Antibodies, kits and testing methods for soybean allergens

Abstract

To provide improved means for detecting soy allergens remaining in food or a food processing apparatus.SOLUTION: Provided is a test kit for quantitatively or qualitatively detecting soy allergens remaining in food or a food processing apparatus by immunoassay, the test kit comprising: an anti-trypsin inhibitor (TI) antibody or an antigen-binding fragment thereof (anti-TI antibody, and the like) having heavy-chain CDR1 to 3 and light-chain CDR1 to 3 consisting of a predetermined amino acid sequence (SEQ ID NO:1 to 6, SEQ ID NO:7 to 12); or an anti-TI antibody or an antigen-binding fragment thereof (anti-TI competitive antibody, and the like) that competes with the anti-TI antibody, and the like for the same or overlap epitope in binding to the trypsin inhibitor protein. Provided is a test method for soy allergens in food, comprising: (1) a step of extracting soy protein remaining in food or a food processing apparatus; and (2) a step of quantitatively or qualitatively detecting trypsin inhibitor protein in the obtained extract by immunoassay using the specific anti-TI antibody, and the like or anti-TI competitive antibody, and the like.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a technique for detecting soy allergens, particularly trypsin inhibitor proteins, contained in food or remaining in food processing equipment. [Background technology]

[0002] Food allergies are a condition in which the body recognizes certain proteins (food allergens) contained in ingested food as foreign substances, and through hypersensitive immunological mechanisms, various symptoms such as skin itching, hives, and coughing are caused. In severe cases, it can lead to loss of consciousness, low blood pressure, and shock symptoms, which can be life-threatening. It is estimated that 1-2% of the total population (about 10% if limited to infants) suffer from food allergies. The Food Sanitation Act requires or recommends that if a food contains ingredients that contain food allergens, that information be displayed on the food to prevent food allergy patients from ingesting foods that contain the food allergen that is the cause of the allergy, especially processed foods that are difficult to identify by appearance. Currently, eight items, "eggs, milk, wheat, shrimp, crab, peanuts, walnuts" (those with a high incidence of onset) and "buckwheat" (which often causes severe symptoms and can be life-threatening), are designated as "specific ingredients" for which labeling is mandatory, while 21 items, "abalone, squid, salmon roe, orange, cashew nuts, kiwi fruit, beef, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, gelatin, almonds" (those that have been reported to cause allergies with a certain frequency in the past), are designated as "substances equivalent to specific ingredients" for which labeling is recommended. The Consumer Affairs Agency (formerly the Ministry of Health, Labor and Welfare) conducts a survey every three years to grasp the nationwide situation of food allergies, and reviews the list of specific ingredients based on the results of the survey.

[0003] In order to operate the food allergen system described above, food manufacturers and public testing organizations use kits to check whether foods containing food allergens are included as ingredients in processed products or whether they remain in food processing equipment. There is also a demand for further improvements in the reliability of kits that can detect soy allergens, which are designated as "equivalent to specific raw materials."

[0004] Generally, the techniques for detecting specific raw materials contained in processed products or remaining in food processing equipment include the ELISA method, which uses an antigen-antibody reaction as a screening test, and the PCR method, which uses a DNA amplification reaction as a confirmatory test. Of these, the ELISA method uses an antibody that recognizes a protein (allergen) specific to soybeans to check whether soybeans are included as an ingredient in processed products.

[0005] As means for detecting soybean allergens in food by antigen-antibody reactions, for example, those described in the following prior art documents are known.

[0006] Patent Document 1 describes a method and a kit for detecting soybean proteins by immunochromatography using several specific monoclonal antibodies targeting soybean 7S globulin (either unmodified or denatured) as immobilizing and labeling antibodies. The Examples in Patent Document 1 show that a strip for immunochromatography was prepared using a specific anti-soybean 7S globulin monoclonal antibody, and 10 ppm or 2 ppm of soybean protein contained in a model meat product was detected and determined as positive (++ or +).

[0007] Non-Patent Document 1 describes a method for detecting soybean protein by ELISA and immunochromatography using monoclonal antibodies targeting trypsin inhibitor (Kunitz trypsin inhibitor: KTI) as immobilizing and labeling antibodies. Non-Patent Document 1 shows that a strip for immunochromatography using a specific anti-KTI monoclonal antibody was prepared and KTI at a concentration of 10 ng / mL or more in a PBS aqueous solution could be detected (however, the 10 ng / mL line was extremely faint (±), and the 25 ng / mL and 50 ng / mL lines were also faint (+)) (Figure 3). On the other hand, Non-Patent Document 1 also shows that the reactivity with the specific anti-KTI monoclonal antibody is greatly reduced (to about 20-25%) when heated KTI is used as the target compared to when non-heated KTI is used as the target (Figure 2). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2016-211967 A (Patent No. 6510307) [Non-patent literature]

[0009] [Non-Patent Document 1] Xu et al., Sandwith ELISA and immunochromatographic strip of Kunitztrypsin inhibitor using sensitive monoclonal antibodies, FOOD AND AGRICULTURAL IMMUNOLOGY, 2016, VOL.27, NO.6, 772-782, http: / / dx.doi.org / 10.1080 / 09540105.2016.1160367 Summary of the Invention [Problem to be solved by the invention]

[0010] Detection kits or detection methods using monoclonal antibodies targeting soybean proteins have the problem that they are difficult to detect (poor sensitivity) when the concentration of soybean proteins in food samples is low. In addition, there is also the problem that the sensitivity to soybean proteins in heated foods is lower than the sensitivity to soybean proteins in non-heated foods due to the effects of denaturation of soybean proteins caused by heating. Furthermore, in kits or measurement methods intended to detect soybean allergens, there remains a possibility that a false positive will be determined due to cross-reaction with other food allergens (e.g., adzuki bean allergens, peanut allergens) (poor specificity), and it is preferable that cross-reactivity with food allergens other than soybean allergens is sufficiently suppressed. In conventional detection kits or detection methods, it has been difficult to achieve a good balance between sensitivity and specificity (cross-reactivity) at a high level.

[0011] The objective of the present invention is to provide an improved means for detecting soy allergens contained in foods or remaining in food processing equipment based on immunological assays, in particular a means that has excellent sensitivity to non-heated and heated soy proteins and preferably also has excellent specificity for soy proteins (having low cross-reactivity to other food allergens). [Means for solving the problem]

[0012] The present inventors have found that by using an anti-trypsin inhibitor antibody (monoclonal antibody) having a specific CDR in a soybean allergen detection kit or detection method based on an immunological assay, soybean allergen proteins in a food sample can be detected even at low concentrations (high sensitivity), that the decrease in sensitivity is suppressed even when the food sample is heated compared to non-heated food samples, and that cross-reactions with food allergens other than soybean allergens are suppressed (high specificity), and have completed the present invention. In addition, compared to the case of targeting soybean 7S globulin (Patent Document 1), when trying to target trypsin inhibitor as a soybean allergen, there is an advantage in that there are fewer proteins highly homologous to closely related legumes (peanuts, walnuts, etc.), but there is a disadvantage in that the amount of target protein contained in food is small, so it is necessary to produce a high-titer antibody. The anti-trypsin inhibitor antibody having a specific CDR according to the present invention has higher titer than conventional anti-trypsin inhibitor antibodies, and is capable of solving the problems described above.

[0013] That is, the present invention includes at least the following: [1] A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:2, and A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:3, and A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:5, and Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:6 or an anti-trypsin inhibitor antibody or an antigen-binding fragment thereof (hereinafter referred to as "first antibody, etc."), A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7; A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:8, and A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:9, and A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10; A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:12 An anti-trypsin inhibitor antibody or an antigen-binding fragment thereof (hereinafter referred to as "second antibody, etc."), [2] An anti-trypsin inhibitor antibody or antigen-binding fragment thereof (hereinafter referred to as the "first competing antibody, etc.") that competes with the first antibody, etc. for the same or overlapping epitope in binding to a trypsin inhibitor protein, or an anti-trypsin inhibitor antibody or antigen-binding fragment thereof (hereinafter referred to as the "second competing antibody, etc.") that competes with the second antibody, etc. for the same or overlapping epitope. [3] A test kit for quantitatively or qualitatively detecting soy allergens remaining in food or food processing equipment by an immunological measurement method, comprising: A testing kit comprising at least one selected from the group consisting of the first antibody etc. and the second antibody etc. described in Item 1, and the first competing antibody etc. and the second competing antibody etc. described in Item 2. [4] Item 4. The test kit according to Item 3, wherein the immunological measurement method is ELISA or immunochromatography, and the kit comprises the first antibody or the first competing antibody or the second antibody or the second competing antibody for enzyme labeling in ELISA or color labeling in immunochromatography. [5] Item 4. The testing kit according to Item 3, wherein the immunological measurement method is ELISA or immunochromatography, and the kit comprises the first antibody or the first competing antibody or the second antibody or the second competing antibody for solid-phase immobilization in ELISA or for immobilization in immunochromatography. [6] the immunoassay is ELISA or immunochromatography; (1) The first antibody or the first competing antibody, etc., is used for solid-phase immobilization in ELISA or immobilization in immunochromatography, and the second antibody or the second competing antibody, etc., is used for enzyme labeling in ELISA or color labeling in immunochromatography, or (2) The second antibody, etc. or the second competing antibody, etc., for use in solid-phase immobilization in ELISA or immobilization in immunochromatography, and an anti-trypsin inhibitor antibody or an antigen-binding fragment thereof other than the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc., for use in enzyme labeling in ELISA or color labeling in immunochromatography, Item 3. The testing kit according to item 3. [7] Item 4. The testing kit according to Item 3, further comprising an extraction reagent. [8] Item 8. The test kit according to Item 7, wherein the extraction reagent is an extraction reagent using a shaking method or a high-speed shear / agitation method without heating. [9] (1) extracting soy protein remaining in food or in food processing equipment; and (2) quantitatively or qualitatively detecting a trypsin inhibitor protein in the obtained extract by an immunological assay using at least one antibody selected from the group consisting of the first antibody or the like and the second antibody or the like described in Item 1 and the first competing antibody or the like and the second competing antibody or the like described in Item 2; A method for testing for soy allergens in food, comprising:

[10] Item 9. The testing method according to Item 8, wherein the immunological measurement method is ELISA or immunochromatography, and the first antibody or the first competing antibody or the second antibody or the second competing antibody or the like is used for enzyme labeling in ELISA or color labeling in immunochromatography.

[11] Item 9. The testing method according to Item 8, wherein the immunological measurement method is ELISA or immunochromatography, and the first antibody or the first competing antibody or the second antibody or the second competing antibody or the like is used for solid-phase immobilization in ELISA or for immobilization in immunochromatography.

[12] the immunoassay is ELISA or immunochromatography; (1) Using the first antibody or the first competing antibody for immobilization in ELISA or immobilization in immunochromatography, and the second antibody or the second competing antibody for enzyme labeling in ELISA or color labeling in immunochromatography, or (2) The testing method according to Item 9, wherein the second antibody, etc. or the second competing antibody, etc. are used for solid-phase immobilization in ELISA or immobilization in immunochromatography, and an anti-trypsin inhibitor antibody or an antigen-binding fragment thereof other than the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. are used for enzyme labeling in ELISA or color labeling in immunochromatography.

[13] Item 10. The method according to item 9, wherein the extraction in step (1) is carried out using an extraction reagent.

[14] Item 14. The testing method according to Item 13, wherein the extraction using the extraction reagent is performed by a shaking method or a high-speed shear / agitation method without heating.

[0014] In addition, a person skilled in the art would be able to convert (reinterpret) the matters relating to the invention described in each of the above paragraphs into matters relating to inventions in other categories based on the technical idea of ​​the present invention, the matters described in this specification, and common technical knowledge.

[0015] For example, the invention described in [3] above can be converted into "use of at least one selected from the group consisting of the first antibody, etc. and the second antibody, etc. described in Item 1 and the first competing antibody, etc. and the second competing antibody, etc. described in Item 2, in the manufacture of a test kit for quantitatively or qualitatively detecting soy allergens remaining in food or in food processing equipment by immunological assay."

[0016] Furthermore, for example, the invention described in [9] above can be converted into "use of a method for testing soy allergens in food, comprising at least one selected from the group consisting of the first antibody, etc. and the second antibody, etc. described in Item 1 and the first competing antibody, etc. and the second competing antibody, etc. described in Item 2, the method comprising: (1) extracting soy protein remaining in food or in food processing equipment; and (2) quantitatively or qualitatively detecting trypsin inhibitor protein in the obtained extract by an immunological assay using at least one selected from the group consisting of the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc." Effect of the Invention

[0017] The test kit and test method of the present invention make it possible to detect soybean allergens with higher reliability (sensitivity and specificity) than conventional methods, even when the soybean allergen is present in a food at a low concentration, even when the food is heated, and in a preferred embodiment, even when other food allergens are present. The antibody of the present invention is suitable for carrying out such a test kit and test method. [Brief description of the drawings]

[0018] [Figure 1]Figure 1 shows the results of the analysis of the minimum detection sensitivity of immunochromatographic test strips described in Test Example 2. [A] Conventional test strip (quoted from Figure 3 in Non-Patent Document 1, modified to include concentrations, arrows, etc.). [B] Test strip of the product of the present invention (Example 4: membrane antibody = 27G4, gold colloid-labeled antibody = 28F2). [Diagram 2] FIG. 2 shows the comparative results of reactivity to native soybean protein (left) and heat-denatured soybean protein (right) in ELISA described in Test Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In this specification, anti-trypsin inhibitor antibodies may be referred to as "anti-TI antibodies."

[0020] In this specification, the anti-TI antibody in the first embodiment (clone "28F2" in the Examples) is referred to as the "first antibody", its antigen-binding fragment is referred to as the "first fragment", and they are collectively referred to as the "first antibody, etc."; the anti-TI antibody in the second embodiment (clone "27G4" in the Examples) is referred to as the "second antibody", its antigen-binding fragment is referred to as the "second fragment", and they are collectively referred to as the "second antibody, etc."

[0021] In this specification, an anti-TI antibody that competes with a first antibody, etc. for the same or overlapping epitope is referred to as a "first competing antibody," and its antigen-binding fragment is referred to as a "first competing fragment," and they are collectively referred to as a "first competing antibody, etc."; an anti-TI antibody that competes with a second antibody, etc. for the same or overlapping epitope is referred to as a "second competing antibody," and its antigen-binding fragment is referred to as a "second competing fragment," and they are collectively referred to as a "second competing antibody, etc."

[0022] In this specification, the "first antibody," "second antibody," "first competing antibody," and "second competing antibody" are collectively referred to as "anti-TI antibodies of the present invention," the "first fragment," "second fragment," "first competing fragment," and "second competing fragment" are collectively referred to as "anti-TI binding fragments of the present invention," and the "first antibody, etc.," "second antibody, etc.," "first competing antibody, etc.," and "second competing antibody, etc." are collectively referred to as "anti-TI antibodies of the present invention, etc.."

[0023] In this specification, anti-TI antibodies other than the anti-TI antibodies of the present invention (e.g., clones "21A12" or "210E11" in the Examples) are referred to as "other anti-TI antibodies", and their antigen-binding fragments are referred to as "other anti-TI binding fragments", and they may be collectively referred to as "other anti-TI antibodies, etc."

[0024] In this specification, when the term "antibody" is used in an appropriate context, the term can be appropriately replaced with "antigen-binding fragment" or "antibody, etc." as a general term for "antibody" and "antigen-binding fragment," i.e., an embodiment using an "antibody" can be replaced with an embodiment using an "antigen-binding fragment" or "antibody, etc."

[0025] -antibody- In a first embodiment of the present invention, an anti-trypsin inhibitor (TI) antibody or an antigen-binding fragment thereof (such as the first antibody) has a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3, as well as a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6.

[0026] In a second embodiment of the present invention, an anti-trypsin inhibitor (TI) antibody or antigen-binding fragment thereof (e.g., second antibody) has a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 9, as well as a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 12.

[0027] The anti-TI antibodies of the present invention have heavy chain CDR1-3 and light chain CDR1-3 consisting of the amino acid sequences represented by specific SEQ ID NOs described herein as three complementarity determining regions (CDRs) in the heavy chain and light chain, respectively. The anti-TI antibodies of the present invention can be produced in the same manner as general antibodies, etc., by grafting appropriate framework regions (FRs), i.e., heavy chain FR1-4 located before and after the heavy chain CDR1-3, and light chain FR1-4 located before and after the light chain CDR1-3, in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus of the heavy chain and light chain, respectively, to construct variable regions, and further combining them with the first to third heavy chain constant regions and the light chain constant region to construct the full-length heavy chain and light chain.

[0028] The anti-TI antibody of the present invention is a specific antibody for IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG 2a , IgG 2b Although it may be any class (subclass) of IgM (IgG1, IgG2, IgG3, IgG4), it is typically IgG (IgG1, IgG 2a , IgG 2b Each class (subclass) of antibodies can be constructed with corresponding heavy and light chains, for example, IgG1, IgG2, and IgG4. 2a , IgG 2b The IgG, IgG3, and IgG4 subclasses can be constructed from γ1, γ2, γ3, γ4, or γ5 heavy chains and λ or κ light chains, respectively.

[0029] The anti-TI binding fragment of the present invention can be prepared by using heavy chain CDR1-3 and light chain CDR1-3 consisting of amino acid sequences represented by specific SEQ ID NOs described herein, and the variable and constant regions of the heavy and light chains containing them. Examples of "antigen-binding fragments" include Fab, Fab', F(ab')2, Fv (a polypeptide in which a single heavy chain variable region and a single light chain variable region are bound), scFv (single-chain Fv), and dsFv (double-chain Fv).

[0030] In one embodiment of the present invention, the first antibody etc. comprises a heavy chain variable region (CDR and FR) consisting of an amino acid sequence having at least 80%, for example, 85% or more, 90% or more, or 95% or more sequence identity (homology) to the amino acid sequence shown in SEQ ID NO:13.

[0031] In one embodiment of the present invention, the first antibody etc. comprises a light chain variable region (CDR and FR) consisting of an amino acid sequence having at least 80%, for example, 85% or more, 90% or more, or 95% or more sequence identity (homology) to the amino acid sequence represented by SEQ ID NO:15.

[0032] In one embodiment of the present invention, the second antibody etc. comprises a heavy chain variable region (CDR and FR) consisting of an amino acid sequence having at least 80%, for example, 85% or more, 90% or more, or 95% or more sequence identity (homology) to the amino acid sequence represented by SEQ ID NO:17.

[0033] In one embodiment of the present invention, the second antibody etc. comprises a light chain variable region (CDR and FR) consisting of an amino acid sequence having at least 80%, for example, 85% or more, 90% or more, or 95% or more sequence identity (homology) to the amino acid sequence represented by SEQ ID NO:19.

[0034] In the heavy chain variable region and / or light chain variable region of the first antibody etc. or second antibody etc. having sequence identity within the above-mentioned range, the difference in amino acid residues (substitution, deletion, insertion, addition, etc.) occurs in the FR other than the CDR, and it is preferable that the amino acid sequence of the CDR is maintained. On the other hand, the first competing antibody etc. or second competing antibody etc. provided in one aspect of the present invention may comprise a heavy chain variable region and / or a light chain variable region consisting of an amino acid sequence having sequence identity (homology) within the above-mentioned range to a variable region (CDR and FR) contained in a heavy chain or light chain consisting of an amino acid sequence represented by a specific SEQ ID NO, and the difference in amino acid residues (substitution, deletion, insertion, addition, etc.) may occur in one or both of the CDR and the FR.

[0035] The CDRs and FRs in the antibodies and the like of the present invention are sites identified by general methods in the technical field, for example, numbering methods such as Kabat, Chothia, IMGT, Aho, etc. In one embodiment of the present invention, the CDRs and FRs are sites identified by the CHOTHIA ANTIBODY NUMBERING method.

[0036] The sequence identity (homology) values ​​in this specification are values ​​determined by well-known and commonly used methods for so-called alignment in the present technical field, and can be calculated using publicly available tools such as BLASTN, BLASTp (see the US National Center for Biotechnology Information (NCBI) website), ClustalW2 (see the European Bioinformatics Institute website), ALIGN, etc., with default parameters, or with parameters customized by selecting an appropriate algorithm as necessary. In one embodiment of the present invention, the sequence identity (homology) values ​​are values ​​calculated with default parameters using BLASTN.

[0037] The anti-TI antibodies, etc. of the present invention can be produced by utilizing a polynucleotide encoding the amino acid sequence of the desired anti-TI antibody, etc., according to a general method. A typical production method is outlined as follows. A DNA encoding the desired polynucleotide is synthesized and cloned into an expression vector. The expression vector is transfected into an appropriate host cell, and the host cell is cultured under appropriate conditions to cause the desired anti-TI antibody, etc. to be secreted into the culture supernatant. The culture supernatant is collected, and the anti-TI antibody, etc. contained therein is isolated and purified.

[0038] In a further aspect of the first embodiment of the present invention, an anti-trypsin inhibitor (TI) antibody or antigen-binding fragment thereof (first competing antibody, etc.) is an anti-TI antibody or antigen-binding fragment thereof that competes for the same or overlapping epitope as the first antibody, etc. in binding to a trypsin inhibitor protein. The amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of the first competing antibody, etc., and further the amino acid sequences of the heavy chain FR1-4 and light chain FR1-4 as necessary, may be amino acid sequences obtained by modifying the amino acid sequence of the first antibody, etc. of the present invention, or may be amino acid sequences obtained without relying on the amino acid sequence of the first antibody, etc. of the present invention.

[0039] In a further aspect of the second embodiment of the present invention, an anti-trypsin inhibitor (TI) antibody or antigen-binding fragment thereof (second competing antibody, etc.) is an anti-TI antibody or antigen-binding fragment thereof that competes for the same or overlapping epitope as the second antibody, etc. in binding to a trypsin inhibitor protein. The amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of the second competing antibody, etc., and further the amino acid sequences of the heavy chain FR1-4 and light chain FR1-4 as necessary, may be amino acid sequences obtained by modifying the amino acid sequence of the second antibody, etc. of the present invention, or may be amino acid sequences obtained without relying on the amino acid sequence of the second antibody, etc. of the present invention.

[0040] The first competing antibody, etc. or the second competing antibody, etc., which competes with the first antibody, etc. or the second antibody, etc. for the same or overlapping epitope, may be any antibody that has the ability to inhibit the binding (interaction) between the first antibody, etc. or the second antibody, etc. and the trypsin inhibitor protein to any detectable extent, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Such a first competing antibody, etc. or second competing antibody, etc., can be selected from candidate clones without excessive trial and error by a general method known to those skilled in the art as so-called "epitope binning", in which the first antibody, etc. or the second antibody, etc. is used as a reference antibody, etc. The binding (interaction) between two specific molecules is generally determined by the binding affinity (K D ), i.e., the ratio of the dissociation rate to the association rate (Koff / Kon). D can be measured using, for example, a measuring device "Biacore" that utilizes surface plasmon resonance. The first competitive antibody etc. or the second competitive antibody etc. preferably has properties that can be evaluated as being equivalent to or superior to the first antibody etc. or the second antibody etc. in the application of detecting soybean allergens in food.

[0041] -Testing kit- The "test kit" of the present invention is a test kit for quantitatively or qualitatively detecting soybean allergens remaining in food or food processing equipment by immunological measurement, and comprises at least one anti-trypsin inhibitor (TI) antibody, etc. of the present invention, i.e., at least one selected from the group consisting of the first antibody, etc., the second antibody, etc., the first competing antibody, etc. and the second competing antibody, etc. of the present invention.

[0042] The "immunological assay" is not particularly limited as long as it is a method that makes it possible to quantitatively or qualitatively detect trypsin inhibitor protein by utilizing the reaction between the trypsin inhibitor protein and an anti-TI antibody, etc., and various known methods can be used. In addition, test kits corresponding to various immunological assay methods are known and commercially available, and the test kit of the present invention can be produced in accordance with known test kits, except that the specific antibody of the present invention is used.

[0043] In one embodiment of the present invention, the immunological measurement method is ELISA (Enzyme-Linked Immunosorbent Assay). ELISA is capable of quantitatively detecting various proteins with high accuracy, and is a suitable measurement method for detecting food allergens from processed foods that have been heated or otherwise processed, and complies with the Consumer Affairs Agency guidelines.

[0044] Anti-TI antibodies and the like for ELISA include, for example, anti-TI antibodies and the like for immobilization and anti-TI antibodies and the like for enzyme labeling. Anti-TI antibodies and the like for immobilization are intended to be immobilized on the surface of a member for performing ELISA, for example, on the bottom of a plate (well). Anti-TI antibodies and the like for enzyme labeling are intended to bind to the TI protein captured by the immobilized antibody and then react with a substrate to develop a color with an intensity according to the amount of trypsin inhibitor protein. Anti-TI antibodies and the like for enzyme labeling may be anti-TI antibodies and the like directly labeled with an enzyme, i.e., anti-TI antibodies and the like to which an enzyme (e.g., horseradish peroxidase (HRP)) has been bound in advance to the antibody and the like by covalent bond (e.g., via a linker), but may also be anti-TI antibodies and the like that are indirectly labeled with an enzyme, such as anti-TI antibodies and the like bound to biotin, which can complete enzyme-labeled anti-TI antibodies and the like by further reacting with an enzyme bound to streptavidin, as exemplified in the procedure described later in relation to the "test method".

[0045] In one embodiment of the present invention, the immunological measurement method is immunochromatography, which is a measurement method capable of qualitatively detecting various proteins in a relatively short period of time with a simple operation.

[0046] Anti-TI antibodies and the like for immunochromatography include, for example, anti-TI antibodies and the like for immobilization and anti-TI antibodies and the like for color-producing labels (e.g., gold colloid labels, latex particle labels, platinum particle labels). Anti-TI antibodies and the like for color-producing labels are generally anti-TI antibodies and the like that are contained in a predetermined position (sample dropping portion) of a test strip and are bound to a color-producing label in advance, and are antibodies that react with a substrate to produce color to indicate the presence of a target protein after being captured by an immobilized antibody or the like in a state of forming a complex with a trypsin inhibitor protein. Anti-TI antibodies and the like for immobilization are generally anti-TI antibodies and the like that are contained in a predetermined position (test line) of a test strip, and are antibodies that capture a complex between a trypsin inhibitor protein that has moved by capillary action and an anti-TI antibody and the like for color-producing labels.

[0047] Such anti-TI antibodies for immunological assays, such as for ELISA or immunochromatography, may be a combination of the anti-TI antibodies of the present invention, or a combination of the anti-TI antibodies of the present invention and other anti-TI antibodies. Anti-TI antibodies may be used in combination with an antibody or an antigen-binding fragment thereof that can recognize and bind to a trypsin inhibitor protein with the specificity required for the application, i.e., the purpose of the test kit. It is preferable that the anti-TI antibodies do not bind to various proteins contained in food and food allergens other than soybean (at least, the binding is limited to a degree that does not affect the test results according to the present invention), but it is acceptable for the antibodies to bind to a certain degree (cross-react) with proteins other than trypsin inhibitor proteins or to non-specifically adsorb to proteins or other substances that are unavoidable due to the nature of the antibody, depending on the application and level.

[0048] Here, trypsin inhibitor proteins in foods such as processed foods may contain variously denatured trypsin inhibitor proteins, for example, aggregates of native trypsin inhibitor proteins and variously denatured trypsin inhibitor proteins, depending on the food production and storage processes (e.g., the presence or absence and processing conditions of kneading, molding, pressurization, heating, drying, enzyme treatment, freezing, thawing, etc.) and on the extraction process from the food (specimen) (e.g., the presence or absence and processing conditions of extraction treatment using an extraction reagent containing an added solubilizing agent). Therefore, it is preferable that the anti-TI antibody, etc. is an embodiment capable of binding to various trypsin inhibitor proteins so as to have a certain detection sensitivity for the aggregates of various trypsin inhibitor proteins in foods (preventing the detection sensitivity from decreasing due to foods).

[0049] From this perspective, in one embodiment of the present invention, the test kit can contain, as the anti-TI antibody, etc., an antibody capable of specifically binding to both native trypsin inhibitor protein and denatured trypsin inhibitor protein (e.g., heat-denatured trypsin inhibitor protein). In such applications, the anti-TI antibody, etc. of the present invention has excellent detection sensitivity for both native trypsin inhibitor protein and trypsin inhibitor proteins variously denatured by heating, etc. It is preferable that other anti-TI antibodies, etc. used in combination with the anti-TI antibody, etc. of the present invention are also capable of specifically binding to both native trypsin inhibitor protein and denatured trypsin inhibitor protein.

[0050] In a first aspect of the present invention, the test kit comprises a first antibody, etc. or a first competing antibody, etc., or a second antibody, etc. or a second competing antibody, etc., as an anti-TI antibody, etc. for enzyme labeling in ELISA or an anti-TI antibody, etc. for color labeling in immunochromatography.

[0051] In a second aspect of the present invention, the testing kit comprises a first antibody, etc. or a first competing antibody, etc., or a second antibody, etc. or a second competing antibody, etc., as an anti-TI antibody, etc. for solid-phase ELISA or an anti-TI antibody, etc. for solid-phase immunochromatography.

[0052] The first and second aspects of the present invention can be combined so that the test kit includes a first antibody, etc., or a first competing antibody, etc., or a second antibody, etc., or a second competing antibody, etc., as an anti-TI antibody, etc. for enzyme labeling in ELISA or an anti-TI antibody, etc. for color-developing labeling in immunochromatography, and includes a first antibody, etc., or a first competing antibody, etc., or a second antibody, etc., or a second competing antibody, etc., as an anti-TI antibody, etc. for solid-phase ELISA or an anti-TI antibody, etc. for solid-phase immunochromatography. Meanwhile, in the first aspect of the present invention, other anti-TI antibodies, etc. (e.g., clones "21A12" or "210E11" in the Examples) may be included as the anti-TI antibody, etc. for solid-phase ELISA or the anti-TI antibody, etc. for solid-phase immunochromatography, and in the second aspect of the present invention, other anti-TI antibodies, etc. may be included as the anti-TI antibody, etc. for enzyme labeling in ELISA or the anti-TI antibody, etc. for color-developing labeling in immunochromatography.

[0053] In one preferred embodiment of the present invention (corresponding to Example 4 described below), the test kit contains a first antibody, etc. or a first competing antibody, etc. as an anti-TI antibody, etc. for solid-phase ELISA or an anti-TI antibody, etc. for immobilization in immunochromatography, and contains a second antibody, etc. or a second competing antibody, etc. as an anti-TI antibody, etc. for enzyme labeling in ELISA or an anti-TI antibody, etc. for color labeling in immunochromatography.

[0054] In one preferred embodiment of the present invention (corresponding to Example 1 described below), the test kit contains a second antibody, etc. or a second competing antibody, etc. as an anti-TI antibody, etc. for solid-phase ELISA or an anti-TI antibody, etc. for immobilization in immunochromatography, and contains another anti-TI antibody, etc. (for example, clone "210E11" in the Examples) as an anti-TI antibody, etc. for enzyme labeling in ELISA or an anti-TI antibody, etc. for color labeling in immunochromatography.

[0055] As for the other anti-TI antibodies, depending on the use and embodiment of the test kit, any one of monoclonal antibodies or their antigen-binding fragments (referred to as "monoclonal antibodies, etc." in this specification) may be used, or two or more types of monoclonal antibodies, etc. may be used in combination, or polyclonal antibodies or their antigen-binding fragments (referred to as "polyclonal antibodies, etc." in this specification) may be used. Monoclonal antibodies, etc. and polyclonal antibodies, etc. differ in specificity (whether they react only with trypsin inhibitor proteins and do not react (cross) with other proteins), reproducibility (whether there are differences in test results depending on the manufacturing lot), stability (whether the binding ability to the antigen is lost due to the solid-phase treatment, labeling treatment, etc. of the antibody in the kit), etc., so that the preferred antibody may vary depending on the use and embodiment of the test kit, and therefore, taking this into consideration, it is possible to select whether to use monoclonal antibodies, etc. or polyclonal antibodies, etc., or to use both in combination.

[0056] In one embodiment of the present invention, the test kit includes polyclonal antibodies as other anti-TI antibodies. In a further embodiment of the present invention, the detection kit includes both polyclonal antibodies and monoclonal antibodies as anti-TI antibodies. For example, in an immunological measurement method of an embodiment described below, a monoclonal antibody can be used as a capture antibody (antibody for solid-phase immobilization in ELISA, antibody for immobilization in immunochromatography) for a trypsin inhibitor protein, and a polyclonal antibody can be used as a detection antibody (antibody for enzyme labeling in ELISA, antibody for color labeling in immunochromatography) for the captured trypsin inhibitor protein. By using a monoclonal antibody as a capture antibody, the specificity for the trypsin inhibitor protein can be increased, and by using a polyclonal antibody as a detection antibody, the captured trypsin inhibitor protein can be detected with high sensitivity.

[0057] Anti-TI antibodies, etc. used in the present invention, particularly anti-TI antibodies, etc. other than the anti-TI antibodies, etc. of the present invention in which the amino acid sequence of the CDR is specified, can be produced as polyclonal antibodies, etc. or monoclonal antibodies, etc., in the same manner as antibodies against other food allergens, according to conventional methods, generally using a food allergen (trypsin inhibitor protein in this invention) that has been extracted from food and usually further purified.

[0058] Polyclonal antibodies are generally produced by a method using immunized animals or a phage display method. The procedure of the method using immunized animals is as follows: An appropriate amount of purified target protein (trypsin inhibitor protein in the present invention) is mixed with an adjuvant as necessary to prepare an immunogen, and the immunogen is injected (immunized) into an animal (rabbit, guinea pig, goat, sheep, rat, mouse, chicken, etc.), thereby producing antibodies in the blood of the animal. After repeated immunization at appropriate intervals and times, blood (plasma, serum) is collected and the antibodies contained therein are purified, for example, by affinity chromatography using an affinity column on which the target protein (trypsin inhibitor protein in the present invention) is immobilized, and further purified by gel filtration chromatography as necessary, to obtain polyclonal antibodies. The procedure of the phage display method is as follows: An antibody gene is introduced into a bacteriophage, and a protein in which the variable regions of the H chain and L chain are linked is expressed (presented) on the coat protein of the bacteriophage. The resulting antibody phage library is used to select antibodies that have affinity for the target protein (trypsin inhibitor protein in this invention). The bacteriophage that produces the antibody are infected into E. coli, grown, and then harvested. The antibodies contained therein are purified to obtain polyclonal antibodies.

[0059] A method using hybridomas is generally used to produce monoclonal antibodies. The outline of the method using hybridomas is as follows. As in the case of producing polyclonal antibodies, a purified product of a target protein (trypsin inhibitor protein in this invention) is injected into an animal as an immunogen multiple times to produce antibodies. Alternatively, according to the "DNA immunization method", an expression vector (plasmid) containing a gene of a target protein is injected into an animal multiple times to express the target protein in the body, thereby producing antibodies. B cells are collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fusion cells). Among the hybridomas, those that produce antibodies with excellent binding affinity and specificity to the target protein (trypsin inhibitor protein in this invention) are selected (screened). The hybridomas are cultured to produce a single antibody in the culture supernatant. The culture supernatant is collected and the antibodies contained therein are purified to obtain monoclonal antibodies. The first and second antibodies of the present invention are also antibodies obtained by the method using hybridomas (DNA immunization method) as described above.

[0060] After an anti-TI antibody (polyclonal or monoclonal) is obtained, its amino acid sequence can be determined, and various antigen-binding fragments (polyclonal or monoclonal) can be produced according to standard methods. The amino acid sequences of the first and second antibodies of the present invention, particularly the amino acid sequences of the heavy chain CDR1-3 and the light chain CDR1-3 that they each possess, were also identified by isolating total RNA from a hybridoma, cloning the cDNA, and performing DNA sequencing according to standard methods.

[0061] The anti-TI antibody etc. can be in a form suitable for quantitatively or qualitatively detecting the trypsin inhibitor protein by immunological assay, for example, can be modified or altered in various ways. If necessary, the anti-TI antibody etc. can be humanized according to a general method. The amino acid sequence of a suitable template human antibody for humanization (particularly the framework regions of the heavy chain variable region and the light chain variable region) can be obtained by using an appropriate database, for example.

[0062] The test kit of the present invention may contain, as necessary or to the extent that the action and effect of the present invention is not inhibited, antibodies, etc. other than anti-TI antibodies, etc. For example, the test kit of the present invention may further contain, as the above-mentioned capture antibodies, etc. and detection antibodies, etc., antibodies, etc. that target proteins other than trypsin inhibitors (e.g., soybean allergens other than trypsin inhibitors, or food allergens other than soybean allergens).

[0063] The test kit of the present invention includes at least an anti-TI antibody, etc., but may include any content such as an extraction reagent, other reagents, and components, if necessary, depending on the embodiment. In the case of an ELISA kit, for example, a plate with wells, a diluent for preparing a solution of a target protein, a washing solution for washing the plate (well) after each step, an enzyme reaction stopping solution in ELISA, BSA for protecting the extracted protein from degradation, and an instruction manual describing the procedure of the test method by ELISA can be included in the kit contents together with the anti-TI antibodies for immobilization and enzyme labeling, etc., and the extraction reagent. In the case of an immunochromatography kit, for example, a test strip that can develop various solutions and reagents by capillary action, a diluent for preparing a solution of a target protein, and an instruction manual describing the procedure of the test method by immunochromatography can be included in the kit contents together with the anti-TI antibodies for immobilization and color labeling, etc., and the extraction reagent.

[0064] "Extraction reagent" refers to a reagent (solution) that is generally used to extract various proteins in food, such as food allergens. Various types of extraction reagents are known, and similar extraction reagents can be used in the present invention.

[0065] The extraction reagent is generally a solution prepared by adding a "solubilizing agent" to a buffer solution having an appropriate pH as an agent for extracting various proteins in food.

[0066] Examples of buffer solutions for preparing the extraction reagent include Tris buffer, phosphate buffer, citrate buffer, EDTA buffer, HEPES buffer, and acetate buffer. Examples of buffer solutions in the present invention are preferably Tris buffer and phosphate buffer. The pH of the buffer solution is generally 4.5 to 8.0 (a pH range that can occur in vivo), for example, 6.0 to 8.0. Those skilled in the art can prepare a buffer solution of a desired concentration and a desired pH by using an appropriate amount of a suitable compound (adding and dissolving each of a plurality of compounds in water in an appropriate amount).

[0067] Examples of solubilizing agents for preparing the extraction reagent include surfactants, chaotropic agents, and reducing agents. The extraction reagent may contain any one of these agents alone or a combination of multiple agents.

[0068] Examples of surfactants include anionic surfactants, cationic surfactants, and nonionic surfactants. Examples of anionic surfactants include alkyl sulfates and alkylbenzene sulfonates. The "alkyl" in alkyl sulfates, alkylbenzene sulfonates, and the like is, for example, dodecyl, decyl, nonyl, and octyl. The "salt" in alkyl sulfates, alkylbenzene sulfonates, and the like is, for example, sodium salt, potassium salt, and ammonium salt. A specific example of an alkyl sulfate is sodium dodecyl sulfate (SDS), and a specific example of an alkylbenzene sulfonate is sodium dodecylbenzene sulfonate. Specific examples of cationic surfactants include hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, and hexadecyltrimethylammonium chloride. Specific examples of nonionic surfactants include "Tween (registered trademark) 20" (polyoxyethylene sorbitan monolaurate), "Tween (registered trademark) 40" (polyoxyethylene sorbitan monopalmitate), "Tween (registered trademark) 60" (polyoxyethylene sorbitan monostearate), and "Tween (registered trademark) 80" (polyoxyethylene sorbitan monooleate).

[0069] When the extraction reagent contains a surfactant, the concentration of the surfactant can be appropriately adjusted taking into consideration the type of surfactant and its action and effect, and the presence, type and concentration of other components (e.g., chaotropic agents, reducing agents) in the extraction reagent as necessary. For example, when the extraction reagent contains an anionic surfactant such as SDS or other surfactant as a surfactant, the lower limit of the concentration can be, for example, 0.005, 0.01 or 0.05% (w / v), and the upper limit can be, for example, 5.0, 3.0 or 2.0% (w / v), and these upper and lower limits can be combined in any way.

[0070] Examples of "chaotropic agents" include urea, formamide, and salts containing anions or cations with a salting-in effect (e.g., guanidine hydrochloride containing a guanidinium ion, sodium chloride containing a sodium ion, potassium chloride containing a potassium ion, etc.).

[0071] When the extraction reagent contains a chaotropic agent, the concentration of the chaotropic agent can be appropriately adjusted taking into consideration the type of chaotropic agent and its action and effect (influence on the action and effect of the present invention), and, if necessary, the presence, type and concentration of other components (e.g., surfactant, reducing agent) in the extraction reagent. For example, when the extraction reagent contains urea as a chaotropic agent, the lower limit of its concentration can be, for example, 0.01, 0.05 or 0.1 (w / v), and the upper limit can be, for example, 10, 5.0 or 3.0% (w / v), and these upper and lower limits can be combined arbitrarily.

[0072] The reducing agent may, for example, be a mercaptoalkanol and / or a sulfite. The mercaptoalkanol may, for example, be thioglycerol, 2-mercaptoethanol, or dithiothreitol. The sulfite may, for example, be an alkali metal sulfite, and the alkali metal may, for example, be sodium or potassium. A specific example of the sulfite may be sodium sulfite.

[0073] When the extraction reagent contains a reducing agent, the concentration of the reducing agent can be appropriately adjusted taking into consideration the type of reducing agent and its action effect, and the presence, type and concentration of other components (e.g., surfactant, chaotropic agent) in the extraction reagent as necessary. When the extraction reagent contains thioglycerol as a reducing agent, the lower limit of its concentration can be, for example, 0.001, 0.005, 0.01 or 0.02% (w / v), and the upper limit can be, for example, 10, 5.0, 3.0, 2.0 or 1.0% (w / v), and these upper and lower limits can be combined arbitrarily. When the extraction reagent contains 2-mercaptoethanol or dithiothreitol as a reducing agent, the lower limit of its concentration can be, for example, 0.05, 0.1 or 0.5% (w / v), and the upper limit can be, for example, 5.0, 2.0 or 1.0% (w / v), and these upper and lower limits can be combined arbitrarily.

[0074] -Testing method- The "testing method" of the present invention is a method for testing soybean allergens remaining in food or food processing equipment, and comprises: (1) a step of extracting soybean proteins remaining in food or food processing equipment (referred to herein as the "extraction step"); and (2) a step of quantitatively or qualitatively detecting trypsin inhibitor proteins in the obtained extract by an immunological assay using at least one anti-trypsin inhibitor (TI) antibody, etc. of the present invention, i.e., at least one selected from the group consisting of the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. of the present invention (referred to herein as the "detection step").

[0075] "Food" may be soybeans themselves as a raw material (e.g., raw soybeans, dried soybeans, roasted soybeans, boiled soybeans), or a processed product that contains soybeans as a raw material (or is the subject of testing for whether it contains soybeans). The form of the food (processed product) is not particularly limited, but may be, for example, a processed product that is manufactured through a process carried out under heating and / or pressure conditions, which generally tends to insolubilize allergen proteins and make them difficult to extract. Food may be, for example, in any of the following forms: solid, semi-solid, jelly, liquid, or emulsified liquid.

[0076] Detection process The "immunological measurement method" is not particularly limited as long as it can detect proteins such as food allergens extracted from food, and can be selected from various known methods.

[0077] In one embodiment of the present invention, the immunological measurement method in the detection step is ELISA. ELISA can be performed, for example, by the following procedure using a target protein (trypsin inhibitor protein in the present invention) in a food (sample) and an antibody that binds to the target protein. 1) A sample solution prepared from food (specimen) is added to a well of a plate equipped with a solid-phase antibody, and the target protein contained in the sample solution is brought into contact with the solid-phase antibody, etc., and bound by an antigen-antibody reaction to form a first complex. 2) After washing the well, a solution of biotin-conjugated antibody is added, and the target protein in the first complex is brought into contact with the biotin-conjugated antibody, etc., and bound by an antigen-antibody reaction to form a second complex. 3) After washing the well, a solution of streptavidin-conjugated enzyme is added, and the biotin-conjugated antibody, etc. in the second complex is brought into contact with the streptavidin-conjugated enzyme, and bound by the biotin-streptavidin reaction to form a third complex. 4) After washing the well, a substrate solution (color developer) is added to react the enzyme in the third complex with the substrate to develop color. 5) After the color reaction has stopped, the absorbance at a specified wavelength is measured using a plate reader. 6) Prepare a standard curve graph from the absorbance obtained by measuring the standard solution separately, and read the amount of target protein (concentration of the solution) from the absorbance measured in 5) and the standard curve graph. Multiply this by the dilution factor used when preparing the sample solution to calculate the amount of target protein in the specimen.

[0078] In one embodiment of the present invention, the immunological measurement method in the detection step is immunochromatography. Immunochromatography can be performed, for example, by the following procedure using a target protein (trypsin inhibitor protein in this invention) in a food (sample) and an antibody that binds to the target protein. 1) A sample solution prepared from food (specimen) is dropped onto a designated area (sample drop area) on a test strip containing a color-labeled antibody, etc., and the target protein contained in the sample solution is brought into contact with the color-labeled antibody, etc., and bound by an antigen-antibody reaction to form a first complex. 2) The sample solution containing the first complex and unreacted color-labeled antibody, etc. spreads over a predetermined area (spreading area) on the test strip, and the first complex, etc. also move due to capillary action. 3) When the first complex reaches a predetermined site (test line) containing immobilized antibodies, etc., and is captured by the immobilized antibodies, etc., a colored line (e.g., a reddish-purple line caused by gold colloid) appears due to the color-producing label. The appearance of the test line indicates that the target protein was contained in the sample solution. 4) When unreacted colloidal gold-labeled antibodies, etc. reach a predetermined area (control line area, downstream of the test line) containing anti-immunoglobulin antibodies and are captured by the anti-immunoglobulin antibodies, a reddish purple line caused by colloidal gold appears. If the control line does not appear, regardless of whether the sample solution contained the target protein or not, it suggests that there was an abnormality in the development of the sample solution (retesting will be necessary).

[0079] ·Extraction process The "extraction process" can be carried out in the same manner as a general extraction process that is usually carried out using an extraction reagent as a process for extracting various proteins in foods, and can further be appropriately modified as necessary to suit the present invention.

[0080] The food specimen to be subjected to the extraction step can be prepared by finely pulverizing or emulsifying the food into a homogeneous state by high-speed shearing and stirring using a food cutter, a mill, a mixer, a homogenizer, etc., depending on the form of the food (high-speed shearing and stirring method). Such finely pulverizing and other treatments may be performed on the food before the extraction step, and the obtained finely pulverized food and the extraction reagent may be mixed, or the mixture of the food and the extraction reagent may be subjected to the finely pulverizing and other treatments and the extraction treatment may be performed simultaneously. The conditions for the finely pulverizing and other treatments (time, temperature, rotation speed (rpm), centrifugal force (×g), etc.) can be appropriately adjusted depending on the selected finely pulverizing and other treatment methods and the treatment equipment used, and taking into consideration the effects of the present invention. In addition, the food to be subjected to the extraction step may be further treated, as necessary, for example, by defatting, taking into consideration the extraction of trypsin inhibitor protein and / or detection after extraction.

[0081] The food, which may or may not be micronized as described above, may be mixed with an extraction reagent, and then the mixture may be shaken to perform extraction (shaking method). The shaking conditions (time, temperature, shaking rate or rotation rate (rpm), etc.) may be appropriately adjusted according to the selected shaking method and the treatment device used, and taking into consideration the effects of the present invention. The shaking time is usually 12 hours or more and less than 24 hours (for example, overnight). The shaking temperature is usually room temperature, but may be a heated temperature if necessary. In the present invention, even without heating in the extraction step (shaking treatment), low concentrations of trypsin inhibitor protein can be detected with high sensitivity, and high concentrations of trypsin inhibitor protein can be detected without false negatives.

[0082] When testing for allergens remaining in food processing equipment, for example, prepare a cotton swab moistened with PBS or saline, wipe the designated areas of the food processing equipment with the swab, and suspend the dirt adhering to the swab in a diluted buffer solution that has been dispensed in advance (e.g., 1 mL) into a test tube, etc., and the resulting suspension can be used as a sample solution equivalent to an "extract." EXAMPLES

[0083] Hereinafter, the embodiments of the present invention will be more specifically disclosed through the examples, but the technical scope of the present invention is not limited to the embodiments disclosed as examples. Those skilled in the art can understand that, in order to adapt to the intended use and effects of the present invention, the embodiments disclosed as examples can be expanded or modified into various other embodiments, or, if necessary, the technical features of the prior art (known inventions) can be further combined, taking into consideration the technical ideas of the present invention and the contents of this specification and drawings as a whole. Matters necessary to implement the present invention other than those described in this specification can be appropriately taken into consideration of the technical common knowledge and prior art in the technical field to which the present invention belongs.

[0084] [Example] Preparation of anti-TI monoclonal antibody Using the DNA immunization method, anti-TI monoclonal antibodies were produced in the following procedure. First, a cDNA sequence encoding a trypsin inhibitor was incorporated into an expression vector according to a general method. Next, the above expression vector was injected into a mouse multiple times according to a general immunization method to produce antibodies. B cells were collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fusion cells). From the hybridomas, those that produce antibodies with excellent binding affinity and specificity to the target protein, trypsin inhibitor protein, in this example, particularly to both native trypsin inhibitor protein and heat-denatured trypsin inhibitor protein, were selected (screened). The hybridomas were cultured to produce a single antibody in the culture supernatant. The culture supernatant was collected, and the antibodies contained therein were purified (adsorbed and eluted) using a Protein G column to obtain monoclonal antibodies.

[0085] The anti-trypsin inhibitor (TI) monoclonal antibodies obtained as described above included those that were confirmed to specifically bind to both native and heat-denatured trypsin inhibitor proteins (data not shown). Among these specific anti-TI antibodies, four clones, "27G4", "28F2", "210E11" and "21A12", were selected and used as immobilized anti-TI antibodies for preparing membrane antibodies or color-labeled anti-TI antibodies for preparing gold colloid-labeled antibodies in the test strips in the following Examples (and Reference Examples). In addition, an antibody against the color-labeled anti-TI antibody (anti-mouse IgG antibody) prepared according to a general method was also used.

[0086] Among the four specific anti-TI antibodies, the amino acid sequences of the heavy and light chains of clones "27G4" and "28F2" were sequenced according to a general method. More specifically, the procedure is as follows: total RNA was isolated from the hybridomas of clones "27G4" and "28F2" using a kit, cDNA was synthesized using a kit, and the cDNA was amplified by PCR using specific primers, and the nucleotide sequence was identified using a sequencer.

[0087] The amino acid sequences of the CDRs of the sequenced clones "27G4" and "28F2", the variable regions including the CDRs and FRs, and the heavy and light chains composed of the variable and constant regions are shown in Table 1. The CDRs and other sites were identified by CHOTHIA ANTIBODY NUMBERING in accordance with a general method.

[0088] [Table 1-1] [Table 1-2]

[0089] [Production example] Manufacture of immunochromatography kit for detecting soybean allergens (trypsin inhibitor protein) The immunochromatography kit was produced according to the following procedure, following the conventional method for producing commercially available kits for detecting soybean allergens (or other food allergens) by immunochromatography. First, the four specific clones were used as anti-TI antibodies for color labeling, and four types of gold colloid-labeled antibodies were produced by binding them to gold colloids according to a general method. Meanwhile, the four specific clones were also used as four types of anti-TI antibodies for immobilization. In general test strip components including a sample dropping section, a reagent-containing section, a development section, an absorption pad, etc., a test strip was produced in which one of the four types of gold colloid-labeled antibodies was placed in the reagent-containing section, one of the four types of anti-TI antibodies for immobilization was placed at the test line appearance position, and an antibody against the anti-TI antibody for color labeling (anti-mouse IgG antibody, common) was placed at the control line appearance position.

[0090] The outlines of the thus prepared Examples 1 to 5 and Reference Examples 1 to 2 are shown in Table 2 below. The test strips of these Examples and Reference Examples were used to construct an immunochromatography kit for detecting soybean protein (allergen) by targeting trypsin inhibitor protein, and were used in the following Test Examples. The results of Test Example 1 are also shown in the table, and the details will be described later.

[0091] [Table 2]

[0092] [Test Example 1] Detection of soybean allergens and other food allergens by immunochromatography In this test example, the following immunochromatographic kits were evaluated: Examples 1 to 5, i.e., immunochromatographic kits using clones "27G4" (first antibody in the present invention) or "28F2" (second antibody in the present invention) as at least one of the anti-TI antibody for color labeling or the antibody for immobilization, which were produced according to the above-mentioned [Production Examples]; and Reference Examples 1 to 2, i.e., immunochromatographic kits using only other anti-TI antibodies (clone "210E11" or "21A12") without using clones "27G4" (first antibody in the present invention) or "28F2" (second antibody in the present invention) as either the anti-TI antibody for color labeling or the antibody for immobilization. These immunochromatographic kits were evaluated for their detection performance against soybean allergens and other food allergens (proteins derived from eggs, milk, wheat, buckwheat, peanuts, crustaceans, and red beans, respectively).

[0093] As the soybean allergen, undenatured soybean protein extracted from unheated soybean powder (defatted) was used, and an aqueous solution prepared to a protein concentration of 25 ng / mL was used as the sample solution. On the other hand, for the other food allergens, a mixture of equal amounts of unheated and heated foods was used as the specimen, and an aqueous solution was prepared by mixing with the specimen extract included in a conventional commercially available kit for detecting each food allergen.

[0094] According to the method of using commercially available kits for detecting soybean allergens or other food allergens by conventional immunochromatography, each sample solution was dropped onto the sample dropping section of the test strip of each immunochromatography kit. After a certain period of time, the reactivity to soybean allergens and other food allergens was evaluated according to the following criteria based on the color of the test line. The results are also shown in Table 2 (see above).

[0095] <Evaluation criteria> ++: A dark reddish purple line appears at the test line location (indicating strong reactivity). +: A reddish purple line appears at the test line location (reactivity confirmed). ±: A faint reddish purple line appears where the test line appears (reactivity is difficult to confirm but cannot be denied). -: No reddish purple line appears at the test line location (no reactivity observed). *In both cases, it was confirmed that a line appeared at the control line appearance location.

[0096] Regarding reactivity to trypsin inhibitor protein, all of the kits (antibody pairs) of Examples 1 to 5, which use at least one of clones "27G4" (first antibody) or "28F2" (second antibody) as the anti-TI monoclonal antibody for immobilization or color labeling, were sufficient (++ or +) even though the soy protein concentration was a low concentration of 25 ng / mL. Furthermore, the kits (antibody pairs) of Reference Examples 1 and 2, which do not use the above clones (first antibody and second antibody) but use clones "210E11" and "21A12" as the anti-TI monoclonal antibody for immobilization or color labeling, also had sufficient reactivity to trypsin inhibitor protein (++ or +) even though the trypsin inhibitor protein concentration was a low concentration of 25 ng / mL.

[0097] On the other hand, with regard to food cross-reactivity, the kits (antibody pairs) of Examples 1 to 5 had generally low cross-reactivity to foods other than soybeans tested, and the kits of Examples 1 and 4 in particular had extremely low cross-reactivity to peanut allergens and adzuki bean allergens (both -), making them excellent. In contrast, the kits (antibody pairs) of Reference Examples 1 and 2 showed some cross-reactivity to wheat and buckwheat allergens (±), and were not as good in cross-reactivity to other food allergens as the kits of the Examples.

[0098] [Test Example 2] Measurement of minimum detection sensitivity by immunochromatography In this test example, the minimum detection sensitivity for soybean allergens was measured using an immunochromatography kit produced in accordance with the above [Production Example], i.e., using clone "27G4" (first antibody) as the immobilized anti-TI antibody and clone "28F2" (second antibody) as the color-labeled anti-TI antibody as in Example 4.

[0099] The sample solutions used were an equal mixture of native trypsin inhibitor protein extracted and purified from unheated soybean powder (purchased as a product) and heat-denatured trypsin inhibitor protein extracted and purified from heated soybean powder (purchased as a product), diluted stepwise from 100 ng / mL as the total of these proteins, or diluted stepwise from 20 ng / mL.

[0100] The results are shown in Figure 1. The detection limit concentration of trypsin inhibitor protein is 5 ng / mL for the conventional product (A) disclosed in Non-Patent Document 1, whereas it is much lower at 0.15 ng / mL for the product of the present invention (B), and a dark test line is visible at 5 ng / mL (arrow). It has been shown that by using the anti-TI antibody of the present invention, it is possible to manufacture a kit for immunochromatography (and other immunological assays) with significantly improved detection sensitivity compared to conventional kits.

[0101] [Test Example 3] Comparison of heat reactivity by ELISA In this test example, clone "28F2" (second antibody) was used as the solid-phase anti-TI antibody, and clone "27G4" (first antibody) was used as the enzyme-labeled TI antibody, and the reactivity against native trypsin inhibitor protein and heat-denatured trypsin inhibitor was compared by ELISA.

[0102] The ELISA kit was manufactured according to the following procedure, following the manufacturing method of a commercial kit for detecting soybean allergens by conventional ELISA. First, clone "27G4" was used as an anti-TI antibody for enzyme labeling, and was bound to biotin according to a general method to prepare a biotin-bound antibody. In addition, to be used in combination with this biotinylated antibody, streptavidin was bound to horseradish peroxidase (HRP) to prepare streptavidin-bound HRP. Meanwhile, clone "28F2" was used as an anti-TI antibody for immobilization, and was bound to the wells of an ELISA plate according to a general method.

[0103] The sample solutions used were an aqueous solution containing only undenatured soybean protein extracted and purified from unheated soybean powder at a concentration of 40 ng / mL, and an aqueous solution containing only heat-denatured soybean protein extracted and purified from heated soybean powder at a concentration of 40 ng / mL.

[0104] According to the method of using a commercial kit for detecting soy allergens by conventional ELISA, each sample solution and standard solution were dropped into the wells of an ELISA plate (primary reaction), and after a certain time, a biotin-bound antibody solution was dropped (secondary reaction), and after a certain time, a streptavidin-bound HRP solution was dropped (tertiary reaction). After a certain time, the color development in the wells was measured using a microplate reader, and the reactivity to native soy protein and heat-denatured soy protein was compared.

[0105] The results are shown in Figure 2. The measured value (right) for heat-denatured soybean protein was about 70% of the measured value (left) for undenatured soybean protein. When a general anti-TI antibody is used for heat-denatured soybean protein (to detect heat-denatured trypsin inhibitor protein contained therein), the sensitivity is significantly lower when the antibody is used for undenatured soybean protein (to detect undenatured trypsin inhibitor protein contained therein) (see Non-Patent Document 1 cited above). However, by using the anti-TI antibody of the present invention, the decrease in sensitivity due to heat denaturation of food allergens is suppressed to some extent (avoiding a significant decrease in sensitivity), and it has been shown that it is possible to manufacture an ELISA kit (and other immunological measurement kits) that can use both heated and unheated foods as samples.

Claims

1. A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:1; A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:2, and A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:3, and A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:4; A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:5, and Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:6 or an anti-trypsin inhibitor antibody or an antigen-binding fragment thereof (hereinafter referred to as "first antibody, etc."), A heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7; A heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:8, and A heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:9, and A light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 10; A light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and Light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:12 An anti-trypsin inhibitor antibody or an antigen-binding fragment thereof (hereinafter referred to as "second antibody, etc."),

2. An anti-trypsin inhibitor antibody or an antigen-binding fragment thereof (hereinafter referred to as the "first competing antibody, etc.") that competes with the first antibody, etc. for the same or overlapping epitope in binding to a trypsin inhibitor protein, or an anti-trypsin inhibitor antibody or an antigen-binding fragment thereof (hereinafter referred to as the "second competing antibody, etc.") that competes with the second antibody, etc. for the same or overlapping epitope.

3. A test kit for quantitatively or qualitatively detecting soy allergens remaining in food or food processing equipment by an immunological measurement method, comprising: A testing kit comprising at least one selected from the group consisting of the first antibody etc. and the second antibody etc. described in claim 1 and the first competing antibody etc. and the second competing antibody etc. described in claim 2.

4. The test kit according to claim 3, wherein the immunological measurement method is ELISA or immunochromatography, and the kit comprises the first antibody or the first competing antibody or the second antibody or the second competing antibody for enzyme labeling in ELISA or color labeling in immunochromatography.

5. The test kit according to claim 3, wherein the immunological measurement method is ELISA or immunochromatography, and the kit comprises the first antibody, etc., or the first competing antibody, etc., or the second antibody, etc., or the second competing antibody, etc., for solid-phase immobilization in ELISA or for immobilization in immunochromatography.

6. the immunoassay is ELISA or immunochromatography; (1) The first antibody or the first competing antibody, etc., for use in solid-phase immobilization in ELISA or immobilization in immunochromatography, and the second antibody or the second competing antibody, etc., for use in enzyme labeling in ELISA or color labeling in immunochromatography, or (2) The testing kit according to claim 3, comprising the second antibody, etc. or the second competing antibody, etc. for solid-phase immobilization in ELISA or immobilization in immunochromatography, and an anti-trypsin inhibitor antibody or an antigen-binding fragment thereof other than the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. for enzyme labeling in ELISA or color labeling in immunochromatography.

7. The test kit according to claim 3 , further comprising an extraction reagent.

8. 8. The test kit according to claim 7, wherein the extraction reagent is an extraction reagent obtained by a shaking method or a high-speed shear / agitation method without heating.

9. (1) extracting soy protein remaining in food or in food processing equipment; and (2) A step of quantitatively or qualitatively detecting a trypsin inhibitor protein in the obtained extract by an immunological assay using at least one antibody selected from the group consisting of the first antibody, etc. and the second antibody, etc. described in claim 1 and the first competing antibody, etc. and the second competing antibody, etc. described in claim 2. A method for testing for soy allergens in food, comprising:

10. The testing method according to claim 9, wherein the immunological measurement method is ELISA or immunochromatography, and the first antibody or the first competing antibody or the second antibody or the second competing antibody is used for enzyme labeling in ELISA or for color labeling in immunochromatography.

11. The testing method according to claim 9, wherein the immunological measurement method is ELISA or immunochromatography, and the first antibody or the first competing antibody or the second antibody or the second competing antibody is used for solid-phase immobilization in ELISA or for immobilization in immunochromatography.

12. the immunoassay is ELISA or immunochromatography; (1) Using the first antibody or the first competing antibody for immobilization in ELISA or immobilization in immunochromatography, and the second antibody or the second competing antibody for enzyme labeling in ELISA or color labeling in immunochromatography, or (2) The testing method according to claim 9, wherein the second antibody, etc. or the second competing antibody, etc. is used for solid-phase immobilization in ELISA or immobilization in immunochromatography, and an anti-trypsin inhibitor antibody or its antigen-binding fragment other than the first antibody, etc., the second antibody, etc., the first competing antibody, etc., and the second competing antibody, etc. is used for enzyme labeling in ELISA or color labeling in immunochromatography.

13. The testing method according to claim 9 , wherein the extraction in the step (1) is carried out using an extraction reagent.

14. The testing method according to claim 13, wherein the extraction using the extraction reagent is carried out by a shaking method or a high-speed shear / agitation method without heating.

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