JP7002101B2 - Anti-glycyrrhetinic acid antibody and its utilization - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act, issued on August 25, 2016, 63rd Annual Meeting of the Japanese Society of Biopharmaceuticals Toyama 2016 Lecture Abstracts, p. 136, Japan Society of Biopharmaceuticals [Publications, etc.] 2016 Held on September 25, 63rd Annual Meeting of the Japanese Society of Biopharmaceuticals Toyama 2016, Toyama International Conference Center (1-2 Otemachi, Toyama City, Toyama Prefecture)

The present specification relates to an anti-glycyrrhetinic acid antibody and its utilization.

Licorice is an important crude drug widely used in medical and general Chinese herbal extract preparations, and glycyrrhizin, which is the main component, is absorbed as glycyrrhetinic acid by hydrolysis by the intestinal flora and exhibits pharmacological action. Glycyrrhetinic acid is known to exert various pharmacological actions such as anti-inflammatory action, anti-allergic action, and antiviral action. On the other hand, glycyrrhetinic acid is said to be involved in the development of pseudoaldosteronism as a side effect.

Although the mechanism of action in these drug effects and the occurrence of side effects is being elucidated, target molecules such as receptors have not been determined.

In order to elucidate the mechanism of action of glycyrrhetinic acid at the molecular level in more detail, an antibody that specifically reacts with glycyrrhetinic acid is required. In addition, monitoring of glycyrrhetinic acid in blood is important for monitoring drug efficacy and the occurrence of side effects. On the other hand, an anti-glycyrrhetinic acid monoclonal antibody has already been reported (Patent Document 1).

Japanese Unexamined Patent Publication No. 5-68584

However, in vivo, for example, in serum, glycyrrhetinic acid binds to albumin. Therefore, in the case of measuring glycyrrhetinic acid in serum, the above-mentioned monoclonal antibody could not specifically bind to glycyrrhetinic acid without deproteinization. Therefore, the operation is complicated.

One object of the present specification is to provide a more practical anti-glycyrrhetinic acid antibody and its utilization.

As a result of diligent studies, the present inventors have been able to obtain an anti-glycyrrhetinic acid antibody that can specifically bind to free glycyrrhetinic acid and also specifically bind to glycyrrhetinic acid in a form that can exist in the living body. rice field. According to the present specification, the following means are provided based on this finding.

(1) An anti-glycyrrhetinic acid antibody,
An antibody having specific binding ability to glycyrrhetinic acid and glycyrrhetinic acid bound to albumin in the living body of a mammal.
(2) The antibody according to (1), which does not have a specific binding ability to glycyrrhizin.
(3) The antibody according to (1) or (2), which is a monoclonal antibody.
(4) Any of (1) to (3), which does not have a specific binding ability to one or more compounds selected from the group consisting of glycyrrhizin, oleanolic acid, β-amyrin, psychogenin-A and hederagenin. The antibody described in Crab.
(5) Described in any of (1) to (4), which does not have a specific binding ability to one or more compounds selected from the group consisting of aldosterone, cholesterol, cortisone, hydrocortisone and corticosterone. Antibodies.
(6) The antibody according to any one of (1) to (5), which is obtained by using a complex having a carrier protein via a carboxy group of glycyrrhetinic acid as an immunogen.
(7) A method for detecting glycyrrhetinic acid in a biological sample.
A step of contacting the antibody according to any one of (1) to (6) with glycyrrhetinic acid in the sample to form a complex of the antibody and the glycyrrhetinic acid.
How to prepare.
(8) The method according to (7), further comprising a step of detecting the complex using a secondary antibody.
(9) A method for searching for a target molecule of glycyrrhetinic acid.
A step of contacting a possible complex of glycyrrhetinic acid and one or more target molecule candidates with the antibody according to any one of (1) to (6) to form a glycyrrhetinic acid-antibody complex. When,
A method comprising the step of separating or identifying the one or more target molecule candidates specifically bound to glycyrrhetinic acid in a glycyrrhetinic acid-antibody complex.
(10) A reagent for detecting glycyrrhetinic acid, which is a reagent for detecting glycyrrhetinic acid.
A reagent containing the antibody according to any one of (1) to (6).
(11) A kit for detecting glycyrrhetinic acid.
A kit containing the antibody according to any one of (1) to (6).
(12) A method for producing an anti-glycyrrhetinic acid antibody.
The process of immunizing an animal with a complex containing a carrier protein via the carboxy group of glycyrrhetinic acid,
A step of obtaining an antibody having a specific binding ability to an albumin-containing complex glycyrrhetinic acid-albumin complex from the animal or a hybridoma derived from the spleen cell of the animal via a carboxy group of glycyrrhetinic acid.
How to prepare.
(13) The antibody acquisition step is a step of separating an antibody having a specific binding ability from the animal to the glycyrrhetinic acid-albumin complex, which is a complex comprising albumin via the carboxy group of glycyrrhetinic acid. The method according to (12).
(14) The antibody acquisition step is a glycyrrhetinic acid-albumin complex which is a complex comprising albumin via a carboxy group of glycyrrhetinic acid from a hybridoma obtained by fusing splenocytes and myeloma cells separated from the animal. The method according to (12), comprising a step of separating a hybridoma having an ability to produce an antibody having a specific binding ability to the same, and a step of producing the antibody using the hybridoma.
(15) An immunogen for obtaining an anti-glycyrrhetinic acid antibody comprising a carrier protein via an amide bond containing a carbonyl derived from the carboxy group of glycyrrhetinic acid.

It is a figure which shows the evaluation result of the antibody titer of the monoclonal antibody produced in an Example. It is a figure which shows the evaluation result of the crossing with glycyrrhetinic acid (18β-GA, 18α-GA) and glycyrrhizin (GL) of a monoclonal antibody. It is a figure which shows the evaluation result of the crossing property with the glycyrrhetinic acid analog of a monoclonal antibody. It is a figure which shows the evaluation result of the crossing with a monoclonal antibody and a steroid compound. It is a figure which shows the evaluation result of the antibody titer of a biotinylated antibody. It is a figure which shows the 18β-GA calibration curve of a biotinylated antibody. It is a figure which shows the search result of the target molecule of 18β-GA by the immunoprecipitation method.

The disclosure herein relates to anti-glycyrrhetinic acid antibodies and their use. According to the anti-glycyrrhetinic acid antibody disclosed herein (hereinafter, also simply referred to as the present antibody), whether it is free glycyrrhetinic acid or glycyrrhetinic acid bound to albumin, it is specific. It can bind and detect or quantify glycyrrhetinic acid. Therefore, it is possible to detect and quantify glycyrrhetinic acid in a biological sample by avoiding the conventionally required deproteinization operation. Therefore, it is possible to search for the mechanism of action of glycyrrhetinic acid at the molecular level as well as the target molecule.

Hereinafter, the antibodies disclosed in the present specification and embodiments relating to their use will be described in detail.

(Anti-glycyrrhetinic acid antibody)
The specific binding ability of this antibody can be listed as follows, for example. This antibody can have at least the specific binding ability of (1). It can further have any one of the following (2) to (4) or two or more specific binding abilities. In addition, in this specification, "specific binding ability" or "specific binding" means the ability to bind and bind based on the antigen recognition ability of an antibody, that is, an immunoglobulin. .. The structure of glycyrrhetinic acid is shown below.

(1) It specifically binds to free glycyrrhetinic acid and glycyrrhetinic acid complexed with albumin. It does not specifically bind to albumin.
(2) Does not specifically bind to glycyrrhizin.
(3) For compounds having an oleanane skeleton other than glycyrrhizin, particularly oleanane-type triterpenes, for example, one or more compounds selected from the group consisting of oleanolic acid, β-amyrin, psychogenin-A and hederagenin. Does not specifically bind.
(4) It does not specifically bind to one or more compounds selected from the group consisting of steroid compounds such as aldosterone, cholesterol, cortisone, hydrocortisone and corticosterone.

Such specific binding ability can be obtained by a cross-reactivity evaluation method based on a well-known antigen-antibody reaction of those skilled in the art such as competitive ELISA.

Regarding the specific binding ability of (1) above, this antibody contains 18α-glycyrrhetinic acid (18α-GA) and 18β-glycyrrhetinic acid, which are stereoisomers of hydrogen atoms that bind to the carbon atom at the 18th position of glycyrrhetinic acid. (18β-GA) may be identified and specifically bound to each of them, or may be specifically bound to any of them without distinguishing between them.

If this antibody recognizes GA regardless of 18α-GA and 18β-GA and does not recognize glycyrrhizin or other compounds having an oleanane skeleton and steroid compounds, the A ring, B ring and C ring in the GA structure. It can be said that the area is identified.

Since this antibody has the specific binding ability of (1) above, glycyrrhetin exists in an in vivo environment (in vivo) existing in a complex bound to albumin or in an in vitro environment similar to the in vivo environment (in vitro). Acids (hereinafter, unless otherwise specified, these forms of glycyrrhetinic acid are collectively referred to as glycyrrhetinic acid or GA) can be detected and quantified. Therefore, glycyrrhetinic acid can be easily detected or quantified by avoiding the deproteinization operation. Conventionally, in order to detect glycyrrhetinic acid, a protein removal operation is required, and therefore, it is extremely difficult to identify the target molecule (protein, etc.) as a target molecule even in the molecular-level action mechanism of glycyrrhetinic acid. However, such inconvenience is eliminated.

The present antibody is an intact antibody having a specific binding ability to an arbitrary antigen (for example, GA in the present specification) or an antigen-binding moiety having the binding ability in the known technique at the time of filing the present application. It can be said that it contains a part. This antibody can take various embodiments based on the following contents in addition to the common general knowledge as of the filing of the present application.

The "antigen binding portion" of an antibody means one or more fragments of an intact antibody that specifically retains the ability to bind to any antigen (eg, GA; the "antigen binding portion" is particularly limited. Monovalent fragment consisting of Fab fragment, _VL , _VH , _CL and CH1 domain; F (ab) ₂ fragment, two Fab fragments linked by a disulfide bridge at the hinge region (generally heavy chain). And a bivalent fragment consisting of one from the light chain); an Fd fragment consisting of the _VH and CH1 domains; an Fv fragment consisting of the _VL and _VH domains of a single arm of the antibody; a single domain antibody consisting of the _VH domain. Fragments of various forms such as (dAb) fragments; and isolated complementarity determination regions (CDRs) or combinations thereof can be included, and antigen binding moieties can be _VL and V using recombinant methods. It can be linked by an artificial peptide linker that can be produced as a single protein chain in which _H regions are paired to form a monovalent molecule.

In addition, the antigen binding moiety may be integrated into a single domain antibody, maxibody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR and bis-scFv.

The species from which this antibody is derived is not particularly limited, but may be a human antibody, a mouse antibody, a goat antibody, or the like, although it varies depending on the organism to which it is applied and the purpose. The term human antibody means that both the framework of the antibody and the CDR regions include an antibody having a variable region derived from a sequence of human origin. Further, when the antibody comprises a constant region, the constant region is also meant to be derived from such a human sequence, eg, a human germline sequence or a mutant human germline sequence. Further, an antibody based on a fragment derived from two or more species can be called a chimeric antibody.

This antibody may be a polyclonal antibody or a monoclonal antibody. If it is a monoclonal antibody, it can exhibit stable binding performance to an antigen. Acquisition of polyclonal antibodies and monoclonal antibodies is well known to those of skill in the art. In addition to the methods described below, for example, human monoclonal antibodies are obtained from transgenic non-human animals fused to immortalized cells (eg, transgenic mice having a genome containing a human heavy chain transgene and a light chain transgene). It is produced by a hybridoma containing B cells.

Further, the present antibody may be a recombinant antibody such as a recombinant human antibody. Recombinant human antibody is, for example, an antibody isolated from an animal (eg, mouse) into which a human immunoglobulin gene has been gene-introduced or chromosome-introduced or a hybridoma produced from it; transformed to express a human antibody. Antibodies isolated from host cells, such as transfectomas; antibodies isolated from recombinant combinatorial human antibody libraries; and splicing of all or part of the human immunoglobulin gene sequence onto other DNA sequences. Includes antibodies produced, expressed, prepared or isolated by other means including. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.

The method for producing this antibody will be described in detail later, but this antibody is a mutant thereof as long as the antibody obtained by a known method has a binding ability to recognize and bind to GA. You may. For example, by making modifications such as introducing a mutation into at least a portion of the antibody as a starting material, eg, a full-length heavy chain and / or light chain sequence, a VE _H and / or _VL sequence or a constant region bound thereto. , It is also possible to obtain a new antibody. It is also possible to introduce a so-called peg chain into the obtained antibody. The method of modifying such an antibody itself is well known to those skilled in the art.

The antibody can be optionally provided with a labeling element. The labeling element is not particularly limited, but conventionally known labeling substances can be appropriately selected and used. The labeling substance is not particularly limited, but typically includes a labeling substance using fluorescence, radioactivity, an enzyme (for example, peroxidase, alkaline phosphatase, etc.), phosphorescence, chemiluminescence, coloring, and the like.

The present antibody may include, as a labeling element, a substance capable of binding to the labeling element. It may be provided with a molecule or substance capable of binding these so that it can be finally identified by the labeling substance. As such substances, protein-protein interactions, small molecule compounds-protein interactions, and the like can be used. For example, antibodies in an antigen-antibody reaction, biotin in an avidin (streptavidin) -biotin system, digoxigenin in an anti-digoxigenin (DIG) -digoxigenin (DIG) system, or haptens represented by FITC in an anti-FITC-FITC system, etc. Can be mentioned. In this case, the labeling substance ultimately used for detection is the other molecule or substance that interacts with the substance that has binding properties to such labeling substance (eg, antigens such as streptavidin, anti-FITC, etc.). Labeling substance Modified to be provided as a site for binding to a binding substance.

Labeling elements of these various embodiments are commercially available, and methods of modifying antibodies with labeling elements are well known to those of skill in the art. Therefore, those skilled in the art can obtain various labeling elements and appropriately use a functional group such as an amino group or a carboxy group for the antibody.

(Manufacturing method of anti-glycyrrhetinic acid antibody)
The method for producing the antibody disclosed herein is a step of immunizing an animal with a complex comprising a carrier protein via the carboxy group of glycyrrhetinic acid, and a hybridoma derived from the animal or the spleen cell of the animal. A step of obtaining an antibody having a specific binding ability to a complex glycyrrhetinic acid-albumin complex comprising albumin via the carboxy group of glycyrrhetinic acid can be provided.

The antibody acquisition step can be a step of separating an antibody having a specific binding ability from the animal to the glycyrrhetinic acid-albumin complex, which is a complex comprising albumin via the carboxy group of glycyrrhetinic acid. According to the present production method, the present antibody, which is a polyclonal antibody, can be efficiently obtained.

Further, in the antibody acquisition step, a hybridoma obtained by fusing splenocytes and myeloma cells separated from the animal is converted into a glycyrrhetinic acid-albumin complex, which is a complex comprising albumin via the carboxy group of glycyrrhetinic acid. On the other hand, a step of separating a hybridoma having an ability to produce an antibody having a specific binding ability and a step of producing the antibody using the hybridoma can be provided. According to the present production method, the present antibody, which is a monoclonal antibody, can be efficiently obtained.

In order to obtain an antibody having a specific binding ability possessed by this antibody, for example, a carrier protein was introduced via the carbonyl of a carboxy group containing the 30-position carbon atom of GA, which is an antigen, for example, GA-KLH (Keyhole limpet). A GA-carrier protein complex such as a hemocyanin (hemocyanin) complex can be used as an immunogen. By using such an immunogen, it is possible to make an immune animal produce an antibody having a specific binding ability to a free GA and also to a GA-albumin complex in a biological environment or the like. Furthermore, a polyclonal antibody or a monoclonal antibody can be obtained by evaluating the antibody-producing ability of the serum obtained from an immune animal or a hybridoma derived from the spleen cell of an immune animal using the specific binding ability to the GA-albumin complex as an index. It can be obtained efficiently.

(Immune process)
Hereinafter, the immune process will be described.

(GA-carrier protein complex)
The GA-carrier protein complex used in this production method can be a complex in which a carrier protein is introduced via the carbonyl of a carboxy group containing the 30-position carbon atom of GA. The GA used here is 3β-hydroxy-11-oxooleana-12-ene-30-acid (IUPAC) ((3β, 20β) -3-hydroxy-11-oxooleana-12-ene-29-acid (CAS)). It may be an 18α form, an 18β form, or a mixture thereof.

The carrier protein is appropriately selected from known carrier proteins such as KLH, bovine serum albumin (BSA), ovalbumin (OVA), rabbit serum albumin (RSA), and bovine thyroglobulin (THY). The carrier protein may be derived from various vertebrate species such as human, mouse, rabbit, goat, etc., or may be a mutant.

The schemes for producing such GA-KLH complex and GA-BSA complex are shown below.

The introduction site of the carrier protein for GA can be a carboxy group containing a carbon atom at the 30-position of GA. By using such a site as a site for introducing a carrier protein, the present antibody having a specific binding ability to GA can be obtained.

When a carboxy group containing a carbon atom at the 30-position of GA is used as an introduction site, a carrier protein can be introduced via the carbonyl of the carboxy group to form a GA-carrier protein complex. Various methods well known to those skilled in the art can be used for such an introduction form. In the carbonyl-mediated introduction form of the carrier protein, for example, the carboxy group and the amino group of the carrier protein are bound to each other using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). be able to. It is also possible to introduce a linker peptide into the carboxy group by an acid amide bond to react the amino group of the linker peptide with the carboxy group activated by the EDC of the carrier protein. Furthermore, the Cys residue in the linker peptide introduced by the acid amide bond and the amino group of the carrier protein can be ligated by reacting with N- (γ-4-maleimidebutylyloxy) succinimide (GMBS). can.

In order to obtain this antibody, it is preferable to directly introduce a carrier protein into the carbonyl of the carboxy group at the 30-position of GA without interposing a linker peptide or the like. That is, as described in the above scheme, by binding a carrier protein to the above carbonyl via a secondary amino group (-NH-), for example, it was closely bound to a protein such as BSA in serum. The GA of the state can also be recognized.

The GA-carrier protein complex thus obtained immunizes vertebrates. The type of vertebrate to be immunized (hereinafter, also referred to as an immune animal) is not particularly limited, and examples thereof include non-human animals that have not been genetically modified or recombinant. Preferred examples include mice, rats, goats, rabbits and the like. As described above, in order to obtain a human antibody, a non-human vertebrate genetically modified so that a humanized antibody can be produced can be used.

The administration of the immunogen to an immunized animal is not particularly limited, but is appropriately selected from well-known methods such as intraperitoneal administration and intravenous administration as needed. In addition, a complete adjuvant or an incomplete adjuvant can be appropriately used for administration of the complex. Administration of the complex can be repeated until fully immunized. Usually, the complex is administered about 2 to 5 times. Whether or not the intended antibody is produced in the immune animal is appropriately determined by collecting blood from the immune animal and evaluating the antibody titer using the GA-albumin complex such as GA-BSA. For the evaluation, a known method such as ELISA can be appropriately used.

By immunizing vertebrates using such immunogens and evaluating the antibody titer against the GA-BSA complex, the production of this antibody in immune animals can be confirmed. Hereinafter, one aspect of the polyclonal antibody acquisition step will be described.

(Acquisition process of this antibody)
To obtain a polyclonal antibody from an immune animal, an antibody having a specific binding ability to the GA-albumin complex, which is a complex containing albumin via the carboxy group of glycyrrhetinic acid, can be separated from the animal. In general, this antibody can be obtained by performing an antibody evaluation in the blood (serum) of an immune animal using the complex. The evaluation of the antibody titer can be carried out by a well-known person skilled in the art, for example, the ELISA method. If the antibody titer can be confirmed, if necessary, after final immunization of the immunized animal, all blood is collected from the immunized animal, the blood is coagulated, and then the serum is collected. Recovery of antibodies from antisera is well known to those of skill in the art.

When obtaining a monoclonal antibody, the spleen was removed from an immune animal whose antibody titer against the GA-BSA complex could be confirmed, splenocytes were prepared, and then mouse myeloma cells such as P3U1 cells and polyethylene glycol were used. Cell fusion is performed by a known cell fusion method to selectively obtain only hybridomas, and then the hybridomas are cloned and screened using a complex such as GA-BSA to obtain a specific hybridoma. .. A monoclonal antibody can be obtained from the culture supernatant by culturing the hybridoma.

In addition to the GA-BSA complex, the polyclonal antibody and the monoclonal antibody (hybridoma) became a complex with various compounds related to the specific binding ability of this antibody described above, that is, free glycyrrhetinic acid and albumin. While it has a specific binding ability to glycyrrhetinic acid, it does not have a binding ability to albumin, a binding ability to glycyrrhetin, a compound having an oleanane skeleton other than glycyrrhetin, especially an oleanane-type triterpene and a steroid compound. Polyclonal antibodies and monoclonal antibodies can be selected based on cross-reactivity.

An immunogen, a hybridoma, and an evaluation complex for producing the antibody used in the production method are also one aspect of the present disclosure. A polynucleotide having a base sequence encoding the amino acid sequence of the present antibody is also an aspect of the present disclosure. Such polynucleotides can be obtained by analysis of the amino acid sequence of the present antibody and / or by base sequence analysis of the antibody coding region obtained from the hybridoma. Further, an expression vector that retains the polynucleotide expressively in the intended host cell is also an aspect of the present invention. As the expression vector, in addition to an appropriate form well known to those skilled in the art according to the type of host, a control region such as a promoter or a terminator can be appropriately selected. The polynucleotide is, for example, DNA and, for example, cDNA.

(Glycyrrhetinic acid detection reagent and glycyrrhetinic acid detection kit)
The GA detection reagent disclosed herein can include the antibody. Also, the kits disclosed herein can include the antibody. According to this reagent and the kit, GA in a biological sample can be detected and quantified without a deproteinization operation.

The kit can further include a reagent for detecting a complex of the antibody with GA, which is an antigen. Such reagents include, for example, a labeling substance and / or a labeling substance binding substance for detecting a GA-antibody complex, as well as a reagent for introducing these labeling substances and / or a labeling substance binding substance, or a labeling substance. Examples thereof include a secondary antibody that may be provided, a reagent for a labeling substance (for example, a substrate when peroxidase is used as a labeling substance, etc.).

In addition, the kit may include blocking reagents, cleaning solutions, buffers, etc. intended for detection by ELISA or antibody chromatography.

(Method for detecting glycyrrhetinic acid in biological samples)
The method for measuring glycyrrhetinic acid in a biological sample disclosed in the present specification is a step of contacting the present antibody with glycyrrhetinic acid in the sample to form a complex of the present antibody and glycyrrhetinic acid. Can be provided. According to this method, glycyrrhetinic acid can be easily detected or measured without a deproteinization operation. For the detection or measurement of glycyrrhetinic acid, a known method such as ELISA or antibody chromatography based on GA-antibody complex formation can be appropriately adopted. In this detection method, a secondary antibody against this antibody may be appropriately used for detecting the GA-antibody complex.

(Method for searching for target molecule of glycyrrhetinic acid)
The method of searching for a target molecule of glycyrrhetinic acid disclosed herein is a possible complex of GA (eg, not bound to a protein) with one or more potential target molecules and the antibody. To provide a step of forming a GA-antibody complex and a step of separating or identifying the one or more target molecule candidates specifically bound to glycyrrhetinic acid in the GA-antibody complex. Can be done. According to this search method, the target molecule of glycyrrhetinic acid can be separated or identified.

A potential complex of one or more potential target molecules with GA is, for example, a free GA and a biological sample in which GA may bind, such as a protein in various organs or tissues such as the liver. A mixed system with an extract or homogenate can be obtained by incubating for an appropriate period of time. The incubation conditions are not particularly limited, but for example, for a few minutes to several hours (typically, about 2 to 3 hours or less, more specifically, about 1 hour or less), stirring as necessary. Incubate at room temperature (for example, about 25 ° C. or higher and 37 ° C. or lower). If necessary, a biocompatible buffer or the like can be used. Thereby, if the target molecule of GA is present in the sample, a GA-target molecule candidate complex can be formed.

This antibody is brought into contact with the possible complex by adding the present antibody to a mixed solution in which GA and the target molecule candidate coexisting which may contain such a complex. The contact conditions are also not particularly limited, but can be, for example, incubation at 4 ° C. for about 8 to 16 hours, or incubation at room temperature (25 ° C. or higher and 37 ° C. or lower) for about 1 to 3 hours. .. As a result, when the complex is present, the first GA-antibody complex in which the present antibody is bound to the GA of the GA-target molecule candidate complex is formed. Further, when the free GA remains, a second GA-antibody complex in which the present antibody is bound to the free GA is formed.

In order to separate or identify the target molecule candidate bound to GA from such a mixed system of GA-antibody complex, the first GA-antibody complex described above is separated and obtained, and the protein is separated from this complex. , Can be identified. In order to efficiently separate the first GA-antibody complex, for example, the antibody is bound to a solid-phase carrier such as a column or beads, and the solid-phase carrier is converted into the first GA-antibody complex. Candidate target molecules to be retained can be isolated or identified.

In order to separate the protein from the first GA-antibody complex, known conditions such as, for example, conditions (heating, alkali, salt) that appropriately denature the protein can be applied. In addition, those skilled in the art can also perform separation, purification and identification of such proteins by applying electrophoresis, column chromatography, and various well-known methods.

Further, even if the second GA-antibody complex coexists, the components of the second GA-antibody complex are GA and the present antibody, which are known, and thus the possibility of such existence is known in advance. By grasping or implementing as a control, the effect of the second GA-antibody complex can be eliminated.

Further, for example, the contact between the target molecule candidate without GA and the present antibody under the same conditions as in the case of containing GA can be used as another control. By doing so, it is possible to eliminate the possibility of a non-specific antibody complex and reliably identify a target molecule candidate that specifically binds in the presence of GA.

As described above, the anti-glycyrrhetinic acid antibody disclosed herein can specifically bind to glycyrrhetinic acid and its form in the biological environment. Therefore, unlike the conventional case, the deproteinization operation is eliminated due to the capture of glycyrrhetinic acid by the antibody, and glycyrrhetinic acid can be easily detected. In addition, it is possible to search for the biodistribution of glycyrrhetinic acid and the target molecule that could not be detected by the deproteinization operation.

Hereinafter, examples as specific examples will be described in order to explain the disclosure of the present specification more specifically. The following examples are intended to illustrate the disclosure herein and are not intended to limit its scope.

(Preparation of anti-glycyrrhetinic acid monoclonal antibody)
(1) Preparation of immunogen Since GA is a small molecule, it cannot be an antigen as it is. Therefore, based on the carbodiimide method, 18β-GA is treated with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS), and then a carrier protein is added to GA. -Protein complex was prepared. Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) were used as carrier proteins to prepare two types of antigens, GA-KLH used for immunization to mice and GA-BSA used for ELISA measurement.

In addition, 20 μmol of glycyrrhetinic acid was added to 400 μl of DMF solution, 5 mg of EDC and 5 mg of Sulfo-NLHS were further added, and the mixture was stirred for 24 hours to prepare a DMF solution of glycyrrhetinic acid. To this solution, 500 μl of KLH 10 mg / ml (PBS) was added, stirred for 4 hours, and dialyzed against the PBS solution to prepare a glycyrrhetinic acid (GA) -KLH complex. The GA-BSA complex was prepared by the same operation using 7.5 mg of BSA instead of 5 mg of KLH.

(2) Immunity to mice Using female BALB / c mice (6 weeks old), the prepared GA-KLH was intraperitoneally administered together with a complete adjuvant (FCA) to perform initial immunization. Then, every other week, four boosters were given with Freund's incomplete adjuvant (FIA). One week after each immunization, blood was collected from the tail vein of the mouse, and the antibody titer in serum against GA-BSA was verified by ELISA. Admitted.

(3) Establishment of hybridoma In order to establish as a monoclonal antibody, the spleen of a mouse showing an increase in antibody titer against GA-BSA was excised, splenocytes were prepared, and then cells using polyethylene glycol with mouse myeloma cell P3U1. Fusion was done. After fusion with P3U1, only hybridoma cells were selected by long-term culture using HAT medium. This is because the de novo pathway is inhibited by aminopterin in the HAT medium, so DNA synthesis proceeds only by the salvage pathway using hypoxanthine and thymidine, but P3U1 provides hypoxanthine guanine phosphoribosyltransferase (HGPRT) required for the salvage pathway. It utilizes the fact that it cannot survive under HAT medium because it is deficient. That is, only hybridoma cells derived from spleen cells, which can utilize the salvage pathway and have infinite proliferation ability derived from P3U1, can survive for a long time in the HAT medium.

After sorting with HAT medium, cloning and screening by ELISA using cell culture, which is the work of isolating clones, were performed according to the limiting dilution method, and single hybridoma cells 6D6 and 6B10 were established. The antibody titer evaluation results of the culture supernatant obtained from these are shown in FIG. As shown in FIG. 1, both monoclonal antibodies specifically bind to the GA-BSA complex, and bind to the GL-BSA complex prepared in the same manner as the GA-BSA complex, and to BSA. I didn't.

ELISA is GA-BSA, GL-BSA and BSA (1 mg / ml each) for coating, Block Ace Powder (DS Pharma Biomedical Co., Ltd.) for blocking agent, and Anti-mouse IgG (whole specific) for secondary antibody. )-Peroxidase antibody produced in goat (1mg / mL) (GE Healthcare), 1% TMB (3,3,5,5,5 Tetramethylbenzidine) (SIGMA / DMF: WAKO): 40 mM citrate / phosphate buffer (pH 5.0): 30% H ₂ O ₂ (50: 5000: 2) was used. The operation was performed as follows. On a 96-well plate, prepare each antigen at 1 μg / ml with PBS, spray 100 μl of each well, allow to stand overnight at 4 ° C., and then use 200 μl of PBS solution (TPBS solution) containing 0.05% Tween 20 wash solution. After washing three times, 200 μl / well of a 1% aqueous solution of the blocking agent was sprayed, incubated at 37 ° C. for 1 hour, and then washed with a TBS solution. Then, the serum or culture supernatant whose antibody titer is to be measured is appropriately diluted with TBS (500-fold diluted, etc.), diluted 3-fold in stages, sprayed with 100 μl / well, and incubated at 37 ° C. for 1 hour. , Washed. Further, 100 μl of a 5000-fold diluted solution of TPSBS of the secondary antibody was sprayed on each well, incubated at 37 ° C. for 1 hour, washed with TBS solution, and 100 μl of a coloring solution was added per well to develop color, and 1N phosphoric acid (1N phosphoric acid (1N phosphate) after 10 minutes. After stopping the color development at 100 μl / well, the absorbance was measured at 450 nm.

(4) Purification of mAb6D6 and 6B10 using a protein G column The culture supernatant of the obtained antibody-producing hybridoma cells contains many bovine serum-derived components such as albumin in addition to the target antibody. In order to prevent unintended reactions caused by them, the antibody was purified by a Protein G column according to a conventional method. Since affinity purification is possible with a Protein G column and the secondary antibody used in ELISA is an antibody that recognizes mouse IgG, the isotype of the antibody produced in this study was expected to be IgG.

After purification of the antibody, BCA assay was performed as appropriate, and mAb6D6 and mAb6B10 were diluted and prepared at a concentration of 1 mg / mL, and cryopreserved at −30 ° C.

(5) Analysis of antibody titer and specificity of mAb6D6 and mAb6B10 In order to proceed with the analysis of GA in vivo using the prepared mAb6D6 and mAb6B10, the specificity of the antibody was analyzed by competing ELISA using 18β-GA and structurally similar compounds. did.

For the competitors, GL, oleanolic acid, β-amyrin, psychogenin-A and hederagenin, which are glycosides of GA, were used as oleanane-type triterpenes other than 18α-GA, 18β-GA, and GA. Further, among steroid compounds, aldosterone, cholesterol, cortisone, hydrocortisone and corticosterone were used as compounds having a structure similar to GA.

An ethanol solution (5 mM) of each competitor was diluted with 0.05% TBSB to prepare a 2 μM solution, which was sequentially diluted 3-fold to obtain a competitor. The ELISA uses only GA-BSA as a coating, and as the primary antibody, 1 mg / ml of each monoclonal antibody is diluted 2000-fold with TBSB, and 50 μl of each of the competitor and the temporary antibody is added per well. It was carried out in the same manner as above. The crossing property with GA and GL is shown in FIG. 2, the crossing property with similar substances is shown in FIG. 3, and the crossing property with steroid compounds is shown in FIG.

As shown in FIG. 2, mAb6D6 was found to exhibit cross-reactivity at 18α-GA and 18β-GA concentrations of 1 nM and above. Further, as shown in FIGS. 3 and 4, no crossing with other analog compounds and steroid compounds was observed. Since mAb6B10 showed the same properties as mAb6D6, the notation here is omitted.

(Quantification of GA in mouse serum)
(1) Preparation of biotinylated anti-GA antibody When GA was quantified using mouse serum, only the serum was detected with an enzyme-labeled anti-mouse IgG antibody, and the absorbance was about 0.4. This is considered to be a value caused by the serum component of mice, and in order to eliminate this effect, mAb6D6 was labeled with biotin, and an ELISA method using a biotin-avidin system was constructed. EZ-Link NHS-LC-Biotin was used to prepare the biotinylated antibody, and biotin was labeled via an amide bond with the primary amine of the antibody. The antibody titers of the biotinylated antibody (0.88 mg / ml) and the pre-biotinylated antibody (1 mg / ml) were measured according to Example 1. The results are shown in FIG.

As shown in FIG. 5, it was found that the sensitivity of biotinylated mAb6D6 was higher than that of prebiotinylated mAb6D6, and the biotinylated antibody was compared with the antibody titer against native-BSA. It was found that a sufficiently high antibody titer against GA-BSA can be obtained. From this, it was found that biotinlation of mAb6D6 reduces the effect of serum. In addition, when a recovery test was performed on GA at known concentrations (100 nM and 10 nM) in serum with a biotinylated antibody, it was confirmed that it was practical.

Considering the absorbance of biotinylated 6D6, the concentration of biotinylated 6D6 used in the GA quantification method was determined to be 0.88 ng / well. As the competitor used, GA ethanol solution (5 mM) was added to mouse serum to prepare a GA 20 μm serum. This was sequentially diluted 3-fold with serum to prepare GA0.2nM serum, incubated at 37 ° C. for 4 minutes, sprayed 50 μl of each on each well, and then biotinylated 6D6 (0.88 mg / ml). Was diluted 50,000 times with 0.05% TPBS, sprayed at 50 μl per well, incubated at 37 ° C. for 1 hour, and the antibody titer was evaluated according to Example 1. The results are shown in FIG.

As shown in FIG. 6, linearity was obtained in the range of serum GA concentration of about 5 to 100 nM. Therefore, it was considered that quantification is possible by using this as a calibration curve.

(Search for GA target molecule using mAb6D6)
GL has an effect of suppressing hepatocellular injury and promoting repair, and is used as a hepatoprotective agent. Therefore, considering the existence of a molecule (GA target protein) involved in the expression of such an action, an attempt was made to identify a protein on which GA directly acts in the liver by an immunoprecipitation method using mouse liver homogenate.

The liver was removed from the mouse, and 1 ml of RIPA buffer and 10 μl of a protease inhibitor mixture were used as a homogenate solvent, and 100 mg of liver tissue was homogenized so as to be 10% by mass. Centrifugation at 4 ° C. and 10000 rpm for 20 minutes was performed to prepare a 1 mg / ml solution by TBST. To 400 μl of this liver homogenate, 20 μl of 5 mM GA ethanol solution and 20 μl of ethanol as a target experiment were added, and the mixture was incubated at room temperature for 60 minutes using a rotator.

Next, 28 μl of mAb6D6 (1 mg / ml) and 42 μl of TBST were added to the beads in 70 μl of the magnetic bead solution, and the beads were incubated at room temperature for 30 minutes using a rotator. The magnetic bead solution was dispensed into equal amounts, added to each liver homogenate, and reacted overnight at 4 ° C. with stirring using a rotator. Then, the magnetic beads were collected, washed with TBST, 30 μl of RIPA buffer and 7.5 μl of 5 × sample buffer were added, respectively, and the mixture was heated at 100 ° C. for 2 minutes, and then the supernatant of the magnetic beads was used as a sample for electrophoresis. did. The composition of the 5 × sample buffer was as follows.
(5 x sample buffer)
SDS (sodium dodecyl sulfate): 0.75 g
Glycerin: 1.25 mL
BPB (bromophenol blue): 0.5 mg
1 M Tris Hydrochloric Acid (pH 6.8): 1.676 mL
Mercaptoethanol: 0.5 mL
After mixing BPB and SDS, glycerin and 1M Tris-hydrochloric acid were added, and the mixture was sufficiently mixed and dissolved. Further, mercaptoethanol was added, and the volume was increased to 5 mL with ultrapure water.

For electrophoresis, 10% gel is dissolved in a migration buffer (2-Amino-2-hydroxymethyl-1,3-propanediol: 30.3 g, Glycine: 144 g, SDS (sodium dodecyl sulfate): 10 g) in ultrapure water. 20 μl of each sample and 3 μl of molecular weight marker were loaded into the wells, and electrophoresed at 200 V and 20 mA for about 120 minutes. Then, it was immersed in 50 mL of a gel fixative (acetic acid: methanol: water = 3.75: 5: 41.25), fixed overnight at room temperature, and visualized by silver staining. The results are shown in FIG.

As shown in FIG. 7, a band observed only in the sample of GA-added mouse liver homogenate was partially confirmed. This suggests that there is a protein to which GA binds in the liver tissue.

Claims (15)

An anti-glycyrrhetinic acid monoclonal antibody
An antibody having specific binding ability to an glycyrrhetinic acid-albumin complex in which 18β-glycyrrhetinic acid and 18β-glycyrrhetinic acid are bound to albumin. The antibody according to claim 1, wherein the glycyrrhetinic acid-albumin complex is a complex in which glycyrrhetinic acid and albumin are bound in vivo. The antibody according to claim 1 or 2, which has cross-reactivity to 18α-glycyrrhetinic acid. The antibody according to any one of claims 1 to 3, which has no crossing property with glycyrrhizin. The antibody according to any one of claims 1 to 4, which has no crossing property with one or more selected from the group consisting of oleanolic acid, β-amyrin, psychogenin-A and hederagenin. The antibody according to any one of claims 1 to 5, which has no cross-reactivity to one or more compounds selected from the group consisting of aldosterone, cholesterol, cortisone, hydrocortisone and corticosterone. The antibody according to any one of claims 1 to 6, which is labeled with biotin. The antibody according to any one of claims 1 to 7, wherein a complex in which a carrier protein is directly bound to an amide group derived from the carboxy group at the 30-position of 18β-glycyrrhetinic acid can be obtained as an immunogen. A method for detecting either or both of 18β-glycyrrhetinic acid and the glycyrrhetinic acid-albumin complex in which 18β-glycyrrhetinic acid and albumin are bound in a biological sample.
A step of contacting the antibody according to any one of claims 1 to 8 with 18β-glycyrrhetinic acid in the biological sample to form a complex of the antibody and the 18β-glycyrrhetinic acid.
How to prepare. The method according to claim 9, wherein the biological sample is a sample that has not been subjected to gradual protein manipulation. A method for searching for a target molecule of 18β-glycyrrhetinic acid.
The 18β-glycyrrhetinic acid-is contacted with a possible complex of the 18β-glycyrrhetinic acid and one or more potential target molecules and the antibody according to any one of claims 1 to 8.
The process of forming a target molecule candidate-antibody complex, and
A method comprising the step of separating or identifying the one or more target molecule candidates specifically bound to the 18β-glycyrrhetinic acid in the 18β-glycyrrhetinic acid-target molecule candidate-antibody complex. 18β-glycyrrhetinic acid and 18β- comprising the antibody according to any one of claims 1 to 8.
A reagent for detecting either or both of the glycyrrhetinic acid-albumin complex in which glycyrrhetinic acid and albumin are bound. 18β-glycyrrhetinic acid and 18β- comprising the antibody according to any one of claims 1 to 8.
A kit for detecting either or both of the glycyrrhetinic acid-albumin complex in which glycyrrhetinic acid and albumin are bound. The method for producing an anti-glycyrrhetinic acid monoclonal antibody according to claims 1 to 8.
An immune process for immunizing non-human animals using a complex in which a carrier protein is directly bound to an amide group derived from the carboxy group at position 30 of 18β-glycyrrhetinic acid.
An antibody having specific binding ability to a complex in which albumin is directly bound to an amide group derived from the carboxy group at position 30 of 18β-glycyrrhetinic acid from the non -human animal or the hybridoma derived from the spleen cell of the non-human animal. And the antibody acquisition process to acquire
How to prepare. The immunogen for obtaining the anti-glycyrrhetinic acid monoclonal antibody according to claims 1 to 8, which comprises a complex in which a carrier protein is directly bound to an amide group derived from the carboxy group at the 30-position of 18β-glycyrrhetinic acid.

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