WO2005116079A1 - Antibody capable of recognizing ligand of allylhydrocarbon receptor and use thereof - Google Patents

Abstract

An antibody capable of specifically recognizing a ligand compound of allylhydrocarbon receptor. The ligand compound of allylhydrocarbon receptor can be specifically assayed with high sensitivity by the use of the antibody in sassy methods such as ELISA. Further, the ligand compound of allylhydrocarbon receptor with immunologically scarce foreign matter can be concentrated at high ratio by the use of this antibody.

Description

 [Technical Field] Antibodies Recognizing Ligands of Arylhydric Carbon Receptors and Their Applications

 The present invention provides an antibody that specifically recognizes a ligand compound of an arylamide carbon receptor, a method of producing the antibody, a method of measuring a ligand compound of an arylamide carbon receptor using the antibody, and the antibody. The present invention relates to a method for enriching ligand compounds for aralno and hydrocarbon receptors, characterized by using Background art

 It is known that the aryl hydrocarbyl receptor is a dioxin receptor and is deeply involved in the expression of toxic effects of dioxin compounds. Dioxin compounds bind to the arylyl mouth carbon receptor and produce various toxicities such as induction of malformation, decreased thyroid function, reduced reproductive organ weight and spermatogenesis, decreased immune function, and accelerated canceration. Therefore, it is important to measure and analyze dioxin compounds present in the environment and living organisms by various methods, and to feed the results to environmental conservation, evaluation of next-generation risk, health management, etc. is there. Methods for measuring dioxin compounds include methods using instruments such as high-performance liquid chromatography (HPLC), gas chromatography (GC), and high-resolution mass spectrometry (HRMS), and recognition of dioxin compounds. An immunological measurement method using an antibody is known (Japanese Patent Application Laid-Open No. 63-14691) 1) Specificity, sensitivity, etc. are not sufficiently satisfactory.

In recent years, indirubin compounds, indigo compounds, and the like, which are metabolites of tributofan, have been discovered as physiological or naturally occurring ligand molecules of allylnoid mouth.carbon receptors. The findings provide clues to elucidate the intrinsic function of the arylhydrocarbon receptor in cells and how dioxin compounds disrupt that function. These Arino Rehydro Given the co-existence of carbon receptor ligand compounds in vivo, accurate measurement and analysis of the individual concentrations of these compounds would be necessary to elucidate the function of the arylhydrocarbon receptor, It is extremely important in elucidating the effects of compounds on living organisms. As methods for measuring indirubin compounds and indigo compounds, methods using devices such as high-performance liquid chromatography (HPLC), gas chromatography (GC), and high-resolution mass spectrometry (HRMS) are known. However, these methods have problems such as using extremely expensive equipment and requiring skill in operating the equipment.

 To solve these problems, a highly sensitive, specific, simple, and inexpensive method for measuring a ligand compound of an aryl hydrocarbon receptor was developed using an immunological method that uses an antibody that specifically recognizes the compound. The development of a dynamic measurement method was desired. However, since indirubin compounds and the like are naturally occurring substances in the body, their immunogenicity is low, and it is extremely difficult to obtain an antibody that specifically recognizes the ligand conjugate of the aryl hydrocarbon receptor. Met.

 In view of the above circumstances, an object of the present invention is to provide an antibody that specifically recognizes a ligand compound of an aryl hydrocarbon receptor. Disclosure of the invention

 The present inventors have conducted intensive studies to achieve the above object. As a result, by immunizing an animal with a conjugate of a ligand compound of an arylhydrocarbon receptor and a protein, the ligand compound of an arylamide-opening carbon receptor can be obtained. It has been found that an antibody that specifically recognizes can be obtained, and the present invention has been completed.

 That is, the present invention relates to the following.

 (1) An antibody that specifically recognizes a ligand compound of an aryl hydrocarbon receptor. (2) The antibody according to the above (1), wherein the ligand compound of an arylhydrin carbon receptor is a compound selected from the group consisting of dioxin compounds, indirubiine compounds and indigo compounds.

(3) The ligand compound of the aryl hydrocarbon receptor is an indirubin compound Or the antibody of (2), which is an indigo compound.

 (4) The indirubin compound has the formula (1)

[Wherein, R ¹ and R ² are the same or different and represent oxygen or NOH, and R ³ , R ⁴ , R ⁵ and R ⁶ are the same or different and represent hydrogen, halogen, SO ₃ H, alkyl, alkoxy, Or a salt thereof, wherein the indigo compound is represented by the formula (2):

[Wherein, R ⁷ and R ⁸ are the same or different and represent oxygen or NOH, and R ⁹ , R ^1Q , R ¹¹ and R ¹² are the same or different and represent hydrogen, halogen, SO ₃ H, alkyl, alkoxy, A group selected from the group consisting of ropoxyalkyl] or a salt thereof.

 (5) The antibody according to any one of the above (1) to (4), which is a monoclonal antibody.

(6) The antibody according to (5), which is DXN-50.

 (7) A hybridoma that produces the monoclonal antibody according to (5).

 (8) Nos, iprids, and karma according to (7) above, which are DXN-50 (FERM B P-08566).

(9) The method for producing an antibody according to the above (1), comprising the following steps: (a) immunizing a mammal with a conjugate of a ligand compound of an aryl hydrocarbon receptor and a protein;

 (b) a step of isolating an antibody that specifically recognizes a ligand compound of an aryl hydrocarbon receptor.

(10) A conjugate of a ligand compound of an arylhydrite carbon receptor and a protein is represented by the formula (3) _:

[Wherein, R ¹ and R ² are the same or different and represent oxygen or NOH, and R ⁴ , R ⁵ and R ⁶ are the same or different and are each a group consisting of hydrogen, halogen, SO ₃ H, alkyl, alkoxy and carboxyalkyl L represents a spacer, Z represents a protein residue] or a salt thereof, or a compound represented by the formula (4):

[Wherein, R ⁷ and R ⁸ are the same or different and represent oxygen or NOH, and R ^1Q , R ¹¹ and R ¹² are the same or different and represent hydrogen, halogen, SO ₃ H, alkyl, alkoxy, and carboxyalkyl. L represents a spacer, and Z represents a protein residue.] Or a salt thereof. (11) The production method according to the above (10), wherein the spacer is a divalent group containing alkylene or polyoxyalkylene. .

 (12) The spacer is the equation (5):

― (CH ₂ ) _n C0NH— (5)

 [In the formula, n means an integer of 1 to 20], Formula (6):

One (CH ₂ CH ₂ 0) _m C0NH- (6)

 [Wherein, m represents an integer of 1 to 20] and equation (7):

— C0 (CH ₂ ) _p C0NH— (7)

 [Wherein, p represents an integer of 1 to 20]. The production method according to the above (11), which is a divalent group represented by a formula selected from the group consisting of:

 (13) A method for measuring a ligand compound of an arylhydrocarbon receptor, comprising using the antibody according to (1).

 (14) A kit for measuring a ligand compound of an aryl hydrocarbon receptor, comprising the antibody according to (1).

 (15) A method for concentrating a ligand compound of an arylhydroforce monobon receptor, comprising using the antibody according to (1).

 (16) A kit for concentrating a ligand compound of an aryl hydrocarbon receptor, comprising the antibody according to (1).

 By using the antibody of the present invention in a measurement method such as ELISA, it is possible to specifically measure or concentrate a ligand compound of an arylinohydrocarpon receptor. Brief Description of Drawings

 FIG. 1 is a standard curve of indirubin created in Example 3 below. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention specifically recognizes an aryl hydrocarbon receptor ligand compound. Antibodies.

 In the present invention, the ligand compound of the aryl hydrocarbon receptor is not particularly limited as long as it can bind to the aryl hydrocarbon receptor, and examples thereof include a dioxin compound, an indirubin compound, and an indigo compound. Can be The ligand compound of the aryl hydrocarbon receptor is preferably an indirubin compound or an indigo compound, and more preferably an indirubiine compound.

 The dioxin compound is not particularly limited. For example, 2,3,7,8-tetrachlorobenzo-P-dioxin, 1,3,7,8-tetrachlorodibenzo-P-dioxin, 1,2,3,7- Polyhalogenated dibenzones such as tetrachlorodibenzo-P-dioxin, 1,2,3,8-tetrata-oral dibenzo-P-dioxin, 1,2,3,7,8-pentaclo-dibenzo-P-dioxin Compounds such as polyhalogenated dibenzofurans such as p-dioxin, 2,3,7,8-tetrac-mouth dibenzofuran, and coplanar-PCB.

 The indirubin compound is not particularly limited. For example, the compound of the formula (1):

[Wherein, R ¹ and R ² are the same or different and represent oxygen or NOH, and R ³ , RR ⁵ and R ⁶ are the same or different and represent hydrogen, halogen, S ₃ H, alkyl, alkoxy and alkoxyalkyl. A group selected from the group consisting of the following] or a salt thereof.

In the formula (1), R ² is preferably oxygen, and R ⁴ , R ⁵ and R ⁶ are preferably hydrogen. The number of carbon atoms of the alkyl is not particularly limited, but is usually 1 to 20, preferably 1 to 10, more preferably 1 to 6, and still more preferably 1 to 4.

 The alkyl may be any of primary, secondary or tertiary alkyl, and is not particularly limited, but is preferably primary alkyl. .

 Examples of the alkyl include methyl, ethyl, propyl, isopropyl, ptinole, sec-puchinole, t-puchinole, pentinole, isopentinole, t-pentinole, hexyl, t-hexyl and the like. Preferably, the alkyl is methyl, ethyl, propyl or butyl.

 The aspect of the alkyl portion of the alkoxy and the carboxyalkyl is the same as the above-mentioned alkyl.

 Examples of the halogen include fluorine, chlorine, bromine, and iodine.

 Examples of the salt include an alkali metal salt (eg, sodium salt, potassium salt, etc.), an alkaline earth metal salt (eg, calcium salt, magnesium salt, etc.), an organic base salt (eg, triethylamine salt, dicyclohexylamine salt, pyridine salt) Tert-butylamine salt) and the like. Preferred are alkali metal salts. Specifically, as the indirubin compound, there are indirubin, 5-methoxy-indinolevin, 5-methinole-indinolevin, 5-potoxyl-methicide Λ ^ -indirubin, 5-butoxyl-propyloxy-indirubin, and indirubin Examples thereof include rubin-1-5-sulfonic acid, indirubin-13,1-monoxime, 5-odoindilvin_3, monomonoxime, indirubin-13,1-monoxime-15-sulfonic acid, and the like.

 The indigo compound is not particularly limited. For example, the compound of formula (2):

[Wherein, R ⁷ and R ⁸ are the same or different and each represents oxygen or Ν Ο, R ⁹ , R ^{1 G} , R ^{1 1} and R ^{1 2} are the same or different and each is hydrogen, and halogen, S 0 ₃ H, alkyl, a compound represented Ru by means a group selected from the group consisting of alkoxy and local port carboxyalkyl], or salts thereof .

In the formula (2), R ⁷ and R ⁸ are preferably oxygen.

^{R 9, R 1 0, R} 1 1 and R ^{1 2} is preferably hydrogen or S 0 ₃ H, a more favorable Mashiku hydrogen.

 Embodiments of the alkyl, alkoxy, carboxyalkyl, halogen and salt are the same as described above.

 Specifically, as the indigo compound, compounds such as indigo, 5,5,7, T-indigotetrasulfonic acid, 5,5,7,7-indigotrisulfonic acid, indigocarmine, and salts thereof are mentioned. No.

Examples of the antibody include natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAb), and recombinant antibodies produced using gene recombination techniques (for example, single-chain Fv fragments (scFv bispecific-chimeric scFV-scFv tandem scFV ( scFv) 2. bispecific-(scFv) 2, disulfide-linked scFv, disulfide- stabilized Fv fragments (dsFv), diabody ^ single-chain diabody (scDb), bivalent diabody ^ bispecific diabody, knob— into- hole stabilized diabody, disu ide-- stabilized diabody, triabody, tetrabody ^ trispecific triabody, CL-dimerized scFv, CHl-CL-dimerized scFv, CH3- dimerized scFv, knob- into-hole CH3-dimerized scFv, CH3-dimerized bivalent diabody Fc-dimerized scFv, Fab-scFv fusions ^ Ig-scFv fusions _s leucine-zipper stabilized scFv dimers ^ he 丄 ix- stabilized scFv dimers, 4 helix-bunde stabilized scFv tetramers ^ streptavidin-scFv _s intrabody _N Fab 'fragments ^ F (ab') fragment Fv fragments (Fv) _N Kimef Antibodies, humanized antibodies, single-chain antibodies, etc.), and human antibodies that can be produced using human antibody-producing transgenic animals, etc., but are not limited thereto. Antibodies are preferably monoclonal antibodies or polyclonal Preferred examples of the monoclonal antibody include DXN-50 shown in the Examples below. Further, in the present invention, an antibody is a concept including a binding fragment thereof. The antibody-binding fragment means a partial region of the above-mentioned antibody, and specifically, for example, F (ab ') ₂ , Fab', Fab, Fv (variable fragment of antibody), scFv, dsfv (disulphide Stabil ised Fv), dAb (single domain antibody), etc. (Exp. Op in. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996), and antibodies produced by Fab expression libraries Fragments and the like are exemplified.

 The present invention also provides a hybridoma that produces the above-mentioned monoclonal antibody. Preferable examples of the hybridoma include DXN-50 (FERMBP-085656) shown in the following examples.

 The antibody of the present invention immunizes a mammal with a conjugate of an aryl hydrocarbon receptor ligand compound and a protein, and isolates an antibody that specifically recognizes a ligand compound of an aryl hydrocarbon opening carbon receptor. By doing, it can be manufactured. The present invention provides a method for producing the antibody.

 The above-mentioned protein is not particularly limited. No. The conjugate between the ligand compound of the aryl hydrocarbon receptor and the protein is not particularly limited, and examples thereof include a compound in which a ligand compound of the aryl receptor at the mouth is directly bonded to the protein, and an aryl hydrocarbon. Compounds in which a ligand compound of a receptor is indirectly bound to a protein (such as a compound in which a derivative (hapten) of a ligand compound of an aryl hydroforce receptor is bound to a protein).

 The conjugate of the ligand compound of the aryl hydrocarbon receptor and the protein is preferably a compound in which the ligand compound of the aryl hydrocarbon receptor is bound to the protein via a spacer.

Preferred examples of the conjugate include, for example, a compound represented by formula (3)

[Wherein, I ¹ , RR ⁴ , R ⁵ and R ⁶ have the same meanings as above, L represents a spacer, and Z represents a protein residue] or a salt thereof, ):

[Wherein, R ⁷ , R ⁸ , R ¹⁰ , R ¹¹ and R ¹² have the same meanings as above, L represents a spacer, and Z represents a protein residue] or a salt thereof. And the like.

 The spacer is not particularly limited as long as the antibody of the present invention can be produced using a conjugate of a ligand compound of an aryl hydrocarbon receptor and a protein via the spacer. Examples include divalent groups including polyoxyalkylene.

 When the spacer is a divalent group containing alkylene, the number of carbon atoms of the alkylene is not particularly limited, but is usually 1 to 20, preferably 1 to 10, more preferably 1 to 6, and still more preferably 1. ~ 4.

When the spacer is a divalent group containing polyoxyalkylene, the polyoxyalkylene is not particularly limited, and examples thereof include polyoxyethylene, polyoxymethylene, and polyoxypropylene. The polyoxygen The degree of polymerization of the alkylene is usually from 1 to 20, preferably from 1 to 10.

 As the spacer, specifically, for example, the formula (5):

― (CH ₂ ) _n C0NH— (5)

 [Where n represents an integer of 1 to 20], formula (6):

— (CH ₂ CH ₂ 0) _m C0NH-1 (6)

 [In the formula, m means an integer of 1 to 20], Formula (7):

— C0 (CH ₂ ) _p C0NH— (7)

 [In the formula, p represents an integer of 1 to 20].

 Here, n and p preferably represent an integer of 1 to 10, more preferably 1 to 6, and even more preferably 1 to 4. M preferably represents an integer of 1 to 10.

 A conjugate of an aryl hydrocarbon receptor ligand compound and a protein can be easily produced by a person skilled in the art from a ligand compound of an aryl hydrocarbon receptor or an analogous compound thereof and a protein by a well-known reaction. be able to.

 For example, equation (8):

[Wherein, n and Z have the same meanings as described above], the conjugate of the ligand compound of the aryl hydrocapone receptor and the protein can be produced by the following production method or the like.

First, as shown in the following formula, a carboxylic acid of formula (9) is reacted with an alcohol in the presence of an acid to obtain an ester of formula (10). _n C00Ri3

[Wherein, R ¹³ represents alkyl or the like, and n has the same meaning as described above]

 The reaction conditions for the esterification reaction are well known in this field. For example, a carboxylic acid conjugate of the formula (9) is dissolved in ethanol, concentrated sulfuric acid is added, and the mixture is heated under reflux to obtain an ethyl ester.

 Next, the benzene ring of the ester of formula (10) is replaced by a nitro group.

 The reaction conditions for nitration are well known in the art. For example, by adding an ester of formula (10) to a mixture of acetic anhydride and fuming nitric acid, —Obtain a mixture of two toro bodies (Equation (11)).

[Wherein, R ¹³ and n are as defined above. Ko

 Subsequently, the ester of the mixture of the nitro compound of the formula (11) is hydrolyzed using a known inorganic base or the like to obtain a carboxylic acid, which is crystallized and separated to obtain a p-nitro compound (formula (12) ) Is isolated.

[Wherein, R ¹³ and n are as defined above. ]

Subsequently, the compound of the formula (12) is esterified again as described above. ) _n C00Ri3

[Wherein, R ¹³ and n are as defined above. ] Subsequently, the nitro group of the compound of the formula (13) is reduced to an amino group. The conditions for the reduction reaction are well known in the art and are not particularly limited. For example, the compound of the formula (13) is reduced by contacting it with hydrogen in the presence of a catalyst such as palladium to obtain a compound of the formula (14) ) Is obtained.

0 „Ν (CH ₂ ) _n C00Ris

 (13) (14)

[Wherein, R ¹³ and n are as defined above. ]

 Subsequently, an isatin compound is obtained from the compound of the formula (14) by the Fischer method. The reaction conditions and the like of the Fischer method are well known in this field. For example, first, the compound of the formula (14) is heated with chloral hydrate and hydroxylamine to give a carpamoyl oxime compound ( Equation (15) is obtained.

[Wherein, R ³ and n are as defined above. ]

 Further, the isapatin compound of the formula (16) is obtained by heating the carpamoyl oxime compound of the formula (15) in concentrated sulfuric acid.

 H0-N0

 (15) (16)

[Wherein, R ¹³ and n are as defined above. ]

Subsequently, the isatin compound of the formula (16) is reacted with indoxylyl acetate in the presence of a base such as sodium carbonate to obtain an indirubin compound of the formula (17).

 (16) (17)

 Wherein n is as defined above. ]

The indolelevine compound of the formula (17) is reacted with N-hydroxysuccinimide to obtain an activated ester (formula (18)), and the activated ester is reacted with a protein to obtain an activated ester. A conjugate of a ligand compound of an arylhydric mouth carbon receptor represented by (8) and a protein can be obtained.

H ₂ N— Z

 [Wherein, n and Z are as defined above. ]

 A conjugate of a ligand compound of an arylhydric carbon receptor other than the compound represented by the formula (8) and a protein can be easily produced by those skilled in the art according to the above-described method.

In the method for producing an antibody of the present invention, a mammal is immunized with a conjugate of a ligand compound of an arylhydrocarbon receptor and a protein obtained by the above method or the like. When the antibody is a monoclonal antibody, the above-mentioned conjugate is administered to a mammal itself or together with a carrier, a diluent and the like, and the mammal is immunized with the conjugate. Upon administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be used to enhance antibody production. The administration can usually be performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, higgs, goats, and chickens, and mice and rats are preferably used.

 Select an individual with an antibody titer from the immunized mammal, collect the spleen or lymph node 2 to 5 days after the final immunization, and fuse the antibody-producing cells contained in them with myeloma cells Thus, a monoclonal antibody-producing hybridoma can be produced. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Myeloma cells include, for example, NS-1, P3U1, SP2Z0, AP-1 and the like. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the PEG (preferably PEG 1000 to PEG 6000) power S is about 10 to 80%. 20-40. Incubation at C, preferably 30 to 37 for 1 to 10 minutes allows efficient cell fusion.

The monoclonal antibody-producing hybridoma is cultured according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As the medium, any medium can be used as long as the hybridoma can grow. For example, R PMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, GIT medium containing 1 to 10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or hybridoma A serum-free culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culture is usually 5% It can be performed under carbon dioxide gas.

 Subsequently, a hybridoma that produces a monoclonal antibody that specifically recognizes a ligand compound of an arylhydrocarbon receptor is selected from the monoclonal antibody-producing hybridomas.

 Various methods can be used for the selection. For example, an anti-immunoglobulin antibody (if the spleen cells used for cell fusion are mouse-derived, an anti-mouse immunoglobulin antibody is used) or a protein A-adsorbed solid Add the hybridoma culture supernatant to the phase (eg, microplate), then add the conjugate of the aryl hydrocarbon receptor ligand compound and the labeling enzyme, and detect the monoclonal antibody bound to the solid phase. Add the hybridoma culture supernatant to a solid phase (eg, microplate) on which the ligand compound of arylhydric carbon receptor is adsorbed, and then add anti-immunoglobulin antibody (cells) labeled with radioactive substances or enzymes. If the spleen cells used for fusion are mouse-derived, an anti-mouse immunoglobulin antibody is used.) A was added, and a method of detecting a monoclonal antibody bound to the solid phase is found using.

 Labeling enzymes include horseradish peroxidase, alkaline phosphatase and the like. The conjugate of the aryl hydrocarbon receptor ligand compound and the labeling enzyme can be produced in the same manner as the conjugate of the aryl hydrocarbon receptor ligand compound and the protein described above.

 As described above, a hybridoma that produces a monoclonal antibody that specifically recognizes a ligand compound of an arylhydric carbon receptor is obtained.

Subsequently, a monoclonal antibody specifically recognizing a ligand compound for arylhydroforce / bonbon receptor was isolated from the culture supernatant of the hybridoma and the ascites obtained by transferring the hybridoma to nude mice treated with pristane. Is done. Monoclonal antibodies can be isolated by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (eg, DEAE ), Ultracentrifugation, gel filtration, antigen-bound solid phase or active adsorbent such as protein A or protein G. Specific purification method in which only the body is collected and the bond is dissociated to obtain an antibody).

 When the antibody is a polyclonal antibody, similarly to the monoclonal antibody, the conjugate of the above-mentioned ligand compound of the aryl hydrocarbon receptor and the protein is administered to a mammal itself or together with a carrier, a diluent, and the like, A mammal is immunized with the conjugate. As the mammal to be used, for example, monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, chickens, and the like are preferably used.

 The polyclonal antibody can be isolated from serum, such as serum or ascites of a mammal immunized by the above method, preferably from serum. Isolation of the polyclonal antibody can be performed according to the same method for separating and purifying immunoglobulin as in the above-described isolation of the monoclonal antibody.

 Preferably, the polyclonal antibody is an antibody that specifically recognizes a ligand compound of an aryl hydrocarbon receptor by affinity chromatography using a column to which a ligand compound of the aryl hydrocarbon receptor is bound. Purified by separating the fractions.

 From the hybridoma producing the monoclonal antibody, a gene encoding the heavy chain, light chain, variable region, etc. of the antibody is cloned, the gene is inserted into an expression vector, and the vector is introduced into a host. By expressing the gene, a recombinant antibody that specifically recognizes a ligand compound of an aryl hydrocarbon receptor can be produced.

The method for producing recombinant antibodies (Recombinant Antibodies) is described in Chapter 2 of RECOMBINANT ANTIBODIES (ed. By F. Breitling, John Wiley & Sons (USA), 1999). Manufacturing method, Cloning method of antibody gene from hybridoma cell line (Hybridoma Cell Line), preparation method of antibody gene library (Antibody Gene Libraries), Selection of recombinant antibody from gene library (Selection of Recombinant Antibodies) From Gene Libraries) method, 遺 遺 遺 遺 操作 (Antibody Engineering) method And the like, and a recombinant antibody can be produced by these methods. Chimeric antibodies are described, for example, in “Experimental Medicine (Temporary Special Issue), Vol. 1.6, No. 10, 1988”, and Japanese Patent Publication No. 3-73280. No. 506458, Japanese Patent Publication No. 62-296890, etc., human antibodies are described in, for example, `` Nature Genetics, Vol. 15, p. 146-156, 1997 '', `` Nature Genetics, Vol. 7, p. 13-21 , 1994, Tokiohei 4-504365, International Application Publication W094 / 25585, Nikkei Science, June, pp. 40-50, 1989, Nature, Vol. 368, p. 856-859, 1994 "and JP-A-6-500233.

F (ab ') ₂ and Fab' can be produced by treating the above-mentioned antibody with pepsin or papain, which are proteases, respectively.

 The antibody of the present invention can be used as a reagent for quantitatively measuring the ligand compound of the aryl hydrocarbon receptor, or can be used to concentrate the ligand compound of the aryl hydrocarbon receptor by immobilizing the ligand compound on various carriers. It can be used for the manufacture of affinity programs. Further, by identifying a compound that binds (ie, cross-reacts) to the antibody of the present invention, the applicable range of the antibody of the present invention can be expanded.

The antibodies of the present invention are described, for example, in the “Metabolism”, bol. 8, 696 (1971), the Promcian method, the darthal aldehyde method, the carpoimide method, the epoxy activation method, the “enzymimnoats” method. The method described on pages 28 to 29, page 26, and in the "Affiliate Chromatography Handbook" (Amersham Pharmacia Biotech Co., Ltd. (published on February 20, 1998)) A solid phase antibody or an immunoadsorbent can be obtained by immobilizing it on a carrier by a known method such as the above method. Examples of the carrier for immobilization include a microplate (eg, a 96-well microplate, a 24 well microplate, a 192-well microplate, a 38 well microplate, etc.), a test tube ( E.g., glass test tubes, plastic test tubes), glass particles, polystyrene particles, modified polystyrene particles, polybutyle particles, latex (e.g. polystyrene 'latex), nitrocellulose membrane, cyanogen bromide activated filter paper, DBM activated Filter paper, granular solid phase (eg, Sepharose , Cefadettas, agarose, cellulose, cefacryl, etc.), iron-containing polycarbonate film, magnet-containing beads, crystal oscillator, and the like.

 These are combined with, for example, a conjugate of the above-mentioned ligand compound of the arylhydric mouth carbon receptor and the labeling enzyme, a standard solution of the ligand compound of the arylhydrocarbon receptor, a washing solution, a coloring substrate, a color stop solution, a buffer solution and the like. Thus, a kit for measuring a ligand compound of an arylhydric carbon receptor can be provided as a kit for concentration. The present invention provides the kit.

 Examples of the standard solution of the ligand compound of the aryl acceptor carbon receptor include a solution containing a known concentration of the ligand compound of the aryl hydrocarbon receptor.

 Examples of the washing solution include a buffer solution containing a surfactant (eg, Tween-20).

 The chromogenic substrate can be appropriately selected according to the type of the labeling enzyme, but when the labeling enzyme is phenol oxidase, TM BZ, ABTS, DAB, AEC, 4-CN, etc. are used. . When the labeling enzyme is alkaline phosphatase, p-2-trophenol, BCIP / NBT and the like are used. The chromogenic substrate may be in the form of a solid such as a powder, or dissolved in a suitable solvent.

 Examples of the color stop solution include an aqueous solution containing an acid such as sulfuric acid.

 The standard solution, color stop solution, washing solution, etc. of the ligand compound of the aryl hydrocarbon receptor may be prepared as a concentrated solution, and may be appropriately diluted before use.

 The above-mentioned kit may contain only one kind of the antibody of the present invention, but may further contain a plurality of different kinds of antibodies recognizing a ligand compound of an aryl hydrocarbon receptor. For example, a ligand containing a specific aryl hydrocarbon receptor can be specifically measured or concentrated by using a kit containing a plurality of antibodies having different cross-reactivity.

As a method for measuring the ligand compound of the aryl hydrocarbon receptor of the present invention, radioisotope immunoassay (RIA), ELISA (Engvall, E., Methods in Enzymol., 70, 419-439 (1980)), fluorescent antibody method, plaque method, spot method, agglutination method, Ouchterlony, etc. Hydridoma Method and Monoclonal Antibody ", published by R & D Plajung, Inc., pp. 30-53, March 5, 1975, and a method using a crystal oscillator (Japanese Patent Application Laid-Open No. 2002-310874). Publication). The ELISA method is widely used from the viewpoints of sensitivity, simplicity, and the like.

 In the method for measuring a ligand compound of an aryl hydrocarbon receptor of the present invention, a sample (for example, environmental water or a biological sample containing a ligand compound of an aryl carbon receptor) is labeled with a known amount of a labeled compound. A so-called competitive method, which is quantified by a quantitative competitive reaction for binding to the antibody of the present invention with a ligand compound of an arylamide-containing carbon receptor, is preferably used. In the competitive method, a sample solution containing an unknown amount of an aryl hydrocarbon receptor ligand compound is mixed with a known amount of a labeled aryl hydrocarbon receptor ligand compound, and the mixed solution is mixed. Is contacted with a fixed amount of the antibody of the present invention held on a carrier. Detect the label retained on the carrier or the label not retained on the carrier.

 Examples of the labeling agent for labeling the ligand compound of the aryl hydrocarbon receptor include the above-mentioned labeling enzyme, radioisotope, enzyme substrate, fluorescent substance, and biotin. The maleimide method [Journal of Ob 'Biochemistry (J. Biochem.), 79, 23 (1976)], and the maleimide method were used to bind the ligand compounds of aryl hydrocarbon receptor to these labeling agents. The activated biotin method [Journal, Op. American, Chemical, Society (J. Am. Chem. Soc.), 100, 358, 5 (1978)] and the like are used.

More specifically, for example, a sample solution containing an unknown amount of a ligand compound of an arylamide-opened carbon receptor is mixed with a known amount of a ligand compound of a labeled aryl hydrocarbon receptor, and the mixture is mixed. The solution is contacted with a fixed amount of the antibody of the present invention held on a carrier. Next, the solid phase is usually washed well, and the activity of the labeling agent bound on the solid phase is measured. If the labeling agent is a radioisotope, measure with a gel counter or liquid scintillation counter. When the labeling agent is a labeling enzyme If this is the case, add the substrate and let it stand, and measure the enzyme activity by colorimetric or fluorescent methods. Even if the labeling agent is a fluorescent substance or the like, it can be measured according to a known method.

 In the method for enriching the ligand compound of the aryl hydrocarbon receptor of the present invention, an antigen-antibody reaction is used by passing a large amount of a sample through an immunoadsorbent force ram or mixing it with immunoadsorbent particles. Then, the ligand compound of an arylhydride carbon receptor is captured by the immunoadsorbent, and then the pH is changed (pH 2.

Lower to 5-3, increase to pH 11; change ionic strength (1M Na

Etc. C 1), change of polarity (1 0% Jiokisan, 50% ethylene glycol, 3M chaotropic salt _{(S CN-, CC l 3 COO-} , I-) , etc.), protein denaturants (8M urea, 6 M hydrochloric acid Guanidine) and elution by a known method such as electrophoretic dissociation, allowing the ligand compound of the aryl hydrocarbon receptor, which is immunologically less contaminated, to have a high magnification of several thousands to tens of thousands of times. Can be concentrated.

 This enrichment method allows for a much higher magnification of ligand compounds of the aryl hydrocarbon receptor, which are present only in trace amounts in the environment, compared to conventional enrichment methods such as solvent extraction and solid phase extraction. It is possible to concentrate and obtain a concentrated liquid having a low content of impurities and the like that interfere with the determination.

 Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples.

 The mouse / mouse-hybridoma DXN-50 manufactured in the following example was purchased on February 10, 2003, under the Pudaest Treaty. Deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, the 6th central administrative agency, under the accession number FE RM BP-0 856 6 6. Example 1

Production of monoclonal antibodies

 (1) Production of hapten (DXN-1)

10 g of 5-phenylpentanoic acid was dissolved in 10 OmL of ethanol, 0.2 mL of concentrated sulfuric acid was added, and the mixture was refluxed for 15 hours. After cooling, concentrated under reduced pressure to remove most of the ethanol. After removal, 10 OmL of a saturated sodium bicarbonate solution was added to the residue, and the mixture was extracted with 10 OmL of ethyl acetate. The organic layer was washed with 5 OmL of saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 11.6 g of ethyl ester as a tan liquid.

 7 OmL of acetic anhydride was cooled on ice, and 7 mL of fuming nitric acid was slowly added at 10 ° C or lower while stirring. To this mixture, a solution in which 11.6 g of ethyl 5-phenylpentanoate was dissolved in 20 mL of acetic anhydride was added dropwise at about 9 ° C. or less for about 20 minutes. After the dropwise addition, the mixture was stirred for 1 hour under ice cooling, and the reaction mixture was poured into 70 OmL of ice water. After stirring for 30 minutes, the mixture was extracted with 30 OmL of ethyl acetate, and the organic layer was washed twice with 15 OmL of saturated aqueous sodium bicarbonate, washed with 10 OmL of saturated saline, and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure to obtain a mixture of the o-nitro compound and the p-nitro compound as 15.7 g of a tan liquid.

 The nitro compound (14.0 g) was dissolved in ethanol (15 OmL), purified water (3 OmL) and potassium hydroxide (6.3 g) were added, and the mixture was stirred at room temperature for 16.5 hours. After evaporating most of the ethanol under reduced pressure, 3 OmL of purified water was added to the residue, and the mixture was made strongly acidic with concentrated sulfuric acid. The precipitated oil was extracted with 100 mL of ethyl acetate, and the organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 11.4 g of a brown liquid. This was dissolved in 3 OmL of diisopropyl ether, seed crystals were added thereto, and then 20 mL of n-hexane was added thereto, followed by ultrasonic irradiation. After the crystals were precipitated, they were allowed to stand at 5 ° C for about 1 hour, and the precipitated crystals were collected by filtration. The crystals were washed with 150 mL of a mixed solution of diisopropylethyl Zn-hexane 1/2 and dried under reduced pressure at room temperature to obtain 3.1 g of 5- (4-nitrophenyl) pentanoic acid.

 3.1 g of 5- (4-nitrophenyl) pentanoic acid was dissolved in 5 OmL of ethanol, 0.7 mL of concentrated sulfuric acid was added, and the mixture was heated under reflux for 15.5 hours. After allowing to cool, the mixture was concentrated under reduced pressure, and the residue was dissolved in 100 mL of ethyl acetate and washed with 50 mL of saturated aqueous sodium hydrogen carbonate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 3.5 g of ethyl ester as a brown liquid.

5-(4-Nitrophenyl) ethyl pentanoate 2.

Dissolve in 10% Pd C (50% water-containing product) 0.9 g Hydrogen reduction was performed. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain 2.9 g of ethyl 5- (4-aminophenyl) pentanoate as a brown liquid.

 Dissolve 19.2 g of chloral hydrate in 25 mL of purified water and, with stirring, 12.3 g of anhydrous sodium sulfate and 2.54 g of ethyl 5- (4-aminophenol) pentanoate in 8 m of 1N hydrochloric acid A solution in which 2.0 g of hydroxylamine hydrochloride was dissolved in 2 mL of purified water was added in order. The mixture was heated and refluxed for about 15 minutes, and maintained for 5 minutes. After allowing to cool to room temperature, the mixture was extracted with black hole form, washed with water, and dried over anhydrous sodium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by a silica gel column (eluent: n-hexane Z: ethyl acetate = 2: 1) to obtain 2.24 g of a carpamoyloxime compound.

 1.719 g of the carpamoyloxime compound was added little by little to 1.1.5 mL of concentrated sulfuric acid over about 5 minutes to dissolve, and then heated to 80 ° C. for 10 minutes. After stirring at the same temperature for another 10 minutes, the mixture was cooled to room temperature and poured into 15 OmL of ice water. After the precipitated orange crystals were collected by filtration, the aqueous layer was extracted with 15 OmL of a 1 oZ1 mixed solution of ethyl acetate / methanol. The extract was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain orange crystals. The crystals obtained by filtration previously were dried, then, combined with the crystals obtained by extraction, suspended in 200 mL of a mixed solution of chloroform-methanol 10/1, and stirred at room temperature. After distilling off the solvent of about 2Z3 under reduced pressure, the crystals were collected by filtration and dried under reduced pressure to obtain 0.96 g of isatin compound. In the reaction, hydrolysis of the ester moiety occurred at the same time, and the isatin compound was obtained as a carboxylic acid.

0.56 g of indoxyl acetate was dissolved in 14 mL of methanol, and a mixture of 0.71 g of the isatin compound and 0.753 g of sodium carbonate was stirred at room temperature for 19 hours. The reaction mixture was diluted with 350 mL of methanol, 20 mL of 1 N hydrochloric acid was added, and the mixture was stirred at room temperature for 30 minutes. The crystals were collected by filtration, washed with methanol, added to 5 OmL of purified water, stirred at room temperature and washed. The crystals were collected by filtration, washed with methanol and then with diisopropyl ether, and dried at room temperature under reduced pressure overnight to obtain the compound of the formula (19):

The indirubin compound (DXN-1) was obtained as a purple crystalline solid (0.995 g).

 (2) Production of conjugate of hapten and KLH

The hapten (DXN-1) 60 ^ mo1, 7-soluble carbodiimide 72-mo1, N-hydroxysuccinimide 72-μmo1 produced in the above (1) was prepared in 1 ml of dimethinole sulfoxide. The reaction produced an activated ester. Then Moshia to Ska sheet shellfish - down (KLH) sodium bicarbonate to 1 0mg 0. 1 3M (N a H C0 3) was dissolved in a solution 3 ml, after addition of the activated ester 3 Q 7 μ L,ー 晚 Reacted at 4 ° C. After dialyzing against Dulbecco's phosphate buffered saline (PBS), the unreacted reagent was removed to obtain a conjugate of hapten and KLH (hereinafter referred to as immunogen). The immunogen was stored frozen.

 (3) Immunization

 The immunogen produced in the above (2) was dissolved in PBS so as to obtain SOO gZmL, and mixed with an equal volume of Freund's adjuvant system to prepare an emulsion. The emulsion was subcutaneously administered to BALBZC mice (female) at a dose of 100 g / mouse, and booster immunization was performed at two-week intervals. After six booster immunizations, the immunogen was intravenously administered to the individual showing the highest serum antibody titer (20-0.2 ml PBSZ mouse, final immunization).

 (4) Cell fusion

The spleen was excised from the mouse that was finally immunized in (3) above (3 days after the final immunization), and the spleen cells were collected. Mouse myeloma cells and spleen cells in a 1:10 ratio And in the presence of polyethylene glycol (average molecular weight 4000) to perform cell fusion. After the fused cells (hypridoma) were suspended in HAT medium, they were seeded in a 96-well 11-microplate and cultured in a carbon dioxide gas incubator (37 ° C, 5% CO ₂ ).

 (5) Hypri-doma screening

 (5)-1 Atsushi plate creation

 A goat anti-mouse Ig AGM antibody (Cappe 1, # 55461) was dissolved in PBS at 5 μg / ml and added to the microplate (100 / X 1 / we 11). Four. After reacting with C, wash with PBS containing 0.05% Tween 20 (T—PBS) (2 times with 300 μl / we 11) and dilute with PBS at 1% Plock Ace (Snow Brand Milk Products Co., Ltd., Tokyo) was added (200 / x lZwe 11). After reacting at -4 ° C for at least 4 hours, the plate was stored at 4 ° C until use.

 (5) -2 Production of conjugate of hapten and HRP

The hapten (DXN-1) 60 / imol, water-soluble lipodimide 72 mo 1, N-hydroxysuccinimide 72 mo 1 prepared in the above (1) was reacted in dimethinole sulfoxide lm 1 with An activated ester was made. Next, 10 mg of horseradish peroxidase (HRP) was dissolved in 3 mL of 0.13 M sodium bicarbonate (NaHCO ₃ ) solution, and 20.8 μL of the activated ester diluted 10-fold with DMF was added. The reaction was performed at −4 ° C. Unreacted reagents were removed by ultrafiltration to obtain a conjugate of hapten and HRP (hereinafter referred to as antigen-enzyme complex). The antigen-enzyme complex was adjusted to a concentration of 3.3 mg / mL and stored refrigerated.

 (5) — 3 Primary screening

In the microplate on which the cells were seeded in (4) above, 100 tL of the culture solution of we11 in which cell growth was confirmed was added to the assay plate prepared in (5) -1 above (before use, PB Washed with S (300 μ 1 / we 11 twice). After reacting at room temperature for 1 hour, the plate was washed with T-PBS (3 times with 300 lZwell) and diluted 5000-fold with T-PBS, and the antibody prepared in (5) -2 above was used. The proenzyme complex was added to the microplate. After reacting at room temperature for 1 hour, the plate was washed with T-PBS (three times with 300 ^ 1 / we 11), and a color developing solution was added (100 µ1 / we 11). After reacting at room temperature for 30 minutes, a reaction stop solution was added (100 ^ 1 / we 11) to stop the color reaction. The absorbance at a wavelength of 450 nm was measured, and the cells in we11 exceeding the absorbance of 0.1 were transferred to a 24 we11 microplate.

 (5) -4 Secondary screening (inhibition test)

 In the 24 we 11 microplate to which the cells were transferred in (5)-3 above, sufficient cell growth was confirmed on the culture medium of we 11. Atsushi plate prepared in (5)-1 above (PBS Or, it was washed with T-PBS (twice with 300 ilZwe 11)) and squeezed (100 1 Zwe 11). After reacting at room temperature for 1 hour, wash with T-PBS (3 times with 300 1 / we 11), and incubate the antigen-enzyme complex (T: 5000-fold diluted with? 33) and 1 mg ZL in indirubin (hereinafter I ND) (dissolved in 10% methanol + 1% acetone solution) or a mixture (1: 1) with 10% MeOH + 1% acetone solution (control) was added. After reacting at room temperature for 1 hour, the plate was washed with T-PBS (3 times with 3001 / we11), and the chromogenic substrate was added at 1001 / we11. After the reaction at room temperature for 30 minutes, 100 µl / we 11 of a reaction stop solution was added to stop the color reaction. The absorbance was read at a wavelength of 450 nm. Cloning was performed by limiting dilution in accordance with a conventional method for cells in we11 in which the absorbance of the 1 mg / L ND solution in the absorbance was less than 50%.

 Table 1 shows the results of the primary screening and the secondary screening.

Primary and secondary screening results

(5) — 5 I ND sensitivity test In the above (5) -4, the absorbance was measured in the same manner as in the above (5) -4, using the culture solution of Hypridoma (strain 6), in which sufficient growth was confirmed among the clawed cells. The concentration of IND that inhibits by 50% (hereinafter referred to as IC50) was determined, and the reactivity to IND was examined (Table 2). Table 2 IND sensitivity test

As shown in Table 2, the antibody produced by the hybridoma (DXN-50) of clone No. 50 had the lowest IC 50 value, indicating that the clone had the highest sensitivity to IND.

 (6) Cross-reactivity test

 Using the culture supernatant of DXN-50, which is the most sensitive clone in (5) -5 above, the cross-reactivity was examined in the same manner as in (5) -4 above. As the cross-reactants, 12 kinds of indirubin-like compounds shown in Table 3 were used. A standard curve was prepared for each of the I ND (control) and the cross-reactive substance, the IC 50 was determined, and the cross-reactivity was determined by the following equation (Table 3).

Cross-reactivity (%) = (IC 50 for ND) / (IC 50 for cross-reactant) x 100

Table 3 Cross reactivity with IC50 of DXN-50

 Indirbin 0.28 100

 5-Methoxy-indirubin 0.19 165

 5-methyl-indirubin 1.1 30

 5-Dipoxymethyl-indilbin 0.51 63

 5-carboxypropyl-indirubin 0.084 362

 Indirubin-5-sulfonic acid, Na 0.54 59

 Indirubin-3'-monoxime 1.4 18

 Indirubin-3'-monoxime, 5-yo

 13 2

 Mode

 Indirubin-3'-monoxime, 5-s

 0.53 46

 Rufonic acid

 2,3-Indolinedione (Isatin)> 100% 0.3

 Indigo 5.0 5

 Indigo carmine> 100% 0.3

As shown in Table 3, the DXN-50 antibody had high cross-reactivity with compounds having an IND backbone.

 (7) Purification of monoclonal antibody

 The cell culture supernatant fractionated with mouse ascites or 45-50% saturated ammonium sulfate by a conventional method was subjected to protein G ab ity chromatography to purify anti- # :.

Example 2

 Production of ELISA kit for measurement of aryl hydrocarbon receptor ligand compounds

(1) Preparation of “immobilized plate”

Goat anti-mouse IgG antibody (ICN, 25 mM B is— Tr is—HC 1 (H5.6.5.7.7) (Dojin Code No. 345-04741) Ca Ppel Co. Co d eNo. 55479) the immobilized plate (C ostar EIA / RIA latestrip 8 Co d eNo. 2592) To dispense _{(5 μ § Ζΐ Ο Ο 1} / we 1 1), at 4 ° C After standing still, wash solution (T-PBS)

Washed twice with 300 μL. Bloking solution (10 g Blo c kAc e (Snow Brand Dairy) + 500 1 Slur off 72Ν (Nippon Environmental Chemicals) _ + 0.58

4 g Na C 1 (Wako Pure Chemical Co., Ltd. Cod No. 191-01665) + 10 g S ucrose (Wako Pure Chemical Co., Ltd. No. 196-00015) + 25 m 1 200 OmM Tr is— HC 1 (pH7. 5) / 1000 ml of ultrapure water to add water (200 / i LZwe 11), let stand at 4 ° C, wash twice with 300 μL of washing solution (T-PBS) . Then, Du 1 becco, s PBS (-) (including Wako Pure Chemical Co., Ltd. Code No. 041-20211, including 0.05% slough off 72 N + 0.1% BSA (Sigma Code No. A- 7638) )), Add the dissolved anti-DXN antibody (DXN-50) (0. O l ^ g / l OO / x 1 / well), allow to stand at 4 ° C for a while, and then wash with washing solution (T-PBS) 300 Washed twice with L. After adding the blocking solution 200 1 / we 11 and allowing it to stand at 4 ° C for a while, aspirate the entire volume with an aspirator, seal the dehydrated and dried solid phase plate in an aluminum bag, and place it in a vacuum dryer. It was further degassed, sealed and stored in a refrigerator at 2-8 ° C.

 (2) Preparation of “IND standard solution”

Preparation of undiluted solution 1 (10 OmgZL): Indirubin (A1 eXis Code No. 270—361—M005) was accurately weighed at 1 Omg for 100% of its content, and placed in a 100-m1 volumetric flask. Then, it was made up with acetone.

Preparation of stock solution 2 (lmg / L) [10% methanol + 1% acetone]: 1 ml of stock solution, 1 OmL of methanol and sloff 72N 50; weigh accurately iL with whole pipette, 100m 1 volumetric flask And made up with distilled water.

Preparation of 10% methanol + 1% acetone solution (containing 0.05N sloughing 72N): 100mL of methanol, 1OmL of acetone and 500L of sloughing 72N were put into a 100OmL measuring flask and made up with distilled water. In addition, the following stock solutions and standard solutions were prepared (Table 4).

Table 4

 (3) Preparation of `` antigen-enzyme complex powder ''

Water soluble carbodiimide (WS C Dojindo Chemical Co., Ltd. for peptide synthesis No. 3 4 8-0 3 6 3 1) 7 2 μmol and N-hydroxysuccinimide (NH SI, Wako Pure Chemical Co., Ltd. No. 0 8 9-04 0 3 2) 72 μmol was dissolved in 1 ml of dimethyl sulfoxide (DMS O Wako Pure Chemicals special grade), and 0.5 ml of the hapten (DXN-1 ) It was mixed with 60 μmol of DMS O solution and reacted at room temperature overnight. A solution obtained by diluting the obtained reaction solution 10-fold with DMF 31.3 1 and 31111 dissolved in 1. l% NaHCO ₃ (Ρ OD Roche EIA Code No. 8 1 4 3 9 3) lOmg was allowed to react under stirring at 4 ° C, and the resulting reaction solution was filtered through an ultrafiltration membrane having a cut-off molecular weight of 30,000 to give a 0.05% slurry. An antigen-enzyme complex solution was obtained by making the final volume 3 ml with PBS containing 72N. After dispensing 200 L each, the mixture was freeze-dried to obtain an antigen-enzyme complex powder.

 (4) Preparation of `` antigen-enzyme complex solution ''

_{N a 2 HP 0 4 - 1} 2H 2 O 1 3. 2 6 N a H 2 PO 4 · 2H 2 0 2. 0 2 g, N a C l 1 4. 6 1 g, Suraofu 7 2 N 2 0 0 After dissolving μ1 in 1 L of distilled water, 8 ml thereof was dispensed into an appropriate container, capped, and stored in a refrigerator at 2 to 8 ° C. (5) Preparation of “6x concentrated washing solution”

 20 ml Tween—Dissolve 20 in 20 Om 1 distilled water, and add 12 bags of PBS, 0.3 ml of Sluroff 7.2 ml, 10% Tween-20 30 ml to 1 L of distilled water. After dissolution, 5 Oml was dispensed into an appropriate container, capped, and stored in a refrigerator at 2 to 8 ° C.

 (6) Preparation of Chromogenic Substrate Solution A

 3, 3,, 5, 5, Tetramethylbenzidine (TMB Z Code No. 346-0404001 for Dojindo Chemical Test Research) 10 mg, dimethylformamide (DMF Wako Pure Chemical Industries, Ltd.) 04 5— 0 2 9 1 6) Dissolve in 1 ml, dispense 2.5 μl each into a brown container, cap and store in a refrigerator at 2-8 ° C did.

 (7) Preparation of Chromogenic Substrate Solution-B

U rea Ji yrogenperoxide (Sigma No. ode No. U— 1 7 5 3) 35 O mg of cunic acid (cunic acid monohydrate Wako Pure Chemicals Reagent Code No. 0 3 5— 0 3 4 9 5) buffer at p H 5. 4 0 mM phosphoric acid prepared in _{0 (N a 2 HPO 4 ·} 1 2H 2 0 Wako pure Chemical reagent grade C ode No. 1 9 6 - by 0 2 8 3 5) buffer It was diluted to 1 L, dispensed in 15 mL portions, capped, and stored in a refrigerator at 2-8 ° C.

 (8) Preparation of "color stop solution"

 1N phosphoric acid solution (Kanto Kagaku Code No. 32 184-08) was dispensed into appropriate containers in 15 ml portions, and the caps were capped. Example 3

Quantification with ELISA kit for measurement of aryl hydrocarbon receptor ligand compounds (preparation of standard curve)

 (1) Preparation of antigen-enzyme complex solution

Add 7 mL of “antigen-enzyme complex solution” to “antigen-enzyme complex powder” and dissolve. "Antigen-enzyme complex solution" was prepared.

 (2) Preparation of “mixture”

 100 μL / we 11 of “IND standard solution” (10% methanol + 1% acetone solution) and “100 μL / we 11 of antigen-enzyme complex solution” in “Mic mouth plate for mixing” Mixed above.

 When a sample is used, the sample is mixed with the “antigen-enzyme complex solution” in the same manner as in “.I ND standard solution”.

 (3) Antigen-antibody reaction (competitive reaction)

 The “mixed solution” prepared in (3) above was dispensed (100 L / we 11) to an “anti-DXN monoclonal antibody-immobilized plate”, and reacted at room temperature for 60 minutes.

 -(4) Preparation of “washing solution”

 During the antigen-antibody reaction time, the “six-fold concentrated washing solution” and distilled water were mixed at a ratio of 1: 5 to prepare a “wash solution”.

 (5) Removal of unreacted substances

 The antigen-antibody reaction solution was discarded, and the inside of we11 was washed three times with 300 L / we11 of "washing solution".

 (6) Color reaction Z reaction stop

 After 100 µL / we 11 of “coloring reagent” was added and the reaction was carried out at room temperature for 30 minutes, 100 µL / we 11 of “color stop solution” was added to stop the reaction.

 (7) Colorimetric and density calculation

 The absorbance was measured at a wavelength of 450 nm using a plate reader to create a standard curve.

 When quantifying the concentration of an arylhydridocarbon receptor ligand compound in a sample, the concentration of the aryl hydrocarbon receptor ligand compound is calculated from the absorbance using the above standard curve.

(8) Result The generated standard curve is shown in FIG. In the figure, the IND concentration is plotted on the X-axis, and the ratio of the absorbance at the IND concentration of 0 g / L to the absorbance at each concentration (inhibition ratio, B / Bo%) is plotted on the Y-axis. Industrial applicability

 By using the antibody of the present invention in a measurement method such as ELISA, a ligand compound of an arylhydrocarbon receptor can be specifically, highly sensitively and simply measured. In addition, the use of the antibody makes it possible to specifically enrich the ligand compound of the aryl acceptor carbon receptor.

Claims

The scope of the claims

1. An antibody that specifically recognizes a ligand compound for aryl hydrocarbon receptor.

2. The antibody according to claim 1, wherein the aryl hydrocarbon receptor ligand compound is a compound selected from the group consisting of dioxin compounds, indirubin compounds, and indigo compounds.

 3. The antibody according to claim 2, wherein the ligand compound for an arylhydric carbon receptor is an indylrubin compound or an indigo compound.

 4. The indirubin compound has the formula (1):

[Wherein, R ¹ and R ² are the same or different and represent oxygen or NOH, and R ³ , R ⁴ , R ⁵ and R ⁶ are the same or different and represent hydrogen, halogen, SO ₃ H, alkyl, alkoxy, Or a salt thereof, wherein the indigo compound is represented by the formula (2):

Wherein the R ⁷ and R ⁸ are the same or different oxygen or ^{NOH, R 9, R 1Q,} R 11 and R ¹² independently represent hydrogen, halogen, S0 ₃ H, alkyl, alkoxy and carboxyalkyl Means a group selected from the group consisting of 4. The antibody according to claim 3, which is a compound or a salt thereof.

 5. The antibody according to any one of claims 1 to 4, which is a monoclonal antibody.

6. The antibody according to claim 5, which is DXN-50.

 7. A hybridoma that produces the monoclonal antibody according to claim 5.

 8. The hybrid dormer according to claim 7, which is DXN-50 (FERM BP-08566).

 9. A method for producing the antibody according to claim 1, comprising the steps of: (a) immunizing a mammal with a conjugate of a ligand compound of an aryl hydrocarbon receptor and a protein. ;

 (b) a step of isolating an antibody that specifically recognizes a ligand compound of an arylinohydrocarbon receptor.

 10. A conjugate of a ligand compound of an arylhydric mouth carbon receptor and a protein is represented by the formula (3):

[Wherein, R ¹ and R ² are the same or different and are oxygen or NOH, and R ⁴ , R ⁵ and R ⁶ are the same or different and are each a group consisting of hydrogen, halogen, SO ₃ H, alkyl, alkoxy and carboxyalkyl L represents a spacer, and Z represents a protein residue] or a salt thereof, or a compound of the formula (4):

Wherein R ⁷ and R ⁸ are the same or different and represent oxygen or NOH, and R ^1G , R ¹¹ and R ¹² are the same or different and represent hydrogen, halogen, SO ₃ H, alkyl, alkoxy, and carboxyalkyl. A group selected from the group consisting of: L represents a spacer, and Z represents a protein residue] or a salt thereof.

 11. The production method according to claim 10, wherein the spacer is a divalent group containing alkylene or polyoxyalkylene.

 1 2. The spacer is the formula (5):

One (CH ₂ ) _n C0NH— (5)

 [In the formula, n means an integer of 1 to 20], Formula (6):

― (CH ₂ CH ₂₀ ) _m C0NH ― (6)

 [Wherein, m represents an integer of 1 to 20] and equation (7):

— C0 (CH ₂ ) _p C0NH— (7)

 12. The production method according to claim 11, wherein the production method is a divalent group represented by a formula selected from the group consisting of: wherein p represents an integer of 1 to 20.

 1 3. A method for measuring a ligand compound of an arylhydrocarbon receptor, comprising using the antibody according to claim 1.

 14. A kit for measuring a ligand compound of an aryl hydrocarbon receptor, comprising the antibody according to claim 1.

 1 5. A method for concentrating a ligand compound of an arylhydrocarbon receptor, comprising using the antibody according to claim 1.

16. An aryl hydrocarbon receptor ligand comprising the antibody of claim 1. Kit for concentration of compound.