阅读说明：本技术 抗雌马酚抗体组合物及其利用 (Anti-equol antibody composition and use thereof ) 是由 泷本阳介 萩原启太郎 于 2017-12-18 设计创作，主要内容包括：本发明公开了一种使用含有以下物质的组合物：作为免疫球蛋白重链可变区,具有SEQ ID NO：1所示的氨基酸排列构成重链超可变区CDR1,SEQ ID NO：2所示的氨基酸排列构成超可变区CDR2及SEQ ID NO：3所示的氨基酸排列构成超可变区CDR3,抗雌马酚抗体或其抗体片段,作为免疫球蛋白轻链可变区,具有SEQ ID NO：4所示的氨基酸排列构成超可变区CDR1′,SEQ ID NO：5所示的氨基酸排列构成超可变区CDR2′及SEQ ID NO：6所示的氨基酸排列构成超可变区CDR3′,作为免疫球蛋白重链可变区,具有SEQ ID NO：1所示的氨基酸排列构成重链超可变区CDR1,SEQ ID NO：2所示的氨基酸排列构成超可变区CDR2及SEQ ID NO：3所示的氨基酸排列构成超可变区CDR3,抗雌马酚抗体或其抗体片段,作为免疫球蛋白轻链可变区,具有SEQ ID NO：7所示的氨基酸排列构成超可变区CDR1′,SEQ ID NO：8所示的氨基酸排列构成超可变区CDR2′及SEQ ID NO：9所示的氨基酸排列构成超可变区CDR3′。(The invention discloses a composition containing the following substances: as an immunoglobulin heavy chain variable region, a light chain variable region having the sequence of SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3, an anti-equol antibody or antibody fragment thereof, as an immunoglobulin light chain variable region, having the amino acid sequence shown in SEQ ID NO: 4 constituting the hypervariable region CDR 1', SEQ ID NO: 5 constitutes the hypervariable region CDR 2' and SEQ ID NO: 6, constituting the hypervariable region CDR 3', which is an immunoglobulin heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3, an anti-equol antibody or antibody fragment thereof, as an immunoglobulin light chain variable region, having the amino acid sequence shown in SEQ ID NO: 7 constitutes the hypervariable region CDR 1', SEQ ID NO: 8, and the amino acid arrangement shown in SEQ ID NO: the amino acid arrangement shown in FIG. 9 constitutes the hypervariable region CDR 3'.)

1. An anti-equol antibody composition comprising:

an anti-equol antibody or antibody fragment thereof, as an immunoglobulin heavy chain variable region, having the amino acid sequence of SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 constitutes the hypervariable region CDR3,

as an immunoglobulin light chain variable region, a light chain variable region having the sequence of SEQ ID NO: 4 constituting the hypervariable region CDR 1', SEQ ID NO: 5 constitutes the hypervariable region CDR 2' and SEQ ID NO: 6 constitutes the hypervariable region CDR 3';

an anti-equol antibody or antibody fragment thereof, as an immunoglobulin heavy chain variable region, having the amino acid sequence of SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 constitutes the hypervariable region CDR3,

as an immunoglobulin light chain variable region, a light chain variable region having the sequence of SEQ ID NO: 7 constitutes the hypervariable region CDR 1', SEQ ID NO: 8, and the amino acid arrangement shown in SEQ ID NO: the amino acid arrangement shown in FIG. 9 constitutes the hypervariable region CDR 3'.

2. The composition according to claim 1, wherein the rate of crossing of S-equol is 100% and the rate of crossing of R-equol is 80% or more, as a whole.

3. The composition according to claim 1 or 2, wherein the rate of crossing for R-equol is above 85%.

4. The composition according to any one of claims 1 to 3, wherein the crossing rate of 1 or 2 or more isoflavones selected from the group consisting of daidzein, genistein and daidzein is 0.01% or less.

5. The composition according to claim 4, wherein the crossing rate of each isoflavone with respect to daidzein, genistein and daidzein is 0.01% or less.

6. The composition according to any one of claims 1 to 5, wherein the crossing rate of 1 or 2 or more selected from the group consisting of ellagic acid dihydrate, catechin monohydrate, and gallic acid is 1% or less.

7. The composition according to claim 6, wherein the crossing rate of ellagic acid dihydrate and catechin monohydrate is 0.01% or less.

8. An equol-detecting reagent comprising the composition according to any one of claims 1 to 7.

9. An anti-equol antibody or an antibody fragment thereof, which has an amino acid sequence represented by SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 constitutes the hypervariable region CDR3,

as an immunoglobulin light chain variable region, a light chain variable region having the sequence of SEQ ID NO: 4 constituting the hypervariable region CDR 1', SEQ ID NO: 5 constitutes the hypervariable region CDR 2' and SEQ ID NO: the amino acid arrangement shown in FIG. 6 constitutes the hypervariable region CDR 3'.

10. An anti-equol antibody or an antibody fragment thereof, which has an amino acid sequence represented by SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 constitutes the hypervariable region CDR3,

as an immunoglobulin light chain variable region, a light chain variable region having the sequence of SEQ ID NO: 7 constitutes the hypervariable region CDR 1', SEQ ID NO: 8, and the amino acid arrangement shown in SEQ ID NO: the amino acid arrangement shown in FIG. 9 constitutes the hypervariable region CDR 3'.

11. A method for measuring equol in a sample derived from a living body,

a method comprising the step of contacting the equol-detecting reagent according to claim 8 with equol in the sample derived from a living body.

12. An equol measurement device, comprising the detection reagent according to claim 8 bound to a solid-phase carrier.

13. An equol assay kit comprising the detection reagent according to claim 8.

14. An expression vector having a polynucleotide encoded by the antibody or antibody fragment thereof according to claim 9.

15. An expression vector having a polynucleotide encoded by the antibody or antibody fragment thereof according to claim 10.

Technical Field

The present specification relates to an anti-equol antibody composition and use thereof.

Background

Equol is known as a metabolite of isoflavone produced by intestinal bacteria such as human beings. It is known that equol can improve various symptoms of women caused by low hormone by an antiestrogenic effect.

Equol is produced by bean products and the like by the action of intestinal bacteria, but in addition to the intake amount of soybean, the amount of equol produced may vary depending on individual differences in intestinal flora or the like. Furthermore, equol may also be taken directly. Equol exerts an antiestrogenic effect, but in addition to the individual's ability to produce (amount of) equol, it is important to monitor the amount of equol in vivo.

Here, a method for measuring equol in urine using an anti-equol antibody has been provided (patent document 1).

Disclosure of Invention

However, the conventional anti-equol antibodies are not necessarily sufficient in terms of their crossability, detection sensitivity, and the like. It is desired to provide a method for easily measuring equol with high sensitivity and high accuracy.

The present specification aims to provide a composition which can be used more practically as an anti-equol antibody, and use thereof.

The present inventors have intensively studied and, as a result, have obtained an anti-equol antibody composition containing equol having more suitable crossability or affinity for equol. According to the present specification, based on this knowledge, the following means are provided.

[1] An anti-equol antibody composition comprising the following:

an anti-equol antibody or antibody fragment thereof, as an immunoglobulin heavy chain variable region, having the amino acid sequence of SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 constitutes the hypervariable region CDR3,

as an immunoglobulin light chain variable region, a light chain variable region having the sequence of SEQ ID NO: 4 constituting the hypervariable region CDR 1', SEQ ID NO: 5 constitutes the hypervariable region CDR 2' and SEQ ID NO: the amino acid arrangement shown in FIG. 6 constitutes the hypervariable region CDR 3',

an anti-equol antibody or antibody fragment thereof, as an immunoglobulin heavy chain variable region, having the amino acid sequence of SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 constitutes the hypervariable region CDR3,

as an immunoglobulin light chain variable region, a light chain variable region having the sequence of SEQ ID NO: 7 constitutes the hypervariable region CDR 1', SEQ ID NO: 8, and the amino acid arrangement shown in SEQ ID NO: the amino acid arrangement shown in FIG. 9 constitutes the hypervariable region CDR 3'.

[2] On the whole, the composition had a crossing rate of 80% or more for R-equol when the crossing rate for S-equol was 100%.

[3] The composition according to [1] or [2], wherein the rate of crossing R-equol is 85% or more.

[4] [1] to [3], wherein the composition has a crossing rate of 0.01% or less with respect to 1 or 2 or more isoflavones selected from the group consisting of daidzein, genistein and daidzein.

[5] [4] the composition according to any one of [1] above, wherein the composition has a daidzein, genistein and daidzein cross-over ratio of each isoflavone of 0.01% or less.

[6] [1] to [5], wherein the composition has a crossing rate of 1% or less for 1 or 2 or more selected from the group consisting of ellagic acid dihydrate, catechin monohydrate, and gallic acid.

[7] [6] the composition according to [6], wherein the intersection rate of ellagic acid dihydrate and catechin monohydrate is 0.01% or less.

[8] An equol-detecting reagent comprising the composition according to any one of [1] to [7 ].

[9] An anti-equol antibody or antibody fragment thereof, as an immunoglobulin heavy chain variable region, having the amino acid sequence of SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 constitutes the hypervariable region CDR3,

as an immunoglobulin light chain variable region, a light chain variable region having the sequence of SEQ ID NO: 4 constituting the hypervariable region CDR 1', SEQ ID NO: 5 constitutes the hypervariable region CDR 2' and SEQ ID NO: the amino acid arrangement shown in FIG. 6 constitutes the hypervariable region CDR 3'.

[10] An anti-equol antibody or antibody fragment thereof, as an immunoglobulin heavy chain variable region, having the amino acid sequence of SEQ ID NO: 1, the amino acid arrangement shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 constitutes the hypervariable region CDR3,

as an immunoglobulin light chain variable region, a light chain variable region having the sequence of SEQ ID NO: 7 constitutes the hypervariable region CDR 1', SEQ ID NO: 8, and the amino acid arrangement shown in SEQ ID NO: the amino acid arrangement shown in FIG. 9 constitutes the hypervariable region CDR 3'.

[11] A method for measuring equol in a sample of biological origin,

a method comprising the step of contacting the equol-detecting reagent described in [8] with equol in the above-mentioned biological sample.

[12] A device for measuring the equol, which comprises a device,

a device for preparing the detection reagent according to [8] in which a solid carrier is bound.

[13] A kit for the determination of equol,

a kit comprising the detection reagent according to [8 ].

[14] An expression vector comprising a polynucleotide encoded by the antibody or antibody fragment thereof according to [9 ].

[15] An expression vector comprising a polynucleotide encoded by the antibody or antibody fragment thereof according to [10 ].

Drawings

FIG. 1 shows the evaluation results of the antibody titer of the anti-equol monoclonal antibody as shown in the figure.

FIG. 2 shows the results of HPLC and immunochromatography measurement of an anti-equol monoclonal antibody.

Detailed Description

The present specification discloses an anti-equol antibody composition and use thereof. According to the composition containing an anti-equol monoclonal antibody (hereinafter, also referred to simply as the present antibody) (hereinafter, simply referred to as the present antibody composition) disclosed in the present specification, equol can be measured with high accuracy and high detection sensitivity. Furthermore, according to the excellent crossability, equol in the biological sample can be accurately and precisely measured.

The antibody composition specifically binds to R-equol, but has high binding ability to S-equol as a whole and high detection sensitivity for S-equol. Even a biological sample having a low equol content or a small amount of a biological sample, equol can be easily measured.

Specific examples, which are representative and non-limiting of the present invention, will be described in detail below with reference to appropriate drawings. The detailed description is intended merely to show the skilled practitioner of the art the details of preferred embodiments of the invention and is not intended to limit the scope of the invention. In addition, additional features and inventions disclosed below are provided for the improved anti-equol antibody composition and use thereof, and may be used in combination with other features or inventions.

Furthermore, the features and process combinations disclosed in the following detailed description are not required to practice the invention in their broadest sense, and are instead taught merely to describe specific examples representative of the invention. In addition, various features of the above-described and below-described representative examples, as well as various features of items described in independent and dependent claims, are not necessarily combined in the order listed according to the specific examples described herein in order to provide additional and useful embodiments of the present invention.

All the features described in the specification and/or the claims are intended to be disclosed individually and independently of each other as limitations of specific matters disclosed and claimed at the beginning of the application, in addition to the features described in the examples and/or the claims. Further, the description of all the numerical ranges and groups or groups is intended to disclose the intermediate structures as the disclosure at the beginning of the application and the limitation of the specific matters required.

Hereinafter, the anti-equol antibody composition disclosed in the present specification and embodiments of use thereof will be described in detail.

(anti-equol antibody composition)

The antibody composition can contain 2 anti-equol monoclonal antibodies. The present antibody composition obtained by combining these antibodies has specific binding ability, and examples thereof include the following. The antibody composition has at least the specific binding ability of (1). And can have specific binding ability of any 1 or 2 or more of the following (2) to (4). In the present specification, "specific binding ability" or "specific binding" refers to the ability and binding of an antibody, that is, an immunoglobulin to bind based on the antigen recognition ability. The structures of R-equol and S-equol are shown below. In addition, the intestinal bacteria can limit the production of S-equol.

[ CHEM 1]

(1) The rate of crossing with S-equol was 100%, and the rate of crossing with R-equol was 80% or more. The rate of intersection of R-equol may be 85% or more, 90% or more, 95% or more, or 98% or more, for example. For example, the concentration may be 105% or less.

(2) The cross-over rate of 1 or 2 or more isoflavones selected from daidzein, genistein and daidzein is 0.01% or less. Further, the crossing rate of daidzein, genistein and daidzein may be 0.01% or less. Further, such an intersection ratio may be 0.005% or less.

(3) The cross-over rate of 1 or 2 or more selected from the group consisting of ellagic acid dihydrate, catechin monohydrate, and gallic acid is 1% or less. For example, the crossing rate of ellagic acid dihydrate and catechin monohydrate may be 0.01% or less, or 0.005% or less.

(4) The crossing rate for apigenin and R-O-angora pterocarpin (DMA) is 0.01%. For example, the rate of homocrossing is 0.005% or less.

The crossability and the crossunder rate can be measured by a method known to practitioners in the art. For example, the measurement can be performed by ELISA, competitive ELISA, or the like.

The crossability of the antibody is not particularly limited, but any concentration range can be measured in the range of 0.1. mu.M to 75. mu.M of each isoflavone concentration. For example, the concentration may be 1. mu.M to 10. mu.M.

The cross rate can be obtained, for example, as follows. First, a standard curve was created using S-equol. S-equol compounds containing 1. mu.M and 10. mu.M were prepared, and each concentration was allowed to react with the antibody, and the concentration of S-equol was calculated from the standard curve. For example, addition of 10. mu.M daidzein was regarded as 100% crossability when the concentration was detected at 10. mu.M from the standard curve, 10% crossability when the concentration was detected at 1. mu.M, and 1% crossability when the concentration was detected at 0.1. mu.M.

The antibody composition can contain 2 monoclonal antibodies. The amino acid arrangement of the hypervariable regions of the heavy and light chains of these 2 monoclonal antibodies is specified.

Variable region (V) of heavy chain (IgG) of antibody 1_HRegions) each having the sequence of SEQ ID NO: 1-3 form the hypervariable regions CDR1, CDR2 and CDR 3. A heavy chain variable region comprising 3 hypervariable regions, e.g., a light chain variable region capable of having the sequence of SEQ id no: 10, or a pharmaceutically acceptable salt thereof. Converting SEQ ID NO: 1 to 3 and 10, each of which is represented by SEQ ID NO: 13 to 15 and 16.

Variable region (V) of the light chain of antibody 1_LRegions) each having the sequence of SEQ ID NO: 4-6 form the hypervariable regions CDR1 ', CDR2 ' and CDR3 '. A light chain variable region comprising 3 hypervariable regions, e.g., a light chain variable region capable of having the sequence of SEQ id no: 11, or a pharmaceutically acceptable salt thereof. Has the sequence shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. In addition, the light chain is a kappa chain. Converting SEQ ID NO: 4 to 6 and 11, each of which is represented by SEQ id no: 18 to 20 and 21.

Variable region (V) of heavy chain of antibody 2_HRegions) each having the sequence of SEQ ID NO: 1-3 form the hypervariable regions CDR1, CDR2 and CDR 3. A heavy chain variable region comprising 3 hypervariable regions, e.g., a light chain variable region capable of having the sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof. Converting SEQ ID NO: 1 to 3 and 10, each of which is represented by SEQ ID NO: 13 to 15 and 16.

Variable region (V) of the light chain of antibody 1_LRegions) each having the sequence of SEQ ID NO: 7-9 form the hypervariable regions CDR1 ', CDR2 ' and CDR3 '. A light chain variable region comprising 3 hypervariable regions, e.g., a light chain variable region capable of having the sequence of SEQ id no: 12, or a pharmaceutically acceptable salt thereof. In addition, the light chain is a kappa chain. Converting SEQ ID NO: 7 to 9 and 12, each of which is represented by SEQ ID NO: 23 to 25 and 26.

The antibody composition of the present invention can contain an intact antibody having a binding ability to a specific arbitrary antigen (in the present specification, for example, S-equol or the like) or a portion containing an antigen-binding portion having the binding ability, by a known technique at the time of filing of the present application. The antibody of the present invention can take various forms in addition to the technical common knowledge at the time of application, as shown below.

The term "antigen-binding portion" of an antibody means any specific antigen (for example, 1 or more fragments of a whole antibody that retains the ability to bind S-equol). "antigen-binding portion", not particularly limited, Fab fragment, V_L、V_H、C_LAnd a monovalent fragment consisting of the domain CH 1; f (ab)₂Fragments, bivalent fragments containing 2 Fab fragments (typically 1 starting from the heavy and light chains) linked in the hinge region by disulfide bridges; v_HAnd Fd fragment consisting of CH1 domain; an FV fragment comprising VL and VH domains of a single two-arm antibody; a single domain antibody (dAb) fragment consisting of the VH domain; and isolated Complementarity Determining Regions (CDRs) or combinations thereof. Furthermore, antigen-binding moieties, methods using recombination, V_LAnd V_HThe monovalent molecules formed by the region pairs can be prepared as a protein chain and can be linked through an artificial peptide bond connector.

Alternatively, the antigen-binding portion may be encoded by a single domain antibody, Maxi Body, Mini Body, internal Body, DIA Body, thoria Body, lipop Body, v-NAR, or Bis-scFv.

The type of organism from which the antibody is derived is not particularly limited, and may be a human antibody, a mouse antibody, a goat antibody, or the like, depending on the organism to be used or the purpose to be used. In the case of a human antibody, it means that both the framework and CDR regions of the antibody contain an antibody having a variable region derived from a human origin sequence. When the antibody contains a constant region, the term "human arrangement" as the constant region means, for example, an arrangement derived from a human germ cell line or an abruptly-modified human germ cell line. Furthermore, the antibody can be chimeric based on a fragment antibody derived from 2 or more organism species.

When the antibody is a monoclonal antibody, it exhibits stable binding properties to an antigen. The acquisition of monoclonal antibodies is well known to practitioners in the industry. In addition to the following methods, for example, human single antibodies are produced by B cell-containing hybrid cells obtained by introducing a genetic gene fused into immortalized cells into a non-human animal (for example, a gene-introduced mouse having a genome containing a human heavy chain-introduced genetic gene and a light chain-introduced genetic gene).

The antibody may be, for example, a recombinant antibody such as a recombinant human antibody. Recombinant human antibodies, for example, antibodies in which genetic genes are introduced into human immunoglobulin genetic genes or into animals (e.g., mice) into which chromosomes are introduced or which are isolated from hybrid cells which are then produced; to find host cells transformed with human antibodies, such as antibodies isolated from transfectomas; antibodies isolated from a recombinant combinatorial human antibody database; and producing, discovering, creating or containing the isolated antibody by other means comprising splicing all or a portion of the human immunoglobulin genetic array with other DNA arrays. The framework and CDR regions of such recombinant human antibodies have variable regions derived from the immunoglobulin arrangement of the human germ cell line.

The second half of the method for producing the antibody is described in detail, and the antibody obtained by a known method may be a variant thereof insofar as it has a binding ability to recognize and bind to RS-equol. For example, as a starting material at least a portion of an antibody, e.g., a full-length heavy and/or light chain arrangement, V_HAnd/or V_LNovel antibodies can also be obtained by aligning or modifying the constant region to introduce a mutation or the like depending on the binding. In other words, a PEG chain or the like can be introduced into the obtained antibody. The techniques for such alteration of antibodies are known per se to practitioners in the art.

The antibody can be provided with a labeling element as required. The labeling element is not particularly limited, and a conventionally known labeling substance can be suitably selected and used. The labeling substance is not particularly limited, and typical examples thereof include labeling elements utilizing fluorescence, radioactivity, enzymes (e.g., peroxidase, alkaline phosphatase, etc.), phosphorescence, chemiluminescence, coloration, and the like.

The antibody may further comprise, as a labeling element, a substance capable of binding to the labeling element. Finally, these bindable molecules or substances can also be identified by means of a labeling substance. Such substances can be used, for example, in protein-protein interactions, low-molecular-weight compound-protein interactions, and the like. For example, there can be exemplified an antibody in an antigen-antibody reaction or a hapten represented by avidin (streptavidin) -biotin in a biotin system, Digoxigenin (DIG) -Digoxigenin (DIG) in an anti-digoxigenin system, FITC in an anti-FITC system, or the like. In this case, the labeling substance to be finally used for detection is modified so as to have a site for binding to the labeling substance, so that molecules or substances (for example, antigens, in other words, streptavidin, anti-FITC, and the like) on the other hand, which interact with the substance having binding properties, are present.

In addition to being commercially available, such various forms of the marker element are also known to practitioners of the art as methods for modifying antibodies with the marker element. Therefore, it is possible for the practitioner of the present invention to obtain various labeling elements and then add functional groups such as amino acids and carbonyl groups to the antibody.

(method for producing anti-equol antibody)

As disclosed in the present specification, the method for producing an antibody, RS-equol (racemic modification) can comprise an animal immunization step using a complex of a complexed carrier protein and a step of obtaining an antibody having a specific binding ability to RS-equol from a hybrid cell derived from a spleen cell of the above-mentioned animal.

The step of obtaining the antibody can include a step of separating a hybrid cell having an antibody-producing ability and having a specific binding ability to RS-equol from a hybrid cell obtained by fusing a spleen cell isolated from the animal and a myeloma cell, and a step of producing the antibody using the hybrid cell. According to the present production method, the present antibody as a monoclonal antibody and the present antibody composition can be efficiently obtained.

(immunization procedure)

Hereinafter, the immunization step will be described. The carrier protein for obtaining the complex is suitably selected from known carrier proteins such as Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA), Rabbit Serum Albumin (RSA), and bovine Thyroglobulin (THY). The carrier protein may be a variant other than those derived from various vertebrate species such as human, mouse, rabbit, and goat.

The complex can be obtained by introducing a carrier protein such as KLH into the carbonyl group having the hydroxyl carbonyl group of RS-equol (racemic body) according to a conventional method. In addition, in order to introduce both carbonyl groups in the hydroxyl groups at the 2-positions, 4 ' -and 7-positions of equol, both of them introduced the 4 ' -complex and the 7-position complex of the carrier protein via the hydroxyl group at the 4 ' -position. In other words, the carrier protein complex of RS-equol can be used by using at least 1 combination of the R-body complex and the S-body complex from among 4 complexes of the R-body 4 '-complex, the R-body 7-complex, the S-body 4' -complex, and the S-body 7-complex. It is desirable to combine these 4 composites and use them. When an animal is immunized with the complex thus obtained, the antibody production ability is evaluated using the specific binding ability as an index for RS-equol (racemic body), and a monoclonal antibody suitable for the antibody composition can be efficiently obtained.

The RS-equol-carrier protein complex thus obtained is used to immunize a vertebrate. The type of the immune vertebrate (hereinafter, also referred to as an immune animal) is not particularly limited, and a transgenic or non-transgenic non-human animal can be exemplified. Suitable examples include mice, goats, rabbits, and the like. As already mentioned, genetically altered non-human vertebrates can be used in order to obtain human antibodies, in order to be able to make human-type antibodies.

Administration to an animal immunized with an immunogen is not particularly limited, and intraperitoneal administration, intravenous administration, and the like are appropriately selected from known methods as needed. Moreover, suitable, complete, incomplete aids can be used in the administration of the complex. Administration of the complex enables sufficiently repeated immunization. Usually, about 2 to 5 times, the complex can be administered.

By immunizing a vertebrate with such an immunogen and evaluating the antibody value for RS-equol, the production of the antibody can be confirmed in the immunized animal. In the immunized animal, blood can be collected from the immunized animal as appropriate, and the antibody value can be evaluated using RS-equol or the like, if the antibody is intended to be produced. The evaluation can be suitably carried out by a known method such as ELISA.

Spleen was excised from an immunized animal in which high-titer RS-equol could be confirmed, spleen cells were prepared, and then mouse myeloma cells such as P3U1 cells and polyethylene glycol were subjected to cell fusion according to a known cell fusion method, and then hybrid cells were requested. Furthermore, the selection of hybrid cells, for example, hybrid cells can be performed by culturing the hybrid cells in a normal medium (HAT medium) using a drug-resistant strain of myeloma 8-azaguanine, for example, for 10 to 14 days. Then, the selected hybrid cells produce antibody titer, and are analyzed by ELISA using RE-equol, and hybrid cells producing antibodies with high antibody titer are separated by limiting dilution or the like. The monoclonal antibody can be purified from the culture supernatant obtained by culturing the separated hybrid cells in a suitable medium by a suitable method such as ammonium sulfate fractionation and affinity chromatography.

Monoclonal antibodies (hybrid cells) to which the antibody composition is applied can be selected by evaluating the cross-reactivity (2) to (4) of various isoflavones as described above, such as precursors of equol, in addition to the cross-reactivity (1) of S-equol and R-equol.

The amino acid sequence of such a monoclonal antibody and antibody fragment thereof can be obtained by analyzing the amino acid sequence of the antibody and/or by the base sequence of the antibody-encoding region obtained from the hybrid cell.

A polynucleotide such as DNA encoding the amino acid sequence of the heavy chain variable region and the light chain variable region of the monoclonal antibody thus obtained is also an embodiment of the present disclosure. Moreover, a host cell that retains such an expression vector is also an embodiment of the present disclosure. The expression vector may be appropriately selected from the control fields of promoters, terminators, and the like, depending on the type of host, in addition to the appropriate forms known to practitioners in the art. Polynucleotides, such as DNA, and, for example, cDNA.

The heavy chain variable region and its hypervariable region, and the light chain variable region and its hypervariable region of a monoclonal antibody capable of comprising the present antibody composition are as described above.

(method of measuring equol in biological sample)

The method for measuring equol disclosed in the present specification can include a step of bringing the antibody into contact with equol in a biological sample. According to this step, the antibody binds to equol based on the specific binding ability of the antibody. This equol-antibody complex can be detected by various methods or by means of a marker element provided to the antibody, and the presence or absence of equol or its concentration (amount) can be determined.

The use of antibodies to detect bound antigen based on its specific binding capacity, the assay itself, is well known to practitioners in the art. The known method can be applied to the present measurement method. Examples of such known methods include ELISA, RIA, and immunochromatography, as well as sedimentation reaction of complex and immunoelectrophoresis based on sedimentation reaction, single-cell immunonucleic acid method, and cross-immunoelectrophoresis method; latex agglutination methods using complex agglutination reaction, western blotting methods (detection methods such as enzyme immunoassay and chemiluminescence), and detection methods in immunohistology.

In the present measurement method, the conditions for measuring the concentration of equol, the concentration of antibody, and the like can be set as appropriate by the practitioner of the industry, depending on the type of the measurement method, and the like. Since the antibody has high antibody-binding property, for example, immunochromatography is used as a carrier of a porous solid phase, and a detection limit of 0.5. mu.M can be ensured as the concentration of equol. The threshold value is detected, and is, for example, 0.4 μ M, and is, for example, 0.3 μ M, and is, for example, 0.2 μ M, and is, for example, 0.1 μ M, and is, for example, 0.05 μ M. And, for example, 0.04 μ M, and for example, 0.03 μ M, and for example, 0.02 μ M, and for example, 0.01 μ M.

In addition, in such immunochromatography, the equol concentration exceeding the detection limit, for example, the equol quantitative value in the range of 0.01. mu.M to 180mM, was confirmed to have a high correlation with the quantitative value by High Performance Liquid Chromatography (HPLC). From the above, it was found that the present antibody can detect equol with high sensitivity and accuracy and can quantify equol even with simple setup such as immunochromatography.

The method can be applied to the determination of equol in various biological samples. The biological sample is not particularly limited, and various samples derived from animals, plants, and microorganisms may be used. For example, in the case of animals such as humans, urine, blood, saliva, tears, serum, plasma, feces, tissue or tissue extract, and the like can be given. In the case of plants, food materials and the like can be exemplified. In the case of microorganisms, culture clarification layers, cell disruptions, cell extracts, and the like can be exemplified.

For detection or measurement of S-equol, a known method such as ELISA or antibody chromatography can be suitably used, based on the formation of an equol-antibody complex. In the present measurement method, when a complex of an equol antibody is detected, a secondary antibody may be used as appropriate for the present antibody.

(Equol measuring apparatus)

The equol measurement device disclosed in the present specification can have the present antibody bound or capable of binding to a solid-phase carrier. According to the present device, equol can be easily measured with high sensitivity and high accuracy in order to obtain the present antibody composition having excellent detection sensitivity.

The measuring apparatus can take various forms, and examples of the solid phase carrier include immunochromatography solid phase carriers such as a rod-like and a long strip-like carrier, latex microspheres, and plate-like solid phase carriers such as glass and plastic.

(Equol determination kit)

Kits for the determination of equol disclosed in the specification can contain the antibody compositions. The kit may further contain a reagent for detecting an equol-antibody complex. The labeling substance may be a labeling substance, a labeling substance conjugate, or a labeling substance conjugate.

In addition to the kit, the kit may contain an intentional detection blocking reagent, a washing solution, a buffer, and the like by ELISA or antibody chromatography.

Hereinafter, examples as specific examples will be described in order to more specifically describe the disclosure of the present specification. The following examples are given for the purpose of illustrating the disclosure of the present specification and are not intended to limit the scope thereof.

[ example 1]

(preparation of anti-equol monoclonal antibody)

(1) Preparation of immunogens

Equol is a low molecular weight, and thus cannot be used as an antigen. Therefore, 66mg of RS-equol (racemate) (Toronto Research Chemicals), benzyl bromoacetate, and 90mg of potassium carbonate were allowed to stand at room temperature for one night, and then contact reduction with palladium on carbon was carried out to hydrolyze equol, and then carboxymethyl groups were introduced into carbon atoms at the 4-and 7-positions to obtain equol. The carboxymethyl groups at the 4-and 7-positions are combined to give 2 kinds of such carboxymethyl equols. After 20mg of these 2 carboxymethylequol, EDC15mg and sulfo-NHS 21mg were reacted in DMF for 24 hours, they were reacted with 3mL of a PBS solution containing 46mg of Keyhole Limpet Hemocyanin (Keyhole Limpet Hemocyanin, KLH) at 25 ℃ for 4 hours to prepare an equol-KLH conjugate bound by amino binding.

(2) Immunization of mice and establishment of hybrid cells

0.5mg/mL PBS of the equol-KLH conjugate and an equal amount of adjuvant were mixed well and 0.15mL was administered intraperitoneally to female BALB/c mice (aged 6 weeks). After 2 additional immunizations every 2 weeks and 3 days of the final immunization, spleens of the mice were excised and fused with myeloma cells (P3U1) to produce hybrid cells. Fusion of these cells, spleen cells and myeloma cells were mixed at a ratio of 1: 5, and fusion was performed using PEG according to a conventional method. After culturing the hybrid cells in HAT medium for 10-14 days, hybrid cells are selected.

The antibody titer of the clarification layer was measured by the above-described ELISA method on the culture clarification layer of the well formed from the cell population of the hybrid cells, and the hybrid cells produced with the antibody having a high antibody titer were separated by the limiting dilution method. In addition, for screening for the isolation of hybrid cells, RS-equol was used as an antigen. After 10% of the separated fused cells were cultured in a DMEM medium, the cells were purified by using an IgG column to obtain a monoclonal antibody (anti-equol antibody).

(3) Evaluation of antibodies

The obtained monoclonal antibody was subjected to amino acid sequence analysis and base sequence analysis. At this time, 2 types (κ and λ) were detected as light chain variable regions, and 1 type was detected as heavy chain variable regions. From this, it was found that the hybrid cells obtained were a mixture of 2 types of hybrid cells, and the antibodies obtained were a mixture (composition) of 2 types of monoclonal antibodies. The hypervariable region and overall amino acid arrangement of the kappa chain light chain variable region represent SEQ ID NO: 4 to 6 and 11, the base sequences of which code for SEQ id no: 18 to 21. And, the hypervariable region and the totality of amino acids of the lambda light chain variable region represent SEQ ID NO: 7 to 9 and 12, the base sequences of which code for SEQ ID NOs: 23-26. And, the hypervariable region and overall amino acid arrangement of the IgG heavy chain variable region represents SEQ ID NO: 1 to 3 and 10, the bases and the sequences of which code for SEQ ID NO: 13 to 16.

[ example 2]

(evaluation of crossability of anti-equol monoclonal antibody composition)

In order to examine the crossability of the obtained antibody composition, the reactivity of 9 kinds of compounds (S-equol, daidzein, genistein, daidzein, ellagic acid dihydrate, catechin monohydrate, gallic acid, apigenin, DMA) containing S-equol was investigated. First, a standard curve was created using S-equol. 9 kinds of S-equol-containing compounds were prepared at 1. mu.M and 10. mu.M, and the antibodies were allowed to react at each concentration, and the crosshatch was calculated from the standard curve. The results are shown in table 1 below.

[ TABLE 1]

As shown in table 1, the resulting antibody compositions, except for S-equol, were specific for R-equol, and similar compounds exhibited only very low crossovers.

[ example 3]

(preparation of equol Standard Curve)

The obtained antibody composition was subjected to stepwise dilution in a dilution buffer using a standard solution of S-equol at a maximum concentration of 3. mu.M, and standard curves in immunochromatography using standard solutions at concentrations of 3, 1, 0.3, 0.1, 0.03, 0.01, and 0. mu.M are shown in FIG. 1. As shown in FIG. 1, the measurement range was 0.01. mu.M to 3. mu.M. The antibody composition obtained in example 1 was found to have very high detection sensitivity.

The immunochromatography was performed under the following conditions using immunoassay (product of Aisin Seikagaku K.K.) as an immunochromatography apparatus. After the standard solution, the buffer and the colloidal gold anti-equol antibody were each set to room temperature, it was assumed that colloidal gold dissolved the antibody solution at a predetermined concentration in 1400. mu.l of the buffer. Mu.l of the antibody solution and equol standard solutions (0, 0.01, 0.03, 0.1, 0.3, 1 and 3. mu.M) were extracted from the microtube, 4. mu.l of the urine specimen and the conditioned urine were added to each of the samples and mixed, 75. mu.l of the mixed solution in the microtube was dropped from a predetermined portion of the immunochromatography apparatus, developed in a humidified chamber and allowed to stand for 20 minutes, and then color development was detected by colloidal gold.

[ example 4]

(evaluation by HPLC according to equol measurement results by immunochromatography)

With respect to the correlation between the method using the previous measurement HPLC and the antibody composition obtained in example 1, and the method of performing an immunostaining assay under the same conditions as in example 3, 30 test materials with respect to human urine were evaluated. FIG. 2 shows the comparative measurement results. As shown in FIG. 2, the assay is recognized as having a straight-line relationship in the middle of the HPLC method, where the similarity curve y is 0.4979x-0.3677 and the correlation coefficient is R²＝0.982。

[ Prior art documents ]

[ patent document ]

[ patent document 1] publication No. 2010-169507