JPH10185918A - Analytic method for grain allergen and grain allergen specific immunological reactant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide analytic methods for grain (or rice) allergen specific immunological reactants and for grain (or rice) allergens. SOLUTION: In a grain allergen specific immunological reactant analytic method, a sample to be inspected is brought into contact with a UPD glucose starch glucosyl transferase. In a rice allergen specific immunological reactant analytic method, a sample to be inspected is brought into contact with at least one kind of salt-insoluble proteins of molecular weights 60kd, 36kd, 33kd, or 13kd. And a reaction result of the allergens with the reactant is analyzed. In a grain allergen analytic method, a sample to be inspected is brought into contact with an immunological reactant against the transferase. And in a rice allergen analytic method, a sample to be inspected is brought into contact with an immunological reactant against the salt-insoluble proteins. And a reaction result of the allergens with the reactant is analyzed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for analyzing a cereal allergen protein, and a method for analyzing a cereal allergen-specific immunological reactant that specifically reacts with the cereal allergen protein. The present invention also relates to a method for analyzing a rice allergen protein and a method for analyzing a rice allergen-specific immunological reactant that specifically reacts with the rice allergen protein. The method of the present invention utilizes a novel cereal allergen protein or rice allergen protein to analyze the reactivity of anti-cereal allergen antibodies or rice allergen antibodies or immunocompetent cells in serum samples or the like, and to diagnose cereal or rice allergic patients It can be used effectively for such purposes. Further, the method of the present invention uses a cereal allergen-specific immunological reactant or a rice allergen-specific immunological reactant that specifically reacts with the cereal allergen protein or the rice allergen protein to prepare a food sample. It can also be effectively used for the analysis of cereal allergen protein or rice allergen protein in the inside.

[0002]

BACKGROUND OF THE INVENTION Food allergy is a deleterious immune response caused by food intake, and presents symptoms such as dermatitis, gastrointestinal disorders, anaphylactic shock or asthma. In recent years, such an increase in the number of patients with food allergies and more severe symptoms have been observed, and at the same time, the types of food allergens that cause causalities have spread, and the characteristics of multiple food allergies that cause symptoms due to more food have been characterized. Approved by patients. In such a situation,
In addition to egg, milk or soy allergy patients, wheat or rice allergy patients are also increasing, and it is necessary to quickly and accurately detect and identify food allergens that cause allergic symptoms in each allergy patient. ,
It has become increasingly important for the treatment of refractory or multiplex allergies.

[0003] As a method for identifying a food allergen, there are many methods such as a doctor's inquiry to a patient, a food removal load test, a skin test, or detection of a specific IgE antibody. In recent years, in addition to these tests, detection of IgG4 antibody, IL-2 (interleukin 2) reactivity test of lymphocytes or histamine release test has also been effective in diagnosis. Detection of specific IgE antibody or IgG4 antibody, IL-2 reactivity test of lymphocytes or histamine release test, etc. are methods for testing the reactivity of a patient's blood components with food allergens in a test tube. However, compared to the food load test, it is less common and has higher safety, so it is becoming the main test method today. In particular, the diagnosis of rice allergy is based on whether IgE antibody or IgG4 antibody against rice is present in the blood, whether IL-2 reactivity is present with rice allergen, or histamine release by rice allergen. It can be determined by whether or not to do.

[0004] A very small amount of IgE antibodies specific to rice allergens necessary for diagnosis of rice allergy, lymphocytes sensitized to rice allergens, or mast cells and basophil cells that release histamine are extremely small in vivo. Because it only contains
It is necessary to detect allergic reactions with high sensitivity.
It is desired that allergies be diagnosed earlier and more reliably. Therefore, the in vitro test method is an extremely effective means. The detection sensitivity of allergen-specific IgE antibody or IgG4 antibody is higher than the specificity of secondary antibody, that is, use of anti-human IgE antibody or anti-human IgG4 antibody, use of radioisotope, enzyme substrate which can be detected with ultra-high sensitivity It can be improved by the use of a fluorescent reagent or a fluorescent reagent. These methods are methods for reacting an allergen molecule with a patient's blood and then amplifying and detecting the reaction, and are effective methods for improving the sensitivity of allergen diagnosis. However, there is a disadvantage that non-specific reactions are also amplified and specificity is reduced even if sensitivity is high. On the other hand, enhancing the binding reaction itself between the allergen and the patient's blood component (IgE antibody, IgG4 antibody or lymphocyte) is considered to be effective in improving both sensitivity and specificity, and was isolated and purified. It is effective to use allergen molecules.

[0005] On the other hand, as a protein as a cereal allergen, particularly a rice allergen, salt-soluble proteins have been mainly studied. Therefore, allergen-reduced rice that has been treated to remove or reduce its salt-soluble protein from rice has been given to rice allergic patients. However, the present inventor has found that there are patients who develop allergies even with treated rice in which the salt-soluble protein as a rice allergen has been removed or reduced, confirming the presence of a plurality of rice allergens other than salt-soluble protein, Furthermore, these allergen proteins were identified, and it was confirmed that among these rice allergens, proteins having high homology to not only rice but also cereals (particularly, corn and wheat) were widely present.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to utilize a rice allergen protein newly discovered by the present inventor, which has been previously undetectable, and which responds to a salt-insoluble rice allergen. Is to be tested in vitro with a small amount of sample.
Furthermore, patients who respond to salt-insoluble cereal cereal allergens, which were previously impossible to detect, can be tested in vitro using a small amount of sample by utilizing allergen proteins that are also present in cereals other than rice. It is to provide a method. In addition, a method capable of testing whether or not the rice allergen protein is present in food by using an immunological reactant (for example, an antibody or an immunocompetent cell) specific to the rice allergen protein. Is to provide. Further, a method capable of testing whether or not the cereal allergen protein is present in a food by using an immunological reactant (for example, an antibody or an immunocompetent cell) specific to the cereal allergen protein. Is to provide.

[0007]

According to the present invention, there is provided a salt-insoluble protein having a molecular weight of 60 kd, a molecular weight of 3 kDa.
A test sample which may contain at least one rice allergen protein selected from the group consisting of a 6 kd salt-insoluble protein, a 33 kd salt-insoluble protein, and a 13 kd molecular weight salt-insoluble protein, and a rice allergen-specific reactant And analyzing the result of the reaction between the rice allergen protein and the rice allergen-specific immunological reactant, wherein the method is achieved by a method for analyzing rice allergen-specific immunological reactants. Can be. Further, the above-mentioned problem is solved by the present invention in that the molecular weight is 60
kd salt-insoluble protein, molecular weight 36 kd salt-insoluble protein, molecular weight 33 kd salt-insoluble protein, and molecular weight 13 kd salt-insoluble protein at least one kind of rice allergen protein specific immunologically A rice allergen-specific immunological reactant that reacts is brought into contact with a test sample that may contain the rice allergen protein, and a reaction between the rice allergen protein and the rice allergen-specific immunological reactant is performed. This can be achieved by a method for analyzing rice allergens, which comprises analyzing the results.

[0008] The object of the present invention is further to provide a method of contacting a UDP-glucose starch glucosyltransferase having allergenicity according to the present invention with a test sample which may contain a cereal allergen-specific immunological reactant. Achieved by a method for analyzing a cereal allergen-specific immunological reactant, which comprises analyzing the result of a reaction between UDP-glucose starch glucosyltransferase having allergenicity and the cereal allergen-specific immunological reactant. Can be. Also, the above-mentioned problem is
According to the present invention, an allergen-specific immunological reactant that specifically immunologically reacts with UDP glucose starch glucosyltransferase having allergenicity, and the possibility of containing the allergenic UDP glucose starch glucosyltransferase described above Contact with a test sample having an allergenic property.
This can be achieved by a method for analyzing cereal allergens, which comprises analyzing the result of the reaction between DP glucose starch glucosyltransferase and the allergen-specific immunological reactant.

In the present specification, the molecular weight [unit: kilodalton (kd)] of a rice protein is determined by measuring a liquid sample containing a protein of interest with a surfactant, sodium dodecyl sulfate (hereinafter, SDS) and a reducing agent (for example, SDS). A sample for electrophoresis obtained by treatment with mercaptoethanol) was subjected to SDS-polyacrylamide electrophoresis (hereinafter referred to as SD).
(S-PAGE). Detailed separation conditions will be described in detail in Examples.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
As used herein, the term “salt-insoluble protein having a molecular weight of 60 kd” refers to a salt-insoluble rice protein having a molecular weight of 6
The present inventors have found for the first time that the protein is a 0 kd protein, and that this protein is an allergen protein. A typical example is a waxy protein, a rice starch synthase [Okagaki et al., Pl.
ant Mol. Biol. (1992) Volume 19 Section 5
13 to 516]. Waxy protein (UDP
Glucose starch glucosyltransferase)
Consists of 532 amino acid residues. In the present specification, the “salt-insoluble protein having a molecular weight of 60 kd” does not need to have the activity as the waxy protein as long as it is a protein having allergenicity. Therefore, in the “salt-insoluble protein having a molecular weight of 60 kd”, in addition to the waxy protein itself, one or more amino acids are added, deleted or substituted in the amino acid sequence of the waxy protein, and Allergenic proteins are included.

[0011] UDP glucose starch glucosyltransferase is a kind of the waxy protein described above, and includes various grains including rice (eg, wheat, corn, barley, rye, oats, millet, millet or millet). Widely distributed. In addition, UDP glucose starch glucosyltransferase has high homology in partial amino acid sequences in various grains including rice (eg, wheat, corn, barley, rye, oats, millet, millet or millet). In this specification, "UDP glucose starch glucosyltransferase" does not need to have enzymatic activity as long as it is a protein having allergenicity. Therefore, the "UDP glucose starch glucosyltransferase" includes, in addition to the UDP glucose starch glucosyltransferase itself,
It includes proteins having one or more amino acids added, deleted or substituted in the amino acid sequence of UDP glucose starch glucosyltransferase, and having the above-mentioned allergenicity.

The term "salt-insoluble protein having a molecular weight of 36 kd" as used herein refers to a salt-insoluble protein having a molecular weight of 36 kd among rice proteins, and is a novel protein discovered by the present inventors for the first time. The present inventors have also found that this novel protein is an allergen protein. N-terminal of this novel protein (positions 1 to 1)
The amino acid sequence at position 0) is as follows. Ser (Ala, Gly) -Lys-Gly-Ala
(Glu) -Gly-Met-Asn-Val-Val
-Phe 1-position "Ser (Ala, Gly)" is used as a 1-position amino acid serine (Ser) when determining the amino acid sequence.
Is detected in a major amount, and alanine (Ala) and glycine (Gly) are also simultaneously detected in small but non-negligible significant amounts, respectively. In addition, alanine (Ala) is also detected as amino acid at position 4 of “Ala (Glu)” at position 4, but glutamic acid (Gl
u) also means that it is simultaneously detected in a small but significant amount that cannot be ignored.

Therefore, in the “salt-insoluble protein having a molecular weight of 36 kd”, the first and fourth positions are serine (Ser).
And proteins that are alanine (Ala),
Also included are proteins in which position 1 is replaced with alanine (Ala) or glycine (Gly) and / or position 4 is replaced with glutamic acid (Glu). Furthermore, in addition to the protein itself having the amino acid sequence at the N-terminus (positions 1 to 10), one or more amino acids in the entire amino acid sequence are added to the “salt-insoluble protein having a molecular weight of 36 kd”. Also included are proteins that have been deleted or substituted and have the allergenic properties described above.

As used herein, the term "salt-insoluble protein having a molecular weight of 33 kd" refers to a salt-insoluble protein having a molecular weight of 33 kd among rice proteins, and is a novel protein discovered by the present inventors for the first time. The present inventors have also found that this novel protein is an allergen protein. N-terminal of this novel protein (positions 1 to 1)
The amino acid sequence at position 5) is as follows. Ser (Ala, Gly) -Thr (Lys) -Gly
-Ala (Glu) -Gly-Met-Asn-Val
-Val-Phe-X-Gly (Thr, Ser) -A
la-Glu-Met

The "Ser (Ala, Gly)" at the 1st position and the "Ala (Glu)" at the 4th position have the same meaning as described above, and the "Thr (Lys)" at the 2nd position also has a major amount of threonine. (Thr) and a significantly small amount of lysine (Lys) are detected.
(Thr, Ser) "also contains a major amount of glycine (G
ly) and significant small amounts of threonine (Thr) and serine (Ser). Also, 11
The "X" at the position means that the type of amino acid cannot be specified.

Therefore, in the “salt-insoluble protein having a molecular weight of 33 kd”, the first, second, fourth and twelfth positions include
Not only are proteins that are serine (Ser), threonine (Thr), alanine (Ala) and glycine (Gly), respectively, but also position 1 is substituted with alanine (Ala) or glycine (Gly) and / or position 2 is lysine. (L
ys) and / or glutamic acid at position 4 (G
lu) and / or at position 12 threonine (T
Also included are proteins substituted with hr) or serine (Ser). Furthermore, one or more amino acids in the entire amino acid sequence are added to the “salt-insoluble protein having a molecular weight of 33 kd” in addition to the protein itself having the N-terminal (positions 1 to 15) amino acid sequence. , Deleted or substituted, and the allergenic protein described above.

In the present specification, the "salt-insoluble protein having a molecular weight of 13 kd" is a salt-insoluble protein having a molecular weight of 13 kd among rice proteins, and the present inventors have first found that this protein is an allergen protein. . A representative example of its active form is prolamin, a rice seed storage protein [Yamagata et al., BioSci. Biotec
hnol. Biochem. (1992) Volume 56 Section 5
37]. Prolamin is expressed by a number of similar multigenes and consists of a population of proteins with slightly different amino acid sequences. In the present specification, the “salt-insoluble protein having a molecular weight of 13 kd” does not need to have the activity as the prolamin protein as long as it is a protein having allergenicity. Therefore,
The “salt-insoluble protein having a molecular weight of 13 kd” includes:
In addition to the prolamin protein group itself, a protein having one or more amino acids added, deleted or substituted in the amino acid sequence of the prolamin protein and having the above-mentioned allergenicity is included.

These salt-insoluble proteins having a molecular weight of 60 kd, salt-insoluble proteins having a molecular weight of 36 kd, and a molecular weight of 3
The salt-insoluble protein of 3 kd and the salt-insoluble protein of molecular weight 13 kd can be extracted and purified from rice grains by a known method. Is UDP glucose starch glucosyltransferase of various cereals commercially available,
Alternatively, it can be extracted and purified from cereal grains such as rice grains, wheat grains or corn grains by a known method.

Hereinafter, the method of the present invention will be mainly described with respect to rice, but unless otherwise specified, it should be understood that the description relating to rice also applies to other cereals. The cereal or rice that can be used for the extraction of the salt-insoluble allergen protein is not particularly limited, and shells are obtained from cereal or rice seeds (ie, grains), for example, to prepare brown rice or white rice. (Eg, pulverized, puffed or gelatinized). If necessary, a degreasing treatment with ethers may be performed. The protein is, for example, a salt-soluble protein-removed cereal preparation or rice preparation obtained by extracting cereal or rice with an aqueous salt solution, an alkaline solution or an acid solution, and further extracted with urea and purified by an appropriate means. Can be obtained. Alternatively, for example, a salt-soluble component can be removed from a liquid extract obtained by extracting cereals or rice with urea by dialysis or the like without performing the salt-soluble protein removal treatment.

Reagents used to obtain a salt-soluble protein-removed cereal preparation or a salt-soluble protein-removed rice preparation include, in addition to various salts, an alkaline aqueous solution, an acidic aqueous solution, a protease, or a microorganism solution. Alternatively, a dispersion can be used. For example, the salt may include a salt of an inorganic acid (eg, hydrochloric acid, sulfuric acid or phosphoric acid) with an alkali metal or an alkaline earth metal (eg, sodium, potassium, calcium, or magnesium), particularly sodium chloride , Potassium chloride, magnesium chloride, calcium chloride, sodium sulfate, sodium hydrogen carbonate, sodium carbonate, sodium polyphosphate and various phosphates are preferred, and these can be used alone or in combination. The concentration of the salt solution is 3
M or less, particularly preferably 1M or less.

As the alkaline aqueous solution, an aqueous solution of an alkali metal hydroxide (such as sodium hydroxide), an alkaline earth metal hydroxide (such as manganese hydroxide), or ammonia can be used. As the acidic aqueous solution, an inorganic acid (hydrochloric acid, sulfuric acid, or phosphoric acid) or an organic acid (for example, acetic acid) can be used. Further, the pressure can be increased or decreased in the extraction step. It should be noted that any of the above processes can be performed in combination. The salt-soluble protein-removed cereal preparation or the salt-soluble protein-removed rice preparation thus obtained is obtained by adding 5 g of these preparations.
It is desirable that the protein in the extract extracted with 0.5 times the volume of 0.5 M aqueous sodium chloride solution be 0.1 mg / ml or less (Kjeldahl method). Therefore, the term “salt-insoluble protein” as used herein means a protein that is not substantially extracted and removed by the salt-soluble protein removal treatment.

Next, when the cereal preparation treated with salt-soluble protein removal or the rice preparation treated with salt-soluble protein is treated with a protein denaturant, a denaturant extract containing UDP glucose starch glucosyltransferase of the various cereals described above, or A denaturant extract of rice containing the salt-insoluble protein having a molecular weight of 60 kd, the salt-insoluble protein having a molecular weight of 36 kd, the salt-insoluble protein having a molecular weight of 33 kd, and / or the salt-insoluble protein having a molecular weight of 13 kd can be obtained. As the protein denaturant, for example, SDS or urea can be used.

Subsequently, the above-mentioned denaturant extract is treated by a method of fractionating the protein according to the molecular weight, and the salt-insoluble protein having a molecular weight of 60 kd (or the U of various cereals) is treated.
DP glucose starch glucosyltransferase), salt-insoluble protein with a molecular weight of 36 kd, molecular weight 3
A 3 kd salt-insoluble protein and a 13 kd molecular weight salt-insoluble protein can be obtained. For example, fractionation can be performed by electrophoresis (particularly SDS-PAGE), filtration with a filter capable of performing molecular weight fractionation, centrifugation, gel filtration chromatography, or the like. When fractionating by SDS-PAGE, the denaturant extract is concentrated as necessary, fractionated using a suitable gradient gel, and appropriately stained to obtain a 60 kd,
Each of the 6 kd, 33 kd and 13 kd bands can be cut out and electrophoretically extracted and recovered. As a carrier for gel filtration chromatography, a wide range of carriers such as inorganic compounds, synthetic resins, or polysaccharides can be used,
For example, any carrier can be used as long as the protein can be fractionated according to the molecular weight by a molecular sieve such as an agarose gel, a polyacrylamide gel, or a chemically modified substance of a polysaccharide.

In addition, the extract of the denaturing agent may be a ketone (eg, acetone), an ether (eg, diethyl ether), a halogenated hydrocarbon (eg, chloroform), or an alcohol (eg, methanol, ethyl). An organic solvent such as alcohol, butanol, or amyl alcohol), an aqueous solution of guanidine hydrochloride, or an aqueous solution of ammonium sulfate are added, and proteins having a specific molecular weight can be selectively precipitated to be fractionated.

In the method for analyzing a cereal allergen-specific immunological reactant according to the present invention or the method for analyzing a rice allergen-specific immunological reactant according to the present invention, the test sample comprises:
The sample is not particularly limited as long as it is a sample that may contain the reactant to be measured, and particularly includes a biological sample such as blood, serum, or plasma. As used herein, the term “cereal allergen-specific immunological reactant” means a reactant capable of immunologically specifically binding to the above-mentioned UDP glucose starch glucosyltransferase having allergenicity. Specifically, it is an antibody (such as IgE or IgG4), an immunocompetent cell (such as T cell, B cell, basophil or mast cell), or platelet. As used herein, the term “rice allergen-specific immunological reactant” refers to the rice allergen, that is, a salt-insoluble protein having a molecular weight of 60 kd, a salt-insoluble protein having a molecular weight of 36 kd, a salt-insoluble protein having a molecular weight of 33 kd, or a molecular weight. It refers to a reactant capable of immunologically specifically binding to any one of the 13 kd salt-insoluble proteins, and specifically, an antibody (Ig)
E or IgG4), immunocompetent cells (such as T cells, B cells, basophils or mast cells), or platelets.

In the method for analyzing a cereal allergen-specific immunological reactant according to the present invention, the above-mentioned UDP glucose starch glucosyltransferase having allergenicity is brought into contact. In the method for analyzing a rice allergen-specific immunological reactant according to the present invention, at least one of the four allergens is brought into contact with the test sample. In general, it is preferable that the allergen is brought into contact with the allergen-specific immunological reactant in an amount that exceeds the allergen-specific immunological reactant contained in the test sample.
The allergen to be brought into contact with the test sample is at least one of UDP glucose starch glucosyltransferase (in the case of a method for analyzing a cereal allergen-specific immunological reactant) or a salt-insoluble protein having a molecular weight of 60 kd.
Species, at least one kind of salt-insoluble protein having a molecular weight of 36 kd, at least one kind of salt-insoluble protein having a molecular weight of 33 kd, and / or at least one kind of salt-insoluble protein having a molecular weight of 13 kd (Method for analyzing rice allergen-specific immunological reactant) Case). In the case of a method for analyzing rice allergen-specific immunological reactants, one salt-insoluble protein having a molecular weight of 60 kd, one salt-insoluble protein having a molecular weight of 36 kd, and one salt-insoluble protein having a molecular weight of 33 kd are used.
Preferably, the species and one salt-insoluble protein with a molecular weight of 13 kd are used together in combination.

In the method for analyzing a cereal allergen-specific immunological reactant according to the present invention or the method for analyzing a rice allergen-specific immunological reactant according to the present invention, the antibody as the allergen-specific immunological reactant and the rice When analyzing the result of the reaction with the allergen protein, the allergen protein and the test sample, the conditions under which an antigen-antibody reaction is possible, that is, the anti-allergen antibody contained in the test sample and the aforementioned The contact is made under conditions that allow the allergen to bind by the antigen-antibody reaction. From the antigen-antibody reaction, a detectable signal can be induced by a well-known method, and the result of the antigen-antibody reaction can be analyzed.

For example, the protein-IgE antibody (or IgG4 antibody) complex formed by the antigen-antibody reaction of the above-mentioned allergen and the above-mentioned anti-allergen antibody (IgE antibody or IgG4 antibody) can be subjected to any known immunology. Can be detected using a quantitative assay. The immunological assay used in the method of the present invention is not particularly limited, but may be an enzyme immunoassay (in particular, an ELISA method) or a radioimmunoassay (RIA). Assays using substances and the like can also be used, and their labels can be detected by known methods.

When the method for analyzing a cereal allergen-specific immunological reactant according to the present invention is carried out by an ELISA method, for example, an allergen solution obtained by dissolving the above-mentioned allergen protein in an appropriate buffer is placed on an ELISA plate. Fixation followed by the test sample (eg, serum sample)
Is added to the plate. When the rice allergen-specific immunological reactant is analyzed by an ELISA method, for example, an allergen solution obtained by dissolving at least one of the allergen proteins in an appropriate buffer is coated on an ELISA plate. And then a test sample (eg, a serum sample) is added to the plate. Subsequently, in any case, after the plate was washed, an anti-human IgE antibody labeled with an enzyme was added, washed, and a substrate of the labeled enzyme was added, and the enzyme activity was measured. The allergen antibody in it can be detected or quantified.

In the method for analyzing an allergen-specific immunological reactant according to the present invention, when the result of the reaction between the immunocompetent cell as the allergen-specific immunological reactant and the allergen protein is analyzed, A test sample (optionally, immunocompetent cells taken out of the test sample) is brought into contact with the allergen protein, and various activities (eg, proliferation activity) of various immunocompetent cells are analyzed, whereby allergen specificity in the test sample is determined. The immunocompetent cells can be detected or quantified.

For example, using interleukin-2 (IL-2) by the following method,
The proliferative activity of the cells can be analyzed. That is, a patient's blood is collected and, in some cases, a peripheral blood leukocyte fraction,
Preferably, the peripheral blood lymphocyte fraction is collected, the allergen molecule is added to the blood sample (or lymphocyte fraction), and the cells are cultured for several days (about 3 to 5 days), and then the IL-2 molecule is added. After further culturing for several days, the number of grown cells is measured.
When the number of T cells is increased as compared to the group without IL-2, it is determined to be positive for IL-2 reactivity, and T cell proliferation activity can be detected. Further, the degree of positiveness can be determined according to the proliferation ratio, and the T cell proliferation activity can be quantitatively analyzed.

Further, the analysis can be performed by a T cell activation test. Collecting the blood of the patient, optionally collecting a peripheral blood leukocyte fraction, preferably a peripheral blood lymphocyte fraction,
After adding an allergen molecule to the blood sample (or lymphocyte fraction) and culturing for several days (about 3 to 5 days), a purine base or a pyrimidine base labeled with a radioisotope, for example, tritium thymidine is added. , About 2
After 4 hours, the cells are washed and collected on a filter using an automatic cell harvester or the like, the cells are disrupted, and the radioactivity (cpm) is counted using a liquid scintillation counter. Radioactivity (cp) compared to the group without allergen
When m) is high, T cell activation test is positive, and T cell activity can be detected. Further, the degree of positiveness can be determined according to the ratio of the activities, and the T cell activity can be quantitatively analyzed.

Furthermore, the action of mast cells or basophils, which are immunocompetent cells, can be analyzed from mediators (eg, histamine or serotonin) generated during the degranulation reaction. For example, a patient's blood is collected, a peripheral blood lymphocyte fraction is collected in some cases, an allergen molecule is added to the blood sample (or lymphocyte fraction), and the cells are centrifuged for several minutes to several hours. After removal, the amount of mediator (eg, histamine or serotonin) in the supernatant is measured. When a mediator (for example, histamine or serotonin) is detected as compared with the case where no allergen is added, a positive mediator release test is performed, and the action of mast cells or basophils can be detected. Further, the degree of positiveness can be determined according to the detected amount, and the action of mast cells or basophils can be quantitatively analyzed.

In the method for analyzing a cereal allergen according to the present invention, the test sample is a food which may be taken by an allergic patient, or the allergen protein to be measured (ie, UDP glucose starch glucosyltransferase having allergenicity). ) Is not particularly limited as long as the sample may contain, and in the method for analyzing rice allergen according to the present invention, the test sample may be a food,
Alternatively, the rice allergen protein to be measured (ie, the salt-insoluble protein having a molecular weight of 60 kd, the salt-insoluble protein having a molecular weight of 36 kd, the molecular weight of 33)
The sample is not particularly limited as long as it is a sample that may contain a kd salt-insoluble protein or at least one of a 13 kd molecular weight salt-insoluble protein). This is because, in general foods, there are foods in which cereals are added in small amounts for the purpose of adjustment, and allergy sufferers are allergic to eating foods containing cereals in small amounts, in other words, small amounts of cereal allergens. This is because symptoms may be exhibited. Accordingly, the test sample may be, for example, a food (including a beverage) or a raw material thereof which may be taken by an allergic patient, or a sample prepared from the food or the raw material for analysis (for example, an extracted sample or Diluted sample), especially cereal preparations, cereal-related foods, or brewing raw materials or brewed products.
Wheat-related foods [eg, bakery products (eg, cakes, confectionery such as cookies, bread), udon, pasta,
Skins of noodles, dumplings, etc.) or corn-related foods [eg, cereal, corn starch, tacos (dough made by kneading corn powder with water or oil or fat)], and cooked rice (eg, cooked rice) And rice-related foods (for example, confectionery products such as rice crackers, rice cakes, cooked products such as pilaf and fried rice) or samples for analysis thereof. Furthermore, feed and its raw materials are included.

In order to prepare an analytical sample from a cooked cereal or a cereal-related food, or a cooked cereal or a rice-related food, the sample is contained in the cooked cereal or a cereal-related food, or a cooked cereal or rice-related food. It is necessary to effectively extract a potential measurement target, that is, the allergen protein. Therefore, the method described above can be applied as it is to the method for extracting the allergen protein. For example, when preparing an analytical sample from cooked rice, first perform a salt-soluble protein removal treatment, and then use a denaturant-treated extract obtained by performing a treatment with a protein denaturant as it is as the analytical sample. Can be. Alternatively, for example, a denaturant-treated extract obtained by directly treating cooked rice with a protein denaturant can be used as an analysis sample as it is.

In the method for analyzing a cereal allergen according to the present invention, the above-mentioned test sample is brought into contact with the above-mentioned UDP glucose starch glucosyltransferase having allergenicity, ie, a specific immunological reactant against the allergen. In the method for analyzing rice allergens according to the present invention, at least one of the specific immunological reactants to any one of the four allergens is brought into contact with the test sample. In general, it is preferable that the allergen-specific immunological reactants are contacted at a ratio such that the amount thereof is in excess of the allergen contained in the test sample. The rice allergen-specific immunological reactant brought into contact with the test sample is a specific immunological reactant against at least one salt-insoluble protein having a molecular weight of 60 kd, and a specific immunological reactant against at least one salt-insoluble protein having a molecular weight of 36 kd. A specific immunological reactant to at least one of the salt-insoluble proteins having a molecular weight of 33 kd, or a specific immunological reactant to at least one of the salt-insoluble proteins having a molecular weight of 13 kd. Preferably, the four immunological reactants are used together in combination.

The allergen-specific immunological reactant can be prepared by a known method. For example, an anti-rice allergen antibody is obtained by suspending the purified rice allergen protein in phosphate buffered saline and mixing it with Freund's complete adjuvant to form an emulsion, and then using the emulsion as an animal (goat, rabbit, sheep, Chicken,
Mouse, rat, etc.), and about 1 week and about 10 days later, immunization was again carried out with an emulsion prepared in the same manner as described above using incomplete adjuvant instead of complete adjuvant, and then about 1 week. Later, blood can be collected and the antibody can be purified from the serum. Alternatively, a monoclonal antibody can be prepared by cell fusion using splenocytes collected from the immunized animal (mouse or rat). As the immunocompetent cells, blood collected from an allergic patient or an immunized animal can be used as it is, or a leukocyte or lymphocyte fraction can be separated and used.

In the method for analyzing a cereal allergen according to the present invention, when the result of the reaction between the anti-UDP glucose starch glucosyltransferase antibody as the allergen-specific immunological reactant and the UDP glucose starch glucosyltransferase is analyzed, or In the method for analyzing rice allergen according to the present invention, when analyzing the result of the reaction between the anti-rice allergen antibody as the rice allergen-specific immunological reactant and the rice allergen protein, the anti-allergen antibody and the test The sample is contacted under conditions that allow an antigen-antibody reaction, that is, under conditions that allow the allergen contained in the test sample and the anti-allergen antibody to bind through the antigen-antibody reaction. From the antigen-antibody reaction, a detectable signal can be induced by a well-known method, and the result of the antigen-antibody reaction can be analyzed.

For example, in the same manner as the method for analyzing a grain allergen-specific immunological reactant and the method for analyzing a rice allergen-specific immunological reactant, the antigen-antibody reaction is carried out. The protein-antibody conjugate to be subjected to any known immunological assay,
For example, it can be detected by an enzyme immunoassay (in particular, an ELISA method), a radioimmunoassay (RIA), or an assay using a fluorescent substance or a chemiluminescent substance as a label.

When the above-mentioned method for analyzing cereal allergen or the above-mentioned method for analyzing rice allergen is carried out by an ELISA method, for example, the above-mentioned anti-allergen antibody (preferably monoclonal antibody) (first antibody) is added to an appropriate buffer. An antibody solution obtained by dissolving the antibody solution in, for example, a phosphate buffered saline is fixed to an ELISA plate, and then a test sample (for example, a food sample) is added to the plate. After washing the plate, another enzyme-labeled anti-allergen antibody (second antibody) is added, washed, and then a substrate of the labeled enzyme is added. The allergen in it can be detected or quantified. Alternatively, the test sample can be fixed by adding the test sample to an ELISA plate, washing, adding an anti-allergen antibody labeled with an enzyme, washing, adding a substrate for the labeled enzyme, and measuring the enzyme activity. Allergen in a sample can be detected or quantified.

In the method for analyzing a cereal allergen according to the present invention or the method for analyzing a rice allergen according to the present invention, when the result of the reaction between the immunocompetent cell as the allergen-specific immunological reactant and the allergen protein is analyzed. Can detect or quantify rice allergen in a test sample by contacting the test sample with immunocompetent cells and analyzing various activities (eg, proliferation activity) of the various immunocompetent cells.

For example, using interleukin-2 (IL-2) by the following method,
The proliferative activity of the cells can be analyzed. For example, a peripheral blood leukocyte fraction, preferably a peripheral blood lymphocyte fraction, is collected from the blood of an allergic patient to the allergen, and a test sample is added to the lymphocyte fraction for several days (3 to 5 days).
After culturing, the IL-2 molecule is added. After further culturing for several days, the number of grown cells is measured by the same operation as described in the above-described method for analyzing allergen-specific immunological reactants, and T cell proliferating activity is detected. Proliferation activity can also be quantitatively analyzed.

Further, the analysis can be performed by a T cell activation test. For example, a test sample is added to a peripheral blood leukocyte fraction, preferably a peripheral blood lymphocyte fraction, prepared in the same manner as described above, cultured for several days (about 3 to 5 days), and labeled with a radioisotope. Purine bases or pyrimidine bases, such as tritium thymidine, are added, followed by performing the same procedure as described above for the method for analyzing allergen-specific immunological reactants to detect T cell activity, or Cellular activity can also be quantitatively analyzed.

Furthermore, the action of mast cells or basophils, which are immunocompetent cells, can be analyzed from mediators (eg, histamine or serotonin) generated during the degranulation reaction. For example, a test sample is added to the peripheral blood lymphocyte fraction prepared in the same manner as described above, and after a few minutes, the cells are removed by centrifugation or the like, and the amount of a mediator (eg, histamine or serotonin) in the supernatant is determined. Is measured.
Subsequently, the same operation as that described in the above-mentioned method for analyzing an allergen-specific immunological reactant can be performed to detect the effect of mast cells or basophils, or to quantitatively analyze the effect.

[0045]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 [Allergenicity of salt-insoluble protein] (1) 1 kg of commercially available Koshihikari polished rice grains were immersed in 5 liters of 0.1N sodium hydroxide aqueous solution, decompressed (20 mmHg) for 40 minutes, and then kept at a constant temperature of water temperature of 40 ° C. Below,
Stir for 2 hours. Next, the rice grains were immersed in 5 liters of 0.1 molar hydrochloric acid aqueous solution for 2 hours, and the rice grains were washed in running water for 24 hours.
By drying, Koshihikari rice grains with reduced salt-soluble protein and reduced allergen were obtained. The obtained salt-soluble protein-removed allergen-reduced rice grains are pulverized, 1 g of the obtained pulverized rice is precisely weighed, and then a 10-fold volume extract (4 M urea aqueous solution is adjusted to pH 7.4 with 100 mM Tris buffer). The mixture was stirred at room temperature for 2 hours with a stirrer, centrifuged (10,000 rpm: 10 minutes), and the supernatant was collected. The supernatant was dialyzed against distilled water and then freeze-dried to obtain a urea extract (sample A) of salt-soluble protein-removed rice.

(2) The reaction of the sample A with the IgE antibody in the serum of a rice allergic patient was measured by ELISA. That is, a phosphate buffer solution (hereinafter referred to as PB
Sample A was dissolved at 10 μg / ml in a solution in which urea was dissolved at 4 M in S), and 100 μl of the resulting solution was added to an ELISA plate Maxisorp (manufactured by Nunc Corporation). Coated at ° C. After washing the plate twice with PBS, 250 μl of 2% bovine serum albumin (hereinafter, BSA) was added,
Blocking was performed at 7 ° C. for 1 hour, followed by washing twice with PBS. Serum X from a patient diagnosed with rice allergy or healthy adult serum (control serum) was diluted 10 times with PBS containing 0.2% BSA and 0.05% Tween 20, and 100 µl of each diluted solution was added. The reaction was added to the plate and allowed to react at room temperature overnight. 0.05% Tween
After washing the plate four times with PBS containing n20 (hereinafter, PBS-T), 2,500 of a sheep anti-human IgE antibody (manufactured by Vector) conjugated with biotin as a secondary antibody was used.
100 μl of a volume-fold diluted solution was added to the plate, and reacted at 37 ° C. for 2 hours. In addition, 0.2% B
PBS-T containing SA was used. After the reaction with the secondary antibody was completed, the plate was washed five times with PBS-T,
Avidin-conjugated peroxidase (manufactured by Boehringer) diluted 4,000 times by volume was added to perform enzyme labeling. Again, the plate was washed 5 times with PBS-T, and 100 μl of a substrate solution (0.1% orthophenylenediamine solution to which 0.034% H ₂ O ₂ was added) was added. 3 at 37 ° C
After correctly react for 10 minutes, 40 [mu a 4N-H ₂ SO ₄
1 to stop the color development, and the absorbance (490 nm) was measured. All measurements were performed in duplicate, and the average was determined.
The ELISA value using control serum was 0.0
The value was 0.267 in the serum X of the rice allergic patient, whereas the serum E was 30
Antibody was detected.

(3) In order to detect the allergen molecules of the sample A, an immunoblot using the serum X of the patient was performed. That is, to the sample A corresponding to a protein amount of 2 mg, SDS was added to a final concentration of 1%, 2-mercaptoethanol was 1%, Tris-HCl was 10 mM, and glycerin was 20%, and each was added with distilled water. The whole was made up to 1 ml to prepare a sample for SDS-PAGE. The sample was heated at 100 ° C. for 2 minutes,
Bromophenol blue (BPB: B
romphenol blue) and centrifugation (10,000 × g; 10 minutes), followed by SDS-PA
Subjected to GE. As the SDS-PAGE gel, a mini gel system manufactured by Daiichi Pure Chemicals was used. SDS gradient mini gel having a polyacrylamide gel concentration of 10 to 20%,
Those having a migration distance of 8 cm and 12 wells were used. Electrophoresis was performed using a vertical cassette electrophoresis system (manufactured by Daiichi Kagaku) at 40 mA per gel at a constant current for 60 minutes. Running buffer is 0.025M-Tris,
PH 6.8 containing 0.19M glycine, 0.1% SDS
Of Tris-Glycine running buffer was used.

Each fractionated protein was transferred to a polyvinylidene fluoride membrane (hereinafter, PVDF membrane) using a semi-dry blotter. After blocking the transfer membrane with PBS-T containing 5% skim milk for 1 hour at 37 ° C., the transfer membrane was mounted on a mini blotter, and then washed with PBS-T containing 0.5% skim milk (hereinafter, PBS-TS). Patient serum X or control serum diluted by a factor of 1 was injected into each lane and allowed to react at room temperature overnight. Biotin-conjugated sheep anti-human IgE antibody (0.5 mg / m2)
l) 500-fold diluted solution (diluted with PBS-TS) 1
0 ml, then avidin-conjugated peroxidase 500 U / ml diluent (diluted with PBS-TS) 10 m
1 was added to perform enzyme labeling of the protein molecule reacted with the IgE antibody. The reacted allergen molecule was detected by adding a chromogenic substrate and staining. For coloring, 0.
05 M diaminobenzidine trihydrochloride / 0.01 M hydrogen peroxide was used. After reacting at 37 ° C. for 1 hour in the absence of light, the transfer film was washed with distilled water to stop the reaction. The reacted band and the intensity of the staining were detected using a densitometer [manufactured by ATTO Corporation: Densitograph]. The molecular weight of the reacted band was compared with a molecular weight marker (Bio-Rad: biotinylated marker) that was electrophoresed simultaneously. As a result, four bands reacting with patient serum X were detected, and their molecular weights were determined from the molecular weight markers by 60 kd, 36 kd, 33 kd, and 13 kd.
kd.

[0049]

Example 2 [Purification of Salt-Insoluble Protein Remaining in Rice Treated with Salt-Soluble Protein] Koshihikari 10 with reduced salt-soluble protein and allergen reduced obtained in Example 1 (1)
g was sufficiently crushed by a coffee mill. 6M urea-0.5% sodium lauryl sulfate solution 1 was added to the obtained rice powder.
After adding 50 ml and suspending well with an ultrasonic device,
Shake at room temperature for 2 hours. This extraction solution is 4000 rpm
And the resulting supernatant was put into a dialysis membrane and concentrated using Sephadex G-150 (manufactured by Pharmacia). The protein was separated from this concentrated solution by SDS-PAGE using a 5 to 20% gradient gel.
Stained with Coomassie Blue R250, 60kd, 36k
The four bands of d, 33 kd, and 13 kd were respectively cut out, put into a dialysis membrane, and the buffer for SDS-PAGE was 5 m each.
and electrophoretically extracted and recovered using a horizontal gel electrophoresis apparatus. This solution was put into a dialysis membrane again, and concentrated using Sephadex G-150 (manufactured by Pharmacia). Each sample was again subjected to SDS-PAGE using a 5 to 20% gradient gel to separate proteins. And it recovered similarly from the acrylamide gel. Thus, 60 kd, 36 kd, 33 kd,
And purified products of the 13 kd protein.

[0050]

Example 3 [Identification of N-terminal amino acid sequence of salt-insoluble protein] 60 kd, 36 kd, 33 k purified in Example 2
d and 13 kd were subjected to SDS-PAGE on a 10-20% gradient gel to separate
Electroblotted onto DF membrane. This membrane was stained with Ponceau S dye, a portion of each protein band of interest was cut out, and the N-terminal amino acid sequence was determined using a protein sequencer (Perkin Elmer; model 492).

For the 60 kd protein, 17 amino acid residues from the N-terminus could be determined as follows. Ala-Thr-Gly-Ala-Gly-Met-A
sn-Val-Val-Phe-Val-Gly-Al
a-Glu-Met-Ala-Pro When the above amino acid sequence (see SEQ ID NO: 1 in the sequence listing) was searched against a protein database, a waxy protein [Okaga], a rice starch synthase, was found.
Ki et al., Plant Mol. Biol. (1992)
19, pp. 513-516].

For the 36 kd protein, 10 amino acid residues from the N-terminus could be determined as follows. Ser (Ala, Gly) -Lys-Gly-Ala
(Glu) -Gly-Met-Asn-Val-Val
-Phe 1st place “Ser (Ala, Gly)” and 4th place “Al
“a (Glu)” has the same meaning as described above. Each amino acid sequence is shown as SEQ ID NO: 2 in the sequence listing.

For the 33 kd protein, 15 amino acid residues from the N-terminus could be determined as follows. Ser (Ala, Gly) -Thr (Lys) -Gly
-Ala (Glu) -Gly-Met-Asn-Val
-Val-Phe-X-Gly (Thr, Ser) -A
la-Glu-Met 1st place “Ser (Ala, Gly)”, 2nd place “Thr
(Lys) "," Ala (Glu) "at position 4," X "at position 11 and" Gly (Thr, Ser) "at position 12
It has the same meaning as above. Each amino acid sequence is shown in SEQ ID NO: 3 in the sequence listing.

The respective amino acid sequences of the 36 kd protein and the 33 kd protein showed high homology to the N-terminal amino acid sequence of the rice waxy protein.
Until now, a completely matching sequence has not been registered in the protein database, and is considered to be a novel protein.

For the 13 kd protein, 30 amino acid residues from the N-terminus could be determined as follows. Phe-Asp-Val-Leu-Gly-Gln-A
sn (Ser) -Ile (Tyr) -Arg-Gln-
Tyr-Gln-Val (Leu) -Gln-Ser-
Pro-Leu (Val) -Leu-Leu-Gln-
Gln-Gln-Val-Leu-Ser-Pro-T
In Asr (Ser) at position 7 of yr-Asn-Glu-Phe, asparagine (Asn) was detected as a major amino acid at position 7 in the determination of the amino acid sequence, but serine (Ser) was also small. It means that it was simultaneously detected in a significant but significant amount. Similarly, “Ile (Tyr)” in the 8th place is also
A major amount of isoleucine (Ile) and a significantly smaller amount of tyrosine (Tyr) were detected, and "Val (Le
u) ”, a major amount of valine (Val) and a significantly small amount of leucine (Leu) were detected, and further,“ Leu ”at position 17 was also detected.
(Val) "also means that a major amount of leucine (Leu) and a significantly smaller amount of valine (Val) were detected. These amino acid sequences are shown in SEQ ID NO: 4 in the sequence listing. As described above, also for the 13 kd protein, a plurality of amino acids were detected, and a mixture of a plurality of proteins, that is, polymorphism was shown.
3kd prolamin [Yamagata et al., BioSc
i. Biotechnol. Biochem. (199
2) Vol. 56, p. 537]. 13kd
A plurality of prolamins are registered as multiple genes having slightly different amino acid sequences.

[0056]

Example 4 [Immunoblot using anti-waxy serum] The method of Sano et al. [Theor. Appl. Genet. (198
4) Vol. 68, pp. 467-473], the waxy protein was partially purified from Koshihikari. The waxy protein and the purified 60 kd protein, 33 kd protein, and 36 kd protein prepared in Example 2 were fractionated by SDS-PAGE using a 10-20% gradient gel (manufactured by Daiichi Kagaku). , P
Electroblotted onto VDF membrane. The membrane was reacted with rabbit anti-waxy serum, POD-labeled anti-rabbit IgG serum was used as a secondary antibody, and color was developed using OPD (ortho-phenylenediamine) as a substrate. The result is shown in FIG. In FIG. 1, lane 1 is a waxy protein partially purified from Koshihikari, lane 2 is a purified 60 kd protein prepared in Experimental Example 2, and lane 3 is a purified 33 kd protein prepared in Experimental Example 2. Lane 4 is the purified 36 kd protein prepared in Experimental Example 2 above, and
d, 66 kd, 45 kd, 31 kd and 22 kd are the positions of the molecular weight markers used. As is clear from FIG. 1, the 60 kd protein and the waxy protein produced color bands of the same size. The 33 kd and 36 kd proteins also reacted with the anti-waxy serum to produce a colored band. Therefore, the 60 kd protein was found to be a waxy protein. Furthermore, it was suggested that the 33 kd protein and the 36 kd protein are proteins related to the waxy protein, although the molecular weights are significantly different from the waxy protein.

[0057]

[Sequence list]

SEQ ID NO: 1 Sequence length: 17 Sequence type: Amino acid Sequence type: Peptide sequence Ala Thr Gly Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro 1 5 10 15

SEQ ID NO: 2 Sequence length: 10 Sequence type: amino acid Sequence type: peptide Sequence characteristics Location: Other information: Xaa is Ser, Ala or Gly Location: 4 Other information: Xaa Is Ala or Glu

SEQ ID NO: 3 Sequence length: 15 Sequence type: amino acid Sequence type: peptide Sequence characteristics Location: Other information: Xaa is Ser, Ala or Gly Location: 2 Other information: Xaa Is Thr or Lys Location: 4 Other information: Xaa is Ala or Glu Location: 11 Other information: Xaa is unknown Location: 12 Other information: Xaa is Gly, Thr or Ser sequence Xaa Xaa Gly Xaa Gly Met Asn Val Val Phe Xaa Xaa Ala Glu Met 1 5 10 15

SEQ ID NO: 4 Sequence length: 30 Sequence type: amino acid Sequence type: peptide Sequence characteristics Location: 7 Other information: Xaa is Asn or Ser Location: 8 Other information: Xaa is Ile Or Tyr Location: 13 Other information: Xaa is Val or Leu Location: 17 Other information: Xaa is Leu or Val Sequence Phe Asp Val Leu Gly Gln Xaa Xaa Arg Gln Tyr Gln Xaa Gln Ser 1 5 10 15 Pro Xaa Leu Leu Gln Gln Gln Val Leu Ser Pro Tyr Asn Glu Phe 20 25 30

[0062]

According to the method for analyzing a cereal allergen-specific immunological reactant according to the present invention or the method for analyzing a rice allergen-specific immunological reactant according to the present invention, an allergen newly discovered by the present inventors. Utilizing proteins, patients who respond to salt-insoluble allergens, which were hitherto undetectable, can be tested in vitro with small samples. Further, according to the method for analyzing a cereal allergen according to the present invention or the method for analyzing a rice allergen according to the present invention, whether or not the allergen protein is present in food is determined by immunology specific to the allergen protein. A method can be provided which can be tested by the target reactant.

[Brief description of the drawings]

FIG. 1 is a drawing showing the results of immunoblotting performed on purified waxy protein, purified 60 kd protein, purified 33 kd protein, and purified 36 kd protein using anti-waxy serum.

Continuing from the front page (72) Inventor Sohei Nojiri 8-4-1 Higashiogu, Arakawa-ku, Tokyo Inside the Allergen-Free Technology Research Institute (72) Inventor Hiroyuki Anzai 8-4-1 Higashiogu, Arakawa-ku, Tokyo Stock Allergen-Free Technology Research Co., Ltd. (72) Inventor Teisuke Shimada 8-4-1 Higashiogu, Arakawa-ku, Tokyo Co., Ltd. (72) Inventor Kazuyuki Mogi 8-4-Higashiogu, Arakawa-ku, Tokyo No. 1 Allergen-Free Technology Research Institute, Inc. (72) Inventor Kazuko Katsumata 8-4-1 Higashi-Oku, Arakawa-ku, Tokyo (72) Inventor Hiroshi Sugiyama 8-Chome Higashi-Oku, Arakawa-ku, Tokyo, Japan No.4-1 Inside the Allergen-Free Technology Research Institute (72) Inventor Saburo Amano 8-4-1 Higashiogu, Arakawa-ku, Tokyo Allergen-Free Technology Research Co., Ltd.

Claims (14)

[Claims] 1. An allergenic UDP-glucose starch glucosyltransferase is brought into contact with a test sample which may contain a cereal allergen-specific immunological reactant, and said UDP-glucose starch glucosyltransferase having allergenicity is brought into contact with said test sample. And analyzing the result of the reaction between the cereal allergen-specific immunological reactant and the cereal allergen-specific immunological reactant. 2. A salt-insoluble protein having a molecular weight of 60 kd,
Salt-insoluble protein with a molecular weight of 36 kd, molecular weight of 33 kd
Contacting at least one type of rice allergen protein selected from the group consisting of a salt-insoluble protein and a salt-insoluble protein having a molecular weight of 13 kd with a test sample that may contain a rice allergen-specific immunological reactant; Analyzing the result of the reaction between the rice allergen protein and the rice allergen-specific immunological reactant,
Method for analyzing rice allergen-specific immunological reactants. 3. The method according to claim 1, wherein the test sample is a blood sample. 4. The method according to claim 3, wherein the allergen-specific immunological reactant is an IgE antibody or an IgG4 antibody specific to an allergen protein. 5. The result of the reaction between the antibody and the allergen protein is analyzed by ELISA.
The method described in. 6. The method according to claim 3, wherein the allergen-specific immunological reactant is platelets contained in a blood sample or immunocompetent cells contained in a leukocyte fraction of the blood sample. 7. The method according to claim 6, wherein the immunocompetent cells are T cells, B cells, mast cells, or basophils. 8. The result of the reaction between the leukocyte fraction of the blood sample and the rice allergen protein is used to determine the rate of proliferation of T cells in the presence of interleukin-2, or the rate of uptake of purine or pyrimidine bases by T cells. The method according to claim 6, wherein the analysis is performed by: 9. The method according to claim 1, wherein a reaction between the platelet contained in the blood sample or mast cells or basophils contained in the leukocyte fraction of the blood sample and the rice allergen protein is performed by a mediator generated in association with the degranulation reaction. 7. The method of claim 6, wherein analyzing. 10. The method according to claim 9, wherein said mediator is histamine or serotonin. 11. An allergen-specific immunological reactant which specifically immunologically reacts with UDP glucose starch glucosyltransferase having allergenicity, and may contain the above-mentioned UDP glucose starch glucosyltransferase having allergenicity. An allergenic UDP glucose starch glucosyltransferase and the allergen-specific immunological reactant, and analyzing the result of the reaction. Method. 12. A salt-insoluble protein having a molecular weight of 60 kd, a salt-insoluble protein having a molecular weight of 36 kd, and a molecular weight of 33.
a rice allergen-specific immunological reactant that specifically immunologically reacts with at least one rice allergen protein selected from the group consisting of a kd salt-insoluble protein and a salt-insoluble protein having a molecular weight of 13 kd; Contacting a test sample which may contain rice allergen protein, and analyzing the result of the reaction between the rice allergen protein and the rice allergen-specific immunological reactant, analyzing rice allergen Method. 13. The method according to claim 11, wherein the test sample is a food, a feed, a raw material thereof, or an extract or diluent thereof. 14. The method according to claim 14, wherein the allergen-specific immunological reactant is an Ig specific for the allergen protein.
12. An immunocompetent cell specific to an E antibody or an IgG4 antibody, or the allergen protein.
Or the method of 12.