JP5737673B2 - Method for detecting epitope of allergen or candidate thereof and use thereof - Google Patents

Description

This application claims priority to Japanese Patent Application No. 2008-316216, which is a Japanese patent application filed on November 10, 2008, and the entire contents of the specification of this Japanese patent application are incorporated herein by reference. Captured in the book.
The present invention relates to a method for detecting an allergen epitope, and more particularly, to a method for detecting a set of at least two epitopes and use thereof.

  For the diagnosis and treatment of allergic diseases against pollen and food-derived proteins, allergen-specific antibody amounts are measured. For example, there is a method (Patent Document 1) for measuring the amount of antibodies such as IgA, IgG, and IgE specific to an allergen in the blood of a patient. In addition, for the same purpose, an array of IgG and IgE peptide fragments of the antigen protein can be created, and using this peptide array, the IgE epitope and IgG epitope to which IgE and IgG specifically bind can be identified. Is also disclosed (Non-Patent Document 1).

JP2008-107154 I. Cerecedo et al., J. Allergy, Clin. Immunol., 122, 589-594

  Basophils and mast cells play an important role in the development of allergic symptoms. Patients with allergic diseases produce IgE antibodies specific for allergens involved in the manifestation of symptoms. This allergen-specific IgE antibody remains on these cells in a state of being bound to an IgE receptor (FcεRI) expressed on the surface of mast cells or basophils. Thereafter, when an allergen specific to IgE binds, cross-linking of the IgE receptor occurs, which triggers a signal transmitted from the IgE receptor and releases granules such as histamine that directly cause allergic symptoms (degranulation). ) Occurs. According to the present inventors, in order for degranulation to occur in this way, it is not sufficient that only one IgE antibody binds to an allergen, and at least two degranulations are caused in a single allergen. Allergen-specific IgE binding sites (epitopes) that are effective for E. coli are required (2-hit theory). Therefore, in the allergen test, it is important to detect at least two epitopes necessary to cause degranulation.

  However, the method described in Patent Document 1 cannot identify epitopes, and the method of Non-Patent Document 1 can detect epitopes in individual patients, but a combination of two or more epitopes that cause degranulation. Cannot be detected.

  As described above, the problem with the current allergen test is that it cannot be detected until there is a combination of epitopes (IgE binding sites) necessary to effectively cause degranulation, and there is simply an IgE binding site on the allergen. Since only the presence is detected, the frequency of false positives is high.

  An object of the present invention is to provide a method for detecting a set of two or more epitopes in one allergen, and to provide a use of the epitope thus obtained and the set.

  The present inventors obtain an overlapping peptide for an antigen protein, create a peptide array, and analyze it by a specific method to detect an epitope set that may be related to induction or exacerbation of allergic reaction The present invention has been completed by finding out what can be done. In addition, the inventors have found a set of epitopes related to the onset and exacerbation of milk allergy and have completed the present invention. According to the present invention, the following means are provided.

According to the disclosure of the present specification, a method for detecting an epitope of an allergen or a candidate thereof, comprising the following steps:
(A) As a test sample, each of a test sample group containing an antibody derived from a plurality of allergy positive individuals and a control sample group containing an antibody derived from a plurality of allergy negative individuals,
A sample of each group is supplied to an array in which a plurality of overlapping peptides of a predetermined length based on a possible allergen protein or a part of the amino acid sequence thereof are immobilized, and the plurality of overlapping peptides and the one kind Or contacting two or more antibodies,
(B) detecting a specific binding of the plurality of overlapping peptides with the one or more antibodies,
(C) An overlap peptide selected from the plurality of overlap peptides, obtained from the control sample group and average control intensity information regarding the intensity of the specific binding of the overlap peptide obtained from the control sample group and the test sample group One or more of the overlapping peptides for which the specific binding is affirmed based on the average test intensity information regarding the strength of the specific binding of the overlapping peptide to be a first epitope in the allergen Or (d) individual test intensities of the overlapping peptides corresponding to the one or more first epitopes or candidates for a test sample selected from the test sample group Based on the information and the mean control intensity information, the overlapping peptide is When it can be a pitope or a candidate thereof, an individual test that is greater than or equal to the critical value obtained based on the mean control intensity information from the first epitope or another overlapping peptide other than the candidate, or exceeds the critical value Using one or more overlapping peptides having intensity information as a second epitope in the allergen or a candidate thereof,
A method is provided comprising:

The detection method further includes the following steps:
(E) For each of the first epitope candidates, a step of obtaining, for each overlapping peptide, the number information of the test sample using the overlap peptide as the second epitope or a candidate thereof, and Furthermore, (f) for each of the first epitope or candidate thereof, when the test sample of a predetermined ratio or more in the test sample group has the specific overlapping peptide as the second epitope or candidate thereof, A step of using the first epitope or candidate thereof and the second epitope or candidate thereof as an epitope set or candidate thereof in the allergen.

  Furthermore, the step (c) is based on the average test intensity information obtained for one of the overlapping peptides of the test sample group and the average control intensity information of the corresponding overlapping peptide. When the specific binding of an overlapping peptide is affirmed, the step of setting the one overlapping peptide as the first epitope or a candidate thereof may include one overlapping peptide of the test sample group. Based on the composite test intensity information obtained for a predetermined number of consecutive overlapping peptides and the composite control intensity information obtained from the corresponding control intensity information for each of the corresponding overlapping peptides, When the specific binding is affirmed as a whole overlapping peptide, The burst peptide may be used as the first epitope or a candidate thereof, and when both of the requirements are satisfied, the one overlapping peptide may be used as the first epitope or a candidate thereof. .

According to the disclosure of the present specification, a method for detecting an epitope of an allergen or a candidate thereof, comprising the following steps:
(A) For each of a test sample containing an antibody derived from an allergy positive individual and a control sample group containing an antibody derived from a plurality of allergy negative individuals,
A sample of each group is supplied to an array in which a plurality of overlapping peptides of a predetermined length based on a possible allergen protein or a part of the amino acid sequence thereof are immobilized, and the plurality of overlapping peptides and the one kind Or contacting two or more antibodies,
(B) for the plurality of overlapping peptides, detecting specific binding with the one or more antibodies, and (c) for the allergy positive individual,
The specific binding is positive based on the individual test strength information obtained for each overlapping peptide and the average control strength information regarding the strength of the specific binding obtained for each overlapping peptide of the control sample group. One or two or more overlapping peptides to be used as a first epitope or a candidate thereof, and obtained from the overlapping peptide other than the first epitope or the candidate based on the average control intensity information A step of using one or two or more overlapping peptides having individual test intensity information that is greater than or equal to a critical value or more than the critical value as a second epitope or a candidate thereof in the allergen,
A method is also provided comprising:

  According to the disclosure of the present specification, there is a method for testing allergic diseases, and there is a possibility of allergic diseases against two or more types of epitope peptides each containing two or more types of epitopes related to expression of allergic reactions. Supplying a test sample containing an antibody derived from an individual, bringing the two or more types of epitope peptides into contact with the antibody, and detecting specific binding of the two or more types of epitope peptides with the antibody A method is provided.

  In the present inspection method, the contact step is preferably a step using a solid phase carrier on which the two or more types of epitope peptides are immobilized. The two or more types of epitope peptides preferably include a first epitope peptide and a second epitope peptide determined by any one of the epitope detection methods described above.

  According to the disclosure of the present specification, a peptide array for testing allergic diseases, wherein two or more types of epitope peptides each containing two or more types of epitopes related to expression of allergic reaction are immobilized on a solid phase carrier. An array is provided.

It is a figure which shows the outline | summary of the determination method of the 1st epitope useful for discrimination of a population. It is a figure which shows the outline | summary of the determination method of the epitope pair useful for discrimination of a population. It is a figure which shows the number of the overlapping peptide used for determination of the epitope of the beta-lactoglobulin protein, or its candidate about a milk allergy positive sample group, each identification number, an amino acid sequence, etc. In addition, arrangement | sequence No. in FIGS. 1 to 51 correspond to the amino acid sequences of SEQ ID NOs: 1 to 51. It is a figure which shows the number of the overlap peptide used for determination of the epitope of a beta-lactoglobulin protein, or its candidate about a milk allergy hypersensitivity test substance group, each identification number, an amino acid sequence, etc. FIG. It is a figure which shows the number of the overlap peptide used for determination of the epitope of the beta-lactoglobulin protein, or its candidate about a milk allergy weak positive sample group, each identification number, an amino acid sequence, etc. It is a figure which shows the number of the overlap peptide used for determination of the epitope of alpha-lactalbumin protein, or its candidate about a milk allergy positive sample group, each identification number, an amino acid sequence, etc. FIG. In addition, arrangement | sequence No. in FIGS. 1 to 40 correspond to the amino acid sequences of SEQ ID NOs: 52 to 91. It is a figure which shows the number of the overlap peptide used for determination of the epitope of alpha-lactalbumin protein, or its candidate about a milk allergy hypersensitivity specimen group, each identification number, an amino acid sequence, etc. FIG. It is a figure which shows the number of the overlap peptide used for determination of the epitope of alpha-lactalbumin protein, or its candidate about a milk allergy weak positive sample group, each identification number, an amino acid sequence, etc. FIG. It is a figure which shows the number of each overlapping peptide used for determination of the epitope of alpha s1-casein protein, or its candidate about a milk allergy positive sample group, each identification number, an amino acid sequence, etc. FIG. In addition, arrangement | sequence No. in FIGS. 1-63 correspond to SEQ ID NOs: 92-154. It is a figure which shows the number of each overlapping peptide used for determination of the epitope of (alpha) s1-casein protein, or its candidate about a milk allergy hypersensitivity specimen group, each identification number, an amino acid sequence, etc. FIG. The figure which shows the number of the overlap peptide used for determination of the epitope of alpha s1-casein protein or its candidate about a milk allergy weak positive sample group, each identification number, an amino acid sequence, etc. It is a figure which shows the number of the overlap peptide used for determination of the epitope of (alpha) s2-casein protein, or its candidate about a milk allergy positive sample group, each identification number, an amino acid sequence, etc. FIG. In addition, arrangement | sequence No. in FIGS. 1 to 66 correspond to SEQ ID NOs: 155 to 220. It is a figure which shows the number of each overlapping peptide used for determination of the epitope of (alpha) s2-casein protein, or its candidate about a milk allergy hypersensitivity specimen group, an individual identification number, an amino acid sequence, etc. FIG. It is a figure which shows the number of each overlapping peptide used for determination of the epitope of alpha s2-casein protein, or its candidate about a milk allergy weak positive sample group, each identification number, an amino acid sequence, etc. FIG. It is a figure which shows the number of the overlap peptide used for determination of the epitope of a beta-casein protein, or its candidate about a milk allergy positive sample group, each identification number, an amino acid sequence, etc. In addition, arrangement | sequence No. in FIGS. 1 to 67 correspond to SEQ ID NOS: 221 to 287, respectively. It is a figure which shows the number of the overlap peptide used for determination of the epitope of (beta) -casein protein, or its candidate about a milk allergy hypersensitivity specimen group, each identification number, an amino acid sequence, etc. FIG. It is a figure which shows the number of the overlap peptide used for determination of the epitope of the beta-casein protein or its candidate about a milk allergy weak positive sample group, each identification number, an amino acid sequence, etc. It is a figure which shows the number of the overlap peptide used for determination of the epitope of (kappa) -casein protein, or its candidate about a milk allergy positive sample group, each identification number, an amino acid sequence, etc. FIG. In addition, arrangement | sequence No. in FIGS. 1 to 53 correspond to SEQ ID NOs: 288 to 340. It is a figure which shows the number of the overlap peptide used for determination of the epitope of (kappa) -casein protein, or its candidate about a milk allergy hypersensitivity test substance group, each identification number, an amino acid sequence, etc. FIG. It is a figure which shows the number of the overlapping peptide used for determination of the epitope of a (kappa) -casein protein, or its candidate about a milk allergy weak positive sample group, each identification number, an amino acid sequence, etc. It is a figure which shows the analysis result of the epitope of (beta) -lactoglobulin protein, or its candidate, A dark gray area shows the combination whose number of applicable patients is 95th percentile or more, and a medium gray area shows the number of applicable patients of 90. Shows combinations above the percentile. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 3 are shown. It is a figure which shows the analysis result of the epitope of (beta) -lactoglobulin protein, or its candidate, A dark gray area shows the combination whose number of applicable patients is 95th percentile or more, and a medium gray area shows the number of applicable patients of 90. Shows combinations above the percentile. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 3 are shown. It is a figure which shows the analysis result of the epitope of alpha-lactoglobulin protein, or its candidate, A dark gray section shows the combination whose number of corresponding patients is 95th percentile or more, and a medium gray section shows the number of corresponding patients 90. Shows combinations above the percentile. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 6 are shown. It is a figure which shows the analysis result of the epitope of (alpha) s1-casein protein, or its candidate, A dark gray area shows the combination whose number of applicable patients is 95th percentile or more, and a medium gray area shows the number of applicable patients 90th percentile. The combination of the above is shown. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 9 are shown. It is a figure which shows the analysis result of the epitope of (alpha) s1-casein protein, or its candidate, A dark gray area shows the combination whose number of applicable patients is 95th percentile or more, and a medium gray area shows the number of applicable patients 90th percentile. The combination of the above is shown. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 9 are shown. It is a figure which shows the analysis result of the epitope of alphas2-casein protein, or its candidate, A dark gray area shows the combination whose number of applicable patients is 95th percentile or more, and a medium gray area shows the number of applicable patients 90th percentile. The combination of the above is shown. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 12 are shown. It is a figure which shows the analysis result of the epitope of alphas2-casein protein, or its candidate, A dark gray area shows the combination whose number of applicable patients is 95th percentile or more, and a medium gray area shows the number of applicable patients 90th percentile. The combination of the above is shown. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 12 are shown. It is a figure which shows the analysis result of the epitope of (beta) -casein protein, or its candidate, A dark gray area shows the combination whose number of applicable patients is 95th percentile or more, and a middle gray area shows the number of applicable patients 90th percentile. The combination of the above is shown. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 15 are shown. It is a figure which shows the analysis result of the epitope of (beta) -casein protein, or its candidate, A dark gray area shows the combination whose number of applicable patients is 95th percentile or more, and a middle gray area shows the number of applicable patients 90th percentile. The combination of the above is shown. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 15 are shown. It is a figure which shows the analysis result of the epitope of (kappa) -casein protein, or its candidate, A dark gray section shows the combination whose number of corresponding patients is 95th percentile or more, and a middle gray section shows the number of corresponding patients 90th percentile. The combination of the above is shown. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 18 are shown. It is a figure which shows the analysis result of the epitope of (kappa) -casein protein, or its candidate, A dark gray section shows the combination whose number of corresponding patients is 95th percentile or more, and a middle gray section shows the number of corresponding patients 90th percentile. The combination of the above is shown. In the vertical and horizontal item columns, the numbers of the overlapping peptides shown in FIG. 18 are shown. Patient # 8 shows a combination of two IgEs that are thought to be involved in the induction of allergic reactions. It is a figure which shows the content of 5 epitope sets among the 10 epitope setters obtained by the analysis of the peptide array of Example 2. It is a figure which shows the content of the remaining 5 epitope sets among the 10 epitope setters obtained by the analysis of the peptide array of Example 2. It is a figure which shows the table | surface of the reactivity with respect to the epitope pair contained in the peak set about the test group of Example 2. FIG. It is a figure which shows the table | surface of the reactivity with respect to the epitope pair contained in the peak set about the learning group of Example 2. FIG. It is a figure which shows the table | surface of the reactivity with respect to the epitope pair contained in the peak set about the learning group of Example 2. FIG. It is a figure which shows the table | surface of the reactivity with respect to the epitope pair contained in the peak set about the learning group of Example 2. FIG. It is a figure which shows the table | surface of the reactivity with respect to the epitope pair contained in the peak set about the learning group of Example 2. FIG.

  The present invention relates to a method for detecting an epitope of an allergen or a candidate thereof. According to the present invention, an epitope important for expression of allergic symptoms or a combination of candidates thereof can be obtained. The first epitope or candidate thereof determined by this detection method is an important epitope that is highly common to the test sample group, that is, allergy-positive individuals, and is an important element for determining the set of epitopes. . Moreover, it can be said that the 2nd epitope determined by this detection method or its candidate is an important epitope set with high possibility of inducing allergic reaction in many allergy positive individuals.

  Moreover, by acquiring the epitope obtained in this way or a combination of candidates thereof, in addition to providing a diagnostic method for allergic diseases in which false positive results are suppressed or avoided, it can be used for the prevention and treatment of allergic diseases.

(Method for detecting allergen epitope or candidate thereof)
(Antigen-antibody reaction execution process)
The epitope or candidate detection method of the present invention first comprises (a) a test sample group containing antibodies derived from a plurality of allergy-positive individuals and an antibody derived from a plurality of allergy-negative individuals as test samples. For each of the control sample groups, each sample of each group is supplied to a solid phase carrier on which a plurality of overlapping peptides of a predetermined length based on a potential allergen protein or a part of the amino acid sequence thereof are immobilized, A step of contacting the plurality of overlapping peptides with an antibody is performed. By this step, the overlapping peptide and the antibody contained in the sample can be brought into contact with each other to cause an antigen-antibody reaction.

  The test sample contains an antibody derived from an allergy positive individual. The test sample is not particularly limited as long as it contains an antibody. For example, blood or serum collected from an allergy positive individual is used. The allergy positive individual may be a human or a non-human animal. Allergy positive individuals are allergy positive individuals for the same allergen. For example, allergy positive individuals for individual foods such as milk, eggs, cedar pollen, cypress pollen, etc. Positive individuals for the same allergen. The same allergen may be limited to the same protein.

  The test sample group is a set of a plurality of test samples. The number of test samples constituting the test sample group is not particularly limited. Moreover, it is preferable that the allergy positive individual which is a collection source of the test sample which comprises a test sample group is limited to a certain range in the grade of an allergic symptom and an IgE antibody level. This is because when the degree of allergic symptoms differs greatly, the epitope recognized by the antibody may be different, and it may be difficult to determine important epitopes and sets. Therefore, a plurality of test sample groups consisting of test samples derived from allergic positive individuals within a range of predetermined allergic symptoms and IgE antibody levels may be prepared.

  A control sample group is a collection of a plurality of control samples. The number of control samples is not particularly limited. The allergy negative individual from which the control data is collected is an allergy negative individual for at least allergen positive allergen. An allergy negative individual can be determined by, for example, allergic symptoms or IgE antibody levels.

  The overlapping peptide has an appropriate length (for example, about 12 to 25 residues) based on the amino acid sequence of the potential allergen protein or a portion thereof, and the overlapping (overlapping region of the sequence) is, for example, 8 to It is designed to be about 17 residues. Preferably, an overlapping peptide is provided over an amino acid sequence that covers all the primary structure of the allergen protein. For example, each amino acid sequence of the overlapping peptide overlaps a predetermined number of residues by shifting the amino acid sequence of a predetermined length from the N-terminal to the C-terminal direction of the allergen protein by a predetermined residue. It can be decided sequentially so that it becomes. The number of overlapping peptides that prepare a certain length of overlapping region for one allergen protein depends on the size of the primary structure to cover, the length of the overlapping peptide, and the number of overlapping residues. When a plurality of proteins such as milk allergy are involved as allergens, overlapping peptides can be prepared for one or more allergen proteins selected from these plurality of allergen proteins.

For example, when detecting an epitope for milk allergy, allergen proteins include six major milk allergen proteins, α-lactalbumin, β-lactoglobulin, α _s1 -casein, α _s2 -casein, β- One type or two or more types selected from casein and κ-casein may be mentioned. In the detection of the epitope, overlapping peptides may be obtained for all of these allergen proteins, or a part thereof.

  The overlapping peptide is immobilized on a suitable solid phase carrier. The solid phase carrier is not particularly limited as long as it is a carrier insoluble in a solvent in a reaction system for 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. Preferably, the solid phase carrier has a flat plate shape such as a test plate shape, a disk shape, or 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 thereof include those having a functional group introduced, glass, polysaccharides, silica gel, porous ceramics, and metal oxides.

  Known methods such as a physical adsorption method, a covalent bond method, an ionic bond method, and a crosslinking method can be used as the method for immobilizing the epitope polypeptide on the solid phase carrier, but it is not particularly limited. A person skilled in the art can appropriately select from known methods to immobilize the overlapping peptide on the solid phase carrier.

  Each test sample of the test sample group and each control sample of the target sample group are supplied to the overlap peptide immobilized on the solid phase carrier, respectively, and conditions for causing an antigen-antibody reaction between the overlap peptide and the antibody are set. Give. Such conditions are appropriately determined.

(Detection process of antigen-antibody reaction)
Next, a step of detecting specific binding with an antibody in each sample is performed for a plurality of overlapping peptides. The antigen-antibody reaction on the solid phase carrier can be detected using a labeling substance used in a conventional immunoassay. Examples of the labeling substance include fluorescent substances, luminescent substances, dyes, enzymes, coenzymes, and radioisotopes. The labeling substance can be labeled by directly binding to an epitope polypeptide or a secondary antibody against an antibody that binds to the epitope polypeptide. Alternatively, indirect labeling can be performed using an antibody recognizing a labeling substance or an avidin-biotin system. Specific binding is detected using a detection device corresponding to the type of signal based on such a labeling substance. The detected signal intensity can be related to the amount of specifically bound antibody.

(Determining the first epitope or its candidate)
Next, the process of determining a 1st epitope or its candidate is implemented. The first epitope or candidate thereof is selected from overlapping peptides. Individual signal intensity information (hereinafter referred to as individual test intensity information) about the overlapping peptide on the solid phase carrier obtained for each test sample is obtained. From these individual test information, it is possible to obtain signal intensity information (referred to as average test intensity information) obtained by averaging all individual test intensity information for the same overlapping peptide for each test sample group. The average test intensity information can be, for example, a simple average of all individual test intensity information.

  On the other hand, individual signal intensity information (hereinafter referred to as individual control intensity information) about the overlapping peptide on the solid phase carrier obtained for each control sample is obtained. From these individual control information, signal intensity information (referred to as average control intensity information) obtained by averaging all the individual control intensity information for the same overlapping peptide can be obtained for the control sample group. For example, the average control intensity information can be, for example, a simple average of all individual control intensity information.

  And about each overlap peptide, based on the obtained average test intensity | strength information and average control intensity | strength information, it is determined whether the specific binding with the antibody of a test sample is affirmed. Specifically, it is determined whether the average test intensity information obtained for a particular overlapping peptide is significantly higher than the average control intensity information. Examples of the determination method include the following methods (1) to (3).

(1) For one specific overlapping peptide, the average test intensity information is higher than the average control intensity information, and p-0.05 is satisfied in the test sample group and the control sample group by performing the t-test with a two-sided test, for example. When p <0.05 is satisfied, the specific binding between a specific overlapping peptide and the antibody of the test sample group is affirmed.

(2) The composite test intensity information obtained by averaging the average test intensity information of a plurality of consecutive overlapping peptides including one specific overlap peptide is higher than the composite control intensity information obtained in the same manner for the control sample. By performing t-test in two-sided test, it is determined whether, for example, p <0.05 is satisfied in the test sample group and the control sample group, and when p <0.05 is satisfied, the test is performed with one specific overlapping peptide. Affirm specific binding to the antibody of the sample group.

  In addition, in the above (2), as the overlapping peptide that includes a predetermined sequence before and after a specific overlapping peptide as a center, for example, 5 consecutive or 7 sequences including 2 sequences before and after or 3 sequences before and after, for example, 7 It is good also as a continuous overlap peptide. The same applies to the composite control intensity information. Also, each of these composite intensity information can be a simple average of predetermined average test intensity information and a simple average of average control intensity information.

(3) When the above (1) and (2) are satisfied, specific binding between a specific one overlapping peptide and the antibody of the test sample group is affirmed.

  Although the above (1) to (3) can be appropriately selected and used, even if selected by any technique, it is an important epitope or a candidate thereof, but (3) is a test sample group. It is suitable for detecting common important epitopes or candidates thereof. In the above (1) to (3), a test method or a significance level other than those used in the above description may be applied.

  As described above, an overlapping peptide in which specific binding to an antibody is affirmed can be used as the first epitope or a candidate for the allergen. Preferably, this step is performed for all overlapping peptides to determine whether each overlapping peptide can affirm specific binding with an antibody of the test sample group and can be the first epitope or a candidate thereof. Determine whether or not. The first epitope thus determined or its candidate is an important epitope that is highly common to the test sample group, that is, allergy positive individuals, and is an important element for determining the set of epitopes.

  The first epitope or its candidate is basically one specific overlapping peptide, but when it is determined that a plurality of consecutive overlapping peptides are the first epitope or its candidate, The overlapping peptide may be the first epitope or a candidate thereof. This is because each overlapping peptide has an overlap, and if the successive overlapping peptides are different first epitopes, the second epitope may not be determined accurately.

(Decision step of second epitope or candidate thereof)
Furthermore, in this detection method, the second epitope or its candidate determination step is performed. This step relates to a test sample selected from the test sample group, and the overlap peptide corresponding to the first epitope or the candidate already determined in the previous step is the individual test strength information and the control strength information of the test sample. To determine whether specific binding to the antibody in the test sample is positive. In other words, it is determined whether the first epitope or its candidate determined in the test sample group can be the first epitope or its candidate in an individual test sample. As the determination method, a method for determining whether or not the test sample group is the first epitope or a candidate thereof can be applied to each test sample. Preferably, the above method (3) is adopted.

  For each test sample, when it can be determined that the first epitope or candidate thereof previously determined for the test sample group is present, these average controls are obtained from other overlapping peptides other than the first epitope or candidate. It is determined whether there is an overlapping peptide having individual test strength information that is greater than or equal to the critical value obtained based on the strength information or exceeds the critical value. Here, the critical value obtained based on the average control intensity information can be obtained by, for example, increasing or decreasing an appropriate multiple of the standard deviation (SD) with respect to the average control intensity information. Specifically, the average control intensity information may be ± 1 to 2SD. Typically, mean control intensity information ± 2SD.

  The determination may be based on the critical value, and is performed based on whether the individual test strength information of the overlapping peptide is equal to or higher than the critical value or exceeds the critical value. “More than” or “exceeds” may be any one, and is appropriately determined according to the type of critical value.

  When a particular overlap peptide has individual test intensity information that is above or above a critical value, the particular overlap peptide is identified as a second epitope for a particular first epitope or candidate thereof in the allergen or The candidate is determined. The second epitope or the candidate thus determined is an epitope that is highly likely to induce an allergic reaction in a pair or pair with the first epitope or the candidate in the test sample, and thus in the allergy positive individual.

  Moreover, you may perform determination of the epitope for each test sample, or its candidate in the determination process of the epitope about the test sample group of the allergy positive individual group already demonstrated. That is, for each test sample, the first epitope or candidate thereof is determined by a method according to the first epitope for the test sample group or its determination step. That is, instead of using the average test intensity information of the test sample group, the first epitope for each test sample or its candidate is determined using the individual test intensity information. And the 2nd epitope which can be combined with this 1st epitope or its candidate, or its candidate is determined like the above.

  In this way, the entire test sample group cannot be determined as the first epitope or a candidate thereof, but can be combined with the unique first epitope or the overlapping peptide determined as a candidate in each test sample. The overlapping peptide which becomes the 2nd epitope can be detected. When the presence of such an individual-specific epitope combination is positive in more individuals, it is considered useful for diagnosis.

  In order to determine the second epitope or a candidate thereof for each test sample group, the number information of the test samples determined to correspond to the second epitope or the candidate is obtained for each overlapping peptide. Is preferably used. This is because an overlapping peptide having a large number information, that is, a second epitope or a large number of test samples as candidates thereof, can be said to be a second epitope common to the test sample group.

  For example, an overlapping peptide that is determined to be a second epitope or a candidate thereof in a test sample of a predetermined ratio or more with respect to the total number of test samples in the test sample group is a second epitope or a candidate common to the test sample group. It can be. The predetermined ratio of the determination criterion can be, for example, preferably 90 percentile or higher, more preferably 95 percentile or higher. The predetermined ratio here is appropriately determined according to the number of overlapping peptides corresponding to the second epitope, the total number of test samples, and the number of test samples.

  In this way, the second epitope or candidate thereof is determined relative to the first epitope or candidate thereof. The first epitope or its candidate and the second epitope or its candidate of the test sample group determined in this way can be said to be an important epitope set that is highly likely to induce an allergic reaction in many allergy positive individuals.

  In the above description, the antigen-antibody reaction process and the detection process for the control sample group are performed. However, if standard average control intensity information is already obtained for the control sample group, the actual process is performed. Alternatively, such information can be used to determine the first epitope or candidate and the second epitope or candidate.

  In the above description, the first epitope common to the test sample group or candidate thereof and the second epitope or candidate thereof have been mainly described. However, as described later, It can also carry out so that each 1st epitope or its candidate and 2nd epitope or its candidate may be detected about each test sample contained in a sample group.

(Method of detecting epitope or candidate of allergy positive individual)
The present invention also relates to a method for detecting allergen epitopes or candidates thereof for individual allergy positive individuals. In the detection method described above, the first epitope or a candidate thereof and the second epitope or a candidate thereof may be determined for each individual test sample included in the test sample group in the detection method described above. Specifically, the step of determining the first epitope or its candidate is performed based on the individual test intensity information and the mean control intensity information, and the determined first epitope or its candidate is determined for the test sample. The two epitopes or their candidates may be determined based on the individual test intensity information and the average control intensity information. Moreover, in order to detect the epitope of an allergy positive individual or its candidate, an antigen antibody reaction and a detection process should just be implemented about an individual test sample, and it is not necessary to implement these processes as a test sample group. By determining such an individual-specific epitope combination, diagnosis with higher accuracy becomes possible.

(Epitope peptide)
According to the present invention, an epitope peptide of an allergen protein such as milk allergy or a set thereof is provided. The epitope peptide and the set thereof are useful for diagnosis and treatment of milk allergy. In particular, the set of epitope peptides identified in the present invention is a combination of epitopes closely related to inducing the development of allergic symptoms. By using this set, false positives (allergic symptoms are not expressed but allergens are not expressed). Therefore, it is possible to make a diagnosis with higher accuracy by avoiding the diagnosis result that the sample has a high IgE.

  For example, a specimen that is positive for a peptide (overlap peptide) detected as a possibility that it corresponds to an epitope peptide or set of epitope peptides of an allergic protein of an allergic disease (especially, a peptide that is positive for both of the epitope peptide sets) (Serum) can be determined to be an allergy positive sample. Moreover, it can be determined that the specimen that does not react with these epitopes is negative, and the specimen that reacts with only one of the epitope peptide sets is also negative. A sample that reacts with only one of the epitope peptide sets corresponds to a sample that has been conventionally determined as a false positive (a sample that is positive in a conventional antigen-antibody reaction test but does not develop allergic symptoms). Therefore, according to the epitope peptide of the present invention or a set thereof, false positive specimens that could not be excluded by the conventional testing method can be surely excluded.

  In addition, by using the epitope peptide of the present invention or a set thereof, in the conventional allergy test, a definitive diagnosis could not be made unless a heavy allergen load test was performed on the patient. Moreover, it is possible to avoid or suppress the determination of false positives and false negatives, and to determine with high accuracy.

  From the above, the allergy test method disclosed in the present specification is directed to an individual having a possibility of allergic disease against two or more epitope peptides each containing two or more epitopes related to the expression of allergic reaction. Providing a test sample containing an antibody derived therefrom, contacting the two or more types of epitope peptides with the antibody, detecting specific binding of the two or more types of epitope peptides with the antibody, and Can be provided. These two or more types of epitope peptides can include a first epitope peptide and a second epitope peptide determined by the determination method described above.

  As will be described later, an epitope peptide or a peptide that may correspond to a set thereof can be used as an allergy test or a kit thereof in various forms, but preferably has a form immobilized on a solid phase carrier. The solid phase carrier is preferably in the form of a plate such as a plate, and more preferably takes the form of a so-called array (peptide array). For example, a preferable form for replacing the food load test is a peptide array in which an epitope peptide detected for all possible allergen proteins for a given allergic disease or an overlapping peptide corresponding to the set is immobilized. Can be mentioned.

  The epitope peptide of the present invention or a peptide that can be used as a set thereof can have the amino acid sequences shown in Tables 1 and 2, respectively. Table 1 shows preferable examples of the first epitope. These first epitopes or candidates thereof are shown by coloring (lightest gray color) in the numbered portions (vertical and horizontal item columns) assigned to each allergen protein in FIGS. Moreover, the set of the epitope peptide of a preferable combination includes the combination corresponding to the section colored in dark gray and medium dark gray in FIGS. Among them, the combination corresponding to the dark gray colored section is a combination of epitope peptides suitable for use as the epitope set of each allergen protein. In addition, these preferred combinations of compartments (dark gray compartments and medium gray compartments) that have the preferred first epitope together (compartments within the area identified by the lightest gray) have more positive patients Is a common and important combination. On the other hand, the dark gray section and the medium gray section outside the lightest gray area are more individual-specific but important combinations.

  The epitope peptide of the present invention is not particularly limited in the number of amino acid residues as long as it contains at least the amino acid sequence represented by the specified SEQ ID NO as a partial sequence. Typically, it consists of a specified amino acid sequence. The epitope peptide of the present invention may be modified by amino acid substitution, deletion or addition by a known method. It is also possible to impart solubility and antigen-antibody reactivity suitable for therapeutic use.

  The epitope peptide of the present invention can be produced by a known peptide synthesis method, for example, a method using gene recombination by a fully automatic peptide synthesizer, yeast, Escherichia coli, mammalian cells or the like.

  The epitope peptide of the present invention may be in the form of a salt, if necessary, preferably in the form of a physiologically acceptable acid addition salt. 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) salts, etc.

  When the epitope peptide of the present invention is used for diagnosis of milk allergy, various immunoassay forms can be adopted. 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 In addition to adopting an immunoassay such as a method of detecting the antigen-antibody reaction by visual observation or turbidity, it can be used by immobilizing on an appropriate solid phase carrier. As the solid phase carrier and the immobilization method, those already described can be used similarly. A peptide array is preferable.

  The epitope peptide of the present invention is useful for peptide immunotherapy such as desensitization therapy because when administered to a milk allergy positive individual (human etc.), it can regulate the allergic response of the individual to the milk-derived allergen. . In particular, the epitope set of the present invention can be used.

  When the epitope peptide of the present invention is used for peptide immunotherapy such as desensitization therapy for a milk allergy positive individual, it can be used in combination with an appropriate pharmaceutically acceptable diluent and carrier. The milk allergy positive individuals include milk allergy positive individuals immunologically cross-reactive with the various allergen proteins described above.

  As a method of administration, simple methods such as injection (subcutaneous, intravenous injection, etc.), eye drops, nasal drop, oral, inhalation, transdermal, etc. can be used. In the case of injection, the peptide can be administered preferably in an amount of about 1 μg to 50 mg per dosage unit. In the case of oral administration, it is preferable that it is absorbed as a peptide as it is or using a DDS technique as necessary.

  Hereinafter, the present invention will be described by way of specific examples. However, the following examples are illustrative of the present invention and are not intended to limit the present invention.

In this example, the entire amino acid sequences of six major milk allergen proteins (α _s1 -casein, α _s2 -casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin) are covered. Using a peptide array consisting of residues, 3-offset (overlapping 13 residues), IgE in the serum of cows with milk allergy was analyzed. Compared with the milk allergy negative specimen group (22 specimens), significant binding of IgE was observed in the milk allergy positive specimen group (61 specimens), the hypersensitivity specimen group (9 specimens), and the milk allergy weak positive group (30 specimens). Identified peptide (IgE binding epitope region). Since the actual allergic manifestation requires a combination of IgE against two epitopes in the patient's serum, analysis of the combination of two peptides involved in the induction of milk allergy in the milk allergy positive sample group (61 samples) did.

The requirements for the individuals making up each group were as follows.
1. Milk allergy positive sample (61 samples)
Confirmation of “milk allergy symptoms” defined below in the past (1) Immediate positive reaction with milk intake of 30 ml or less in milk load test (2) Milk of 30 ml or less or equivalent dairy products ( In milk for childcare, corresponding to 60 ml) Immediate type reaction by ingestion (3) There is an immediate type reaction in processed products containing milk, and allergies of other ingredients contained therein are denied (4) Symptoms are confirmed within 2 hours after ingestion

2. Milk "super" hypersensitivity sample (9 samples)
(1) History of milk allergy from early childhood (induced symptoms) and diagnosis of milk allergy has been confirmed (2) Immediate allergy by ingestion, contact, or inhalation of milk after age 5 years Have experience with symptoms. Regardless of current age (3) Symptoms include widespread skin symptoms (erythema / wheal / edema), respiratory symptoms (cough, wheezing, dyspnea), digestive symptoms (abdominal pain, vomiting), circulatory system Includes one or more symptoms (excluding exacerbation of eczema, oral mucosal symptoms only, hives and diarrhea limited to contact areas)
(4) Symptoms appear within 2 hours after ingestion (5) The milk-containing food that induced the symptoms falls under any of the following (regardless of whether it is a stress test or a misfeed)
Milk of 10ml or less or equivalent dairy products, processed products containing a small amount of milk (although allergies to other ingredients (eg eggs and wheat) contained at the same time are denied), contamination of milk ingredients and milk Contact with foods ・ Inhalation of steam heated dairy products, pharmaceuticals known to contain milk components (Rack B, Enteronone, Meiact)
(6) Milk-specific IgE antibody positive (Class 1 or higher)
(7) Complete removal of milk from infancy to the present. However, lactose and therapeutic hydrolyzed milk (Milphy, MA-1, MA-mi, pepdiet) may be ingestible.

3. Milk allergy weak positive group (30 samples)
Meets the definition of “(1) Milk allergy positive specimen” (2) Milk IgE (CAP-RAST) class 3 or lower

(1) Preparation of Peptide Array The peptide array is composed of 6 types of allergen proteins, β-lactoglobulin, α-lactalbumin, α _s1 -casein, α _s2 -casein, β-casein, and κ-casein, respectively. 1 to 51, SEQ ID NOs: 52 to 91, SEQ ID NOs: 92 to 154, SEQ ID NOs: 155 to 220, SEQ ID NOs: 221 to 287, and SEQ ID NOs: 288 to 340 were chemically synthesized to prepare peptide arrays. Immobilization to the array to the peptide was as follows. A glass substrate was used as the solid support. Thereafter, immunoassay (specimen supply and fluorescence detection) was performed, and the detected fluorescence intensity was digitized. In addition, Agilent scanner model G2505B and software G2565BA / DA were used for the fluorescence scanner, and Gene Pix.Pro made by Axon was used as numerical analysis software.

(2) Immobilization of peptide a) Heat treatment at 80 ° C. for 1 hour b) Immersion in (2 × SSC, 0.2% SDS) solution for 15 minutes (room temperature)
c) Immersion in (2 × SSC, 0.2% SDS) solution for 5 minutes (95 ° C.)
d) Shake about 10 times in sterile water (3 times)
e) Centrifugal drying

(3) Immunoassay a) (50mMethanolamine, 0.1% SDS, 0.1M Tris (hydroxymethyl) aminomethane) soaked for 90 minutes (room temperature)
b) Immersion in PBS-T (1 × PBS, 0.1% Tween20) solution for 5 minutes (room temperature, 3 times)
c) A carrier applied with 200 μL of patient serum (1:10) diluted with (1% OVA, PBS-T) solution is covered with a micro cover glass (size 24 × 60 mm, Thickness No. 4 manufactured by Matsunami Glass Co., Ltd.), and a Humid chamber ( (37 ° C.)
d) The carrier in the reaction in c) was transferred to an environment of 4 ° C. and left overnight. e) The micro cover glass was removed in the PBS-T solution.
f) Immersion in PBS-T solution for 5 minutes (room temperature, 3 times)
g) Goat anti-human IgE-Alexa 647 polyantibodies (1: 500) (200 μL) diluted with (1% OVA, PBS-T) solution was reacted in the same procedure as in c) and allowed to stand in the dark for 3 hours. (room temperature)
h) Remove micro cover glass in PBS-T solution i) Immerse in PBS-T solution for 5 minutes (room temperature, 3 times)
j) Shake about 10 times in sterile water (3 times)

(4) Epitope analysis (a) Determination of first epitope or candidate thereof Using the obtained fluorescence intensity information, compared to a milk allergy negative sample group, a milk allergy positive sample group (61 samples), a hypersensitivity sample Epitope regions in which IgE binding was significantly recognized in the group (9 samples) and the milk allergy weak positive group (30 samples) were identified by the following procedure (see FIG. 1).

  First, the average fluorescence intensity value of the patient group is higher than that of the negative group at a single peak (one overlapping peptide), and p <0.05 is satisfied between the patient group and the negative group by performing a two-sided test. The peptides were extracted (group a). Next, the average fluorescence intensity value of the patient group of 5 sequence peaks (5 consecutive overlapping peptides consisting of 2 overlapping peptides before and after including one overlapping peptide) is higher than that of the negative group, and t-test is two-sided. In step (b), peptides satisfying p <0.05 were extracted in the patient group and the negative group. Overlapping peptides that were a group and b group were extracted. These overlapping peptides correspond to the first epitope or candidate thereof.

(B) Regarding a milk allergy positive specimen group (61 specimens), FIG. 2 shows a scheme for determining a second epitope or a candidate thereof and a combination of two epitopes involved in the induction of a milk allergic reaction. First, for each sample in the positive sample group, it is the first epitope determined in (a) or a candidate thereof and has significance for the patient group in both five consecutive cases or alone. Overlapping peptide (second epitope) that extracts an overlapping peptide, and shows an intensity that is not equal to the extracted overlapping peptide but is equal to or greater than the value obtained by adding 2SD to the average fluorescence intensity value of the negative group (22 samples) Or candidate). This operation was repeated for all the specimens in the positive specimen group, and the number of specimens having the second epitope or its candidate overlap with respect to the specific first epitope or its candidate was counted for each overlapping peptide. And the combination of two peptides recognized by 90th percentile or more of the number of applicable samples was extracted.

  In addition, based on the fluorescence intensity value of each specimen according to the above-described determination method of the first epitope, the first epitope is determined for each specimen included in the milk allergy positive specimen group, and the first epitope is determined. In the same manner as described above, the second epitope to be combined with the epitope of the negative group is added with 2SD of the mean fluorescence intensity value of the negative group, and the epitope set is determined as an overlapping peptide having an intensity higher than the value, and counted. did.

(result)
(1) Results of the above (a) The whole amino acid sequences of the six major milk allergen proteins (β-lactalbumin, α-lactoglobulin, α _s1 -casein, α _s2 -casein, β-casein, κ-casein) were covered. Comparison of the average fluorescence intensity value between the patient group and the negative group for the overlapping peptides consisting of 16-mer and 3-offset, two-sided test of t-test, and extraction of peptides in which significant IgE binding was observed in the patient group The results obtained are shown in FIGS.

As shown in FIGS. 3 to 20, the patient group is a milk allergy positive sample group (61 samples), a hypersensitivity sample group (9 samples), and a milk allergy weak positive group (30 samples). Sample). Peptides having a single peak, 5 sequence peaks, single peak and 5 sequence peaks that are significantly different from each other are very important sequence candidates as IgE recognition sites involved in the induction of milk allergy. When compared by protein, the IgE binding region was most widely distributed throughout the protein for α _s1 -casein, which is particularly important as a major allergen in milk, whereas almost no IgE binding region was observed for α-lactalbumin. In addition, the 1st epitope detected about the allergy positive sample group or the overlap peptide which is the candidate was as showing in Table 1.

(2) Result of the above (b) Since the combination of IgE with two epitopes in patient serum is necessary for the actual expression of allergic symptoms, it is recognized next in the milk allergy positive specimen (61 cases) 2 Two IgE binding peptide combinations were analyzed. The results of counting the number of positive patients having a combination of two IgE-binding peptides are shown in FIGS. In FIG. 21 to FIG. 26, the number of milk allergy positive samples having a combination of IgE recognizing two peptides, respectively shown on the vertical axis and the horizontal axis, is displayed in the grids that intersect. The cut-off value is a value obtained by adding 2SD to the average fluorescence intensity value of the milk allergy negative sample group (22 cases). In addition, the axis indicates peptide No. in the order of N-terminal to C-terminal amino acid sequence of the same protein. Was written. In these figures, the first epitope detected for an allergy positive specimen group or an overlapping peptide that is a candidate (see Table 1) is colored in the lightest gray color in the item column indicating the sequence number for each figure. Showed. In addition, in these figures, in addition to the combination of the first epitope and the second epitope determined for the sample group, the first epitope and the second epitope determined for individual samples included in the sample group The combination is also described.

  As shown in FIG. 21 to FIG. 26, it was found that this analysis can determine the distribution of epitopes and their combinations that are highly important in the patient group for each allergen protein (the darkest gray section in the figure and the combination thereof). (Shown in lighter shades of medium gray). Moreover, it was found that these can be easily grasped visually by displaying in two dimensions. In addition, the number of positive specimens having a first epitope or a candidate thereof having significance in both 5 and 1 sequences and a second epitope or a candidate thereof having a peak equal to or higher than the cut-off value is calculated as follows. This corresponds well to the result of counting in the area where there is a significant difference in the binding at (1 seq window, 5 seq window). From the above, it was found that the combination with the number of corresponding patients of 90th percentile or more is a particularly important candidate as a combination of IgE recognition sites that can explain the induction of milk allergy in more patients. It was also easily understood that these specific epitopes can be selected and used for diagnosis and treatment.

The dark gray compartment and the medium gray compartment are both important combinations, but among the compartments in the figure, the peptide compartment determined to be the first common epitope in the whole allergic positive specimen group ( The section in the lightest gray section) is an important combination in terms of higher commonality, and the section outside the lightest gray section is a more specific but important combination. For example, when an array is produced using these, it is preferable to use a more common and important combination when fewer peptides need to be immobilized. Tables 2 to 11 show preferred epitope peptide sets for each of β-lactoglobulin, α _s1 -casein, α _s2 -casein, β-casein, and κ-casein obtained from these results.

(3) Analysis at individual patient level Finally, the combination of two IgEs that are considered to be involved in the induction of allergic reactions when viewed in individual patients is shown (FIG. 27). The combination of IgE (fluorescence intensity value: a vertical axis | shaft) which recognizes peptide ID (the order of N-> C end: horizontal axis) of (alpha) _s1- casein which exists in the serum of the patient 8 is shown. In this patient, in α _s1 -casein, the average fluorescence intensity value of the peptide (No. 52-56) having significance in both 5 and 1 sequences and the negative group (22 samples) plus 2SD is greater than the value. It was found that the combination with the peptide having the peak (No. 2) is most likely to be involved in the induction of allergic symptoms.

From the above results, it is possible to predict the induction of milk allergy symptoms by analyzing a combination of two peptides (epitope) to which serum IgE binds using a peptide array, and these can be diagnosed and treated. Found to be useful.

  In this example, a peptide consisting of 16-residues and 3-offsets covering the entire amino acid sequences of six milk allergen proteins prepared in the same manner as in Example 1 for 160 patients suspected of having milk allergies. Epitope analysis of IgE in serum was performed using arrays and immunoassays. About these patients, the diagnosis about milk allergy has already been confirmed clinically (Table 12), and based on the confirmed diagnosis, a test group (total of 20; milk allergy positive 10; milk allergy negative) 10) were randomly selected, and the rest were set as a learning group (total 140; milk allergy positive 117, milk allergy negative 23), and multiple epitopes suitable for diagnosis obtained from epitope analysis of the learning group The presence or absence of reactivity to the set (epitope pair) was applied to the test group and the learning group as the basis of diagnosis, and the diagnostic accuracy was evaluated. In addition to these epitope sets, the presence or absence of reactivity to multiple single epitopes (single epitopes) obtained from epitope analysis of the learning group is applied to the test group and learning group as the basis of diagnosis, and the diagnostic accuracy Was also evaluated. A negative patient (healthy person) is a patient who was suspected of having milk allergy according to the CAP-RAST class determination, but had no symptoms in the milk load test and was negative. In addition, patients (positive patients) are load-positive specimens (all may be referred to as negative and positive in the figure) that have allergic reactions when a milk load test is performed, and positive medical history that has symptoms at home. It was set as the sample (it may be called a disease positive in the figure).

  In the epitope analysis, the average fluorescence intensity value of the learning group is higher than that of the negative group with a single peak (one overlapping peptide) for all overlapping peptides, and the t-test is performed by a two-sided test. The overlapping peptide satisfying p <0.01 (P <0.05 may be used) was extracted (Condition 1). In the two-sided test (Bonferroni), the number of peptide sequences was integrated with the previous t-test, and at that time, the overlapping peptide with p <0.01 was extracted (condition 2). Furthermore, the average fluorescence intensity value of the positive patient group / the average fluorescence intensity value of the negative patient was calculated, and the overlapping peptide that became 2.0 or more was extracted (condition 3).

  In addition, the average fluorescence intensity value of the patient group of 5 sequence peaks (5 consecutive overlapping peptides consisting of 2 overlapping peptides before and after including one overlapping peptide) is higher than that of the negative group, and t-test is a two-sided test. By performing, a peptide satisfying p <0.01 (P <0.05 may be used) was extracted in the patient group and the negative group (Condition 4). In the two-sided test (Bonferroni), the number of peptide sequences was integrated with the previous t-test, and at that time, the peptide having p <0.01 (P <0.05 may be used) was extracted ( Condition 5). Furthermore, the average fluorescence intensity value of the positive patient group / the average fluorescence intensity value of the negative patient was calculated, and peptides having a value of 2.0 or more were extracted (condition 6).

The selection criteria for epitope candidates were as follows.
(1) In 5 sequence peak analysis, when at least condition 4 is satisfied, it is preferable as one of the epitopes of one epitope pair, more preferably, condition 5 or condition 6 is satisfied, and more preferably conditions 5 and 6 An overlapping peptide satisfying
(2) In addition to satisfying the conditions in the 5-sequence peak analysis, it is more preferable to satisfy at least the condition 1 in the single peak analysis. More preferably, an overlapping peptide that satisfies Condition 2 or Condition 3 and more preferably satisfies Conditions 2 and 3 can be used.
(3) One candidate of the epitope pair is extracted based on the above (1) and (2).

(4) Next, for each overlap peptide of at least one side candidate of the epitope pair thus selected, the average fluorescence intensity value + 2SD or more for both the overlap peptide and the overlap peptide other than the overlap peptide The positive patients were counted when they had a fluorescence value. Thus, for all positive patients in the learning group, the number of positive patients reactive to possible epitope pairs was obtained.

(5) Possible epitope pairs are displayed as one grid or grid on a two-dimensional matrix. The number of positive patients that can be counted in this grid or first is displayed, and when there is a certain count or more, or a certain percentage of the total number of positive patients (for example, 70% or more, preferably 80% or more, more preferably 90%). % Or more) When there are more counts, epitope pairs corresponding to the grids or grids can be more preferable epitope pair candidates. That is, for a possible epitope pair, a more preferred epitope pair candidate is selected when the number of positive patients in the learning group meets a certain criterion.

  Such more preferred epitope pair candidates are usually a grid or grid (region) or a set of two or more of these that displays different epitope pairs on the matrix (a set of such epitope pairs is referred to as an epitope set). It will be found. That is, for example, an epitope pair may be composed of one region (a combination of a pair of epitopes), may be composed of two regions, or may be composed of more regions. Various aspects are mentioned, such as the case where the epitope set corresponding to this region may consist of a region within the range of 2 × 2 and a region within the range of 3 × 3.

Among preferred epitope pair candidates, a more preferred epitope pair candidate is an epitope pair that is actually used for diagnosis. For example, the following points can be selected as a reference.
(1) Whether one of the epitopes constituting the pair satisfies the requirements of the 5 sequence peak such as Condition 4 at a higher level (2) One of the epitopes constituting the pair satisfies the conditions such as Condition 1 at a higher level Whether the single peak requirement is met (3) Whether more positive patients (absolute number or percentage of all positive patients) are responding to the epitope pair

  As a result of searching for epitope pairs based on the above selection criteria, more epitope pairs were found for αS1-casein, β-casein, and κ-casein than other allergen proteins.

  Through these operations, ten epitope sets shown in FIGS. 28A and 28B were selected in this example. There were 3 sets per αs1 casein, 5 sets per β casein and 2 sets per kappa casein. The results of single peak analysis and 5-sequence peak analysis are shown on the right side of each peptide number. In FIG. 28A, for example, αS1 casein peak set 1 is composed of a total of nine peptide pairs. That is, the peak numbers 23, 24, and 25 are the first epitopes, 37, 38, and 39 are the second epitopes, respectively, and there are 9 combinations of these first and second epitopes. It is. (9 pairs in total). Therefore, the 10 epitope sets include a total of 80 epitope pairs. Any of these epitope pairs is an effective epitope pair for diagnosis of milk allergy. In particular, in FIGS. 28A and 28B, peak sets 1 to 3 and peak sets 5 and 6 including epitopes marked with double circles in the overall determination are preferable for the determination, and more preferably, the double circles in the overall determination. An epitope pair containing an epitope marked with is a more preferred epitope pair.

The test group (20 samples) and the learning group for each epitope pair of the 10 epitope sets (a total of 80 epitope pairs included in these sets) obtained from the learning group as described above. It was examined whether each of (140 specimens) had reactivity. That is, for all patients included in these groups, the reactivity is affirmed when the selected individual epitope pair has a fluorescence intensity value equal to or greater than the average value of healthy subjects + 2SD, and any one of them When the reactivity was positive for the epitope pair, it was determined that the milk allergy was positive. The summary of reactivity for the test group is shown in FIG. 29, and the summary of reactivity for the learning group is shown in FIGS. 30A to 30D. The determination results are shown in Table 13. Note that the coverage was calculated based on the numerical value in the column of “1 if detected even once in all areas covered by the peak set, and 0 otherwise” in the table.

  In addition, in the learning group positive patients, 47 overlapping peptides satisfying the conditions of both the single peak and the 5-sequence peak described above were selected. About these 47 overlapping peptides, it was investigated whether each patient of a test group (20 samples) and a learning group (140 samples) had reactivity. That is, for all patients included in these groups, the reactivity was affirmed when the selected epitope peptide had a fluorescence intensity value equal to or higher than the average value of healthy subjects + 2SD. Table 14 shows the determination results based on the epitope obtained by single peak analysis.

  Further, Table 15 shows the results of the CAP-RAST method. In addition, since 8 patients in the learning group had not measured the CAP-RAST method, these patients were excluded from the determination.

  As shown in Table 13, in the judgment by the peptide pair, the false positive rate (coverage rate (healthy person) in the table) that judges the healthy person (negative patient) as positive is the test group and the learning group. (The test group: 40.0%, the learning group: 17.4%). In addition, the false positive rate (coverage rate (healthy person class 4 or higher) in the table is 0.0% in the test group, and the learning group determines that a healthy person of class 4 or higher is positive by the CAP-RAST method) On the other hand, the rate of positive milk allergy positive patients (coverage rate (patient)) in the test group was 60.0% in the test group and the learning group in the learning group. In addition, the rate of positive results for patients of class 4 or higher (in the table, the coverage rate (class 4 or higher)) was 100% in the test group and 91.7% in the learning group. It was.

  On the other hand, as shown in Table 14 which shows the determination result by the epitope obtained by the single peak analysis, the false positive rate (coverage rate (healthy person) in the table) which erroneously determines the healthy person as positive is tested. It was 80% in the group and 87% in the learning group. Moreover, the false positive rate (coverage rate in the table (healthy person class 4 or higher) in the table) is 100.0% in the test group and 100.0% in the learning group. there were.

  Further, as shown in Table 15, according to the CAP-RAST method, if it is determined that class 1 or higher is positive, it is determined that the test group and the learning group are negative but positive, respectively. %, 95.7%.

  From the above, it was found that the determination by the epitope pair can reduce the false positive rate that determines a healthy person as allergy positive. In addition, it was found that allergic patients can be determined as positive even if the class is low, as determined by non-paired epitopes (single epitopes) by single peak analysis. Furthermore, it can be said that an allergy diagnosis with a high patient positive rate and a low false positive rate can be performed by combining the epitope pair and the determination by single epitope.

Claims (12)

A test method for allergic diseases to milk,
An antibody derived from an individual having a possibility of allergic disease against two or more types of epitope peptides containing a combination of two or more types of allergen-specific epitopes effective for inducing degranulation against a single allergen Supplying a test sample containing, and bringing the two or more types of epitope peptides into contact with the antibody;
Detecting the specific binding with the antibody for the two or more types of epitope peptides constituting the combination of the allergen-specific epitopes;
With
The single allergen is one or more selected from the group consisting of β-lactoglobulin, αs1-casein, αs2-casein, β-casein and κ-casein,
The two or more types of epitope peptides constituting the combination of allergen-specific epitopes are related to the single allergen,
The inspection method which is a combination selected from the following Tables 1 to 9 .
  The method according to claim 1, wherein the contacting step is a step of using a solid phase carrier on which the two or more types of epitope peptides are immobilized. An array for use in the inspection method according to claim 1 or 2,
The two or more types of epitope peptides constituting the combination of allergen-specific epitopes are arrays in which epitope peptides of combinations selected from Table 1 to Table 9 are immobilized as probes relating to the single allergen . Used in allergy testing method according to claim 1 or 2, a valid combinations of two or more allergen-specific epitope or a method for detecting the candidate to elicit degranulation to a single allergen The following steps;
(A) As a test sample, each of a test sample group containing an antibody derived from a plurality of allergy positive individuals and a control sample group containing an antibody derived from a plurality of allergy negative individuals,
The samples of each group are respectively supplied to an array on which a plurality of overlapping peptides of a predetermined length based on the protein of a single allergen or a part of the amino acid sequence is immobilized, and the plurality of overlapping peptides and the 1 Contacting the species or two or more antibodies,
(B) a step of detecting specific binding of the plurality of overlapping peptides with the one type or two or more types of antibodies;
(C) An overlap peptide selected from the plurality of overlap peptides, obtained from the control sample group and average control intensity information regarding the intensity of the specific binding of the overlap peptide obtained from the control sample group and the test sample group The one or more overlapping peptides whose specific binding is affirmed based on the average test intensity information on the strength of the specific binding of the overlapping peptide to be determined, the allergen in the single allergen A step of making a first epitope constituting a combination of specific epitopes or a candidate thereof; and (d) a test sample selected from the test sample group, the one or more types of first epitopes or candidates thereof Individual test intensity information and the average of the overlap peptide corresponding to Based on the illumination intensity information, when the overlapping peptide can be the first epitope or a candidate thereof, the average control intensity can be obtained from the overlapping peptide other than the first epitope or the candidate. A second epitope or a candidate thereof comprising a combination of allergen-specific epitopes with one or two or more types of overlapping peptides having individual test intensity information that is greater than or equal to the critical value obtained based on the information or exceeds the critical value The process of
A method comprising: And the following steps:
(E) For each of the first epitope or its candidate, obtaining the number information of the test sample with the overlap peptide as the second epitope or its candidate for each overlapping peptide;
The method of claim 4 comprising: And the following steps:
(F) For each of the first epitope or a candidate thereof, when the test sample of a predetermined ratio or more in the test sample group uses the specific overlapping peptide as the second epitope or a candidate thereof, The epitope or candidate thereof and the second epitope or candidate thereof as an epitope set in the allergen or candidate thereof,
The method of claim 5 comprising:   The step (c) is based on the average test intensity information obtained for one of the overlap peptides of the test sample group and the average control intensity information of the corresponding overlap peptide. The method according to any one of claims 4 to 6, which is a step of using the one overlapping peptide as the first epitope or a candidate thereof when the specific binding is positive.   In the step (c), the composite test intensity information obtained for a plurality of consecutive overlapping peptides including one overlapping peptide of the test sample group and the corresponding control intensity for each overlapping peptide Based on the composite control intensity information obtained from the information, when the specific binding is affirmed as a whole of the predetermined number of overlapping peptides as a whole, the one overlapping peptide is the first epitope or its The method in any one of Claims 4-6 which is a process made into a candidate. The step (c) is based on the average test intensity information obtained for one of the overlap peptides of the test sample group and the average control intensity information of the corresponding overlap peptide. And the specific binding of
Compound test intensity information obtained from a plurality of overlapping peptides including the one overlapping peptide and a predetermined number of overlapping peptides, and composite control intensity information obtained from the corresponding average control intensity information for each of the plurality of overlapping peptides; 5. The step of setting the one overlapping peptide as the first epitope or a candidate thereof when the specific binding is affirmed as a whole of the predetermined number of overlapping peptides as a whole based on The method in any one of -6. A method for detecting a combination of two or more allergen-specific epitopes effective for inducing degranulation with respect to a single allergen or a candidate thereof, which is used in the allergy test method according to claim 1 or 2. The following steps:
(A) For each of a test sample containing an antibody derived from an allergy positive individual and a control sample group containing an antibody derived from a plurality of allergy negative individuals,
The samples of each group are respectively supplied to an array on which a plurality of overlapping peptides of a predetermined length based on the protein of a single allergen or a part of the amino acid sequence is immobilized, and the plurality of overlapping peptides and the 1 Contacting with two or more kinds of antibodies,
(B) for the plurality of overlapping peptides, detecting specific binding with the one or more antibodies, and (c) for the allergy positive individual,
The specific binding is positive based on the individual test strength information obtained for each overlapping peptide and the average control strength information regarding the strength of the specific binding obtained for each overlapping peptide of the control sample group. One or two or more overlapping peptides to be used as the first epitope constituting the allergen-specific epitope combination or a candidate thereof, from the first epitope or the other overlapping peptide other than the candidate, A second or more of the overlapping peptides having individual test intensity information greater than or equal to the critical value obtained based on the average control intensity information or exceeding the critical value constitutes a combination of allergen-specific epitopes. An epitope or a candidate for it,
A method comprising: The method according to claim 10, wherein the step (c) affirms the specific binding for the one overlapping peptide when the following (1) and (2) are satisfied.
(1) per said one overlapping peptidase de, the average test intensity information is higher than the mean control level information, the control sample group test sample group from carrying out the t-test two-sided, satisfy at least p <0.05 When.
(2) The composite test intensity information obtained by averaging the average test intensity information of a plurality of consecutive overlapping peptides including the one overlapping peptide is higher than the composite control intensity information obtained in the same manner for the control sample, When the test sample group and the control sample group satisfy at least p <0.05 by performing the two-sided t-test.   The method according to claim 10 or 11, wherein the critical value in the step (c) is a value obtained by adding a multiple of 1 or 2 of a standard deviation (SD) to the average control intensity information.