JP2012073114A - Allergy diagnostic kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and accurate method for early diagnosis of allergic diseases, especially to enable early diagnosis of the onset of allergic symptoms caused by Hypsizigus marmoreus.SOLUTION: The allergy diagnostic kit includes at least one kind of peptide selected from peptides having at least a amino acid sequence of serine-glycine-leucine-proline-valine. The peptide preferably has a sequence repeatedly containing the amino acid sequence of serine-glycine-leucine-proline-valine.

Description

  The present invention relates to an allergy diagnostic kit equipped with a peptide useful for diagnosing Bunashimeji allergy.

  Allergies continue to increase and become a social problem. Currently, as a method for diagnosing type I allergic disease, the CAP-RAST method (diagnosis based on the presence of allergen-specific IgE antibodies in patient serum) Radioallergen adsorption test method) is used. However, allergens that can be diagnosed by the CAP-RAST method are limited, and are not suitable for "mushroom allergy" that mushrooms allergens. In addition, there is no in vitro test method that can appropriately diagnose factors of type III and type IV allergies other than type I. For this reason, methods such as precipitating antibodies are used for type III allergies, and methods such as macrophage migration inhibition and lymphocyte blastogenesis are used for type IV allergies. I can't.

  In addition, the CAP-RAST method and the precipitated antibody method use the allergen protein molecule itself as an antigen, but specify the epitope part involved in allergic symptoms on the allergen protein molecule, cut out only that part, For example, by using an antigen, it is considered that the accuracy of allergy diagnosis is improved and precise diagnosis is possible.

  Regarding the diagnosis of allergic symptoms as described above, several examples of diagnosis of mushroom allergy have been reported (Non-patent Documents 1 and 2).

Takahiro Takeuchi, Mari Tsujibayashi, Kenji Tsushima, Kazutoshi Urushibata, Masayuki Hanaoka, Shinshu Medical Journal 53 (2): 73-75, 2005 1 Example Yuji Tanaka, Nobuyuki Tanaka, Journal of the Japan Society of Internal Medicine 98 (12): 108-114, 2009 Subtypes / special types of asthma Occupational asthma

  These reports show the results of diagnosis by lymphocyte stimulation test and sedimentation antibody test, and are not related to precise diagnosis of allergic patients. That is, it does not enable highly sensitive diagnosis by a simple method. In addition, there is no description regarding the identification of epitopes that can confirm the onset of allergic symptoms at an early stage.

  In view of the above situation, the present inventors have developed a simple and highly accurate diagnostic method. That is, an object of the present invention is to provide a diagnostic method for allergic diseases that can be easily and precisely diagnosed. Therefore, an object of the present invention is to enable early diagnosis of mushroom allergy, in particular, onset of allergic symptoms caused by Bunashimeji.

  The present inventors first used a patient serum (positive serum) diagnosed as positive by an interview and various symptoms, and a standard serum (negative serum) as a screening test. The structure of the protein was analyzed. As a result of this analysis, the amino acid sequence of Bunashimeji, which is considered to be an epitope, is estimated, and the reactivity of the peptide containing the amino acid sequence estimated as this epitope with patient sera (positive and negative) is studied, thereby completing the present invention. It came to.

  The present invention includes the following inventions. [1] A kit for allergy diagnosis in which a peptide having at least the amino acid sequence of serine-glycine-leucine-proline-valine is mounted on a solid phase carrier. [2] A diagnostic kit, wherein the peptide has a sequence that repeatedly contains the amino acid sequence of serine-glycine-leucine-proline-valine. [3] A diagnostic kit, wherein the peptide consists of 15 to 30 amino acid sequences. [4] On a carrier coated with a nonspecific 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 per molecule. A diagnostic kit comprising a peptide loaded with an immobilizing agent. [5] 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. [6] A diagnostic kit in which the photoreactive group is an azide group or a diazirine group. [7] A diagnostic kit wherein the functional group reactive with an amino group at room temperature is an epoxy group or a succinimide ester. [8] The diagnostic kit, wherein the water-soluble polymer is a nonionic polymer. [9] The diagnostic kit, wherein the nonionic polymer is a vinyl polymer of polyethylene glycol mono (meth) acrylate.

Amino acid sequence of Bunashimeji. 6 kinds of immobilized peptides in Examples. Results of IgE measurement of positive sera against standard sera with Peptides # 1-4 IgE measurement result of positive serum with respect to standard serum by peptide # 4-6 Results of IgE measurement of sera from Bunashimeji factory employees using peptide # 4 Results of IgG measurement of positive sera against standard sera by peptide # 1-4 IgG measurement result of standard serum with peptide # 4 and standard error

  The peptide having at least the serine-glycine-leucine-proline-valine sequence mounted in the diagnostic kit of the present invention has the number of amino acid residues as long as it contains at least the amino acid sequence represented by the above sequence as a partial sequence. 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.

  In the present specification, a peptide having at least the sequence of serine-glycine-leucine-proline-valine may be simply referred to as “epitope peptide”.

  "The sequence containing the amino acid sequence of serine-glycine-leucine-proline-valine (herein referred to as SGLPV)" refers to a sequence containing a plurality of units when SGLPV is one structural unit. For example, when 2 units are included, SGLPVSGPLPV is included, and when 3 units are included, the array includes SGLPVSGLPVSGPLV. The epitope peptide preferably contains 3 or more units of SGLPV. In this case, the epitope peptide tends to be suitable for both IgE measurement and IgG measurement.

  The above-described epitope peptide has a function of recognizing and binding to an anti-Bunashimeji antibody present only in a patient who develops Bunahimeji 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 Bunashimeji allergy.

  According to the diagnostic kit of the present invention, diagnosis of Bunashimeji allergy can be performed by contacting the epitope peptide 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 any sample that can detect antibodies against Buna shimeji, such as blood, plasma, serum, synovial fluid, lymph fluid, joint fluid, ascites, saliva, spinal cord isolated from the subject. Liquid, and fraction components obtained therefrom.

  In the present invention, “diagnosis” refers to the determination of whether or not the subject suffers from beech shimeji allergy, the determination of whether there is a risk of suffering from beech shimeji allergy in the future, the determination of the effect of treatment, And the determination of whether or not there is a risk of recurrence of Bunashimeji allergy after treatment. Diagnosis also includes measuring the severity of the allergic disease and its risk.

  The diagnostic kit of the present invention is used in a method for diagnosing beech shimeji 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. 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 preferable method for producing a protein chip, as described in JP-A-2010-101661, one molecule is formed on a carrier coated with a nonspecific adsorption inhibitor composed of an electrically neutral water-soluble polymer as a whole 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 bipolar 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 inhibitor according to the present invention is used by containing an appropriate solvent such as water and a buffer solution such as phosphate buffered saline (PBS). 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 photocrosslinking agent contained in the nonspecific adsorption inhibitor effectively crosslinks the carrier and the water-soluble polymer of the nonspecific adsorption inhibitor, and the immobilizing agent according to the present invention effectively nonspecifically adsorbs. Crosslink with 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 nonspecific 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 an immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule on the non-specific adsorption inhibitor. After spotting and light irradiation, the substance to be immobilized may be spotted thereon and then reacted at room temperature.

  The film thickness of the nonspecific adsorption inhibitor is not particularly limited, but is usually 0.01 to 10 μm, preferably 0.05 to 1 μm. 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 the non-specific adsorption inhibitor with light, 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 the non-specific adsorption inhibitor, and after light irradiation, 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 peptide immobilization
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). Thereafter, 20 nL of a 0.1% aqueous solution of bunashimedipeptide was spotted and reacted and immobilized at 37 ° C. for 30 minutes to prepare an allergy diagnostic kit.

In the immobilized peptide , 5 amino acids of serine-glycine-leucine-proline-valine estimated as epitopes by the screening test by Western blot method in the amino acid sequence of Bunashimeji shown in FIG. Three types of 15-residue peptides (hereinafter referred to as peptides # 1 to 3) in the region were used. In addition, three types of 15-residue, 20-residue, and 25-residue peptides (hereinafter referred to as peptides # 4 to 6) containing 5 amino acid sequences of serine-glycine-leucine-proline-valine were used. . The amino acid sequences of peptides # 1-6 are shown in FIG.

Measurement After washing the above allergy diagnostic kit with PBS (0.1% Tween20 (registered trademark)), the serum of Buna Shimeji positive serum (Sapporo Medical University), negative standard serum (Rockland Immunochemical, Inc.) or Buna Shimeji factory employees The reaction was carried out at 20 ° 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.

  In the IgE measurement, anti-human IgE GE-80P (commercial product) diluted 100-fold with PBS (10% Wako RIA grade BSA added) was used as the secondary antibody. In the IgE measurement, patient serum was used as it was without being diluted. The results are shown in FIGS. Next, in IgG measurement, anti-human IgG GE Healthcare NA933 diluted 800 times with PBS (Wako RIA grade BSA added 10%) was used as a secondary antibody. For IgG measurement, patient serum was diluted 128-fold with PBS (4% Wako RIA grade BSA added). The result is shown in FIG. Moreover, the error bar (standard error) of IgG measurement by peptide # 4 is shown in FIG.

  From the results shown in FIG. 3, in the IgE measurement, peptide # 1 having serine-glycine-leucine-proline-valine amino acid sequence at the C-terminus and serine-glycine-leucine-proline-valine 5 residue repeating sequences According to peptide # 4 containing the unit, no luminescence was observed in the standard serum, and only the positive serum was luminescence. Therefore, these peptides are considered suitable for IgE measurement.

  FIG. 4 shows the results of comparing the luminescence intensities of positive serum and standard serum different from those in FIG. 3 using peptides # 4, 5 and 6. In any of the peptides, no luminescence was observed in the standard serum, but luminescence was seen in the positive serum, indicating that it was suitable for IgE measurement.

  Next, FIG. 5 relating to IgE measurement shows the measurement result of the luminescence intensity in the case of using the serum (Sapporo Medical University) of the Buna-Shimeji factory employee with peptide # 4. Table 1 shows whether the serum numbers in FIG. 5 are positive or negative depending on the presence or absence of cough symptoms.

  From the results shown in FIG. 5, the luminescence intensity of the serum of allergy positive employees with cough symptoms is higher than the luminescence intensity of the sera of allergy negative employees. It was shown to match well.

  Here, the positive serum is considered to contain not only type I allergy derived from IgE type immunoglobulin but also factors of type III and type IV allergy. For this reason, not only IgE measurement but also IgG measurement is considered to be useful for an epitope peptide that exhibits high luminescence intensity of positive serum relative to standard serum.

  FIG. 6 shows that all of peptides # 1 to # 4 have higher luminescence intensity of positive serum than that of standard serum, and are suitable for IgG measurement. In particular, peptide # 4 having serine-glycine-leucine-proline-valine amino acid sequence at the center of the amino acid sequence, and peptide # 4 containing 3 units of repeating sequence of 5 residues of serine-glycine-leucine-proline-valine Is considered suitable for IgG measurement since the difference in luminescence intensity between the standard serum and the positive serum was relatively large when either of the two types of positive sera was used. Further, from the results of the error bar (standard error) of IgG measurement with peptide # 4 shown in FIG. 7, when peptide # 4 is used, the luminescence intensity of the positive serum can be sufficiently allergic to the standard serum. It was shown that it was a value.

As described above, by measuring IgE and IgG using a peptide having at least the amino acid sequence of serine-glycine-leucine-proline-valine, it is possible to comprehensively diagnose Buna shimeji allergy. In particular, a peptide having a sequence containing a repetitive sequence of serine-glycine-leucine-proline-valine can be precisely diagnosed when examined by any measurement of IgE and IgG, and is considered to be optimal for allergy diagnosis of Bunashimeji.

  When diagnosing beech shimeji allergy using the allergy diagnosis kit of the present invention, it is possible to quickly diagnose shimeji allergy quickly and with high reliability without giving a physical burden to the subject. By performing allergy diagnosis with a high degree of reliability, an earlier diagnosis than the current diagnosis becomes possible.

Claims (9)

  A kit for allergy diagnosis in which a peptide having at least the amino acid sequence of serine-glycine-leucine-proline-valine is mounted on a solid phase carrier.   The kit according to claim 1, wherein the peptide has a sequence that repeatedly contains the amino acid sequence of serine-glycine-leucine-proline-valine.   The kit according to claim 1 or 2, wherein the peptide consists of 15 to 30 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 composed of an electrically neutral water-soluble polymer as a whole molecule The kit according to any one of claims 1 to 3, wherein the agent is loaded with a peptide.   The non-specific adsorption inhibitor 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 4.   6. The kit according to claim 1, wherein the photoreactive group is an azide group or a diazirine group.   The kit according to any one of claims 1 to 6, wherein the functional group reactive with the amino group at room temperature is an epoxy group or a succinimide ester.   The kit according to any one of claims 1 to 7, wherein the water-soluble polymer is a nonionic polymer.   The kit according to claim 8, wherein the nonionic polymer is a vinyl polymer of polyethylene glycol mono (meth) acrylate.

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