Device, kit, and method for cow's milk allergy test

Abstract

The present invention relates to a device and a kit for a cow's milk allergy test which each comprise an epitope peptide mixture including at least six peptides selected from the group consisting of peptides that are included in the amino acid sequences indicated in SEQ ID NOs: 10-12, and zero to two peptides selected from the group consisting of peptides that are included in the amino acid sequences indicated in SEQ ID NOs: 13 and 14. The present invention also relates to a method for measuring cow's milk allergen-specific antibody and a cow's milk allergy test method which each use the epitope peptide mixture.

Description

Device, kit, and method for milk allergy testing

[0001] The present invention relates to a milk allergy testing technique.

[0002] Milk is the second most common allergen in food allergies among infants, after chicken eggs. The causative substances of milk allergy are proteins derived from milk, such as casein and β-lactoglobulin. Milk allergy often occurs in infants, but it is said that it often resolves naturally after the age of three. However, the onset of milk allergy has a serious impact on the health of infants, and restricting milk intake to avoid the onset of milk allergy may also have a negative impact on the growth of infants.

[0003] Oral immunotherapy (OIT), in which a patient receives a small amount of an allergen followed by a gradual increase, is known as a 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 efficacy of oral immunotherapy varies greatly from individual to individual, and there is a risk of anaphylaxis. Therefore, oral immunotherapy is performed under advanced medical supervision. Predicting the efficacy of oral immunotherapy for individual patients would reduce the burden on patients and medical professionals, but no reliable prediction method has been established.

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

[0005] Patent Document 1 discloses a method for detecting milk allergy and a diagnostic composition for the detection. Patent Document 1 also 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 the milk allergen-specific IgE antibody level, which is related to the patient's milk allergy state, or an allergy testing method based thereon.

[0006] There is a need for the development of a milk allergy testing technique that can measure the milk allergen-specific antibody levels associated with a patient's milk allergy status.

[0007] Japanese Patent Application Laid-Open No. 2022-157411

[0008] An objective of the present invention is to provide a milk allergy testing technique that can measure the level of milk allergen-specific IgE antibodies that are related to a patient's milk allergy state.

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

[0010] That is, the present invention includes the following.

[0011] [1] A device for testing for a milk allergy, comprising an epitope peptide mixture comprising: at least six peptides selected from the group consisting of (a) to (l) below: (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2, (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3, (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4, (e) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5, (f) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 6, (g) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 7, (h) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 8, (i) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 9, (j) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10, (k) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11, and (l) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and zero to two peptides selected from the group consisting of (m) and (n) below: (m) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13, and (n) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0012] [2] The device according to [1] above, wherein the epitope peptide mixture contains all of the peptides (a) to (n).

[0013] [3] The device according to [1] or [2] above, wherein the peptide is a peptide having a length of 17 to 21 amino acids.

[0014] [4] The device according to any one of [1] to [3] above, wherein the peptide contains a biotin-binding lysine added to the N-terminus or C-terminus of the amino acid sequence.

[0015] [5] The device described in [4] above, wherein the peptide is complexed with streptavidin or avidin.

[0016] [6] The device according to any one of [1] to [5] above, which is an immunochromatography strip in which the epitope peptide mixture is immobilized on a test line.

[0017] [7] A milk allergy testing kit comprising the device according to any one of [1] to [6] above.

[0018] [8] The milk allergy testing kit according to [7] above, for ELISA or dot blot analysis.

[0019] [9] A milk allergy testing kit for immunochromatography analysis, comprising the device described in [6] above.

[0020]

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

[0021]

[11] A biological sample derived from a subject is contacted with an epitope peptide mixture comprising at least six peptides selected from the group consisting of the following (a) to (l): (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2, (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3, (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4, (e) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5, (f) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 6, (g) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 7, (h) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 8, (i) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 9, (j) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10, (k) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11, and (l) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and 0 to 2 peptides selected from the group consisting of the following (m) and (n): (m) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13, and (n) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 14, A method for measuring cow's milk allergen-specific antibodies in a biological sample derived from a subject, comprising measuring the amount of binding between the antibodies in the biological sample and the epitope peptide mixture.

[0022]

[12] The method according to

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

[0023]

[13] The method according to

[11] or

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

[0024]

[14] The method according to any one of

[11] to

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

[0025]

[15] The method according to any one of

[11] to

[14] above, carried out using the device according to any one of [1] to [6] above or the kit according to any one of [7] to

[10] above.

[0026]

[16] The method according to any one of

[11] to

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

[0027]

[17] A method for obtaining a test indicator for milk allergy in a subject, comprising measuring milk allergen-specific IgE antibodies in a biological sample derived from the 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 the milk allergen.

[0028]

[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 undergoing or planning to undergo 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 responsiveness to oral immunotherapy.

[0029]

[19] The method described in

[17] above, further comprising measuring the amount of binding between IgG4 antibodies 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 antibodies to the measured amount of milk allergen-specific IgG4 antibodies.

[0030]

[20] The method described in

[18] above, further comprising measuring the amount of binding between IgG4 antibodies 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 antibodies to the measured amount of milk allergen-specific IgG4 antibodies.

[0031] This specification includes the disclosure of Japanese Patent Application No. 2024-154124, from which the present application claims priority.

[0032] The present invention is capable of measuring milk allergen-specific antibody levels that correlate with a patient's milk allergy status.

[0033] Figure 1 shows the IgE-binding epitope profiles for 12 epitope peptides obtained for serum samples from three cow's milk allergy patients (#101, #108, #ID-03). "ALL (No. 1-12)" indicates the IgE level measured using a plate bound with a biotinylated epitope peptide mixture (12 epitopes-ALL). Figure 2 shows the IgE-binding epitope profiles for 14 epitope peptides obtained for serum samples from three cow's milk allergy patients. "ALL (No. 1-14)" indicates the IgE level measured using a plate bound with a biotinylated epitope peptide mixture (14 epitopes-ALL). #2814 (x30)_IgE: IgE level measured using a 30-fold dilution of the serum sample from patient #2814. #2910 (x150)_IgE: IgE level measured using a 150-fold dilution of a serum sample from patient #2910. #3018 (x300)_IgE: IgE level measured using a 300-fold dilution of a serum sample from patient #3018. Figure 3 shows the results of measuring epitope-binding IgE antibodies (Figure 3A) and epitope-binding IgG4 antibodies (Figure 3B) in diluted serum samples from patients with milk allergy using a plate bound to 14 epitope-ALL. α-human IgE x5: IgE antibodies in a 5-fold dilution of a serum sample from a patient with milk allergy. α-human IgE x15: IgE antibodies in a 15-fold dilution of a serum sample from a patient with milk allergy. α-human IgE x50: IgE antibodies in a 50-fold dilution of a serum sample from a patient with milk allergy. α-human IgG4 x 15: IgG4 antibody in a 15-fold dilution of a serum sample from a human patient with cow's milk allergy, α-human IgG4 x 50: IgG4 antibody in a 50-fold dilution of a serum sample from a human patient with cow's milk allergy, α-human IgG4 x 150: IgG4 antibody in a 150-fold dilution of a serum sample from a human patient with cow's milk allergy. Figure 4 shows standard curves based on the measured values ​​of IgE antibody and IgG4 antibody obtained by ELISA using solutions of known concentrations of recombinant human IgE antibody A and recombinant human IgG4 antibody B on a streptavidin-coated plate bound to 14 epitope-ALL.FIG. 5 shows the 12 epitope-ALL specific IgE antibody content (vertical axis) and ImmunoCAP for serum samples from milk allergy patients. （Ｒ） 6 shows a correlation diagram plotted based on IgE values ​​(horizontal axis). （Ｒ） The correlation diagram is plotted based on IgE levels (horizontal axis). The class on the horizontal axis is ImmunoCAP. （Ｒ） The IgE value (horizontal axis) is based on the IgE value. Figure 7 shows the distribution of 14 epitope-ALL specific IgE antibody content (pg / mL) in milk allergy patients and non-allergic individuals. An example of the threshold (5 ng / mL) for distinguishing between the milk allergy patient group and the non-allergic group is shown by the dotted line. Figure 8 shows the milk allergy patients undergoing oral immunotherapy, compared with the allowable milk intake amount and ImmunoCAP. （Ｒ） Figure 9 is a graph plotting the milk intake tolerance and 14 epitope-ALL specific IgE antibody content in cow's milk allergy patients undergoing oral immunotherapy. Figure 10 is a photograph showing the results of immunochromatographic analysis of antibody samples using an immunochromatographic strip with 14 epitope-ALL. Figure 10A: Recombinant human IgE antibody A, 10 ng (left lane) and 100 ng (right lane) (Lipidure （Ｒ） -BL206), Figure 10B: Recombinant human IgE antibody B, 10 ng (left lane) and 100 ng (right lane) (Lipidure （Ｒ） -BL802), Figure 10C: IgG4 antibodies (left lane) and IgE antibodies (right lane) in serum samples from patient #2900 (Lipidure （Ｒ） -BL206 and -BL802), Fig. 10D: IgG4 antibodies (left lane) and IgE antibodies (right lane) in a serum sample from patient #2910 (Lipidure （Ｒ） -BL206 and -BL802 added).

[0034] The present invention will be described in detail below.

[0035] The present invention relates to a milk allergy testing technique, and in particular to a milk allergy testing technique capable of measuring the level of milk allergen-specific antibodies associated with a milk allergy state.

[0036] Specifically, the present invention relates to a milk allergy testing device and kit, a method for measuring milk allergen-specific antibodies, a method for testing milk allergy, a method for obtaining a test indicator for milk allergy, and a method for evaluating the effectiveness of oral immunotherapy for milk allergy. In the methods of the present invention, the measurement is preferably performed in vitro. In the present invention, "oral immunotherapy" includes not only OIT conducted as part of clinical research, but also dietary therapy and the like conducted in general medical practice.

[0037] In the present invention, the binding of a milk allergen-specific antibody to a mixture (mixture) 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 binding amount. In the present invention, the term "allergen-specific antibody" refers to an antibody capable of specifically binding to a specific allergen.

[0038] The multiple epitope peptides (sometimes simply referred to as peptides) used in the present invention derived from cow's milk allergens, specifically 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 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, (l) a peptide comprising the amino acid sequence shown in SEQ ID NO: 12, (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.

[0039] In a typical embodiment, the epitope peptide mixture used in the present invention comprises at least six, preferably seven, more preferably eight, nine, ten, eleven, or twelve peptides selected from the group consisting of the following (a) to (l): (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2, (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3, (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4, (e) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5, (f) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 6, (g) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 7, (h) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 8, (i) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 9, (j) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10, (k) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11, and (l) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and the following (m) and (n): (m) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13, and (n) 0 to 2 peptides (0 to 1, or 2 of these peptides) selected from the group consisting of peptides comprising the amino acid sequence set forth in SEQ ID NO: 14; and

[0040] The peptides 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.

[0041] In one embodiment, the epitope peptide mixture used in the present invention contains all of the above peptides (a) to (l), and also contains one or both of the peptides (m) and (n).

[0042] In one embodiment, the epitope peptide mixture used in the present invention contains all of the above peptides (a) to (n).

[0043] In a preferred embodiment, the epitope peptide mixture consists of the peptides (a) to (l) above. In the present invention, this epitope peptide mixture corresponds to the 12 epitopes-ALL described in the Examples below.

[0044] In another preferred embodiment, the epitope peptide mixture consists of the peptides (a) to (n) above. In the present invention, this epitope peptide mixture corresponds to the 14 epitopes-ALL described in the Examples below.

[0045] In one embodiment, the epitope peptide used in the present invention may be, but is not limited to, a peptide of 17 to 30 amino acids in length, preferably a peptide of 17 to 25 amino acids in length, and more preferably a peptide of 17 to 21 amino acids in length. In one embodiment, the epitope peptide used in the present invention may be a peptide of 17 to 20 amino acids in length. In one embodiment, the epitope peptide used in the present invention may be a peptide of 18 to 21 amino acids in length. In the present invention, the term "peptide" refers to one having an amino acid sequence of 2 to 50 amino acids in length.

[0046] In one embodiment, the epitope peptide used in the present invention may comprise one to several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acids added to the N-terminus or C-terminus of the amino acid sequence set forth in any of SEQ ID NOs: 1 to 14. In one embodiment, the epitope peptide used in the present invention may comprise a lysine added to the N-terminus or C-terminus of the amino acid sequence set forth in any of SEQ ID NOs: 1 to 14. Examples include, but are not limited to, peptides consisting of the amino acid sequences set forth in any of SEQ ID NOs: 15 to 28. In one embodiment, the epitope peptide used in the present invention may be chemically modified, for example, to which biotin, streptavidin, or avidin may be attached. Alternatively, the epitope peptide used in the present invention may be labeled with any labeling substance, such as a fluorescent dye, an enzyme, a radioactive substance, or colloidal gold particles. The epitope peptides used in the present invention can be labeled with a labeling substance by any method, for example, by binding a labeling substance to a functional group, such as an amino group, a sulfhydryl group, a carboxyl group, or an aldehyde group, that the epitope peptide originally has or that has been introduced into the epitope peptide. The epitope peptides used in the present invention can be synthesized by standard methods using a peptide synthesizer or the like.

[0047] In one embodiment, the epitope peptide used in the present invention may be biotinylated, for example, it may contain a biotin-binding lysine attached to the N-terminus or C-terminus. For example, the epitope peptide used in the present invention may be a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 15 to 28 (not containing biotin), or it may be a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 15 to 28 with biotin bound to the C-terminal lysine (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 (e.g., biotin-binding lysine).

[0048] In a preferred embodiment, the epitope peptide mixture may be a mixture of multiple epitope peptides at equal weight ratios relative to the total amount. In the present invention, "equal weight ratios" refers to a difference in weight ratio between the epitope peptide with the highest weight ratio and the epitope peptide with the lowest weight ratio of 10% or less, preferably 5% or less, and more preferably 1% or 0.5% or less.

[0049] The present invention provides a device produced using the epitope peptide mixture used in the present invention. Specifically, the device of the present invention comprises the epitope peptide mixture. In a preferred embodiment, the device of 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, plastic plates, etc. In one embodiment, the device of the present invention may be a device for ELISA, immunochromatography, or dot blot analysis, but is not limited to these.

[0050] In one embodiment, the device of the present invention may be a test strip, such as an immunochromatography strip, in which the epitope peptide mixture is immobilized on a test line provided on the above-described substrate. The immunochromatography strip may include, for example, a sample pad (which may include a conjugate pad) for applying a test sample, a substrate (e.g., a nitrocellulose membrane) to which the epitope peptide mixture is bound, and an absorbent pad for absorbing the test sample solution, with at least a portion of the underside of the sample pad contacting one end of the substrate (e.g., the nitrocellulose membrane) and at least a portion of the underside of the absorbent pad contacting the other end of the substrate (e.g., the nitrocellulose membrane), with the test line to which the epitope peptide mixture is bound located between the sample pad and the absorbent pad on the substrate (e.g., the nitrocellulose membrane). The immunochromatography strip may further include a control line located on the substrate (e.g., the nitrocellulose membrane) between the test line and the absorbent pad (downstream of the test line). In one embodiment, the immunochromatographic strip is arranged so that the upstream lower surface of the sample pad does not contact one end of the substrate (e.g., nitrocellulose membrane), and the downstream lower surface of the absorbent pad does not contact one end of the substrate (e.g., nitrocellulose membrane). In another embodiment, the immunochromatographic strip is arranged so that the entire lower surface of the sample pad contacts one end of the substrate (e.g., nitrocellulose membrane), and the entire lower surface of the absorbent pad contacts one end of the substrate (e.g., nitrocellulose membrane). The immunochromatographic strip may be fixed on a solid support such as a plastic sheet. In the test strip of the present invention, "upstream" refers to the side to which the sample is applied in the sample migration axis, and "downstream" refers to the side to which the sample migrates in the sample migration axis.

[0051] The epitope peptide mixture can be immobilized on a device (typically, a substrate of a device) by a conventional method, such as by using streptavidin, avidin, and / or biotin, or by binding via a linker.

[0052] The present invention also provides a kit, such as a milk allergy test kit, comprising the epitope peptide mixture or a device comprising the epitope peptide mixture. In one embodiment, the kit of the present invention is a milk allergy test kit for immunochromatography analysis, and includes, for example, a test strip (such as an immunochromatography strip) on which the epitope peptide mixture is immobilized on a test line. In one embodiment, the device of the present invention may be, but is not limited to, a milk allergy test kit for ELISA or dot blot analysis.

[0053] The kit of the present invention may include other components. In one embodiment, the kit of the present invention may include instructions for use of the kit in a milk allergy test. In one embodiment, the kit of the present invention may further include an anti-immunoglobulin antibody, such as an anti-IgE antibody, an anti-IgG antibody (e.g., an anti-IgG4 antibody), an anti-IgA antibody, or an anti-IgD antibody, and such an anti-immunoglobulin antibody may be labeled. In one embodiment, the kit of the present invention may further include an anti-IgE antibody and / or an anti-IgG4 antibody, for example, a labeled anti-IgE antibody and / or an anti-IgG4 antibody. Such an anti-immunoglobulin antibody is a secondary antibody for detecting antibodies bound to the epitope peptide. For example, the anti-IgE antibody and / or the anti-IgG4 antibody is a secondary antibody for detecting IgE antibodies and / or IgG4 antibodies bound to the epitope peptide mixture. The label of the secondary antibody is not particularly limited. The secondary antibody may be labeled with any labeling substance, such as a fluorescent dye, an enzyme, a radioactive substance, or colloidal gold particles. In one embodiment, the kit of the present invention may include a colloidal gold-labeled secondary antibody. In one embodiment, the kit of the present invention may include a fluorescently labeled secondary antibody. In one embodiment, the kit of the present invention may include an enzyme-labeled secondary antibody and a detection substrate for the enzyme (e.g., a chromogenic substrate, a fluorogenic substrate, or a chemiluminescent substrate). Examples of enzymes and their detection substrates used to label antibodies include, but are not limited to, peroxidases such as horseradish peroxidase and tetramethylbenzidine, alkaline phosphatase and p-nitrophenyl phosphate, etc.

[0054] The device or kit according to the present invention can be advantageously used in the methods of the present invention described below, such as a method for measuring cow's milk allergen-specific antibodies (e.g., IgE antibodies and IgG4 antibodies) and a method for testing for cow's milk allergy. The device or kit according to the present invention can also be a companion diagnostic device or kit for evaluating the effectiveness of treatments such as oral immunotherapy for cow's milk allergy.

[0055] The present invention also provides a method for measuring cow's milk allergen-specific antibodies. More specifically, the present invention provides a method for measuring cow's milk allergen-specific antibodies in a subject-derived biological sample, comprising contacting the subject-derived biological sample with the epitope peptide mixture and measuring the amount of binding between the antibody in the biological sample and the epitope peptide mixture.

[0056] In a typical embodiment, the present invention provides at least six, preferably seven, more preferably eight, nine, ten, eleven, or twelve peptides selected from the group consisting of the following (a) to (l): (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2, (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3, (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4, (e) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5, (f) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 6, (g) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 7, (h) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 8, (i) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 9, (j) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10, (k) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11, and (l) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 12; and the following (m) and (n): (m) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13, and (n) A method for measuring a cow's milk allergen-specific antibody in a biological sample derived from a subject, comprising contacting the biological sample with an epitope peptide mixture containing 0 to 2 peptides (0 to 2 peptides, or 1 or 2 peptides) selected from the group consisting of peptides comprising the amino acid sequence shown in SEQ ID NO: 14, and measuring the amount of binding between the antibody in the biological sample and the epitope peptide mixture.

[0057] The epitope peptide mixture used in this method may contain all of the above peptides (a) to (n).

[0058] This method can detect and measure milk allergen-specific antibodies. The antibodies that can be detected in this method include any antibody (immunoglobulin), such as IgE antibody, IgG antibody (e.g., IgG1, IgG2, IgG3, IgG4 antibody), IgA antibody, IgD antibody, and IgM antibody. In one embodiment, this method can be a method for measuring milk allergen-specific IgE and / or IgG4 antibody. In one embodiment, this method can be a method for measuring milk allergen-specific IgE antibody. According to the method of the present invention, it is possible to examine the qualitative level of milk allergen-specific antibodies, which indicates a correlation with a patient's milk allergy status.

[0059] In one embodiment, the method can be carried out using the device or kit of the present invention. The epitope peptides contained in the epitope peptide mixture used in the method are as described above with respect to the device or kit of the present invention, regardless of whether the device or kit of the present invention is used.

[0060] Alternatively, this method can be performed using a test strip (e.g., an immunochromatography strip) capable of binding the epitope peptide mixture to a test line. For example, a biotinylated epitope peptide mixture can be added to a test sample containing a biological sample from a subject, which can then be applied to an immunochromatography strip with streptavidin alone immobilized on the test line and developed by immunochromatography. In this case, by further adding a labeled secondary antibody (an anti-immunoglobulin antibody such as an anti-IgE antibody or an 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, complexes between the biotinylated epitope peptides bound to streptavidin on the test line and milk allergen-specific antibodies from the biological sample can be easily detected. For example, the type of antibody that can be detected depends on the secondary antibody used. For example, if an anti-IgE antibody is used as the secondary antibody, milk allergen-specific IgE antibodies can be detected, and if an anti-IgG4 antibody is used, milk allergen-specific IgG4 antibodies 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 as appropriate based on known immunological detection techniques and are not particularly limited. The method of the present invention can also be suitably carried out using such a measurement method instead of the method using the above-mentioned device or kit.

[0061] In the present invention, the subject is a mammal, preferably a human, more preferably a human suffering from or suspected of having a cow's milk allergy, or having a genetic or environmental predisposition to a cow's milk allergy. In one embodiment, the subject is an infant, for example a human under the age of 5 or under the age of 3. In one embodiment, the subject is a human undergoing or planning to undergo oral immunotherapy for cow's milk allergy (see above; same below). In one embodiment, the subject is administered ImmunoCAP （Ｒ）The human is classified into classes 1 to 6, preferably classes 1 to 3, more preferably class 3, by testing.

[0062] In the present invention, the biological sample derived from a subject may be any biological sample, but is preferably a body fluid sample (blood sample, saliva sample, tear sample, nasal secretion sample, etc.), and more preferably a blood sample. The blood sample may be, but is not limited to, a whole blood sample, a serum sample, or a plasma sample. A test sample (preferably a liquid sample) containing a biological sample derived from a subject can be used in this method.

[0063] In this method, the amount of binding between the antibody in the biological sample and the epitope peptide mixture can be measured by a conventional method, but can be suitably measured 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 allowed to bind to the antibody that specifically binds to the epitope peptide mixture, and the signal derived from the label of the secondary antibody is detected, thereby detecting and measuring the antibody that specifically binds to the epitope peptide mixture. Examples of labels for secondary antibodies include, but are not limited to, fluorescent dyes, enzymes, radioactive substances, and colloidal gold particles. For example, when an enzyme-labeled secondary antibody is used, a detection substrate for the enzyme (such as a chromogenic, fluorescent, or chemiluminescent substrate) is added, and the label signal (such as a chromogenic, fluorescent, or chemiluminescent signal) generated by the reaction between the enzyme and the substrate is measured, thereby detecting and measuring the antibody that specifically binds to the epitope peptide mixture. In another embodiment, when a labeled epitope peptide mixture is used, the amount of binding between the antibody in the biological sample and the epitope peptide mixture 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 binding between the antibody in the biological sample and the epitope peptide mixture may be measured using a non-immunological detection method such as surface plasmon resonance (SPR).

[0064] This method can be used to measure cow's milk allergen-specific antibodies, such as cow's milk allergen-specific IgE antibodies, in a biological sample. The amount of IgE antibodies specific to the epitope peptide mixture (antibody titer) measured by the method for measuring cow's milk allergen-specific IgE antibodies in the biological sample can be determined by ImmunoCAP. （Ｒ） The amount of milk allergen-specific IgE antibodies measured by this method correlates well with the amount of total milk allergen-specific IgE antibodies obtained using a milk-specific IgE (milk) measurement kit. 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. The amount of milk allergen-specific IgE antibodies measured by this method correlates well with the subject's milk allergy status.

[0065] Therefore, the present invention also provides a milk allergy testing method that utilizes the above-mentioned method for measuring milk allergen-specific antibodies in a biological sample.

[0066] The present invention provides a method for testing (diagnosing) milk allergy in a subject, which comprises measuring milk allergen-specific IgE antibodies in a biological sample derived from the subject using the above-mentioned method for measuring milk allergen-specific IgE antibodies in a biological sample, and the measured amount of milk allergen-specific IgE antibodies indicates the subject's tolerance level to milk allergens.

[0067] In one embodiment, the method of the present invention comprises measuring milk allergen-specific IgE antibodies in a biological sample derived from a subject using the above-mentioned method for measuring milk allergen-specific IgE antibodies in a biological sample, and is a method for obtaining a test indicator of milk allergy in a subject, in which the measured amount of milk allergen-specific IgE antibodies indicates the subject's tolerance level to milk allergens.

[0068] In a preferred embodiment, when the amount of cow's milk allergen-specific IgE antibodies measured as the amount of IgE antibodies specific to the epitope peptide mixture is 5 ng / mL or more, it can be determined that the tolerance level to cow's milk allergens is low and that the subject is susceptible to developing cow's milk allergy. However, this threshold value is not limited to 5 ng / mL, as it may vary depending on conditions such as the subject population.

[0069] The present invention also relates to a method for evaluating (predicting) the effectiveness of oral immunotherapy for cow's milk allergy in a subject, using the above-mentioned method for measuring cow's milk allergen-specific IgE antibodies in a biological sample. The present invention provides a method for evaluating (predicting) the effectiveness of oral immunotherapy for cow's milk allergy in a subject, comprising measuring cow's milk allergen-specific IgE antibodies in a biological sample from a subject who is undergoing or planning to undergo oral immunotherapy for cow's milk allergy, using the above-mentioned method for measuring cow's milk allergen-specific IgE antibodies in a biological sample, wherein the measured amount of cow's milk allergen-specific IgE antibodies indicates the subject's milk intake tolerance and therapeutic responsiveness to oral immunotherapy. The "cow's milk intake tolerance" in the present invention may be the intake tolerance (amount that can be safely ingested) of cow's milk determined by an oral food challenge (OFC) test.

[0070] In one embodiment, the method of the present invention comprises measuring milk allergen-specific IgE antibodies in a biological sample from a subject who is undergoing or planning to undergo oral immunotherapy for milk allergy using the above-mentioned method for measuring milk allergen-specific IgE antibodies in a biological sample, and the measured amount of milk allergen-specific IgE antibodies indicates the subject's milk intake tolerance and therapeutic responsiveness to oral immunotherapy, thereby providing an evaluation index for the effectiveness of oral immunotherapy for milk allergy in a subject.

[0071] In a preferred embodiment, when the amount of cow's milk allergen-specific IgE antibodies measured as the amount of IgE antibodies specific to the epitope peptide mixture is high (e.g., 5 ng / mL or more), it can be determined that the acceptable intake of milk is low and that the subject will have a poor therapeutic response to oral immunotherapy. For a population of cow's milk allergy patients, a correlation diagram between the content of epitope peptide mixture-specific IgE antibodies measured by the method of the present invention and the acceptable intake of milk, as shown in Figure 9, can be created, and the acceptable intake of milk for the subject can be determined based on the correlation diagram from the amount of cow's milk allergen-specific IgE antibodies measured by the method of the present invention.

[0072] Furthermore, in any of the above methods of the present invention, in addition to the above method for measuring cow's milk allergen-specific IgE antibodies in a biological sample, 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 cow's milk allergen-specific IgE antibodies measured above to the amount of cow's milk allergen-specific non-IgE antibodies measured in this way (e.g., IgG4 antibody amount, total IgG antibody amount, IgA antibody amount, etc.) may be determined.

[0073] The ratio of the amount of cow's milk allergen-specific IgE antibody measured above to the amount of cow's 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 a cow's milk allergen, the tolerance level to a cow's milk allergen based thereon, or the effectiveness of oral immunotherapy. For example, the ratio of the amount of cow's milk allergen-specific IgE antibody (epitope ALL-IgE) measured above to the amount of cow's milk allergen-specific IgG4 antibody (epitope ALL-IgG4) based on binding to the 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 cow's milk allergen-specific IgE antibody (14 epitope ALL-IgE) measured above to the amount of cow's milk allergen-specific IgG4 antibody (14 epitope ALL-IgG4) based on binding to 14 epitope-ALL can be expressed as the 14 ALL-IgE / 14 ALL-IgG4 ratio.

[0074] The present invention also provides a method for treating milk allergy in a subject, comprising the steps of the above-mentioned method for measuring milk allergen-specific antibodies, the steps of the above-mentioned method for testing for milk allergy, or the steps of the above-mentioned method for evaluating (predicting) the effectiveness of oral immunotherapy for milk allergy. The method of the present invention may include a step of treating milk allergy after the steps of measuring milk allergen-specific antibodies, the steps of the above-mentioned milk allergy testing, or the steps of evaluating (predicting) the effectiveness of oral immunotherapy for milk allergy. Treatment of milk allergy can be performed by, but is not limited to, oral immunotherapy for milk allergy, feeding milk (hydrolyzed milk, amino acid milk, etc.) that has been removed from milk allergens, or administering antiallergic drugs such as antihistamines to a subject in need of such treatment.

[0075] In one embodiment, the present invention also provides oral immunotherapy, which comprises performing each step of the above-described method for evaluating (predicting) the effectiveness of oral immunotherapy for cow's milk allergy in a subject (patient) undergoing oral immunotherapy and determining whether to continue or discontinue oral immunotherapy based on the results of the efficacy evaluation. In the method of the present invention, after determining whether to continue or discontinue oral immunotherapy based on the results of the evaluation of the effectiveness of oral immunotherapy for cow's milk allergy, if oral immunotherapy can be continued, oral immunotherapy can be further performed in the subject. According to the method of the present invention, the effect (therapeutic response) of oral immunotherapy for cow's milk allergy in a subject undergoing oral immunotherapy can be assessed, and a decision to continue or discontinue oral immunotherapy can be made effectively based on the assessment results. Oral immunotherapy is a treatment that carries the risk of causing serious side effects, such as anaphylaxis, in patients. In light of this, the ability to accurately determine whether to discontinue oral immunotherapy using the method of the present invention is particularly useful for patients in avoiding the risk of side effects. The method of the present invention can be used to evaluate the efficacy of oral immunotherapy for cow's milk allergy, and by determining whether to continue or discontinue oral immunotherapy based on the results, it is possible to appropriately change the treatment options for a subject (patient) undergoing oral immunotherapy. Therefore, the method of the present invention can be used as a so-called companion diagnostic method for oral immunotherapy for cow's milk allergy.

[0076] The present invention also provides use of the epitope peptide mixture in the manufacture of the device or kit for testing for milk allergy.The present invention provides the epitope peptide mixture for use in the method for testing for milk allergy.The present invention provides the epitope peptide mixture for use in the method for evaluating (predicting) the effectiveness of oral immunotherapy (including dietary therapy, etc.) for milk allergy.

[0077] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0078] [Test Samples] Serum samples were obtained from the blood of cow's milk allergy patients (whether or not receiving oral immunotherapy) who participated in clinical trials with informed consent, stored at -80°C, and used as test samples in the following examples.

[0079] 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 use in identifying epitopes of milk allergens. For the milk allergen proteins αS1-casein, αS2-casein, and β-casein, peptides consisting of 15 amino acids were designed, each containing overlapping sequences of 10 amino acids, with shifts to cover the full-length amino acid sequences of each. Furthermore, peptides 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 175 designed peptides were synthesized and immobilized on a glass array plate for each peptide sequence to create a peptide array.

[0080] Serum samples from patients with milk allergy were appropriately diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and incubated with the peptide array. DyLight-labeled anti-human IgE or DyLight-labeled anti-human IgG antibodies (PEPperPRINT) were added as secondary antibodies and incubated. The fluorescent signal from DyLight was detected using an image analyzer, Odyssey CLx (LI-COR, Nebraska, USA). Twenty-nine patients tested positive for milk allergen-specific IgE antibodies, and the peptide sequences recognized by the detected milk allergen-specific IgE antibodies were identified. In contrast, 13 patients showed low IgE antibody titers but high IgG antibody titers, and the peptide sequences recognized by the detected milk allergen-specific IgG antibodies were further identified.

[0081] Twenty-one 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, including the peptide sequences identified in this manner, were prepared.

[0082] 2) Selection of cow's milk allergen epitopes by peptide-ELISA (enzyme-linked immunosorbent assay) analysis. Candidate epitope peptides were synthesized and biotinylated by adding a biotin-binding lysine to their C-termini. The biotinylated candidate epitope peptides were purified and their molecular weights confirmed by mass spectrometry. The biotinylated candidate epitope peptides were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions, which were then diluted with 1% BSA-containing PBS and added to each well of a 96-well plate precoated with 300 ng of streptavidin (Fujifilm) at a concentration of 30 ng / 100 μL / well.

[0083] After washing the streptavidin-coated plate with the biotinylated candidate epitope peptide bound thereto, a dilution of a serum sample from a patient with milk allergy was added and incubated in a refrigerator for one day. To the plate, a horseradish peroxidase (HRP)-conjugated anti-human IgE monoclonal antibody (mAb) (MCA E-09-HRP) and an HRP-conjugated anti-human IgG4 monoclonal antibody (mAb) (MCA DG-01-HRP) (both from Yamasa) were added. The plate was then washed four times with PBS-T (Phosphate Buffered Saline with Tween 20), and a TMB (3,3',5,5'-tetramethylbenzidine) chromogenic substrate (E-101; Bethyl Laboratories) was added. The color developed by the enzyme reaction was measured by measuring the absorbance at 450 nm using a microplate reader iMark (Bio-Rad, Hercules, US). Based on the binding of IgE and IgG4 antibodies in patient-derived serum samples to each candidate epitope peptide, IgE-binding epitope profiles and IgG4-binding epitope profiles were obtained. The IgE-binding epitope profiles differed between patients. Furthermore, within serum samples from the same patient, the IgG4-binding epitope profile differed from the IgE-binding epitope profile.

[0084] For each candidate epitope peptide, the number of IgE-positive patients to which IgE antibodies in the serum sample bound, and the number of IgG4-positive patients to which IgG4 antibodies in the serum sample bound were counted, and based on the total numbers, epitopes that were frequently recognized by IgE antibodies or IgG4 antibodies among patients were selected. Twelve epitopes were selected: eight epitopes derived from αS1-casein and αS2-casein, two epitopes derived from β-casein, and two epitopes derived from κ-casein. Two epitopes derived from β-lactoglobulin were also selected and added.

[0085] In this way, 14 epitopes derived from each of the five major cow's milk allergens, αS1-casein, αS2-casein, β-casein, κ-casein and β-lactoglobulin, were obtained (Table 1).

[0086]

[0087] The set of 14 selected epitopes (Table 1; Nos. 1 to 14) is hereinafter referred to as 14 epitopes-ALL. The set of 12 epitopes (Nos. 1 to 12) excluding the epitopes derived from β-lactoglobulin is hereinafter referred to as 12 epitopes-ALL. Furthermore, 14 epitopes-ALL and 12 epitopes-ALL are collectively referred to as epitope-ALL herein.

[0088] Example 2: Obtaining epitope filings by ELISA using epitope-ALL. ELISA was performed on serum samples from each patient using 12 epitope-ALL or 14 epitope-ALL. Streptavidin-coated plates (30 ng of epitope peptide per well for each peptide) were prepared in the same manner as in Example 1, to which the biotinylated epitope peptides (single peptides) shown in Table 1 were bound. Furthermore, the biotinylated epitope peptide solutions prepared in Example 1 were mixed to prepare a mixture of the 12 epitope peptides constituting 12 epitopes-ALL or a mixture of the 14 epitope peptides constituting 14 epitopes-ALL, and this was added to each well of a 96-well plate precoated with 300 ng of streptavidin so that the total epitope peptide concentration was 100 ng / well (8.3 ng / each epitope / well for 12 epitopes-ALL, 7.1 ng / each epitope / well for 14 epitopes-ALL), thereby producing a streptavidin-coated plate to which the biotinylated epitope peptide mixture was bound.

[0089] After washing the streptavidin-coated plate with the biotinylated epitope peptide bound thereto, a dilution of a serum sample from a patient with milk allergy was added and incubated in a refrigerator for one day. To the plate, a horseradish peroxidase (HRP)-conjugated anti-human IgE monoclonal antibody (mAb) (MCA E-09-HRP) and an HRP-conjugated anti-human IgG4 monoclonal antibody (mAb) (MCA DG-01-HRP) (both from Yamasa) were added. The plate was then washed four times with PBS-T (Phosphate Buffered Saline with Tween 20), and a 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic substrate (E-101; Bethyl Laboratories) was added. The color produced by the enzyme reaction is iMark ＴＭ The 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 and IgG4 antibodies in patient-derived serum samples to the epitope peptides.

[0090] Figure 1 shows the IgE-binding epitope profiles (using 12 epitopes-ALL) of serum samples from three cow's milk allergy patients (#101, #108, #ID-03). Figure 2 shows the IgE-binding epitope profiles (using 14 epitopes-ALL) of serum samples from three other cow's milk allergy patients (#2814, #2910, #3018, undergoing oral immunotherapy). As shown in Figures 1 and 2, the IgE-binding epitope profiles varied significantly among patients. Furthermore, as shown in Figures 1 and 2, the amount of epitope-binding IgE antibodies measured using plates bound with biotinylated epitope peptide mixtures (12 epitopes-ALL or 14 epitopes-ALL) corresponded to the sum of the amounts of individual epitope-binding IgE antibodies measured using plates bound with a single biotinylated epitope peptide.

[0091] Figure 3 shows the results of measuring epitope-binding IgE antibodies (Figure 3A) and epitope-binding IgG4 antibodies (Figure 3B) in diluted serum samples from patients with milk allergy using a plate bound to a biotinylated epitope peptide mixture (14 epitopes-ALL). Serum samples from nine patients with milk allergy (#3154, #3103, #3091, #2814, #2782, #2780, #2774, #2714, and #2701) were used. Serum samples diluted 5-fold, 15-fold, and 50-fold were used to measure IgE antibodies (Figure 3A). Serum samples diluted 15-fold, 50-fold, and 150-fold were used to measure IgG4 antibodies (Figure 3B).

[0092] The OD values ​​on the vertical axes of the graphs in Figures 1 to 3 can be converted into quantitative values ​​(pg / mL) based on the calibration curve.

[0093] Comparison of the levels of IgE antibodies (Figure 3A) and IgG4 antibodies (Figure 3B) against 14 epitope-ALL in serum samples from the same patient indicates whether IgE or IgG4 antibodies are dominant, which differs between patients. For example, IgE antibodies are dominant over IgG4 antibodies in patient #3154, whereas IgG4 antibodies are dominant over IgE antibodies in patient #3103. The ratio of IgE antibodies to IgG4 antibodies against epitope-ALL may be useful for assessing the degree of tolerance to cow's milk allergens and the responsiveness to oral immunotherapy in cow's milk allergy patients.

[0094] Example 3 Quantitative Values ​​of Cow's Milk Allergen-Specific IgE Antibodies and IgG4 Antibodies in Serum Samples and Their Evaluation Based on measurements using streptavidin-coated plates bound to biotinylated epitope peptide mixtures (12 epitopes-ALL or 14 epitopes-ALL), calibration curves were prepared to quantify the epitope-ALL-specific IgE antibody and IgG4 antibody contents in serum samples.

[0095] Recombinant human IgE antibody A and recombinant human IgG4 antibody B, each specific for a single epitope peptide, were used as standard samples. Recombinant human IgE antibody A is a recombinant human-mouse chimeric IgE antibody specific for a single epitope peptide derived from a specific cow's milk allergen shown in Table 1, and 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 for a single epitope peptide derived from the same specific cow's milk allergen as above, and was prepared by fusing the Fab of a mouse monoclonal antibody against the cow's 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.

[0096] Recombinant human IgE antibody A and recombinant human IgG4 antibody B were each diluted to a known concentration and added to a streptavidin-coated plate bound to 12 epitope-ALL or 14 epitope-ALL, and the IgE antibody and IgG4 antibody bound to epitope-ALL were measured by ELISA in the same manner as in Example 2. Based on the measured values, a standard curve was prepared with the antibody concentration on the horizontal axis and the absorbance at 450 nm on the vertical axis, and this was used as the calibration curve. An example of the standard curve is shown in Figure 4.

[0097] According to this calibration curve, the contents (antibody titers) of 14 epitope-ALL-specific IgE antibodies and IgG4 antibodies in serum samples from each cow's milk allergy patient were determined from the absorbance at 450 nm of epitope-ALL-binding IgE antibodies and IgG4 antibodies measured by ELISA using streptavidin-coated plates to which biotinylated epitope peptide mixtures (12 epitope-ALL or 14 epitope-ALL) were bound in Example 2.

[0098] In addition, the same serum samples were analyzed by ImmunoCAP. （Ｒ） ImmunoCAP using a specific IgE (milk) measurement kit (Thermo Fisher Scientific) （Ｒ）The test measured the total content of milk allergen-specific IgE antibodies (antibody titer) in serum samples according to the manufacturer's instructions.

[0099] FIG. 5 shows the 12-epitope-ALL-specific IgE antibody titers (vertical axis) measured using the 12-epitope-ALL binding plate for serum samples from each milk allergy patient (40 patients; pediatric patients undergoing oral immunotherapy), and the ImmunoCAP （Ｒ） The milk allergen-specific IgE antibody titer measured by the test (horizontal axis; ImmunoCAP （Ｒ） The correlation diagram in FIG. 5 is plotted based on the values ​​of the vertical axis ( Ａ The concentration (U / L) can be converted into a quantitative value by preparing a calibration curve and using the calibration curve.

[0100] FIG. 6 shows the 14 epitope-ALL specific IgE antibody content (vertical axis) measured using the 14 epitope-ALL binding plate for serum samples from each milk allergy patient (39 patients; pediatric patients undergoing oral immunotherapy) and the ImmunoCAP （Ｒ） The milk allergen-specific IgE antibody titer measured by the test (horizontal axis; ImmunoCAP （Ｒ） The correlation diagram plotted based on the IgE level is shown.

[0101] As shown in Figures 5 and 6, the 12-epitope-ALL or 14-epitope-ALL specific IgE antibody content and the current standard total IgE measurement method, ImmunoCAP, were compared. （Ｒ） The milk allergen-specific IgE antibody titers measured by the test showed a good correlation overall. That is, the 12-epitope-ALL-specific IgE antibody content and the 14-epitope-ALL-specific IgE antibody content were significantly correlated with the ImmunoCAP. （Ｒ） It was shown that the test can be used as an alternative indicator of milk allergen-specific IgE antibody titers.

[0102] Here, ImmunoCAP （Ｒ） ImmunoCAP using specific IgE (milk) measurement kit （Ｒ） In the test, the milk allergen-specific IgE antibody titer (ImmunoCAP) in the sample was （Ｒ）Samples are classified into classes 0 to 6 according to the IgE level, with class 0 being negative, class 1 being suspected positive, and class 2 or higher being positive.

[0103] As shown in Figures 5 and 6, the above-mentioned ImmunoCAP （Ｒ） All serum samples classified as Class 4 or higher (ImmunoCAP IgE values ​​≥ 17.5 kU / mL) by the test showed high epitope-ALL-specific IgE antibody contents (≥ 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 contents in serum samples from 38 cow's milk allergy patients and 34 non-allergic subjects (Figure 7).

[0104] On the other hand, in Figs. 5 and 6, the above ImmunoCAP （Ｒ） Serum samples that were tested classified as Class 3 (ImmunoCAP IgE values ​​= 3.5-17.49 kU / mL) and showed intermediate ImmunoCAP IgE values ​​showed relatively high inter-sample variability in epitope-ALL specific IgE antibody content. （Ｒ） It is known that in cow's milk allergy patients classified as Class 3 by testing, the frequency of onset of allergic symptoms with oral immunotherapy, as shown by the probability curve, ranges from approximately 30 to 70%. Therefore, it is thought that the fluctuations in the epitope-ALL-specific IgE antibody content shown in Figures 5 and 6 are related to the frequency of onset of allergic symptoms.

[0105] From the above, ImmunoCAP （Ｒ） It was shown that milk allergy patients classified as Class 3 or lower by the test can be divided into high-risk and low-risk groups, who are more likely to develop allergic symptoms, depending on the content of epitope-ALL specific IgE antibodies.

[0106] ImmunoCAP （Ｒ） For milk allergy patients who were classified as Class 3 by the test and were receiving oral immunotherapy, Figure 8 shows the milk intake tolerance and ImmunoCAP for each patient. （Ｒ）The data are plotted based on the IgE values, and in Figure 9, the milk intake tolerance of each patient and the epitope-ALL specific IgE antibody content are plotted. The milk intake tolerance of each patient was determined by oral food challenge (OFC) testing. As shown in Figure 8, the ImmunoCAP （Ｒ） Many patients with relatively high IgE levels also had a high tolerance for milk intake (see the dotted circle in Figure 8). （Ｒ） This indicates that it is difficult to use IgE levels as an indicator of acceptable milk intake. 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 acceptable milk intake.

[0107] Example 4 Immunochromatographic Analysis Using the 14 epitope-ALL selected in Example 1, immunochromatographic analysis was carried out on serum samples from patients with cow's milk allergy.

[0108] First, the 14 biotinylated epitope peptides that make up 14 Epitope-ALL (Table 1; Nos. 1 to 14) were mixed and then mixed with streptavidin solution in an equimolar ratio to prepare a 14 epitope-streptavidin complex mixture. 0.5 μL of the resulting 14 epitope-streptavidin complex mixture was applied to a line (test line) on a nitrocellulose membrane and allowed to dry, 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 adhered (bound) became the test line.

[0109] The nitrocellulose membrane with the 14 epitope-streptavidin complexes attached (bound) was blocked with a 0.1% BSA solution in Tris-buffered saline (TBS) for 1 hour at 37°C, washed with TBS, and dried. Next, a sample pad, the 14 epitope-streptavidin complex-bound nitrocellulose membrane, and an absorbent pad were assembled in this order so that the sample pad and absorbent pad were fixed on both ends of the nitrocellulose membrane, respectively, to prepare an immunochromatography strip. Specifically, this immunochromatography strip comprises a sample pad for applying a test sample solution, a nitrocellulose membrane bound with the 14 epitope-streptavidin complex prepared above, and an absorbent pad for absorbing the test sample solution, and is arranged so that the underside of the sample pad contacts one end of the nitrocellulose membrane and the absorbent pad contacts the other end, and a test line bound with the 14 epitope-streptavidin complex is present between the sample pad and the absorbent pad on the nitrocellulose membrane.

[0110] 1% Lipidure （Ｒ） Serum samples from milk-allergic patients (#2900, #2910) or recombinant human IgE antibody A were added to PBS containing BL206 and / or BL802 (Nichiyu). Then, gold (Au) colloid-labeled secondary antibodies, i.e., mouse anti-human IgE monoclonal antibody or mouse anti-human IgG4 monoclonal antibody, were added. After allowing to stand for 5 minutes, the mixture was applied to the immunochromatography strip prepared above.

[0111] To pass the applied sample solution up to the line of 14 epitope-streptavidin complex (test line) on the immunochromatography strip, a washing solution (1% Lipidure （Ｒ） After adding PBS containing gold twice and drying, band images of gold colloid accumulation were obtained.

[0112] The colloidal gold-conjugated 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 colloidal gold solution (Sigma-Aldrich, 0.2 M in potassium carbonate solution, pH 9.0) and allowing the mixture to react at room temperature for 30 minutes. Next, 635 μL of 10% BSA solution was added to the mixture, and the reaction was continued for an additional 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 used as the colloidal gold-conjugated antibody solution and stored at 4°C before use.

[0113] The results are shown in Figure 10. As shown in Figures 10A and 10B, the surfactant Lipidure （Ｒ） The recombinant human IgE antibody A supplemented with BL206 or BL802 showed a band corresponding to the colloidal gold-labeled IgE antibody at the 14-epitope-streptavidin complex line on the immunochromatography strip. Furthermore, as shown in Figures 10C and 10D, the serum samples at 10 μL / lane (Figure 10C) or 20 μL / lane (Figure 10D) also showed a band corresponding to the colloidal gold-labeled IgE antibody or IgG4 antibody at the 14-epitope-streptavidin complex line on the immunochromatography strip.

[0114] Thus, it was demonstrated that epitope-ALL-specific IgE antibodies and IgG4 antibodies can be detected in serum samples from milk-allergic patients by immunochromatography using epitope-ALL.

[0115] According to the present invention, it is possible to easily test the amount of milk allergen-specific antibodies in milk allergy patients, and also to more accurately evaluate the milk allergen tolerance level of milk allergy patients and the risk of developing milk allergy through oral immunotherapy. The device and kit of the present invention can be used as a companion diagnostic for evaluating the effectiveness of treatments such as oral immunotherapy for milk allergy.

[0116] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A milk allergy testing device comprising an epitope peptide mixture comprising: at least six peptides selected from the group consisting of (a) to (l) below: (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2, (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3, (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4, (e) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5, (f) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 6, (g) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 7, (h) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 8, (i) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 9, (j) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10, (k) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11, and (l) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and zero to two peptides selected from the group consisting of (m) and (n) below: (m) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13, and (n) a peptide comprising the amino acid sequence set forth in SEQ ID NO:

14.

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

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

4. The device of claim 1, wherein the peptide comprises a biotin-binding lysine added to the N-terminus or C-terminus of the amino acid sequence.

5. The device of claim 4, wherein the peptide is conjugated to streptavidin or avidin.

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

7. A milk allergy testing kit comprising the device of claim 1.

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

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

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

11. A biological sample derived from a subject is contacted with an epitope peptide mixture comprising at least six peptides selected from the group consisting of (a) to (l) below: (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2, (c) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3, (d) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4, (e) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5, (f) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 6, (g) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 7, (h) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 8, (i) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 9, (j) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 10, (k) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11, and (l) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and 0 to 2 peptides selected from the group consisting of (m) and (n) below: (m) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13, and (n) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 14, A method for measuring cow's milk allergen-specific antibodies in a biological sample derived from a subject, comprising measuring the amount of binding between the antibodies in the biological sample and the epitope peptide mixture.

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

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

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

15. The method according to claim 11, which is carried out using the device according to claim 1 or the kit according to claim 7.

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

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

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 undergoing or planning to undergo oral immunotherapy for milk allergy using the method described in claim 13, wherein the measured amount of milk allergen-specific IgE antibodies indicates the subject's milk intake tolerance and therapeutic responsiveness to oral immunotherapy.

19. The method of claim 17, further comprising measuring the amount of binding between IgG4 antibodies 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 antibodies to the measured amount of milk allergen-specific IgG4 antibodies.

20. The method of claim 18, further comprising measuring the amount of binding between IgG4 antibodies 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 antibodies to the measured amount of milk allergen-specific IgG4 antibodies.

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