Devices, kits, and methods for testing for milk allergy

Abstract

Providing a milk allergy testing technology that can measure the level of milk allergen-specific IgE antibodies associated with the patient's milk allergy status. [Solution] A device and kit for testing milk allergies, comprising an epitope peptide mixture comprising at least six peptides selected from the group consisting of (a) a peptide containing the amino acid sequence shown in SEQ ID NO: 1, (b) a peptide containing the amino acid sequence shown in SEQ ID NO: 2, (c) a peptide containing the amino acid sequence shown in SEQ ID NO: 3, (d) a peptide containing the amino acid sequence shown in SEQ ID NO: 4, (e) a peptide containing the amino acid sequence shown in SEQ ID NO: 5, (f) a peptide containing the amino acid sequence shown in SEQ ID NO: 6, (g) a peptide containing the amino acid sequence shown in SEQ ID NO: 7, (h) a peptide containing the amino acid sequence shown in SEQ ID NO: 8, (i) a peptide containing the amino acid sequence shown in SEQ ID NO: 9, (j) a peptide containing the amino acid sequence shown in SEQ ID NO: 10, (k) a peptide containing the amino acid sequence shown in SEQ ID NO: 11, and (l) a peptide containing the amino acid sequence shown in SEQ ID NO: 12, and 0 to 2 peptides selected from the group consisting of (m) a peptide containing the amino acid sequence shown in SEQ ID NO: 13 and (n) a peptide containing the amino acid sequence shown in SEQ ID NO: 14, as well as a method for measuring milk allergen-specific antibodies and a method for testing milk allergies using the epitope peptide mixture.

Description

[Technical Field]

[0001] This invention relates to a milk allergy testing technology. [Background technology]

[0002] In infants and young children, milk is the second most common allergen after eggs. The causative substances of milk allergies are proteins derived from milk, such as casein and β-lactoglobulin. Milk allergies frequently develop in infants and young children, but are often said to resolve spontaneously after the age of three. On the other hand, the development of milk allergies can have serious health consequences for infants and young children, and restricting milk intake to avoid developing milk allergies may also negatively affect their growth.

[0003] Oral immunotherapy (OIT), which involves gradually increasing the amount of allergen ingested, is a known treatment for food allergies. Oral ingestion of small amounts of allergen induces the production of allergen-specific IgG antibodies such as IgG4, which inhibit the binding of allergen-specific IgE antibodies to the allergen, thereby suppressing allergic symptoms. However, the effectiveness of oral immunotherapy varies greatly from person to person, and there is a risk of causing anaphylaxis, so oral immunotherapy is performed under high medical supervision. If the effectiveness of oral immunotherapy in individual patients could be predicted in advance, the burden on patients and healthcare professionals could be reduced, but a reliable prediction method has not yet been established.

[0004] Allergen-specific IgE antibodies are known to play an important role in inducing allergic symptoms, including anaphylaxis, but not all IgE antibodies that bind to allergens are involved in allergic symptoms. One method for measuring milk allergen-specific IgE antibodies in a sample using the ELISA method is ImmunoCAP from Thermo Fisher Scientific. (R) The test is used as a standard testing technique. However, ImmunoCAP (R)The test determines the total IgE antibody titer against all milk allergen molecules and is not suitable for qualitative evaluation of IgE antibodies involved in allergic symptoms.

[0005] Patent Document 1 discloses a method for detecting milk allergy and a diagnostic composition for that detection. Patent Document 1 discloses a number of antigens to which IgE antibodies in the serum of milk allergy patients specifically bind. However, Patent Document 1 does not disclose a method for easily measuring milk allergen-specific IgE antibody levels related to a patient's milk allergy status, nor does it disclose an allergy test method based on that.

[0006] There is a need for the development of milk allergy testing technologies that can measure the level of milk allergen-specific antibodies associated with the patient's milk allergy status. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2022-157411 [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to provide a milk allergy testing technology that can measure the level of milk allergen-specific IgE antibodies associated with a patient's milk allergy status. [Means for solving the problem]

[0009] As a result of diligent research to solve the above problems, the present inventors have found that a predetermined set of epitope peptides derived from milk allergens is useful for measuring antibody titers useful as indicators for milk allergy testing, such as IgE antibody titers and IgG4 antibody titers, and have completed the present invention.

[0010] In other words, the present invention encompasses the following: [1] The following (a) to (l): (a) A peptide comprising the amino acid sequence shown in SEQ ID NO: 1, (b) A peptide comprising the amino acid sequence shown in SEQ ID NO: 2, (c) A peptide comprising the amino acid sequence shown in SEQ ID NO: 3, (d) A peptide comprising the amino acid sequence shown in SEQ ID NO: 4, (e) A peptide comprising the amino acid sequence shown in SEQ ID NO: 5, (f) A peptide comprising the amino acid sequence shown in SEQ ID NO: 6, (g) A peptide comprising the amino acid sequence shown in SEQ ID NO: 7, (h) A peptide comprising the amino acid sequence shown in SEQ ID NO: 8, (i) A peptide comprising the amino acid sequence shown in SEQ ID NO: 9, (j) A peptide comprising the amino acid sequence shown in SEQ ID NO: 10, (k) A peptide comprising the amino acid sequence shown in SEQ ID NO: 11, and (l) A peptide comprising the amino acid sequence shown in SEQ ID NO: 12 At least 6 peptides selected from the group consisting of: The following (m) and (n): (m) A peptide comprising the amino acid sequence shown in SEQ ID NO: 13, and (n) A peptide comprising the amino acid sequence shown in SEQ ID NO: 14 0 to 2 peptides selected from the group consisting of: An epitope peptide mixture containing the above, for use in a milk allergy test device. [2] The device according to [1] above, wherein the epitope peptide mixture contains all of the peptides (a) to (n). [3] The device according to [1] or [2] above, wherein the peptide is a peptide 17 to 21 amino acids in length. [4] The device according to any one of [1] to [3] above, wherein the peptide contains biotin-binding lysine added to the N-terminus or C-terminus of the amino acid sequence. [{5}] The device according to [4] above, wherein the peptide is complexed with streptavidin or avidin. [6] The device according to any one of [1] to [5] above, wherein the epitope peptide mixture is immobilized on an immunochromatography strip on a test line. [7] A milk allergy testing kit including one of the devices described in [1] to [6] above. [8] The milk allergy test kit described in [7] above for ELISA or dot blot analysis. [9] A kit for testing for milk allergy for immunochromatographic analysis, including the device described in [6] above.

[10] A kit according to any one of [7] to [9] above, further comprising a labeled anti-IgE antibody and / or an anti-IgG4 antibody.

[11] Biological samples derived from the subject were taken as follows: (a) to (l): (a) A peptide containing the amino acid sequence shown in Sequence ID No. 1, (b) A peptide containing the amino acid sequence shown in Sequence ID No. 2, (c) A peptide containing the amino acid sequence shown in Sequence ID No. 3, (d) A peptide containing the amino acid sequence shown in SEQ ID NO: 4, (e) A peptide containing the amino acid sequence shown in Sequence ID No. 5, (f) A peptide containing the amino acid sequence shown in Sequence ID No. 6, (g) A peptide containing the amino acid sequence shown in Sequence ID No. 7, (h) A peptide containing the amino acid sequence shown in Sequence ID No. 8, (i) A peptide containing the amino acid sequence shown in Sequence ID No. 9, (j) A peptide containing the amino acid sequence shown in Sequence ID No. 10, (k) A peptide containing the amino acid sequence shown in Sequence ID No. 11, and (l) Peptide containing the amino acid sequence shown in SEQ ID NO. 12 At least six peptides selected from the group consisting of, The following (m) and (n): (m) A peptide containing the amino acid sequence shown in SEQ ID NO: 13, and (n) Peptide containing the amino acid sequence shown in SEQ ID NO: 14 0 to 2 peptides selected from the group consisting of, Contact with an epitope peptide mixture containing, This includes measuring the amount of binding between the antibody in the biological sample and the epitope peptide mixture. A method for measuring milk allergen-specific antibodies in biological samples derived from test subjects.

[12] The method according to

[11] , wherein the epitope peptide mixture comprises all of the peptides (a) to (n).

[13] The method according to

[11] or

[12] above, wherein the antibody is an IgE antibody.

[14] The method according to any one of the above

[11] to

[13] , wherein the peptide is a peptide having a length of 17 to 21 amino acids.

[15] The method described in any of

[11] to

[14] above, using the device described in any of [1] to [6] above or the kit described in any of [7] to

[10] above.

[16] The method according to any of the above

[11] to

[15] , wherein the biological sample is a blood sample.

[17] A method for obtaining a test index for a subject's milk allergy, comprising measuring milk allergen-specific IgE antibodies in a biological sample derived from a subject using the method described in

[13] above, wherein the measured amount of milk allergen-specific IgE antibodies indicates the subject's tolerance level to milk allergens.

[18] A method for obtaining an evaluation index for the effectiveness of oral immunotherapy for milk allergy in a subject, comprising measuring milk allergen-specific IgE antibodies in a biological sample from a subject who is receiving or is scheduled to receive oral immunotherapy for milk allergy using the method described in

[13] above, wherein the measured amount of milk allergen-specific IgE antibodies indicates the subject's milk intake tolerance and therapeutic response to oral immunotherapy.

[19] The method according to

[17] , further comprising measuring the amount of IgG4 antibody in a biological sample contacted with the epitope peptide mixture and the amount of the epitope peptide mixture, and determining the ratio of the amount of milk allergen-specific IgE antibody to the measured amount of milk allergen-specific IgG4 antibody.

[20] The method according to

[18] , further comprising measuring the amount of IgG4 antibody in a biological sample contacted with the epitope peptide mixture and the amount of the epitope peptide mixture, and determining the ratio of the amount of milk allergen-specific IgE antibody to the measured amount of milk allergen-specific IgG4 antibody. [Effects of the Invention]

[0011] This invention can measure the level of milk allergen-specific antibodies associated with a patient's milk allergy status. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the IgE-bound epitope profiles for 12 epitope peptides obtained from serum samples from three milk allergy patients (#101, #108, #ID-03). "ALL (No. 1-12)" indicates the IgE value measured using a plate bound with a biotinylated epitope peptide mixture (12 epitopes-ALL). [Figure 2] Figure 2 shows the IgE-bound epitope profiles for 14 epitope peptides obtained from serum samples from three milk allergy patients. "ALL (No. 1-14)" indicates the IgE value measured using a plate bound with a biotinylated epitope peptide mixture (14 epitopes-ALL). #2814(×30)_IgE: IgE value measured using a 30-fold dilution of serum sample from patient #2814. #2910(×150)_IgE: IgE value measured using a 150-fold dilution of serum sample from patient #2910. #3018(×300)_IgE: IgE value measured using a 300-fold dilution of serum sample from patient #3018. [Figure 3]Figure 3 shows the measurement results of epitope-conjugated IgE antibodies (Figure 3A) and epitope-conjugated IgG4 antibodies (Figure 3B) in dilutions of serum samples from human patients with milk allergy, using a plate conjugated with 14 epitopes-ALL. α-human IgE ×5: IgE antibodies in a 5-fold dilution of serum samples from human patients with milk allergy, α-human IgE ×15: IgE antibodies in a 15-fold dilution of serum samples from human patients with milk allergy, α-human IgE ×50: IgE antibodies in a 50-fold dilution of serum samples from human patients with milk allergy. α-human IgG4 ×15: IgG4 antibodies in a 15-fold dilution of serum samples from human patients with milk allergy, α-human IgG4 ×50: IgG4 antibodies in a 50-fold dilution of serum samples from human patients with milk allergy, α-human IgG4 ×150: IgG4 antibodies in a 150-fold dilution of serum samples from human patients with milk allergy. [Figure 4] Figure 4 shows standard curves based on measured values ​​of IgE antibody and IgG4 antibody obtained by ELISA measurement on a streptavidin-coated plate conjugated with 14 epitope-ALL, using recombinant human IgE antibody A and recombinant human IgG4 antibody B solutions of known concentrations. [Figure 5] Figure 5 shows a correlation diagram plotting serum samples from milk allergy patients based on 12-epitope-ALL specific IgE antibody content (vertical axis) and ImmunoCAP(R) IgE value (horizontal axis). [Figure 6] Figure 6 shows a correlation diagram plotting serum samples from milk allergy patients based on 14-epitope-ALL specific IgE antibody content (vertical axis) and ImmunoCAP(R) IgE value (horizontal axis). The class indication on the horizontal axis is based on the ImmunoCAP(R) IgE value (horizontal axis). [Figure 7] Figure 7 shows the distribution of 14-epitope-ALL-specific IgE antibody content (pg / mL) in milk allergy patients and non-allergy patients. An example of a threshold (5 ng / mL) used to distinguish between the milk allergy patient group and the non-allergy group is shown by the dotted line. [Figure 8]Figure 8 plots milk allergy patients receiving oral immunotherapy based on their milk intake tolerance and ImmunoCAP(R) IgE levels. [Figure 9] Figure 9 plots milk allergy patients receiving oral immunotherapy based on their milk intake tolerance and 14-epitope-ALL specific IgE antibody content. [Figure 10] Figure 10 is a photograph showing the results of immunochromatographic analysis of antibody samples using an immunochromatographic strip with 14 epitopes-ALL. Figure 10A: Recombinant human IgE antibody A, 10 ng (left lane) and 100 ng (right lane) (with Lipidure(R)-BL206 added), Figure 10B: Recombinant human IgE antibody B, 10 ng (left lane) and 100 ng (right lane) (with Lipidure(R)-BL802 added), Figure 10C: IgG4 antibody (left lane) and IgE antibody (right lane) in a serum sample from patient #2900 (with Lipidure(R)-BL206 and -BL802 added), Figure 10D: IgG4 antibody (left lane) and IgE antibody (right lane) in a serum sample from patient #2910 (with Lipidure(R)-BL206 and -BL802 added). [Modes for carrying out the invention]

[0013] The present invention will be described in detail below. This invention relates to a milk allergy testing technique, and more particularly to a milk allergy testing technique that can measure the level of milk allergen-specific antibodies associated with the state of milk allergy.

[0014] Specifically, the present invention relates to a device and kit for testing milk allergies, a method for measuring milk allergen-specific antibodies, a method for testing milk allergies, a method for obtaining indicators for milk allergies, and a method for evaluating the effectiveness of oral immunotherapy for milk allergies.

[0015] In this invention, the binding of milk allergen-specific antibodies to a mixture (mixture system) of multiple epitope peptides derived from milk allergens, specifically α-casein (αS1-casein, αS2-casein), β-casein, κ-casein, and / or β-lactoglobulin, can be measured, and milk allergy can be evaluated based on the total amount of binding. In this invention, "allergen-specific antibody" refers to an antibody that can specifically bind to a predetermined allergen.

[0016] In the present invention, the milk allergens used, specifically, multiple epitope peptides (sometimes simply referred to as peptides) derived from α-casein (αS1-casein, αS2-casein), β-casein, κ-casein, and / or β-lactoglobulin, can be selected from the following peptides (a) to (l): (a) A peptide containing the amino acid sequence shown in Sequence ID No. 1, (b) A peptide containing the amino acid sequence shown in Sequence ID No. 2, (c) A peptide containing the amino acid sequence shown in Sequence ID No. 3, (d) A peptide containing the amino acid sequence shown in SEQ ID NO: 4, (e) A peptide containing the amino acid sequence shown in Sequence ID No. 5, (f) A peptide containing the amino acid sequence shown in Sequence ID No. 6, (g) A peptide containing the amino acid sequence shown in Sequence ID No. 7, (h) A peptide containing the amino acid sequence shown in Sequence ID No. 8, (i) A peptide containing the amino acid sequence shown in Sequence ID No. 9, (j) A peptide containing the amino acid sequence shown in Sequence ID No. 10, (k) A peptide containing the amino acid sequence shown in Sequence ID No. 11, (l) A peptide containing the amino acid sequence shown in Sequence ID No. 12, (m) A peptide containing the amino acid sequence shown in SEQ ID NO: 13, and (n) A peptide containing the amino acid sequence shown in Sequence ID No. 14.

[0017] In a typical embodiment, the epitope peptide mixture used in the present invention is as follows (a) to (l): (a) A peptide containing the amino acid sequence shown in Sequence ID No. 1, (b) A peptide containing the amino acid sequence shown in Sequence ID No. 2, (c) A peptide containing the amino acid sequence shown in Sequence ID No. 3, (d) A peptide containing the amino acid sequence shown in SEQ ID NO: 4, (e) A peptide containing the amino acid sequence shown in Sequence ID No. 5, (f) A peptide containing the amino acid sequence shown in Sequence ID No. 6, (g) A peptide containing the amino acid sequence shown in Sequence ID No. 7, (h) A peptide containing the amino acid sequence shown in Sequence ID No. 8, (i) A peptide containing the amino acid sequence shown in Sequence ID No. 9, (j) A peptide containing the amino acid sequence shown in Sequence ID No. 10, (k) A peptide containing the amino acid sequence shown in Sequence ID No. 11, and (l) Peptide containing the amino acid sequence shown in SEQ ID NO. 12 At least six, preferably seven, more preferably eight, nine, ten, eleven, or twelve peptides selected from the group consisting of, The following (m) and (n): (m) A peptide containing the amino acid sequence shown in SEQ ID NO: 13, and (n) Peptide containing the amino acid sequence shown in SEQ ID NO: 14 A group consisting of 0 to 2 peptides (either containing none of these peptides, or containing one or two of them), Includes.

[0018] The peptide selected from the group consisting of (m) and (n) above may or may not be included in the epitope peptide mixture used in the present invention.

[0019] In one embodiment, the epitope peptide mixture used in the present invention contains all of the peptides (a) to (l) above, and also contains one or both of the peptides (m) and (n).

[0020] In one embodiment, the epitope peptide mixture used in the present invention includes all of the peptides (a) to (n) above.

[0021] In a preferred embodiment, the epitope peptide mixture consists of the peptides (a) to (l) described above. In the present invention, this epitope peptide mixture corresponds to the 12 epitopes-ALL described in the examples below.

[0022] In another preferred embodiment, the epitope peptide mixture comprises the peptides (a) to (n) described above. In the present invention, this epitope peptide mixture corresponds to the 14 epitope-ALL described in the examples below.

[0023] In one embodiment, the epitope peptide used in the present invention is not limited to the following, but may be a peptide with a length of 17 to 30 amino acids, preferably a peptide with a length of 17 to 25 amino acids, and more preferably a peptide with a length of 17 to 21 amino acids. In one embodiment, the epitope peptide used in the present invention may be a peptide with a length of 17 to 20 amino acids. In one embodiment, the epitope peptide used in the present invention may be a peptide with a length of 18 to 21 amino acids. In the present invention, "peptide" refers to an amino acid sequence having a length of 2 to 50 amino acids.

[0024] In one embodiment, the epitope peptide used in the present invention may contain one to several amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9) attached to the N-terminus or C-terminus of the amino acid sequence shown in any of SEQ ID NOs: 1 to 14. In one embodiment, the epitope peptide used in the present invention may contain lysine attached to the N-terminus or C-terminus of the amino acid sequence shown in any of SEQ ID NOs: 1 to 14. An example of this is a peptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 15 to 28, but it is not limited to these. In one embodiment, the epitope peptide used in the present invention may be chemically modified, for example, by adding biotin, streptavidin, or avidin. Alternatively, the epitope peptide used in the present invention may be labeled with any labeling substance such as a fluorescent dye, enzyme, radioactive material, or gold colloid particles. The epitope peptides used in this invention can be labeled with a labeling substance in any way. For example, the epitope peptides can be labeled by attaching a labeling substance to functional groups such as amino groups, sulfhydryl groups, carboxyl groups, or aldehyde groups that are originally present in the epitope peptides or introduced into the epitope peptides. The epitope peptides used in this invention can be synthesized by conventional methods using a peptide synthesis apparatus or the like.

[0025] In one embodiment, the epitope peptide used in the present invention may be biotinylated, for example, containing biotin-bound lysine attached to the N-terminus or C-terminus. For example, the epitope peptide used in the present invention may be a peptide (without biotin) consisting of the amino acid sequence shown in any of SEQ ID NOs: 15 to 28, or it may be a peptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 15 to 28 with biotin bound to the lysine at the C-terminus (biotinylated epitope peptide). In another embodiment, the epitope peptide used in the present invention may be bound to a labeling substance, such as biotin, via a linker. For example, the epitope peptide used in the present invention may be bound to biotin via glycine and lysine attached to the N-terminus or C-terminus of the epitope peptide. In one embodiment, the epitope peptide used in the present invention may be complexed with streptavidin or avidin via biotin attached to the N-terminus or C-terminus (for example, biotin-bound lysine).

[0026] In a preferred embodiment, the epitope peptide mixture may be a mixture of multiple epitope peptides in equal weight ratios relative to the total amount. In the present invention, "equal weight ratios" means that the difference in weight ratio between the epitope peptide with the highest weight ratio and the epitope peptide with the lowest weight ratio is 10% or less, preferably 5% or less, more preferably 1% or 0.5% or less.

[0027] The present invention provides a device made using an epitope peptide mixture used in the present invention. Specifically, the device according to the present invention comprises the epitope peptide mixture. In a preferred embodiment, the device according to the present invention is a device in which the epitope peptide mixture is immobilized on a substrate (solid phase). Examples of substrates include, but are not limited to, insoluble membranes such as nitrocellulose membranes and polyvinylidene fluoride (PVDF) membranes, filters, and plastic plates. In one embodiment, the device according to the present invention may be a device for ELISA, immunochromatography, or dot blot analysis, but is not limited to these.

[0028] In one embodiment, the device according to the present invention may be a test strip, for example, an immunochromatography strip, on which the epitope peptide mixture is fixed on a test line provided on a substrate as described above. The immunochromatography strip may be a test strip comprising, for example, a sample pad (which may include a conjugate pad) to which a test sample is applied, a substrate (for example, a nitrocellulose membrane) to which the epitope peptide mixture is bound, and an absorbent pad for absorbing a solution of the test sample, wherein at least a portion of the lower surface of the sample pad is in contact with one end of the substrate (for example, a nitrocellulose membrane), and at least a portion of the lower surface of the absorbent pad is in contact with the other end of the substrate (for example, a nitrocellulose membrane), and a test line to which the epitope peptide mixture is bound is located between the sample pad and the absorbent pad on the substrate (for example, a nitrocellulose membrane). The immunochromatography strip may further have a control line on the substrate (for example, a nitrocellulose membrane) located between the test line and the absorbent pad (downstream of the test line). In one embodiment, the immunochromatography strip is arranged such that the upstream lower surface of the sample pad does not contact one end of the substrate (e.g., a nitrocellulose film), and the downstream lower surface of the absorption pad does not contact one end of the substrate (e.g., a nitrocellulose film). In another embodiment, the immunochromatography strip is arranged such that the entire lower surface of the sample pad contacts one end of the substrate (e.g., a nitrocellulose film), and the entire lower surface of the absorption pad contacts one end of the substrate (e.g., a nitrocellulose film). The immunochromatography strip may be fixed on a solid support such as a plastic sheet. In the test strip of the present invention, "upstream" means the side on the sample movement axis to which the sample is applied, and "downstream" means the side on the sample movement axis to which the sample moves.

[0029] The epitope peptide mixture can be immobilized onto a device (typically the device's substrate) by conventional methods. This immobilization may be carried out using streptavidin, avidin, and / or biotin, or by linker-mediated binding.

[0030] The present invention also provides kits, such as a milk allergy test kit, that include the epitope peptide mixture or a device containing the epitope peptide mixture. In one embodiment, the kit according to the present invention is a milk allergy test kit for immunochromatographic analysis, and includes, for example, a test strip (such as an immunochromatographic strip) on which the epitope peptide mixture is immobilized on a test line. In one embodiment, the device according to the present invention may be, but is not limited to, a milk allergy test kit for ELISA or dot blot analysis.

[0031] The kit according to the present invention may include other components. In one embodiment, the kit according to the present invention may include instructions for use in a milk allergy test. In one embodiment, the kit according to the present invention may further include anti-immunoglobulin antibodies such as anti-IgE antibodies, anti-IgG antibodies (such as anti-IgG4 antibodies), anti-IgA antibodies, and anti-IgD antibodies, and such anti-immunoglobulin antibodies may be labeled. In one embodiment, the kit according to the present invention may further include anti-IgE antibodies and / or anti-IgG4 antibodies, for example, labeled anti-IgE antibodies and / or anti-IgG4 antibodies. Such anti-immunoglobulin antibodies are secondary antibodies for detecting antibodies bound to the epitope peptide, for example, anti-IgE antibodies and / or anti-IgG4 antibodies are secondary antibodies for detecting IgE antibodies and / or IgG4 antibodies bound to the epitope peptide mixture. The labeling of the secondary antibodies is not particularly limited. The secondary antibodies may be labeled with any labeling substance such as fluorescent dyes, enzymes, radioactive materials, or gold colloid particles. In one embodiment, the kit according to the present invention may include gold colloid-labeled secondary antibodies. In one embodiment, the kit according to the present invention may include a fluorescently labeled secondary antibody. In one embodiment, the kit according to the present invention may include an enzyme-labeled secondary antibody and a detection substrate for the enzyme (a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, etc.). Examples of enzymes used for labeling the antibody and their detection substrates include, but are not limited to, horseradish peroxidase and tetramethylbenzidine, alkaline phosphatase and p-nitrophenyl phosphate, etc.

[0032] The devices and kits according to the present invention can be advantageously used in the methods of the present invention described later, such as methods for measuring milk allergen-specific antibodies (such as IgE antibodies and IgG4 antibodies) and milk allergy testing methods, and therefore may be intended for use in those methods.

[0033] The present invention also provides a method for measuring milk allergen-specific antibodies. More specifically, the present invention involves contacting a biological sample derived from a subject with the above-mentioned epitope peptide mixture, This includes measuring the amount of binding between the antibody in the biological sample and the epitope peptide mixture. This invention provides a method for measuring milk allergen-specific antibodies in biological samples derived from test subjects.

[0034] In a typical embodiment, the present invention relates to the following (a) to (l): (a) A peptide containing the amino acid sequence shown in Sequence ID No. 1, (b) A peptide containing the amino acid sequence shown in Sequence ID No. 2, (c) A peptide containing the amino acid sequence shown in Sequence ID No. 3, (d) A peptide containing the amino acid sequence shown in SEQ ID NO: 4, (e) A peptide containing the amino acid sequence shown in Sequence ID No. 5, (f) A peptide containing the amino acid sequence shown in Sequence ID No. 6, (g) A peptide containing the amino acid sequence shown in Sequence ID No. 7, (h) A peptide containing the amino acid sequence shown in Sequence ID No. 8, (i) A peptide containing the amino acid sequence shown in Sequence ID No. 9, (j) A peptide containing the amino acid sequence shown in Sequence ID No. 10, (k) A peptide containing the amino acid sequence shown in Sequence ID No. 11, and (l) Peptide containing the amino acid sequence shown in SEQ ID NO. 12 At least six, preferably seven, more preferably eight, nine, ten, eleven, or twelve peptides selected from the group consisting of, The following (m) and (n): (m) A peptide containing the amino acid sequence shown in SEQ ID NO: 13, and (n) Peptide containing the amino acid sequence shown in SEQ ID NO: 14 A group consisting of 0 to 2 peptides (either containing none of these peptides, or containing one or two of them), Contact with an epitope peptide mixture containing, This includes measuring the amount of binding between the antibody in the above biological sample and the above epitope peptide mixture. This invention relates to a method for measuring milk allergen-specific antibodies in biological samples derived from test subjects.

[0035] The epitope peptide mixture used in this method may contain all of the peptides listed in (a) to (n) above.

[0036] This method can detect and measure antibodies specific to milk allergens. The antibodies detectable in this method include any antibodies (immunoglobulins) such as IgE antibodies, IgG antibodies (IgG1, IgG2, IgG3, IgG4 antibodies, etc.), IgA antibodies, IgD antibodies, and IgM antibodies. In one embodiment, this method may be a method for measuring milk allergen-specific IgE or IgG4 antibodies. In another embodiment, this method may be a method for measuring milk allergen-specific IgE antibodies. According to the method of the present invention, it is possible to examine the qualitative level of milk allergen-specific antibodies that indicate a correlation with the patient's milk allergy status.

[0037] In one embodiment, the method can be carried out using a device or kit according to the present invention. The description of the epitope peptides contained in the epitope peptide mixture used in the method is as described above with respect to the device or kit according to the present invention, regardless of whether or not the device or kit according to the present invention is used.

[0038] Alternatively, this method can also be performed using a test strip (such as an immunochromatography strip) on which the epitope peptide mixture can be bound to the test line. For example, a biotinylated epitope peptide mixture can be added to a test sample containing a biological sample derived from the subject, and this can be applied to an immunochromatography strip on which streptavidin alone is immobilized on the test line, and then developed by immunochromatography. In this case, by further adding a labeled secondary antibody (anti-immunoglobulin antibody such as anti-IgE antibody or anti-IgG4 antibody), for example, a gold colloid-labeled secondary antibody, to the test sample to which the biotinylated epitope peptide mixture has been added, the complex of the biotinylated epitope peptide bound to streptavidin on the test line and the milk allergen-specific antibody derived from the biological sample can be easily detected. For example, the type of antibody can be detected depending on the secondary antibody used; for example, if an anti-IgE antibody is used as the secondary antibody, milk allergen-specific IgE antibody can be detected, and if an anti-IgG4 antibody is used, milk allergen-specific IgG4 antibody can be detected. The combination of streptavidin and biotin can be interchanged, or streptavidin can be replaced with avidin. The order of addition to the test sample and the order of application to the immunochromatography strip can also be changed as appropriate. Such changes can be made based on known immunological detection techniques and are not particularly limited. The method of the present invention can also be suitably performed by such measurement methods instead of the methods using the above-mentioned devices or kits.

[0039] In the present invention, the subject is a mammal, preferably a human, more preferably a human who has, is suspected of having, a milk allergy, or has a genetic or environmental predisposition to milk allergy. In one embodiment, the subject is an infant, for example, a human under 5 years of age or under 3 years of age. In one embodiment, the subject is a human who is receiving or is scheduled to receive oral immunotherapy for milk allergy. In one embodiment, the subject is ImmunoCAP (R)The human beings are classified into classes 1-6, preferably classes 1-3, and more preferably class 3, based on the tests.

[0040] In the present invention, the biological sample derived from the subject may be any biological sample, but it is preferably a fluid sample (blood sample, saliva sample, tear sample, nasal secretion sample, etc.), and more preferably a blood sample. The blood sample may be a whole blood sample, serum sample, or plasma sample, but is not limited to these. A test sample (preferably a liquid sample) containing a biological sample derived from the subject can be used in this method.

[0041] In this method, the amount of antibody bound to the epitope peptide mixture in a biological sample can be measured by conventional methods, but it can be suitably carried out using immunological detection methods such as ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, and immunochromatography. In a typical immunological detection method, a labeled anti-immunoglobulin antibody, which is a secondary antibody, is bound to an antibody specifically bound to the epitope peptide mixture, and the antibody specifically bound to the epitope peptide mixture can be detected and measured by detecting the signal derived from the label of the secondary antibody. Examples of labeling for the secondary antibody include, but are not limited to, fluorescent dyes, enzymes, radioactive materials, and gold colloid particles. For example, when using an enzyme-labeled secondary antibody, an enzyme detection substrate (chromogenic substrate, fluorescent substrate, chemiluminescent substrate, etc.) is added, and the label signal (e.g., color development, fluorescence, or chemiluminescence signal, etc.) produced by the reaction between the enzyme and the substrate is measured to detect and measure the antibody specifically bound to the epitope peptide mixture. In another embodiment, when using a labeled epitope peptide mixture, the amount of antibody bound to the epitope peptide mixture in the biological sample can be measured by detecting the signal derived from the label of the epitope peptide mixture bound to the antibody in the biological sample. Alternatively, the amount of antibody bound to the epitope peptide mixture in the biological sample may be measured using a non-immunological detection method such as surface plasmon resonance (SPR).

[0042] This method allows for the measurement of milk allergen-specific antibodies, such as milk allergen-specific IgE antibodies, in biological samples. The amount of IgE antibodies (antibody titer) specific to the epitope peptide mixture, measured by this method for measuring milk allergen-specific IgE antibodies in biological samples, is expressed as ImmunoCAP. (R)The measured values ​​of total IgE antibodies specific to milk allergens obtained using a specific IgE (milk) measurement kit generally correlate well with the results obtained using this method. Therefore, the amount of milk allergen-specific IgE antibodies in a subject's biological sample can be evaluated and measured based on the amount of IgE antibodies specific to the epitope peptide mixture described above. The amount of milk allergen-specific IgE antibodies measured by this method correlates well with the subject's milk allergy status.

[0043] Therefore, the present invention also provides a method for testing for milk allergies that utilizes the above-described method for measuring milk allergen-specific antibodies in a biological sample.

[0044] The present invention provides a method for testing (diagnosing) a subject's milk allergy, which includes measuring milk allergen-specific IgE antibodies in a biological sample derived from a subject using the above-described method for measuring milk allergen-specific IgE antibodies in a biological sample, and wherein the measured amount of milk allergen-specific IgE antibodies indicates the subject's tolerance level to milk allergens.

[0045] In one embodiment, the present invention includes measuring milk allergen-specific IgE antibodies in a biological sample derived from a subject using the above-described method for measuring milk allergen-specific IgE antibodies in a biological sample, and obtaining a test index for the subject's milk allergy, in which the measured amount of milk allergen-specific IgE antibodies indicates the subject's tolerance level to milk allergens.

[0046] In a preferred embodiment, if the amount of milk allergen-specific IgE antibody, measured as the amount of IgE antibody specific to the epitope peptide mixture, is 5 ng / mL or higher, it can be determined that the tolerance level to milk allergens is low and that the individual is prone to developing a milk allergy. However, this threshold is not limited to 5 ng / mL, as it may vary depending on conditions such as the subject population.

[0047] The present invention provides a method for evaluating (predicting) the effectiveness of oral immunotherapy for milk allergy in a subject, which includes measuring milk allergen-specific IgE antibodies in a biological sample derived from a subject who is currently receiving or is scheduled to receive oral immunotherapy for milk allergy, using the above-described method for measuring milk allergen-specific IgE antibodies in a biological sample, and where the measured amount of milk allergen-specific IgE antibodies indicates the subject's milk intake tolerance and therapeutic response to oral immunotherapy.

[0048] In one embodiment, the present invention includes measuring milk allergen-specific IgE antibodies in a biological sample derived from a subject who is currently receiving or is scheduled to receive oral immunotherapy for milk allergy, using the above-described method for measuring milk allergen-specific IgE antibodies in a biological sample, and obtaining an evaluation index for the effectiveness of oral immunotherapy for milk allergy in a subject, where the measured amount of milk allergen-specific IgE antibodies indicates the subject's milk intake tolerance and therapeutic response to oral immunotherapy.

[0049] In a preferred embodiment, if the amount of milk allergen-specific IgE antibody, measured as the amount of IgE antibody specific to the epitope peptide mixture, is high (for example, 5 ng / mL or higher), it can be determined that the tolerance for milk intake and the response to oral immunotherapy are poor. For a population of milk allergy patients, a correlation diagram can be created between the epitope peptide mixture-specific IgE antibody content measured by the method of the present invention and the tolerance for milk intake, as shown in Figure 9, for example. Based on this correlation diagram, the tolerance for milk intake of a subject can also be determined from the amount of milk allergen-specific IgE antibody measured by the method of the present invention.

[0050] Furthermore, in any of the above methods according to the present invention, in addition to the method for measuring milk allergen-specific IgE antibodies in a biological sample as described above, the amount of binding between non-IgE antibodies (e.g., IgG4 antibodies, total IgG antibodies, IgA antibodies, etc.) in a biological sample that has been contacted with the epitope peptide mixture and the epitope peptide mixture may be measured as the amount of non-IgE antibodies specific to the epitope peptide mixture, and then the ratio of the amount of milk allergen-specific IgE antibodies measured above to the amount of milk allergen-specific non-IgE antibodies measured in this way (e.g., amount of IgG4 antibodies, amount of total IgG antibodies, amount of IgA antibodies, etc.) may be determined.

[0051] The ratio of the amount of milk allergen-specific IgE antibody measured above to the amount of milk allergen-specific non-IgE antibody (e.g., IgG4 antibody amount, total IgG antibody amount, IgA antibody amount, etc.) determined in this manner is also useful as a further indicator of the risk of developing anaphylaxis due to milk allergens, the level of tolerance to milk allergens based on this risk, or the effectiveness of oral immunotherapy. For example, the ratio of the amount of milk allergen-specific IgE antibody (epitope ALL-IgE) measured above to the amount of milk allergen-specific IgG4 antibody (epitope ALL-IgG4) based on binding to an epitope peptide mixture (epitope-ALL) can be expressed herein as the ALL-IgE / ALL-IgG4 ratio. As an example, the ratio of the amount of milk allergen-specific IgE antibody (14-epitope ALL-IgE) measured above to the amount of milk allergen-specific IgG4 antibody (14-epitope ALL-IgG4) based on binding to 14-epitope-ALL can be expressed as the 14ALL-IgE / 14ALL-IgG4 ratio. [Examples]

[0052] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0053] [Test sample] Serum samples were obtained from the blood of milk allergy patients (with or without oral immunotherapy) who participated in clinical trials with informed consent, stored at -80°C, and used as test samples in the following examples.

[0054] [Example 1] Identification of epitopes of milk allergens 1) Identification of milk allergen-specific IgE and IgG using peptide arrays Peptide arrays were prepared for the identification of epitopes of milk allergens. For the milk allergen proteins αS1-casein, αS2-casein, and β-casein, peptide groups consisting of 15 amino acids were designed, including sequences that overlap by 10 amino acids while shifting to cover the full-length amino acid sequences of each protein. Furthermore, peptide groups consisting of 15 amino acids were designed to cover most of the full-length sequences of the milk allergen proteins κ-casein and β-lactoglobulin, focusing on the regions containing the major epitopes. The peptide arrays were constructed by synthesizing and immobilizing the 175 designed peptides on glass array plates, sequence by sequence.

[0055] Serum samples from patients with milk allergy were appropriately diluted with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and incubated with a peptide array. DyLight-labeled anti-human IgE antibody or DyLight-labeled anti-human IgG antibody (PEPperPRINT) was added as a secondary antibody and incubated. Fluorescent signals from DyLight were detected using an Odyssey CLx image analyzer (LI-COR, Nebraska, USA). Milk allergen-specific IgE antibody detection was positive in 29 patients, and the peptide sequences recognized by the detected milk allergen-specific IgE antibodies were identified. In contrast, in 13 patients, the detected IgE antibody titer was low, while the IgG antibody titer was high; therefore, the peptide sequences recognized by the detected milk allergen-specific IgG antibodies were further identified.

[0056] In this way, we created 21 consecutive sequences (candidate epitope peptides) of approximately 20 amino acids in length, derived from αS1-casein, αS2-casein, β-casein, κ-casein, or β-lactoglobulin, containing the identified peptide sequences.

[0057] 2) Selection of milk allergen epitopes by peptide-ELISA (enzyme-linked immunosorbent assay) analysis Candidate epitope peptides were synthesized and biotinylated by adding biotin-binding lysine to the C-terminus of each peptide. After purification, the molecular weight of the biotinylated candidate epitope peptides was determined by mass spectrometry. The biotinylated candidate epitope peptides were dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, which was diluted with 1% BSA-containing PBS and added to each well of a 96-well plate pre-coated with 300 ng streptavidin (Fujifilm) at a concentration of 30 ng / 100 μL / well.

[0058] After washing streptavidin-coated plates to which biotinylated candidate epitope peptides were bound, diluted serum samples from milk allergy patients were added and incubated in a refrigerator for 1 day. Horseradish peroxidase (HRP)-conjugated anti-human IgE monoclonal antibody (mAb) (MCA E-09-HRP) and HRP-conjugated anti-human IgG4 monoclonal antibody (mAb) (MCA DG-01-HRP) (both from Yamasa Co., Ltd.) were added to the plates. The plates were then washed four times with PBS-T (Phosphate Buffered Saline with Tween 20) and TMB (3,3',5,5'-tetramethylbenzidine) chromogenic substrate (E-101; Bethyl Laboratories Co., Ltd.) was added. The enzymatic color development was measured by absorbance at 450 nm using a microplate reader iMark (Bio-Rad, Hercules, US). IgE-binding epitope profiles and IgG4-binding epitope profiles were obtained based on the binding of IgE antibodies and IgG4 antibodies in patient-derived serum samples to each candidate epitope peptide. The IgE-binding epitope profiles differed among patients. Furthermore, within serum samples from the same patient, the IgG4-binding epitope profiles differed from the IgE-binding epitope profiles.

[0059] For each candidate epitope peptide, the number of IgE-positive patients to which IgE antibodies bound in serum samples, and the number of IgG4-positive patients to which IgG4 antibodies bound in serum samples were counted. Based on these totals, epitopes that were frequently recognized by IgE or IgG4 antibodies among patients were selected. Twelve epitopes were selected: eight from αS1-casein and αS2-casein, two from β-casein, and two from κ-casein. In addition, two epitopes from β-lactoglobulin were selected and added.

[0060] In this way, we obtained 14 epitopes derived from each of the five major milk allergens: αS1-casein, αS2-casein, β-casein, κ-casein, and β-lactoglobulin (Table 1).

[0061] [Table 1]

[0062] The set of 14 selected epitopes (Table 1; No. 1-14) will be hereinafter referred to as 14-epitope-ALL. The set of 12 epitopes (No. 1-12), excluding the epitope derived from β-lactoglobulin, will be hereinafter referred to as 12-epitope-ALL. Furthermore, 14-epitope-ALL and 12-epitope-ALL will be collectively referred to as epitope-ALL here.

[0063] [Example 2] Acquisition of epitope filing by ELISA measurement using epitope-ALL ELISA analysis was performed on serum samples from each patient using either 12-epitope-ALL or 14-epitope-ALL. Streptavidin-coated plates (30 ng / well for each epitope peptide) conjugated with the biotinylated epitope peptides (single peptides) shown in Table 1 were prepared in the same manner as in Example 1. Furthermore, the biotinylated epitope peptide solutions prepared in Example 1 were mixed to prepare a mixture of 12 epitope peptides constituting 12-epitope-ALL, or a mixture of 14 epitope peptides constituting 14-epitope-ALL. This mixture was then added to each well of a 96-well plate pre-coated with 300 ng of streptavidin so that the total epitope peptide concentration was 100 ng / well (8.3 ng / each epitope / well for 12-epitope-ALL, and 7.1 ng / each epitope / well for 14-epitope-ALL), thereby creating streptavidin-coated plates to which the biotinylated epitope peptide mixture was bound.

[0064] After washing streptavidin-coated plates bound with biotinylated epitope peptides, diluted serum samples from milk allergy patients were added and incubated in a refrigerator for 1 day. Horseradish peroxidase (HRP)-conjugated anti-human IgE monoclonal antibody (mAb) (MCA E-09-HRP) and HRP-conjugated anti-human IgG4 monoclonal antibody (mAb) (MCA DG-01-HRP) (both from Yamasa Co., Ltd.) were added to the plates. The plates were then washed four times with PBS-T (Phosphate Buffered Saline with Tween 20) and TMB (3,3',5,5'-tetramethylbenzidine) chromogenic substrate (E-101; Bethyl Laboratories Co., Ltd.) was added. Enzymatic color development was performed using iMark. TM Absorbance at 450 nm was measured using a microplate reader (Bio-Rad). IgE-binding epitope profiles and IgG4-binding epitope profiles were obtained based on the binding of IgE antibodies and IgG4 antibodies in patient-derived serum samples to epitope peptides.

[0065] Figure 1 shows the IgE-binding epitope profiles (using 12 epitopes-ALL) for serum samples from three milk allergy patients (#101, #108, #ID-03). Figure 2 shows the IgE-binding epitope profiles (using 14 epitopes-ALL) for serum samples from three other milk allergy patients (receiving oral immunotherapy, #2814, #2910, #3018). As shown in Figures 1 and 2, the IgE-binding epitope profiles differed significantly among the patients. Furthermore, as shown in Figures 1 and 2, the amount of epitope-binding IgE antibodies measured using plates conjugated with biotinylated epitope peptide mixtures (12 epitopes-ALL or 14 epitopes-ALL) was shown to correspond to the sum of the individual epitope-binding IgE antibody amounts measured using plates conjugated with a single biotinylated epitope peptide.

[0066] Figure 3 shows the results of measuring epitope-conjugated IgE antibodies (Figure 3A) and epitope-conjugated IgG4 antibodies (Figure 3B) in dilutions of serum samples from milk allergy patients using plates conjugated with a biotinylated epitope peptide mixture (14 epitopes-ALL). Serum samples were used from nine milk allergy patients (#3154, #3103, #3091, #2814, #2782, #2780, #2774, #2714, #2701). Serum samples diluted 5-fold, 15-fold, and 50-fold were used for IgE antibody measurement (Figure 3A). Serum samples diluted 15-fold, 50-fold, and 150-fold were used for IgG4 antibody measurement (Figure 3B).

[0067] Note that the OD values ​​on the vertical axis of the graphs in Figures 1 to 3 can be converted to quantitative values ​​(pg / mL) based on the calibration curve.

[0068] By comparing the amount of IgE antibody (Figure 3A) and IgG4 antibody (Figure 3B) against 14 epitopes-ALL in serum samples from the same patient, it is possible to indicate which antibody is dominant, IgE or IgG4; however, this varies from patient to patient. For example, in patient #3154, IgE antibody is dominant over IgG4 antibody, while in patient #3103, IgG4 antibody is dominant over IgE antibody. The ratio of IgE antibody to IgG4 antibody levels against epitope-ALL is considered useful for evaluating the degree of tolerance to milk allergens and responsiveness to oral immunotherapy in patients with milk allergy.

[0069] [Example 3] Quantitative values ​​of milk allergen-specific IgE antibodies and IgG4 antibodies in serum samples and their evaluation A calibration curve was created to quantify the epitope-ALL-specific IgE and IgG4 antibody content in serum samples based on measurements using streptavidin-coated plates conjugated with a biotinylated epitope peptide mixture (12-epitope-ALL or 14-epitope-ALL).

[0070] Recombinant human IgE antibody A and recombinant human IgG4 antibody B, both specific to a single epitope peptide, were used as standard samples. Recombinant human IgE antibody A is a recombinant human-mouse chimeric IgE antibody specific to a single epitope peptide derived from a specific milk allergen shown in Table 1. It was prepared by fusing the Fab of a mouse monoclonal antibody against the epitope with the Fc portion of a human IgE antibody. Recombinant human IgG4 antibody B is a recombinant human-mouse chimeric IgG4 antibody specific to the same single epitope peptide derived from a specific milk allergen. It was prepared by fusing the Fab of a mouse monoclonal antibody against the milk allergen epitope with the Fc portion of a human IgG4 antibody. Recombinant human IgE antibody A and recombinant human IgG4 antibody B have the same Fab derived from the same α-casein-specific mouse monoclonal antibody.

[0071] Recombinant human IgE antibody A and recombinant human IgG4 antibody B were diluted to known concentrations and added to streptavidin-coated plates conjugated with 12-epitope-ALL or 14-epitope-ALL. The IgE and IgG4 antibodies conjugated to epitope-ALL were measured using the same ELISA method as in Example 2. Based on the measured values, a standard curve was created with antibody concentration on the x-axis and absorbance at 450 nm on the y-axis, and this was used as a calibration curve. An example of a standard curve is shown in Figure 4.

[0072] Following this calibration curve, the content (antibody titer) of 14-epitope-ALL specific IgE antibody and IgG4 antibody in serum samples from each milk allergy patient was determined from the absorbance at 450 nm of epitope-ALL-conjugated IgE antibody and IgG4 antibody measured by ELISA using streptavidin-coated plates to which a biotinylated epitope peptide mixture (12-epitope-ALL or 14-epitope-ALL) was conjugated in Example 2.

[0073] Furthermore, ImmunoCAP was used for the same serum sample. (R) ImmunoCAP using a specific IgE (milk) measurement kit (Thermo Fisher Scientific)(R) By testing, in accordance with the instructions for use, the total content (antibody titer) of milk allergen-specific IgE antibodies in the serum sample was measured.

[0074] In Figure 5, the 12-epitope-ALL-specific IgE antibody titers (vertical axis) measured using a 12-epitope-ALL binding plate for serum samples from each milk allergy patient (40 patients; pediatric patients undergoing oral immunotherapy), and ImmunoCAP (R) The correlation diagram plotted based on the milk allergen-specific IgE antibody titers (horizontal axis; ImmunoCAP (R) IgE values) measured by the test is shown. The values on the vertical axis in Figure 5 ( A U / L) can be converted to quantitative values by creating a calibration curve and using it.

[0075] In Figure 6, the 14-epitope-ALL-specific IgE antibody content (vertical axis) measured using a 14-epitope-ALL binding plate for serum samples from each milk allergy patient (39 patients; pediatric patients undergoing oral immunotherapy), and ImmunoCAP (R) The correlation diagram plotted based on the milk allergen-specific IgE antibody titers (horizontal axis; ImmunoCAP (R) IgE values) measured by the test is shown.

[0076] As shown in Figures 5 and 6, the 12-epitope-ALL or 14-epitope-ALL-specific IgE antibody content and the milk allergen-specific IgE antibody titers measured by the test with ImmunoCAP (R) which is the current standard total IgE measurement method, showed a generally good correlation. That is, the 12-epitope-ALL-specific IgE antibody content and the 14-epitope-ALL-specific IgE antibody content were shown to be (R) usable as alternative indicators for the milk allergen-specific IgE antibody titers measured by the ImmunoCAP test.

[0077] Here, ImmunoCAP (R) ImmunoCAP using a specific IgE (milk) measurement kit (R)In the test, the milk allergen-specific IgE antibody titer (ImmunoCAP) in the sample was measured. (R) Samples are classified into classes 0-6 according to their IgE levels, with class 0 being negative, class 1 being suspected positive, and class 2 or higher being positive.

[0078] As shown in Figures 5 and 6, the above ImmunoCAP (R) All serum samples classified as Class 4 or higher (ImmunoCAP IgE value ≥ 17.5 kU / mL) by the test showed high levels of epitope-ALL specific IgE antibodies (≥ 5 ng / mL in Figure 6). The reference value (cutoff value) of "≥ 5 ng / mL" in Figure 6 was determined based on the epitope-ALL specific IgE antibody content in serum samples from 38 milk allergy patients and 34 non-allergic individuals (Figure 7).

[0079] On the other hand, in Figures 5 and 6, the above ImmunoCAP (R) The test classified serum samples into Class 3 (ImmunoCAP IgE levels = 3.5-17.49 kU / mL), and it was shown that serum samples with moderate ImmunoCAP IgE levels exhibited relatively large variations in epitope-ALL specific IgE antibody content between samples. (R) In patients with milk allergy classified as Class 3 by testing, the incidence of allergic symptoms with oral immunotherapy, as shown by the probability curve, is known to range from approximately 30% to 70%. Therefore, it is possible that the changes in epitope-ALL specific IgE antibody content shown in Figures 5 and 6 are related to the incidence of allergic symptoms.

[0080] Based on the above, ImmunoCAP (R) The study showed that in patients with milk allergies classified as class 3 or lower, the content of epitope-ALL-specific IgE antibodies could be divided into high-risk and low-risk groups that are more likely to trigger allergic symptoms.

[0081] immunoCAP (R)Figure 8 shows the milk intake tolerance and ImmunoCAP levels for each patient receiving oral immunotherapy, for milk allergy patients classified as Class 3 in the trial. (R) The data was plotted based on IgE levels, and in Figure 9, it was plotted based on each patient's milk intake tolerance and epitope-ALL specific IgE antibody content. As shown in Figure 8, ImmunoCAP (R) Among patients with relatively high IgE levels, there are many who have a high tolerance for milk intake (see the dashed circle in Figure 8), and ImmunoCAP (R) It was shown that it is difficult to use IgE levels as an indicator of milk intake tolerance. On the other hand, Figure 9 shows that patients with relatively high epitope-ALL specific IgE antibody levels generally had low milk intake tolerance (see the dashed circle in Figure 9), suggesting that epitope-ALL specific IgE antibody levels can be used as an indicator of milk intake tolerance.

[0082] [Example 4] Immunochromatography Analysis Immunochromatographic analysis was performed on serum samples from milk allergy patients using the 14 epitopes-ALL selected in Example 1.

[0083] First, the 14 biotinylated epitope peptides constituting 14-epitope-ALL (Table 1; No. 1-14) were mixed, and this mixture was then mixed in equimolar ratio with streptavidin solution to prepare a 14-epitope-streptavidin complex mixture. 0.5 μL of the obtained 14-epitope-streptavidin complex mixture was applied to a line (test line) on a nitrocellulose membrane and dried, thereby attaching the 14-epitope-streptavidin complex (approximately 0.5 μg / strip) to the nitrocellulose membrane. The line to which the 14-epitope-streptavidin complex was attached (bound) became the test line.

[0084] A nitrocellulose membrane to which a 14-epitope-streptavidin complex was attached (bound) was added, and a 0.1% BSA solution in Tris-buffered saline (TBS) was blocked at 37°C for 1 hour. The membrane was then washed with TBS and dried. Next, an immunochromatography strip was prepared by constructing the sample pad, the 14-epitope-streptavidin complex-bound nitrocellulose membrane, and the absorption pad in this order, with the sample pad and absorption pad fixed to the ends of the nitrocellulose membrane, respectively. Specifically, this immunochromatography strip comprises a sample pad to which the test sample solution is applied, a nitrocellulose membrane conjugated to the 14-epitope-streptavidin complex prepared as described above, and an absorption pad to absorb the test sample solution. The lower surface of the sample pad is in contact with one end of the nitrocellulose membrane, and the absorption pad is in contact with the other end. A test line to which the 14-epitope-streptavidin complex is conjugated exists between the sample pad and the absorption pad on the nitrocellulose membrane.

[0085] 1% Lipidure (R) Serum samples from milk allergy patients (#2900, #2910) or recombinant human IgE antibody A were added to PBS containing BL206 and / or BL802 (Nichiyu Co., Ltd.), and then gold (Au) colloid-labeled secondary antibodies, which are mouse anti-human IgE monoclonal antibodies or mouse anti-human IgG4 monoclonal antibodies, were added. After standing for 5 minutes, the mixture was applied to the immunochromatography strips prepared above.

[0086] To allow the applied sample solution to pass through to the 14 epitope-streptavidin complex line (test line) on the immunochromatography strip, use a washing solution (1% Lipidure). (R) After adding PBS (containing the gold) twice and drying, band images of the gold colloid accumulation were obtained.

[0087] The gold colloid-labeled secondary antibody was prepared by mixing 500 μL of mouse anti-human IgE monoclonal antibody solution or mouse anti-human IgG4 monoclonal antibody solution (1 mg / mL in 2 mM borate buffer, pH 9.0) with 5 mL of gold colloid solution (Sigma-Aldrich, 0.2 M in potassium carbonate solution, pH 9.0) and reacting at room temperature for 30 minutes. Then, 635 μL of BSA solution (10%) was added to the mixture and the reaction was continued for another 15 minutes. After centrifugation, the supernatant was collected and adjusted to an OD value (525 nm) of 1.0 using 1% BSA-containing PBS solution. This solution was stored at 4°C before use as the gold colloid-labeled antibody solution.

[0088] The results are shown in Figure 10. As shown in Figures 10A and 10B, the surfactant Lipidure (R) Recombinant human IgE antibody A, to which -BL206 or BL802 was added, showed a band due to gold colloid-labeled IgE antibody on the 14 epitope-streptavidin complex line on the immunochromatography strip. Also, as shown in Figures 10C and 10D, serum samples at 10 μL / lane (Figure 10C) or 20 μL / lane (Figure 10D) also showed a band due to gold colloid-labeled IgE antibody or IgG4 antibody on the 14 epitope-streptavidin complex line on the immunochromatography strip.

[0089] Thus, it has been shown that immunochromatography using epitope-ALL can also detect epitope-ALL-specific IgE antibodies and IgG4 antibodies in serum samples from patients with milk allergies. [Industrial applicability]

[0090] According to the present invention, not only can the amount of milk allergen-specific antibodies in milk allergy patients be easily tested, but the level of milk allergen tolerance in milk allergy patients and the risk of developing milk allergy during oral immunotherapy can be evaluated more accurately. The devices and kits of the present invention can also be used as companion diagnostics to evaluate the effectiveness of treatments such as oral immunotherapy for milk allergy.

Claims

1. (a) to (l) below: (a) A peptide containing the amino acid sequence shown in SEQ ID NO: 1, (b) A peptide containing the amino acid sequence shown in SEQ ID NO: 2 (c) A peptide containing the amino acid sequence shown in SEQ ID NO: 3, (d) A peptide containing the amino acid sequence shown in SEQ ID NO: 4, (e) A peptide containing the amino acid sequence shown in Sequence ID No. 5, (f) A peptide containing the amino acid sequence shown in SEQ ID NO: 6, (g) A peptide containing the amino acid sequence shown in SEQ ID NO: 7, (h) A peptide containing the amino acid sequence shown in SEQ ID NO: 8, (i) A peptide containing the amino acid sequence shown in Sequence ID No. 9, (j) A peptide containing the amino acid sequence shown in SEQ ID NO: 10, (k) A peptide containing the amino acid sequence shown in SEQ ID NO: 11, and (l) Peptide containing the amino acid sequence shown in SEQ ID NO: 12 At least six peptides selected from the group consisting of, The following (m) and (n): (m) A peptide containing the amino acid sequence shown in SEQ ID NO: 13, and (n) Peptide containing the amino acid sequence shown in SEQ ID NO: 14 A group consisting of 0 to 2 peptides, A device for testing milk allergies, containing an epitope peptide mixture.

2. The device according to claim 1, wherein the epitope peptide mixture comprises all of the peptides (a) to (n).

3. The device according to claim 1, wherein the peptide is a peptide having a length of 17 to 21 amino acids.

4. The device according to claim 1, wherein the peptide comprises biotin-bound lysine attached to the N-terminus or C-terminus of the amino acid sequence.

5. The device according to claim 4, wherein the peptide is complexed with streptavidin or avidin.

6. The device according to claim 1, wherein the epitope peptide mixture is immobilized on an immunochromatography strip on a test line.

7. A kit for testing for milk allergy, comprising the device described in claim 1.

8. A milk allergy testing kit according to claim 7, for ELISA or dot blot analysis.

9. A kit for testing for milk allergy for immunochromatographic analysis, comprising the device described in claim 6.

10. A kit according to any one of claims 7 to 9, further comprising a labeled anti-IgE antibody and / or an anti-IgG4 antibody.

11. The biological samples derived from the subject were (a) to (l) below: (a) A peptide containing the amino acid sequence shown in SEQ ID NO: 1, (b) A peptide containing the amino acid sequence shown in SEQ ID NO: 2 (c) A peptide containing the amino acid sequence shown in SEQ ID NO: 3, (d) A peptide containing the amino acid sequence shown in SEQ ID NO: 4, (e) A peptide containing the amino acid sequence shown in Sequence ID No. 5, (f) A peptide containing the amino acid sequence shown in SEQ ID NO: 6, (g) A peptide containing the amino acid sequence shown in SEQ ID NO: 7, (h) A peptide containing the amino acid sequence shown in SEQ ID NO: 8, (i) A peptide containing the amino acid sequence shown in Sequence ID No. 9, (j) A peptide containing the amino acid sequence shown in SEQ ID NO: 10, (k) A peptide containing the amino acid sequence shown in SEQ ID NO: 11, and (l) Peptide containing the amino acid sequence shown in SEQ ID NO: 12 At least six peptides selected from the group consisting of, The following (m) and (n): (m) A peptide containing the amino acid sequence shown in SEQ ID NO: 13, and (n) Peptide containing the amino acid sequence shown in SEQ ID NO: 14 A group consisting of 0 to 2 peptides, Contact with an epitope peptide mixture containing, This includes measuring the amount of binding between the antibody in the biological sample and the epitope peptide mixture. A method for measuring milk allergen-specific antibodies in biological samples derived from test subjects.

12. The method according to claim 11, wherein the epitope peptide mixture comprises all of the peptides (a) to (n).

13. The method according to claim 11 or 12, wherein the antibody is an IgE antibody.

14. The method according to claim 11, wherein the peptide is a peptide having a length of 17 to 21 amino acids.

15. The method according to claim 11, performed using the device described in claim 1 or the kit described in claim 7.

16. The method according to claim 11, wherein the biological sample is a blood sample.

17. A method for obtaining a test index for a subject's milk allergy, comprising measuring milk allergen-specific IgE antibodies in a biological sample derived from a subject using the method described in claim 13, wherein the measured amount of milk allergen-specific IgE antibodies indicates the subject's tolerance level to milk allergens.

18. A method for obtaining an evaluation index for the effectiveness of oral immunotherapy for milk allergy in a subject, comprising measuring milk allergen-specific IgE antibodies in a biological sample from a subject who is receiving or scheduled to receive oral immunotherapy for milk allergy using the method of claim 13, wherein the measured amount of milk allergen-specific IgE antibodies indicates the subject's milk intake tolerance and therapeutic response to oral immunotherapy.

19. The method according to claim 17, further comprising measuring the amount of binding between IgG4 antibody in a biological sample contacted with the epitope peptide mixture and the epitope peptide mixture, and determining the ratio of the amount of milk allergen-specific IgE antibody to the measured amount of milk allergen-specific IgG4 antibody.

20. The method according to claim 18, further comprising measuring the amount of binding between IgG4 antibody in a biological sample contacted with the epitope peptide mixture and the epitope peptide mixture, and determining the ratio of the amount of milk allergen-specific IgE antibody to the measured amount of milk allergen-specific IgG4 antibody.

Images