JP2011247618A - Allergy diagnosis kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and accurate method for early diagnosis of an allergic disease.SOLUTION: An allergy diagnosis kit has at least one type of peptide selected from: peptides comprising at least the sequence of glycine-lysine-valine-methionine-valine-leucine-cysteine-asparagine-arginine-alanine-phenylalanine-asparagine-proline-valine-cysteine (Gly-Lys-Val-Met-Val-Leu-Cys-Asn-Arg-Ala-Phe-Asn-Pro-Val-Cys); and peptides comprising at least the sequence of asparagine-phenylalanine-cysteine-asparagine-alanine-valine-valine-glutamate-serine-asparagine-glycine-threonine-leucine-threonine-leucine (Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu).

Description

  The present invention relates to a diagnostic kit equipped with a peptide useful for diagnosis of egg allergy.

  Food allergies continue to increase, and growth disorders due to extreme dietary restrictions are becoming social issues. The most reliable method for diagnosing type I allergic disease is a food load test (feeding test). Although the food load test is a highly reliable test used for definitive diagnosis, it is not widespread because it places a heavy physical burden on the subject and few institutions can perform it. Therefore, at present, the CAP-RAST method (radioallergen adsorption test method) is frequently used in which diagnosis is performed based on whether or not an allergen-specific IgE antibody is present in patient serum. The CAP-RAST method is a simple method but cannot be used for early diagnosis.

  The CAP-RAST method uses the allergen protein molecule itself as an antigen, but by identifying the epitope part involved in the development of allergic symptoms on the allergen protein molecule and cutting out only that part, for example, by using it as an antigen Therefore, early diagnosis of allergic symptoms will be possible.

  Regarding the IgE epitope on the allergen, since the binding of the IgE epitope and IgE and the formation of a cross-linked structure are indispensable for the development of allergic symptoms, many reports have been made on various allergens as models. There have been some reports on IgE epitopes of ovalbumin and ovomucoid which are allergens of egg allergy (Non-patent Document 1 and Patent Document 1).

JP 2007-217303 A

Yoshinori Mine and Prithy Rupa “Fine mapping and structural analysis of immunodominant IgEallergenic epitopes in chicken egg ovalbumin” Protein Engineering vol.16 no.10pp.747-752,2003

  These reports are intended to identify epitopes on allergens by PIN-ELISA method using the serum of egg allergy patients, and did not enable early diagnosis. That is, the epitope which can confirm the onset of an egg allergy symptom early by a simple method was not specified. Moreover, ovumcoid, which is an egg allergen, is not coagulated by heating, and considering that the allergic incidence does not decrease by heating, there is a case where allergy caused by ovomucoid is particularly desired to be diagnosed early.

  In view of the above situation, the present inventors have developed a simple and accurate early diagnosis method. That is, an object of the present invention is to provide a simple and accurate method for early diagnosis of allergic diseases. In particular, the onset of allergic symptoms caused by ovomucoid, an egg allergen, can be diagnosed early.

  The present inventors have completed the present invention as a result of studies using various levels of serum from patients with various levels of allergen-specific IgE, that is, those diagnosed with egg allergy by the CAP-RAST method. It came to do.

  The present invention includes the following inventions. [1] A peptide having at least the sequence of glycine-lysine-valine-methionine-valine-leucine-cysteine-asparagine-arginine-alanine-phenylalanine-asparagine-proline-valine-cysteine, asparagine-phenylalanine-cysteine-asparagine-alanine-valine A kit for allergy diagnosis equipped with at least one peptide selected from peptides having at least the sequence of valine-glutamic acid-serine-asparagine-glycine-threonine-leucine-threonine-leucine. [2] A diagnostic kit, wherein the peptide consists of an amino acid sequence of 15 to 25. [3] On a carrier coated with a non-specific adsorption inhibitor composed of an electrically neutral water-soluble polymer as a whole molecule, at least a photoreactive group, an amino group, and a room temperature-reactive functional group in one molecule. A diagnostic kit comprising a peptide loaded with an immobilizing agent. [4] Diagnosis characterized in that the non-specific adsorption inhibitor comprises a photocrosslinking agent having at least two photoreactive groups in one molecule and an electrically neutral water-soluble polymer as a whole molecule. For kit. [5] A diagnostic kit in which the photoreactive group is an azide group or a diazirine group. [6] A diagnostic kit in which an amino group and a room temperature-reactive functional group are epoxy groups or succinimide esters. [7] The diagnostic kit wherein the water-soluble polymer is a nonionic polymer. [8] The diagnostic kit, wherein the nonionic polymer is a vinyl polymer of polyethylene glycol mono (meth) acrylate.

Amino acid sequence of chicken egg white ovomucoid. 36 kinds of immobilized peptides in Examples. Results of luminescence reaction between human serum and ovomucoid peptide. The figure which shows the correlation with the result of the luminescent reaction using human serum, and the result of CAP-RAST method.

  Glycine-lysine-valine-methionine-valine-leucine-cysteine-asparagine-arginine-alanine-phenylalanine-asparagine-proline-valine-cysteine peptide, asparagine-phenylalanine-, which is mounted in the diagnostic kit of the present invention As long as the peptide having at least the sequence of cysteine-asparagine-alanine-valine-valine-glutamic acid-serine-asparagine-glycine-threonine-leucine-threonine-leucine contains at least the amino acid sequence represented by the above sequence as a partial sequence The number of amino acid residues is not particularly limited. Typically, it is a peptide having 15 to 30 amino acid residues, and more typically a peptide having 15 to 25 amino acid residues.

  As used herein, a peptide having at least the sequence of glycine-lysine-valine-methionine-valine-leucine-cysteine-asparagine-arginine-alanine-phenylalanine-asparagine-proline-valine-cysteine, asparagine-phenylalanine-cysteine-asparagine-alanine A peptide having at least the sequence of -valine-valine-glutamic acid-serine-asparagine-glycine-threonine-leucine-threonine-leucine may be simply referred to as "epitope peptide".

  The above-described epitope peptide has a function of recognizing and binding to an anti-egg white ovomucoid antibody present only in a patient who develops egg allergy in a body fluid (eg, serum) of a subject. Therefore, the diagnostic kit of the present invention loaded with these epitope peptides can be used as an early diagnostic agent for egg allergy.

  According to the diagnostic kit of the present invention, egg allergy can be diagnosed by bringing an epitope peptide into contact with a test sample and detecting and quantifying the antibody present in the test sample.

  The epitope peptide can be produced by a known peptide synthesis method, for example, a method using a fully automatic peptide synthesizer.

  The epitope peptide may be in the form of a salt, preferably a physiologically acceptable acid addition salt, if necessary. Such salts include salts of inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, Citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The test sample may be anything as long as antibodies against egg white ovomucoid can be detected. For example, blood, plasma, serum, synovial fluid, lymph fluid, joint fluid, ascites, saliva, Examples thereof include cerebrospinal fluid and fractional components obtained therefrom.

  In the present invention, “diagnosis” means determining whether or not a subject suffers from egg allergy, whether or not there is a risk of suffering from egg allergy in the future, determination of the effect of treatment, And the determination of whether there is a risk of recurrence of egg allergy after treatment. Diagnosis also includes measuring the severity of egg allergy and the risk.

  The diagnostic kit of the present invention is used in a method for diagnosing egg allergy, and is used in a method capable of detecting a change in physical quantity based on an antigen-antibody reaction between an epitope peptide and an antibody in a test sample. If it is a diagnostic kit, the form will not be specifically limited. For example, a method of detecting the antigen-antibody reaction as color development, luminescence, and fluorescence using a labeling substance as is done with a labeled immunoassay reagent, and aggregation of an insoluble carrier as is done with an immunological agglutination reagent A diagnostic kit used for immunoassay such as a method for detecting the antigen-antibody reaction by visual observation or turbidity can be employed.

Specific examples of immunoassays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), chemiluminescent immunoassay (CLIA), in-gel Sedimentation reaction (Ouchterlony method, immunoelectrophoresis, single radial
immunodiffusion method), immunoturbidimetric method, particle agglutination method and the like.

  In the above method, as a diagnostic kit, an epitope peptide immobilized on a solid phase carrier can be adopted. At this time, the solid phase carrier that can be used is not particularly limited as long as it is a carrier insoluble in a solvent in the reaction system of the antigen-antibody reaction, and a known solid phase carrier can be used. As the shape of the solid phase carrier, an appropriate shape may be selected according to the purpose of use, and examples thereof include a test plate shape, a bead shape, a spherical shape, a disk shape, a tube shape, and a filter shape. In addition, as the material thereof, those used as usual carriers for immunoassays, for example, synthetic resins such as polypropylene, polystyrene, polyacrylamide, etc., or reactive properties such as sulfonic acid groups and amino groups by known methods are used. Examples include those into which functional groups are introduced, glass, polysaccharides, silica gel, porous ceramics, metal oxides, erythrocytes and the like.

  Known methods such as physical adsorption, covalent bonding, ionic bonding, and cross-linking can be used as the method for immobilizing the epitope peptide on the solid phase carrier, but from the viewpoint of ease of operation, reproducibility, sensitivity, etc. It is preferable to use a covalent bond method.

  Examples of the labeling substance used in the immunoassay include fluorescent substances, luminescent substances, dyes, enzymes, coenzymes, and radioisotopes. Among them, enzyme labels such as alkaline phosphatase and peroxidase are preferable because they are excellent in safety and economy and can achieve the necessary sensitivity relatively easily. The labeling substance can be labeled by directly binding to an epitope peptide or a secondary antibody against an antibody that binds to the epitope peptide. Alternatively, indirect labeling can be performed using an antibody recognizing a labeling substance or an avidin-biotin system.

  The diagnostic kit of the present invention can be made into a kit together with necessary reagents. In the case of a reagent kit for ELISA, the constituent reagents include, for example, the epitope peptide of the present invention, an enzyme-labeled secondary antibody, a substrate solution, and the like. The epitope peptide may be preliminarily immobilized on a solid phase, or may be in a form immobilized on a solid phase at the time of use. In this case, the reagent kit may include a solid phase for immobilizing the epitope peptide. In addition, the reagent kit for agglutination reaction contains carrier particles on which an epitope peptide is immobilized.

  In addition to the above-described constituent reagents, a standard sample, a buffer solution, a lysis solution, a washing solution, a reaction stopping solution, an instruction manual, and the like may be included. Each of the above constituent reagents can be in the form of a suspension, a solution, or a lyophilized product.

  The epitope peptide is most preferably used as a protein chip (protein array) in which a plurality of these types are affixed at a high density, and such a protein chip is also included in the diagnostic kit of the present invention. In this case, the kit may include a mass spectrometer, software necessary for measurement / analysis, a computer into which the software is introduced, and the like.

As a production method of the preferred protein chip, as described in JP 2010-101661, the electrically neutral comprising a water-soluble polymer for preventing unspecific adsorption material The coated on the carrier as a whole molecule, one molecule In this method, the epitope peptide is immobilized with an immobilizing agent having at least a photoreactive group, an amino group, and a room temperature-reactive functional group.

  Examples of water-soluble polymers that can be used as non-specific adsorption inhibitors include ambipolar polymers such as phosphorylcholine-containing polymers and nonionic polymers. Nonionic polymers include polyalkylene glycols such as polyethylene glycol (PEG) and polypropylene glycol; vinyl alcohol, methyl vinyl ether, vinyl pyrrolidone, vinyl oxazolidone, vinyl methyl oxazolidone, 2-vinyl pyridine, 4-vinyl pyridine, N-vinyl. Succinimide, N-vinylformamide, N-vinyl-N-methylformamide, N-vinylacetamide, N-vinyl-N-methylacetamide, 2-hydroxyethyl methacrylate, polyethylene glycol methacrylate, polyethylene glycol acrylate, acrylamide, methacrylamide, N, N-dimethylacrylamide, N-iso-propylacrylamide, diacetoneacrylamide, methylolacrylamide, acryloylmorpholine, acryloylpi Loridine, acryloylpiperidine, styrene, chloromethylstyrene, bromomethylstyrene, vinyl acetate, methyl methacrylate, butyl acrylate, methyl cyanoacrylate, ethyl cyanoacrylate, n-propyl cyanoacrylate, iso-propyl cyanoacrylate, n-butyl cyanoacrylate, Single or mixture of monomer units such as iso-butyl cyanoacrylate, tert-butyl cyanoacrylate, glycidyl methacrylate, ethyl vinyl ether, n-propyl vinyl ether, iso-propyl vinyl ether, n-butyl vinyl ether, iso-butyl vinyl ether, tert-butyl vinyl ether Nonionic vinyl-based polymer containing as a constituent: gelatin, casein, collagen, gum arabic, xanthan gum, tragacanth gum , Guar gum, pullulan, pectin, sodium alginate, hyaluronic acid, chitosan, chitin derivatives, carrageenan, starch (carboxymethyl starch, aldehyde starch), dextrin, cyclodextrin and other natural polymers, methyl cellulose, viscose, hydroxyethyl cellulose, hydroxy Natural polymers such as water-soluble cellulose derivatives such as ethyl methyl cellulose, carboxymethyl cellulose, and hydroxypropyl cellulose can be exemplified, but are not limited thereto. It is also possible to use a commercially available product of a photocrosslinking water-soluble polymer based on these polymers, for example, “AWP” manufactured by Toyo Gosei Co., Ltd. based on polyvinyl alcohol. Of these, particularly preferred are polyethylene glycol polymers, and vinyl polymers of polyethylene glycol mono (meth) acrylate.

  The water-soluble polymer used in the present invention is a water-soluble polymer that is electrically neutral as a whole molecule. Here, “electrically neutral as a whole molecule” means that it does not have or does not have a group that becomes ionized by ionization in an aqueous solution having a pH near pH (pH 6 to 8). It has an ion and an anion, and it means that the total charge becomes substantially zero. Here, “substantially” means that the total charge becomes 0 or is small enough not to adversely affect the effect of the present invention even if it does not become 0.

  The water solubility of the water-soluble polymer used in the present invention (the number of grams dissolved in 100 g of water) is preferably 5 or more.

  “Nonionic” means having substantially no group that becomes ionized by ionization in an aqueous solution having a pH close to neutrality (pH 6 to 8). Here, “substantially” means that such a group is not contained at all, or even if such a group is contained, it is so small that the effect of the present invention is not adversely affected (for example, the number of such groups is the number of carbon atoms). 1% or less).

  The molecular weight (average molecular weight, the same applies hereinafter) of the nonionic water-soluble polymer is not particularly limited and is usually about 1000 to 50 million. However, if the molecular weight of the nonionic water-soluble polymer becomes too large, the water solubility is limited. Will arise. Moreover, since immobilization cannot be performed efficiently when the molecular weight is small, about 10,000 to 50 million is preferable. The concentration at the time of using the water-soluble polymer is not particularly limited.

  When used as a non-specific adsorption inhibitor, a photoreactive group may be introduced into the water-soluble polymer, but a photocrosslinking agent having at least two photoreactive groups in one molecule It is convenient and desirable to add to an electrically neutral water-soluble polymer.

  The addition amount of the photocrosslinking agent having at least two photoreactive groups in one molecule is not particularly limited, but is preferably 0.1 to 50% by weight based on the water-soluble polymer, More preferably, it is 1-30 weight%, More preferably, it is 1-20 weight%.

Preferable examples of the photoreactive group possessed by the photocrosslinking agent include an azide group (—N ₃ ), but are not limited thereto. As the photocrosslinking agent, a diazide compound having two azide groups is preferable, and a water-soluble diazide compound is particularly preferable. Preferable examples of the photocrosslinking agent used in the present invention include diazide compounds represented by the following general formulas [I] and [II].

In general formula [I], R represents a single bond or an arbitrary group. Since -R- has a structure that only connects two phenyl azide groups, it is not particularly limited as long as the diazide compound has the necessary water solubility (described later). Examples of preferred —R— include a single bond (that is, two phenyl azide groups are directly linked), an alkylene group having 1 to 6 carbon atoms (including one or two carbon-carbon unsaturated bonds). 1 or 2 carbon atoms may be bonded to oxygen to form a carbonyl group, particularly preferably a methylene group.), —O—, —SO ₂ —, —SS -, - S -, - R 2 ··· Y ··· R 3 - ( where ... represents a single bond or a double bond, Y is a cycloalkylene group having 3 to 8 carbon atoms, R ₂ and R ₃ independently represents an alkylene group having 1 to 6 carbon atoms (one or two carbon-carbon unsaturated bonds may be included, and the bond between the carbon atom at the base of the alkylene group and Y is doubled). The cycloalkylene group may be a bond, or one or two carbon atoms may form a carbonyl group by a double bond with oxygen), May be substituted with one or more arbitrary substituents (when substituted, preferably one or two of the carbon atoms constituting the cycloalkylene group are double-bonded to oxygen). A carbonyl group and / or substituted with one or two alkyl groups having 1 to 6 carbon atoms, and each benzene ring in the general formula [I] is One or more optional substituents (preferably a hydrophilic group such as halogen, an alkoxyl group having 1 to 4 carbon atoms, a sulfonic acid or a salt thereof) may be substituted. .-, there can be mentioned the following ones as examples _{- CH 2 -, - O -} , - SO 2 -, - SS -, - S -, - CH = CH -, - CH = CH-CO-, -CH = CH-CO-CH = CH-, -CH = CH-, the substituent shown in Chemical Formula 2.

  N in the above formula [II] is 1-1000, preferably 1-500. If the molecular weight is too large, the photocrosslinking efficiency is deteriorated.

  Among the photocrosslinking agents used in the present invention, particularly preferred are water-soluble ones. “Water-soluble” for the photocrosslinking agent means that an aqueous solution having a concentration of 0.5 mM or more, preferably 2 mM or more can be provided. The concentration at the time of using the photocrosslinking agent is not particularly limited.

  In addition, the nonspecific adsorption | suction prevention material which concerns on this invention is used including appropriate solvents, such as buffer solutions, such as water and phosphate buffered saline (PBS), in the case of the use. This is to facilitate application to the carrier. At this time, the concentration of the water-soluble polymer is not particularly limited, but is preferably 0.005% by weight to 20% by weight, and preferably 0.04% by weight to 10% by weight with respect to the entire solution (suspension). % Is more preferable. As described above, the concentration of the photocrosslinking agent in this case is preferably 0.1 to 50% by weight, more preferably 1 to 30% by weight, and still more preferably 1 to 20% by weight with respect to the water-soluble polymer. It is.

  The immobilizing agent according to the present invention may include a surfactant such as polyoxymonolaurate (Tween 20 (registered trademark)). In particular, by including both the buffer solution and the surfactant, an effect of reducing the background value is obtained as a synergistic effect, which is particularly useful when detecting using serum or the like. The addition amount of the surfactant is preferably 0.1 to 20% with respect to the entire solution (suspension). Furthermore, an organic solvent may be included. As this organic solvent, a lower alcohol (preferably ethanol) or the like mixed with water at an arbitrary ratio can be used.

  As the immobilizing agent having a photoreactive group, an amino group and a room temperature reactive group in one molecule used together with the substance to be immobilized, the following are preferable. That is, the photoreactive group is preferably an azido group or a diazirine group, and the room temperature reactive group is preferably an active ester such as an epoxy group or a succinimide ester. Specifically, N- [4- [3- (trifluoromethyl) -3H-diazirine-3-yl] phenyl] oxirane, N- [4- [3- (trifluoromethyl) -3H-diazirine-3 -yl] benzoxyl] succinimide, 5-azido-2-nitrobenzoic acid N-hydroxysuccinimide ester and the like are preferably used.

  When used in combination with a substance to be immobilized, the amount of the immobilizing agent having a photoreactive group, amino group, and room temperature reactive group in one molecule is not particularly limited. On the other hand, 0.001% by weight to 100% by weight is preferable, and 0.01% by weight to 10% by weight is particularly preferable. The concentration of the immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule is not particularly limited.

  The immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule according to the present invention is applied including a solvent. As the solvent, water, lower alcohol mixed with water at an arbitrary ratio, dimethylformamide (DMF), and a mixture thereof can be used.

  The photocrosslinking agent, the water-soluble polymer, and the immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule are known per se. These can be produced by known production methods, and some are commercially available.

  The azide group or diazirine group used as the photoreactive group of the present invention is a nitrogen radical or a carbon radical generated by irradiating with light, and a nitrogen radical or a carbon radical is generated. This nitrogen radical or carbon radical is an amino group or a carboxyl group. It is possible to bond not only to functional groups such as, but also to carbon atoms constituting organic compounds, so that it can crosslink with most organic substances. That is, the photo-crosslinking agent contained in the non-specific adsorption inhibitor effectively cross-links the carrier and the water-soluble polymer of the non-specific adsorption inhibitor, and the immobilizing agent according to the present invention effectively non-specific adsorption. Crosslinks with the inhibitor.

  On the other hand, the epoxy group or active ester in the immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule selectively reacts with the amino group of the substance to be immobilized, on the carrier. This makes it possible to fix the orientation of the material to be immobilized.

  According to the present invention, immobilization of a desired substance on a carrier can be performed as follows. First, a non-specific adsorption inhibitor is coated on a carrier, and a mixture of a substance to be immobilized thereon and a fixing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule is spotted. Then, after the reaction at room temperature, the entire surface of the support may be irradiated with light, or a fixing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule on the nonspecific adsorption preventing material is firstly prepared. After spotting and light irradiation, the substance to be immobilized may be spotted thereon and then reacted at room temperature.

  Although there is no restriction | limiting in particular in the film thickness of a nonspecific adsorption | suction prevention material, Usually, 0.01-10 micrometers, Preferably it is 0.05-1 micrometer. The concentration of the substance to be immobilized is not particularly limited, but is usually 0.01 to 10%, preferably 0.05 to 1%.

The light to be irradiated may be any light as long as the photoreactive group used can generate radicals. When an azide group or a diazirine group is used as the photoreactive group, an ultraviolet ray of 300 to 400 nm is preferable. The dose of the irradiated light is not particularly limited, but is usually about 1 mW to 100 mW per 1 cm ² .

  By irradiating light to the non-specific adsorption preventing material, the photoreactive group generates a radical, and the carrier and the water-soluble polymer are covalently bonded. On top of that, a mixture of a substance to be immobilized and a fixing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule is spotted, and a photoreactive group is contained in one molecule by reacting at room temperature. And a fixing agent having an amino group and a room temperature reactive group and a substance to be immobilized are covalently bonded, and then irradiated with light, whereby a photoreactive group, an amino group, and a room temperature reactive group are contained in one molecule. The photoreactive group of the immobilizing agent having a radical generates a radical and is covalently bonded to the water-soluble polymer. As a result, the substance to be immobilized on the carrier is oriented and fixed. In addition, an immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule is first spotted on a non-specific adsorption preventing material, and after irradiation with light, a substance to be immobilized is spotted thereon. And may be allowed to react at room temperature. In particular, the two-step spot method is effective when a buffer solution or the like is included in the solution of the substance to be immobilized. In the normal one-step spot method, the sensitivity decreases when a buffer solution or the like is included, but the sensitivity does not decrease in the two-step spot method. In the method of the present invention, the substance to be immobilized is oriented and fixed because it binds to a room temperature reactive group of a fixing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule. It will be. Therefore, the sensitivity can be increased as compared with a random immobilization method in which a substance to be immobilized is not bonded to a specific site that is immobilized with a radical by a conventional photoreactive group.

Preparation of water-soluble polymer 26.3 g of polyethylene glycol monomethacrylate (polyethylene glycol part molecular weight 350) was dissolved in 200 ml of ethyl acetate, 61.6 mg of AIBN was added as an initiator, and the mixture was reacted for 6 hours under reflux. After removing the solvent with an evaporator, the reaction solution was dissolved in water and subjected to ultrafiltration to remove unreacted monomers. Polymer formation was confirmed by gel filtration chromatography (GPC).

Formation of non-specific adsorption inhibitor and immobilization of peptides
Sodium 4,4′-diazidostilbene-2,2′-disulfonate (commercially available) was used as a photocrosslinking agent. Mixing was performed so that the concentration of the photocrosslinking agent was 0.15% and the concentration of the water-soluble polymer was 3%. The obtained aqueous solution was spin-coated on a high impact polystyrene resin carrier, dried (film thickness: about 0.1 μm), and then irradiated with UV (black light for 7 minutes). Next, DMF-water (1: 5) in which the fixing agent N- [4- [3- (trifluoromethyl) -3H-diazirine-3-yl] phenyl] oxirane (Taika Pharmaceutical Co., Ltd.) was mixed to 0.001%. 7) 20 nL of the solution was spotted, dried, and then irradiated with UV (black light for 7 minutes). Then, 20 nL of an ovomucoid 0.1% aqueous solution and the following immobilized peptide 0.1% DMF-water (1: 5) solution was spotted, reacted and immobilized at 37 ° C. for 30 minutes to prepare an allergy diagnostic kit.

Immobilized peptides In the amino acid sequence of chicken egg white ovomucoid shown in Fig. 1, 36 types of 15-residue overlapping peptides (Sigma) (Fig. 2) shifted by 5 residues (hereinafter referred to as peptide #) 1 to 36).

Measurement After washing the above allergy diagnostic kit with PBS (0.1% Tween 20 (registered trademark)), human serum (International Diagnostic Resources, Inc.) showing the results shown in Table 1 by the egg white CAP-RAST method; The reaction was allowed to proceed at 37 ° C for 20 minutes. After washing with PBS (0.1% Tween20 (registered trademark)), it was reacted with a horseradish peroxidase (HRP) -labeled secondary antibody (commercially available) at 37 ° C. for 20 minutes. After washing with PBS (0.1% Tween20 (registered trademark)), a chemiluminescent reagent was added, and the luminescence intensity was measured. The results are shown in FIGS. In addition, the description of the numbers that did not emit light was omitted.

  From the results of FIG. 3, peptide # 17 (amino acid sequence: Gly-Lys-Val-Met-Val-Leu-Cys-Asn-Arg-Ala-Phe-Asn-Pro-Val-Cys) and peptide # 34 (amino acid sequence) : Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu) is considered an epitope peptide.

  According to the results of the luminescence reaction in FIG. 3, the ovomucoid of the protein emitted light for the first time when the CAP value was considerably high (RS-14474 having a CAP value of 65.60), compared with the case of peptide # 17. Even a low value (21776-CJ with a CAP value of 0.45 kU / L) emitted light. Furthermore, according to FIG. 4 showing the correlation with the results of the CAP-RAST method, peptide # 17 has a peak of luminescence intensity around a CAP value of 10 kU / L and is negative (CAP value of 0.34 kU / L or less). ), False positive (CAP value of 0.35 to 0.69 kU / L), positive (CAP value of 0.70 kU / L or more), the rise in the vicinity of the boundary was abrupt. For this reason, according to peptide # 17, the boundary region can be diagnosed accurately.

  Peptide # 34 also emits light with a low CAP value (14674 with a CAP value of 1.05 kU / L). Peptide # 34 does not have a peak value, and the luminescence intensity increases in proportion to the CAP value. Also in this case, early diagnosis can be performed more accurately in the vicinity of the negative, false positive, and positive boundaries than when the allergen protein ovomucoid is used. Furthermore, since peptide # 17 and peptide # 34 have different luminescence intensity rises as described above, comprehensive diagnosis is possible by using both peptides in combination.

  If egg allergy is diagnosed using the allergy diagnostic kit of the present invention, egg allergy can be diagnosed quickly and with high reliability without subjecting a subject to a physical burden in a food load test. By performing allergy diagnosis with high reliability, earlier diagnosis is possible than diagnosis by the current CAP-RAST method.

Claims (8)

  Peptide having at least the sequence of glycine-lysine-valine-methionine-valine-leucine-cysteine-asparagine-arginine-alanine-phenylalanine-asparagine-proline-valine-cysteine, asparagine-phenylalanine-cysteine-asparagine-alanine-valine-valine- A kit for allergy diagnosis in which at least one peptide selected from peptides having at least the sequence of glutamic acid-serine-asparagine-glycine-threonine-leucine-threonine-leucine is mounted on a solid phase carrier.   The kit according to claim 1, wherein the peptide consists of 15 to 25 amino acid sequences.   Immobilization having at least a photoreactive group, an amino group, and a room temperature-reactive functional group in one molecule on a carrier coated with a nonspecific adsorption inhibitor made of an electrically neutral water-soluble polymer as a whole molecule The kit according to claim 1 or 2, wherein the peptide is loaded with an agent.   The non-specific adsorption preventing material comprises a photocrosslinking agent having at least two photoreactive groups in one molecule and a water-soluble polymer that is electrically neutral as a whole molecule. 4. The kit according to any one of 3.   The kit according to any one of claims 1 to 4, wherein the photoreactive group is an azide group or a diazirine group.   The kit according to any one of claims 1 to 5, wherein the room temperature reactive functional group is an epoxy group or a succinimide ester.   The kit according to any one of claims 1 to 6, wherein the water-soluble polymer is a nonionic polymer.   The kit according to claim 7, wherein the nonionic polymer is a vinyl polymer of polyethylene glycol mono (meth) acrylate.

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