat antibody (250 × dilution) was added to the plate, allowed to stand at 37 ° C for 1 hour, and PBST washing. The luminosity (illuminance meter CT-9000D, Diatron) was read after adding the anti-biotin protein and the luminescent substrate of the conjugated alkali phosphatase and allowing to stand at 37 ° C for 30 minutes. The results are shown in Figure 2. As shown in Figure 2, serum that was confirmed to be free of heated egg antigen (shown as 〇 in Figure 2) specifically reacted with native egg antigens (left panel of Figure 2), but serum could not react with heated antigens. (Right figure in Figure 2). At the same time, serum that has been shown to contain no natural egg antigen (shown in Figure 2) can specifically react with heated egg antigens (right panel in Figure 2), but serum cannot react with native antigens (left of Figure 2) Figure). As a result of this, it was confirmed that patients with food allergy had IgG antibodies that clearly recognized both natural and heated eggs. Therefore, methods for detecting food allergens should be able to detect both heated and natural allergens" Example 9 (Enhanced ELISA strength using antibodies prepared by immunization with heated antibodies) 26 326\总档\92\92101099VTF948034 1342784 Even if the anti-allergen partial antibody as described in Example 4 is assayed, the ELISA intensity (optical density, 0 D value) obtained by the reaction between the antibody and the heated sample may be compared with that of the antibody. The reaction between natural samples is low. This may be due to (1) the concentration of protein extracted from the heated sample is lower than that obtained from the natural sample; and (2) the reactivity between the heated sample and the antibody is greater than the reaction between the natural sample and the antibody. Low in sex, even if the protein concentration of the sample is evenly adjusted. Therefore, the preparation of antibodies with enhanced reactivity was investigated.

The egg allergen fraction as described in Example 4 (1) was treated in an autoclave at 120 ° C for 30 minutes, cooled, homogenized with P B S to which 4 Μ urea was added, and centrifuged. The supernatant was freeze-dried and finely ground to prepare an antigen, and the preparation of the rabbit antibody as described in Example 3 (1) (hereinafter referred to as an antibody against the autoclave-treated egg antigen) was carried out. The antibody was reacted with a plate coated with natural egg antigen as described in Example 8 and a plate coated with heated egg antigen according to the method as described in Example 6. The results are shown in Table 4. Table 4 shows that the natural and heated egg antigens were detected to have almost the same E L I SA intensity as the antibody prepared by immunizing the extract from the autoclave-treated food allergen. Therefore, the aforementioned problem was solved. 27 326V total file \92\92101099\TF94S034 1342784 Table 4. Increased antibody by 使 经由 经由 EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL EL The plate anti-egg allergen antibody against the egg antigen-antibody plate of the hot-pressed dad 36 118 1〇〇, ~ 1〇〇 will pass through the anti-egg allergen part of the anti- τ τ 0 and the standard egg antigen plate Between the reaction and the ELISA intensity (0D value) today 10 〇. Example 1 0 (food allergens and the determination of foods causing eight & allergy foods -1) k test the identification of a variety of antigen mountains +, ', the antibody (as described in Example 3) and whether the food containing the food allergen can be detected as described in Example 7. The calibration curve prepared as described in Example 7 > θ was used.疋$ and calculate the quantitative index (measured value * value / actual value χΙΟΟ). Table 5 shows the results of egg yolk 'protein, mayonnaise and whole mayonnaise. When the antibody prepared from the single protein in the protein is determined When '(1) could not detect the egg yolk, And 4 detectable proteins, and (2) the actual value of the standard egg antigen measured by the calibration curve is two to 60 times more. When the identification of the plurality of antigens of the present invention is made, (3) the egg yolk and protein can be detected. The amount is almost the same as the actual value. From the results, it is noted that the anti-standard egg antigen #anti-standard egg antigen antibody of the present invention is identified, and (4) the standard egg antigen is detectable and quantifiable. Processed Egg Allergy Foods Eggs or ingredients in food 326\总档\92\92101099VTF948034 28 1342784 Sample determination Ϊλ^Ϊλ. λ ij- ' Table 5. Quantification of the possibility of egg quantification using various antibodies Identification of a variety of antibodies against the identification of a single anti-identification single antigen (antigen antibody (anti-antibody (anti-egg standard egg antigen egg mucin protein antibody) antibody) antibody yolk detection possibility 〇 XX quantitative index 74-106 The possibility of protein detection 〇〇〇 quantitative index 105-170 240-310 6, 330-13, 150 possibility of mayonnaise detection 〇 XX whole egg test Possibility of Determination of Yellow Sauce Example 1 1 (Measurement of Food Allergens and Foods That Cause Allergies - 2) Detection of serum, casein, lactoferrin and hydrolyzed casein as described in Example 10. Table 6 shows (1) the anti-standard milk antigen of the present invention for identifying multiple antigens can detect and quantify milk and its components causing food allergy; but (2) by placing it in the serum It is impossible to quantify the identification of the antigen prepared by the substance immunization. The symbol of △ in Table 6 shows that the quantification can be completed, but the quantitative index is lower than 5. 29 326\ί® file\92\92101099\TF94S034 1342784 Table 6. Identification of various milk antigen samples by various antibody detection and/or quantification Identification of multiple anti-identification antibodies against a single antigen (antigen antibody (antigen antibody (anti-antibody) Standard milk antigen casein antibody)/5-lactoglobulin antibody) anti-chain) Possibility of detection of 〇X 〇Quantitative index 160-230 320-1, 080 S Possibility of detection of each protein 〇〇X Quantitative index 38 -47 170-180 Possibility of detection of lactoferred eggs 〇Δ X Possibility of detection of white hydrolyzed cheese 〇X Δ protein

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the reaction between a standard antigen and a patient I g E antibody, and a reaction between a standard antigen and an antibody against a standard antigen antibody of the present invention. Figure 2 shows the patient's serum with an Ig E antibody specific for both native and denatured egg proteins. 326\总档Λ92\92 Η) 1099VTF948034 30

Claims (1)

1342784 DEC 1 〇 2010 Replacement 1 · A food allergen mixture consisting essentially of a variety of undenatured and denatured substances recognized by the I g Ε antibody of a food allergy patient, obtained by the following steps: Using conventional SDS-PAGE for food ingredients and transferring to a film, (ii) using the film, pooling patient serum, conjugated anti-human Ig E antibodies, and color reagents for Western blotting, (iii) Similarly, Western blotting is carried out using a film, animal serum prepared by conventionally immunizing food components, conjugated anti-animal I g G and coloring reagents, and then (iv) comparing two western ink dots Distribution, the measurement does not occur in the former, but the molecular size of the band in the latter, and (v) the various food allergens identified by the I g E antibody of food allergy patients by conventional methods according to the above results Food ingredients are separated, And the food allergens are (1) egg egg protein, egg mucin, lysozyme and egg transfer protein; (2) milk casein,/5-lactoglobulin and α-lactalbumin; 3) wheat gliadin and α-houser enzyme inhibitor; (4) Cai Maizhi 132-, 84-, 27- and ΙΙ-kDa substances; or (5) peanuts 107-, 72-, 35- And 28-kDa substances. 2. A method of preparing a food allergen mixture of claim 1 comprising the steps of: (i) performing a conventional SDS-PAGE using a food ingredient and transferring to a thin film of 31 94135351 1342784, (ii) Western blotting method using a film, pooling patient serum, conjugated anti-human I g E antibody, and coloring reagent, (iii) Similarly, using a film, an animal blood prepared by immunizing a food component by a conventional method, Combining the anti-animal I g G and the coloring reagent to carry out the western ink dot method, and then (iv) comparing the distribution of the two western ink dots, the measurement does not appear in the former, but the molecular size of the band appearing in the latter, and (v) Depending on the results described above, various food allergens identified by the Ig E antibody of a food allergy patient are separated from the food component by a conventional method, and the food allergen is (1) egg egg protein , egg mucin, lysozyme and egg transferrin; (2) milk casein, yS-lactoglobulin and α-lactalbumin; (3) wheat gliadin and α-amylase inhibitor; 4) 132-, 84-, 27- and ΙΙ-kDa substances of buckwheat; or (5) 107-, 72-, 35- and 28-kDa substances of peanuts 32 94135351