WO2005116080A1 - Antibody specific for alkylphenol and use thereof - Google Patents

Abstract

An antibody which specifically recognizes alkylphenol compounds and does not recognize alkylphenol ethoxylate compounds. By using the antibody in an examination method such as ELISA, an alkylphenol compound can be specifically determined even in the presence of an alkylphenol ethoxylate compound without being influenced by the compound. Use of this antibody enables an alkylphenol compound to be specifically concentrated even in the presence of an alkylphenol ethoxylate compound without being influenced by the compound.

Description

 Description Alkylphenol-specific antibodies and their uses

 The present invention relates to an antibody that specifically recognizes an alkylphenol compound and does not recognize an alkylphenol ethoxylate compound, a method for producing the antibody, a method for measuring an alkylphenol compound using the antibody, and a method using the antibody. The present invention relates to a method for concentrating an alkylphenol compound and the like, characterized by the above. Background art

 Noel phenol (hereinafter may be referred to as NP) Alkyl phenol (hereinafter sometimes referred to as AP) such as octyl phenol (hereinafter sometimes referred to as OP) Compounds are found in the environment, especially in drinking water, river water, and lakes. It exists in environmental waters such as water, seawater or sewage, has endocrine disrupting effects on living organisms such as mammals and fish, and has serious effects on ecosystems. Therefore, it is necessary to measure and analyze alkylphenolic compounds in the environment, and to use the results for environmental conservation.

 Various methods for measuring an alkylphenol compound have been conventionally known. For example, methods for measuring these alkylphenol compounds in environmental water include high performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography—high-resolution mass spectrometry (GC-HRMS), and high-performance liquid chromatography. Resolution mass spectrometry (HPLC—H

RM S) is known.

However, these methods do not have the sensitivity required for quantifying alkylphenol conjugates having only a trace amount, and the quantification by these methods requires high-magnification concentration such as extraction with a solvent. The necessity, the use of very expensive equipment, and the need to master the operation of the equipment are problems. Because of these issues, more sensitive, specific, convenient, and low-cost alkyl It is desired to develop a method for measuring one Luich compound.

 Means for solving the above problems include an immunological measurement method using an antibody or the like that specifically recognizes the alkylphenol compound.

 However, until now, various attempts have been made to create antibodies that specifically recognize alkylphenol compounds, but the structure of alkylphenol compounds is very similar to that of alkylphenol ethoxylate compounds. Therefore, any of the known antibodies that recognize an alkylphenol enol compound have cross-reactivity with an alkylphenol ethoxylate compound (WO99 / 43997). No. bread fret). Therefore, it was difficult to measure only an alkylphenol compound by the immunoassay using the antibody under the influence of an alkylphenol ethoxylate compound present in a large amount in the environment. Therefore, it has been desired to obtain an antibody that specifically recognizes an alkylphenol compound and does not recognize an alkylphenol ethoxylate compound.

 In view of the above circumstances, an object of the present invention is to provide an antibody that specifically recognizes an alkylphenol phenol compound and does not recognize an alkylphenol ethoxylate compound. Disclosure of the invention

 Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and specifically recognize an alkylphenol compound by immunizing an animal with a conjugate of an alkylphenol compound and a protein, thereby obtaining an alkylphenol ethoxylate. The present inventors have found that an antibody that does not recognize a compound can be obtained, and have completed the present invention.

 That is, the present invention relates to the following.

 (1) An antibody that specifically recognizes an alkyl phenol compound and does not recognize an alkyl phenol ethoxylate compound.

(2) When the molar concentration of the alkylphenol-conjugated compound required to inhibit the binding to the alkylphenol / Reich compound by 50% is set to 1, the binding is inhibited by 50%. The required molar concentration of the alkylphenol ethoxylate compound is 5 The antibody according to (1) above,

 (3) The alkylphenol compound is represented by the formula (1):

[Wherein, RR ² and R ³ are the same or different and each represent H or alkyl]. The alkylphenol ethoxylate compound is represented by the formula (2):

[Wherein R ⁴ , R ⁵ and R ⁶ are the same or different and are each H or alkyl, and R ⁷ is (OC ₂ H ₄ ) _m OH or (OC ₂ H ₄ ) _m COOH (wherein m represents an integer of 1 to 70), which is the compound represented by the above (1) or

The antibody according to (2).

 (4) The antibody according to any one of (1) to (3) above, which is a monoclonal antibody.

(5) The antibody according to (4), which is AP-215.

 (6) A hybridoma that produces the monoclonal antibody according to (4). (7) The hybrid according to (6) above, which is AP-215 (FERM BP-08567).

 (8) The method for producing an antibody according to the above (1), comprising the following steps:

(a) immunizing a mammal with a conjugate of an alkylphenol compound and a protein;

 (b) a step of isolating an antibody that specifically recognizes the alkylphenol compound and does not recognize the alkylphenolethoxylate compound.

(9) The conjugate of the alkylphenol conjugate and the protein is represented by the formula (3):

 L—Z

[Wherein RR ² and R ³ are the same or different and are each H or alkyl, L is a spacer, Z is a protein residue] or a compound represented by the formula (4) :

Wherein RR ² and R ³ are the same or different and each represents H or alkyl, L represents a spacer, and Z represents a protein residue. 8. The production method according to 8).

 (10) The method according to (9), wherein the spacer is a divalent group containing alkylene or a divalent group derived from a hydrophilic polymer.

 (11) The production method according to the above (10), wherein the hydrophilic polymer is a polyoxyalkylene glycol.

 (12) The spacer is the equation (5):

— X— Y— 0C0 (CH ₂ ) _n C0NH— (5) [where X is a bond or alkylene, Y is a bond or 1 (Z) _p — (where Z is a repeating hydrophilic polymer) A unit, p represents an integer of 1 to 20), and n represents an integer of 1 to 10.] or a divalent group represented by the following formula (6):

 —— X——Y——C0NH—— (6)

[Wherein, X is a bond or alkylene, Y is a bond or 1 (z) _p — (wherein Z represents a repeating unit of a hydrophilic polymer, and p represents an integer of 1 to 20).] The production method according to the above (10), wherein the divalent group is represented by the following formula:

 (13) A method for measuring an alkylphenol compound, comprising using the antibody according to (1).

 (14) A kit for measuring an alkylphenol compound comprising the antibody described in (1) above.

 (15) A method for concentrating an alkylphenol compound, comprising using the antibody according to (1).

 (16) A kit for concentrating an alkylphenolic compound comprising the antibody according to (1).

 By using the antibody of the present invention in a measurement method such as ELISA, even in the presence of an alkyl phenol ethoxylate compound, the alkyl phenol conjugate can be specifically measured without being affected by the compound. Can be. Further, by using the antibody of the present invention, even in the presence of an alkylphenol ethoxylate compound, the alkylphenol compound can be specifically concentrated without being affected by the presence thereof. Brief Description of Drawings

 FIG. 1 is a standard curve of an alkylphenol compound (noelphenol) prepared in ^ S Example 4 described below.

 FIG. 2 is a diagram showing the pretreatment of seawater in Example 5.

 FIG. 3 is a graph showing the correlation between the value measured by the ELISA method and the value measured by GC-MSZMS for the alkylphenol compound concentration in the sample in Example 5. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention provides an antibody that specifically recognizes an alkylphenol conjugate and does not recognize an alkylphenol ethoxylate compound.

"An antibody that specifically recognizes an alkylphenol compound and does not recognize an alkylphenol ethoxylate compound" means that it specifically recognizes an alkylphenol compound. The antibody is not particularly limited as long as it is an antibody that does not substantially recognize the alkylphenol ethoxylate compound. More specifically, the molar concentration of the alkylphenol compound required to inhibit binding to an alkylphenol compound (for example, 4-noylphenol, 4-octylphenol, etc.) by 50% ( When IC 50) is set to 1, the molar concentration of an alkylphenol ethoxylate compound (for example, noni ^^ phenenoleethoxylate, octi ^ / phenenoleethoxylate, etc.) required to inhibit the binding by 50% is obtained. Antibodies with 5 or more, preferably 10 or more are exemplified.

 In the present invention, the alkylphenol compound is not particularly limited. For example, the compound represented by the formula (1)

R ² R ³

[Wherein, RR ² and R ³ are the same or different and each represent H or alkyl].

In the formula (1), preferably, any one group selected from the group consisting of RR ² and R ³ is alkyl, and the other two groups are H. More preferably, R ¹ is alkyl and R ² and R ³ are H.

 In the formula (1), the number of carbon atoms of the alkyl is not particularly limited, but is usually 1 to 20, preferably 3 to 16, and more preferably 5 to 14.

Examples of the alkyl include methyl, ethyl, propyl, isopropyl, butyl, sec-butynole, t-pentyl, pentynole, isopentinole, t-pentynole, hexinole, t-hexynole, heptinole, t-hexinole, and octynole. , T-octyl, noel, _t -nonyl, dodecyl, t-dodecyl, and the like, but are not limited thereto.

The alkyl may be a primary, secondary or tertiary alkyl, and is not particularly limited, but is preferably a tertiary alkyl. Specific examples of the above alkylphenol compounds include DP (4-dodecylphenol), EP (4-ethylphenol), HP (heptylphenol), IPP (4-isopentylphenol), and 2-OP (2-octylphenol). Phenol), NP (noyulphenore), 4-NP (4-nonylphenol), 4-OP (4-octylphenol), 4-sBP (4-sec-butylphenol), 4- Examples thereof include t BP (4-t-ptinolephenol), 4-t PP (4-t-pentinolephenol), and 4-t OP (4-t-octylphenol).

 In the present invention, the alkylphenol ethoxylate compound is not particularly limited. For example, the compound represented by the formula (2):

[Wherein, R ⁴ , R ⁵ and R ⁶ are the same or different and each is H or alkyl, and R ⁷ is (OC ₂ H ₄ ) _m OH or (OC ₂ H ₄ ) _m COOH, Represents an integer of 1 to 70].

 In the formula (2), the number of carbon atoms of the alkyl is not particularly limited, but is usually 1 to 20, and preferably 1 to 12.

Examples of the alkyl include methyl, ethyl, and phenyl. Mouth pill, isopropyl, butyl, sec-butyl, t-butyl, pentyl, isopentyl, t-pentyl, hexinole, t-hexinole, heptinole, t-heptizole, octinole, t-octyl 7, _t- nonyl, _t Examples include, but are not limited to, one noel, dodecyl, t-dodecyl, and the like.

 In the formula (2), m is preferably an integer of 1 to 30, more preferably 1 to 15. m means the average number of ethylene oxide chains.

As the alkylphenol ethoxylate compound, specifically, NPE (noylphenol ethoxylate, for example, NP 1 EO (the number of ethylene oxide chains = 1), NP 2 EO (the number of ethylene oxide chains = 2), NP 5EO (Average number of ethylene oxide chains = 5), NP 6 EO (Roh loose phenol to hexa ethoxylate: ethylene oxide chain number = 6), NP 10EO (average ethylene oxide chain number = 10), NP 15 EO (average acid modified styrene chain number _{= 1 5)} NP 1EC (average number of ethylene oxide chains = 1), NP 2EC (average number of ethylene oxide chains = 2), NP4EC (average number of ethylene oxide chains = 4), NP 7 EC (average number of ethylene oxide chains = 7), NP 10EC (Average number of ethylene oxide chains = 10, terminal OH → carboxylic acid), OPE (octylphenol ethoxylate) and the like.

Examples of the antibody include natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAb), recombinant antibodies produced using genetic recombination techniques (eg, single-chain Fv fragments (scFv), bispecific-chimeric scFV (% -scFv), tandem scFV (scFv) 2. bispecif ic-(scFv) 2, disulfide-linked scFv, disulfide-stabilized Fv fragments (dsFv) _N diabody, single-chain diabody (scDb) bivalent diabody ^ bispecif ic diabody _N knob -into-hole stabilized diabody, disulfide-stabilized diabody ^ triabody ^ tetrabody ^ trispecific triabody _N CL-- dimerized scFv, CHI-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, leucine-zipper staoilized scFv dimers _N helix-stabilized scFv dimers, 4 helix-bundle stabilized scFv tetramers ^ streptavidin-scFv, intrabody, Fab 'fragment F ( ab) fragment s _x Fv fragments (Fv), Kimefu antibodies, humanized antibodies, single chain antibodies, etc.), but are not limited to force human antibodies and the like that can be produced using human antibody-producing transformer diethyl nick animals like. The antibody is preferably a monoclonal antibody or a polyclonal antibody, more preferably a monoclonal antibody. Preferable examples of the monoclonal antibody include AP-215 shown in Examples below.

In the present invention, “antibody” is a concept including a binding fragment thereof. The antibody-binding fragment refers to a partial region of the above-mentioned antibody, specifically, for example, F ^ ab) ₂ , Fab, Fab, Fv, variab 丄 e fragment of antibody), scFv, dsFv ^ disulfide stabilised Fv), dAb (single domain antibody), etc. (Exp.Op in.Ther.Patents, Vol. 6, No. 5, p. 441-456, 1996), and antibody fragments produced by Fab expression libraries.

 The present invention also provides a hybridoma that produces the above-mentioned monoclonal antibody. Preferable examples of the hybridoma include AP-215 (FERM BP-08567) shown in the following examples.

 The antibody of the present invention immunizes mammals with a conjugate of an alkylphenol compound and a protein, specifically recognizes an alkylphenol compound, and does not recognize an alkylphenol ethoxylate compound. By separating, it can be manufactured. The present invention provides a method for producing the antibody.

 The above-mentioned protein is not particularly limited, and examples thereof include Pseudoserum albumin (BSA), squash shellfish hemosyanin (KLH), ovalbumin (OVA), Peagle serum albumin (RSA), and Psitide log mouth purine (BTG). Can be The conjugate of the alkylphenol compound and the protein is not particularly limited as long as the alkylphenol compound is a compound in which the alkylphenol compound is directly or indirectly bound to the protein. Preferably, the conjugate is an alkylphenol compound. A compound bound to a protein via a spacer.

 Preferred examples of the conjugate include, for example, formula (3):

R ² R ³

L-Z

Wherein, ^1, R ² and R ³ are as defined above, L is a spacer, Z is meant protein residue] and the compound represented by the formula (4):

[Wherein, I ¹ , RR ³ , L and Z have the same meanings as described above].

 From the viewpoint of increasing the specificity of the obtained antibody to the alkylphenol compound, or by specifically detecting the alkylphenol-conjugated compound, the antibody that does not recognize the alkylphenol ethoxylate compound can be obtained with higher efficiency. From the viewpoint of obtaining the compound, the conjugate of the alkylphenol compound and the protein is preferably a compound represented by the above formula (3).

 The spacer is not particularly limited as long as the antibody of the present invention can be produced using a conjugate of an alkylphenol compound and a protein via the spacer. And a divalent group derived from a hydrophilic polymer. The spacer is preferably a divalent group derived from a hydrophilic polymer.

If spacers one is a _divalent group containing an alkylene, the carbon number of the alkylene is not particularly limited, usually 1-2 0, for example, 1-1 0, preferably because at 1-6.

 Examples of the hydrophilic polymer include, but are not limited to, polyoxyalkylene daricol (for example, polyoxyethylene glycol, polyoxymethylene glycol, polyoxypropylene glycol, etc.), polybutyl alcohol, polyacrylamide, and polybutylpyrrolidone. , Polyhydroxyethyl acrylate, polymethyl ether, polyacrylic acid, poly (isobutylene monomalenic acid), poly (2-acrylamide 2-methylpropane monosulfonic acid), poly (acryloxypropane sulfonic acid) ), Polybutylphosphonic acid, poly (methacryloyloxyethyl) quaternized ammonium chloride, polybutylpyridine, N-N-dimethyl-N- (3-acrylamidepropyl) -1-N- (carboxymethyl) ammonium, Polydiene O carboxymethyl SORUN, polyethylene I Min, poly (N- Byuruasetoami de), and the like. Preferably, the hydrophilic polymer is a polyoxyalkylene glycol, more preferably polyoxyethylene dalicol.

Although the degree of polymerization of the hydrophilic polymer is not particularly limited, it is usually 1 to 20, for example, 1 110, preferably 1-6.

As the spacer one, more specifically, for example, the formula (5): A _{X- Y- 0C0 (CH 2) n} C0NH- (5)

[Wherein, X is a bond or alkylene, Y is a bond or one (z) _p — (wherein, Z is a repeating unit of a hydrophilic polymer, and p is an integer of 1 to 20)] , N represents an integer of 1 to 10], a divalent group represented by the formula (6):

— X— Y— CO 丽 一 (6)

[Wherein, X and Y have the same meanings as described above]. The spacer is preferably a divalent group represented by the formula (5).

 When X is alkylene, the carbon number of the alkylene is not particularly limited, but is usually 1 to 20, for example, 1 to 10, preferably 1 to 6, more preferably 1 or 2. p represents an integer of preferably 1 to 10, more preferably 1 to 6. N is preferably an integer of 1 to 6, more preferably 2 to 4.

 Specific examples of the alkylene include, for example, methylene, ethylene, propylene, isopropylene, butylene, sec-butylene, t-butylene, pentylene, isopentylene, t-pentylene, hexylene, t-hexylene, heptylene, t- Butylene, octylene, t-octylene, nonylene, t-nonylene, dodecylene, 't-dodecylene and the like.

A conjugate of the alkylphenol compound and the protein can be easily produced by those skilled in the art from the alkylphenol compound and the protein by a well-known reaction. For example, equation (7) _:

[Wherein RR ² , R ³ s Z and p are as defined above], and a conjugate of an alkylphenol compound and a protein can be produced by the following production method or the like.

First, the Friedel-Crafts reaction, and alkylphenol torquecontrol compound of formula (1), is reacted with C 1 ₂ CHOCH _3, by formylating the alkyl Hue no Louis匕合of formula (1), wherein The compound of (8) is obtained.

 (1) (8)

[Wherein, RR ² and R ³ are as defined above. ]

The Friedel-Crafts reaction is usually dichloromethane, black hole Holm, in a solvent that does not inhibit the reaction such as carbon _{tetrachloride, A 1 C 1 3, T} i C l 4, F e C 1 3 , etc. Le chair acid Done in the presence of Reaction conditions such as reaction temperature and reaction time are well known in this field.

 Next, a protecting group is introduced into the hydroxyl group of the compound of the formula (8).

 (8) (9)

[Wherein, R ¹ R ² and R ³ are as defined above, and R ⁸ represents a protecting group. The protecting group is not particularly limited as long as it is a protecting group for a phenolic hydroxyl group. For example, in DMF compound of formula (8), i-by reaction with C 1 CH ₂ OC H ₃ with P r NE t, protected by a hydroxyl group one CH ₂ OCH ₃ of a compound of formula (8) Is done.

Subsequently, the aldehyde of the compound of the formula (9) is reduced.

[Wherein, RR ² , R ³ and R ⁸ are as defined above. ]

Known reducing agents such as Na BH ₄ and Li A 1 H ₄ are used.

For example, a compound of formula (10) is obtained by adding a solution of Na BH ₄ in ethanol to a compound of formula (9) dissolved in ethanol.

 Subsequently, the compound of the formula (10) is halogenated.

[Wherein, RR ² , R ³ and R ⁸ are as defined above, and Ha 1 represents halogen (F, C 1, Br, I, etc.). ]

For example, in dichloromethane compound of formula (10), by reaction with CB r ₄ with PPh _3, to obtain the bromide compound of formula (10).

 Subsequently, the compound of formula (11) is reacted with polyethylene glycol in the presence of a base to introduce a polyethylene dalicol chain.

[Wherein, RR ² , R ³ , R ⁸ , Ha 1 and p are as defined above. ] Examples of the base include metal hydride compounds (eg, sodium hydride, hydrogen hydride). Well-known inorganic bases such as calcium hydroxide and calcium hydride are used.

For example, to obtain a compound of formula (11), in DMF, under NaH presence, is reacted with _{H (OC 2 H 4) p} OH, a compound of formula (12).

 Subsequently, the compound of the formula (12) is reacted with succinic anhydride to obtain a succinic ester of the compound of the formula (12) (the compound of the formula (13)).

n0C0C ₂ H ₄ C00H

[Wherein, RR ² , R ³ , R ⁸ and p are as defined above. ]

 Further, the compound of the formula (14) is obtained by removing the protecting group of the formula (13) with an acid.

[Wherein, RR ² , R ³ , R ⁸ and p are as defined above. ]

The compound of the formula (14) is reacted with N-hydroxysuccinimide to obtain an activated ester (formula (15)), and the activated ester is reacted with the protein to obtain a compound represented by the formula (7). A conjugate of the alkylpheno / Reich compound and the protein can be obtained.

(15)

 0

¾N— Z

)

^{Wherein, 1, R 2, R 3} , Z and n are as defined above. ]

 A conjugate of an alkylphenol compound other than the compound represented by the formula (7) and a protein can be easily produced by a person skilled in the art according to the method described above or according to the method described in Examples described later. Can be.

 In the method for producing an antibody of the present invention, a mammal is immunized with a conjugate of the alkylphenol compound obtained by the above-described method and a protein.

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. Dosing usually once every 2 to 6 weeks It can be performed about 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, 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)]. As the fusion promoter, for example, a force S such as polyethylene glycol (PEG) or Sendai virus, preferably PEG is used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0 _s AP-1, and the like. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells to be used is about 1: 1 to 20: 1, and about 10 to about 80% of PEG (preferably PEG 1000 to PEG 6000). Concentrated, 20-40. Incubation with 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, 1 to 20%, preferably 10 to 20%. /. RPMI 1640 medium containing fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM—10) 1, Nissui Pharmaceutical Co., Ltd.) 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. The culture can be usually performed under 5% carbon dioxide gas.

Subsequently, a hybridoma that produces a monoclonal antibody that specifically recognizes the alkylphenol compound 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 a phase (eg, microplate), then add a conjugate of an alkylphenol compound and a labeling enzyme, and detect the monoclonal antibody bound to the solid phase. Adsorb the alkylphenol compound. A hybridoma culture supernatant is added to a solid phase (eg, microplate), and then an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the spleen cells used for cell fusion are mouse-derived, anti-mouse Or immunoglobulin antibody) or monoclonal with protein A bound to solid phase A method of detecting the body is used.

 Labeling enzymes include horseradish peroxidase, alkaline phosphatase and the like. The conjugate of the alkylphenol compound and the labeling enzyme can be produced in the same manner as the above-mentioned conjugate of the alkylphenol compound and the protein.

 Further, from the hybridomas selected above, a hybridoma that produces a monoclonal antibody that does not recognize the alkylphenol ethoxylate compound is selected. The selection can be carried out using a conjugate of an alkylphenol ethoxylate compound and a labeled enzyme, and the like, in the same manner as described above. The conjugate of the alkylphenol ethoxylate compound and the labeling enzyme can be produced, for example, by the method described in JP-A-10-155484.

 As described above, a hybridoma producing a monoclonal antibody that specifically recognizes an alkylphenol compound and does not recognize an alkylphenol ethoxylate compound is obtained.

Subsequently, the alkylphenol compound was specifically recognized from the culture supernatant of the hybridoma and the ascites obtained by transferring the hybridoma to a pristine-treated nude mouse, and the alkylphenol ethoxylate compound was recognized. No monoclonal antibodies are isolated. Monoclonal antibodies can be isolated by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, Isoelectric focusing, electrophoresis, adsorption / desorption using an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase, or active antibody such as protein A or protein G. Specific Purification Method of Collecting and Dissociating the Bonds to Obtain Antibodies].

 When the antibody is a polyclonal antibody, the conjugate of the above-described alkylphenol compound and the protein is administered to the mammal itself or together with a carrier, a diluent, etc., and the mammal is treated with the conjugate in the same manner as the monoclonal antibody. Immunize. Examples of mammals to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and rabbits, sheep, goats, 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 obtained by separating an antibody fraction specifically recognizing an alkylphenol compound by affinity chromatography using a column to which an alkylphenol compound is bound, and further comprising the alkylphenol ethoxylate compound. Purification is performed by removing the antibody fraction recognizing the alkylphenol ethoxylate compound using a column to which is coupled. '

 In addition, a gene encoding the monoclonal antibody is cloned from the hybridoma producing the monoclonal antibody, and the gene is used to specifically recognize the alkylphenol-conjugated product. A recombinant antibody that does not recognize the phenol ethoxylate compound 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). Production method, Cloning method of antibody gene from hybridoma cell line (Hybridoma Cell Line), Preparation method of antibody gene library (Antibody Gene Libraries), Gene It describes how to select recombinant antibodies from libraries (Selection of Recombinant Antibodies From Gene Libraries) and how to manipulate antibodies (antibody engineering). it can. The chimeric antibody is described, for example, in “Experimental Medicine (Extra Extra Number), Vol. 1.6, No. 10, 1988”, and Japanese Patent Publication No. 3-73280. No. 506458, Japanese Patent Publication No. 62-296890, etc., and human antibodies are described, for example, in fNature Genetics, Vol. 15, p. 146-156, 1997, iNature Genetics, Vol. 7, p. 13-21, 1994. Japanese Patent Application Publication No. H4-1504504365, International Application Publication No. TO94 / 25585, "Nikkei Science, June Issue, pp. 40-50, 1995," Nature, Vol. 368 , p. 856-859, 1994, and Japanese Patent Application Publication No. 6-500233.

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

 The antibody of the present invention can be used as a reagent for quantitatively measuring an alkylphenol compound or an affinity column for concentrating the alkylphenol compound by immobilizing it on various carriers. It can be used for the production of and the like. 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 antibody of the present invention can be produced, for example, by the methods described in “Metabolism”, Vol. 8, 696 (1971), the Promcian method, the Daltaraldehyde method, the carbodiimide method, the epoxy activation method, and the “Enzymimnoassay” "Affinity Chromatography Handbook" (Amersham Fanoremasia Biotech Co., Ltd. (published on Feb. 20, 1998)). A solid phase antibody or an immunoadsorbent can be obtained by solid-phase immobilization on a carrier by a known method such as this. Examples of the carrier for immobilization include microplates (eg, 96-well microplate, 24 well microplate, 1992 well plate, 38 well microplate, etc.), test tubes (eg, , Glass test tubes, plastic test tubes), glass particles, polystyrene particles, modified polystyrene particles, polybutyle particles, latex (eg, polystyrene 'latex), nitrocellulose Membrane, cyan bromide-activated filter paper, DBM-activated filter paper, granular solid phase (eg, Sepharose, Sephadex, Agarose, Senorelose, Cefacryl etc.), iron-containing poly-ponate membrane, magnet-containing beads, quartz oscillator, etc. Can be

 These are combined with, for example, the above-mentioned conjugate of an alkylphenol compound and a labeling enzyme, a standard solution of the alkylphenol compound, a washing solution, a coloring substrate, a coloring stop solution, a buffer solution, and the like to measure the alkylphenol compound. Kit ゃ Concentration kit can be used. The present invention provides these kits.

 The standard solution of the alkylphenol compound includes, for example, a solution containing a known concentration of the alkylphenol compound.

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

 The chromogenic substrate can be appropriately selected according to the type of the labeling enzyme.If the labeling enzyme is peroxidase, TMB Z, TMB, ABTS, DAB, AEC, 4-CN, etc. are used. Can be When the labeling enzyme is alkaline phosphatase, p-2-trophenol, BCIP / NBT and the like are used. The coloring substance may be in a solid state such as a powder or a state dissolved in an appropriate 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 and the like of the alkylphenol compound may be prepared as a concentrated solution, and may be appropriately diluted before use.

 The above-described 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 the alkenyl phenol conjugate. For example, a specific alkylphenol compound can be specifically measured or concentrated by using a kit containing a plurality of antibodies having different cross-reactivity.

Examples of the method for measuring the alkylphenol compound of the present invention include a radioisotope immunoassay (RIA method), an ELISA method (Engvall, Ε ·, Methods in Enzymol., 70, 419-439 (1980)), and a fluorescent antibody method. , Plaque method, spot method, agglutination method, Octerlony, etc. Ridoma Method and Monoclonal Antibody ", published by R & D Brajung, p. 30 — p. 53, March 5, 1982, and a method using a crystal oscillator (Japanese Patent Application Laid-Open No. 2002-31008). No. 74 gazette). The ELISA method is widely used from the viewpoints of sensitivity, simplicity, and the like.

 In the method for measuring an alkylphenol compound of the present invention, the amount of binding of a sample (for example, environmental water containing an alkylphenol compound) to a known amount of a labeled alkylphenol compound to the antibody of the present invention is determined. The so-called competitive method, which is quantified by a competitive reaction, is preferably used. In the competitive method, a sample solution containing an unknown amount of an alkylphenol compound is mixed with a known amount of a labeled alkylphenol compound, and the mixed solution is mixed with a fixed amount of the present invention held on a carrier. Contact with antibody. Detects label retained on carrier or label not retained on carrier.

 Examples of the labeling agent for labeling the alkylphenol compound include the above-mentioned labeling enzyme, radioisotope, enzyme substrate, fluorescent substance, biotin, and the like. Alkylphenolic compounds and these labeling agents can be bound by the maleimide method [Journal of Biochem (J. Biochem.), 79, 23 (1976)], the active hibitin method. [Journal 'ob' American Chemical Chemical Society (J · Am. Chem. Soc.), 100, 358 (1978)]. More specifically, for example, a sample solution containing an unknown amount of an alkylphenol compound is mixed with a known amount of a labeled alkylphenol compound, and the mixed solution is held in a fixed amount held on a carrier. Of the antibody of the present invention. 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. If the labeling agent is a labeling enzyme, add the substrate and allow to stand. Measure the enzyme activity by a colorimetric or fluorescent method. 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 concentrating the alkylphenol aldehyde compound of the present invention, a large amount of a sample is passed through an immunoadsorbent ram or mixed with immunoadsorbent particles. Using the antigen-antibody reaction, the alkylphenol compound is captured by the immunoadsorbent, and then the pH is changed (for example, lowered to pH 2.5 to 3, or increased to pH 11), and the ionic strength is changed (1 MN such as a C 1), change of polarity (10% Jiokisan, 50% ethylene glycol, 3M chaotropic salt _{(SCN-, CC l 3 COO-,} I I), etc.), protein denaturants (8M urea, 6M hydrochloric acid guanidine etc. ) And elution by a known method such as electrophoretic dissociation, it is possible to concentrate an alkylphenol-enriched compound containing few immunologically contaminants at a high magnification of thousands to tens of thousands of times.

 With this concentration method, it is possible to concentrate alkylphenol compounds, which are present only in trace amounts in the environment, at a much higher magnification than conventional concentration methods such as solvent extraction and solid phase extraction. In addition, it is possible to obtain a concentrated solution having a low content of impurities and the like that hinder 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.

 In addition, mouse / mouse-hybridoma AP-215 manufactured in the following examples was established on December 10, 2003 under the Budapest Treaty under the Deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology under the accession number F ERM BP-08567. Example 1

Production of monoclonal antibodies

 (1) Production of no and putten

 (1) Manufacture of 1 AP-1

4-Nourphenol (Tokyo Kasei, Lot. FGE01) 4.4 g (20 mm o 1) and glutaric anhydride 2.28 g (2 Ommo 1) at 100 ° C for 22 hours under nitrogen atmosphere The reaction was carried out with stirring. After cooling, purification with a silica gel column (silica 100 g, hexane: ethyl acetate = 1: 1) was repeated twice to obtain a pale yellow oily substance giving a single spot by TLC. This is diluted with ether and re-concentrated to obtain equation (17):

1.6 g of a compound (AP-1) represented by the following formula was obtained (purity: 99.0%, analysis by HPLC).

 (1) Manufacture of 1 2 AP-3

4 one nonyl Hue no Honoré (Tokyo Kasei, L ot. FGE 01) 1 2. 5 g (56. 8mmo 1) a was dissolved in CH ₂ C 1 ₂ 60 mL, one 30. Cooled to C. It was added dropwise T i C l ₄ 12. 5mL, was added dropwise C 1 ₂ CHOCH ₃ 5mL. The cooling bath was removed, and the reaction solution was returned to room temperature (about 30 minutes). After introducing the reaction mixture into ice water 10 OML, and the mixture is separated, and the aqueous layer was extracted with CH ₂ C 1 ₂ 5 OmL, the combined organic layers were washed with brine 10 OML. After drying over Na ₂ SO ₄ , the mixture was concentrated under reduced pressure to obtain 12.9 g of a brown oil. The oil was applied to a silica gel column (150 g of silica, hexane: ethyl acetate 100:

(0, 50: 1, 10: 1 elution) to obtain 1-1.5 g (46.4 mmo 1) of 2-formyl-41-noylphenol (yellow oil) (process).

1, yield 82%) o

After dissolving 2.48 g (1 Ommo 1) of 2-formyl-1-noerphenol in 2 OmL of DMF, the mixture was cooled on ice. After adding 0.42 g (10.5 mMol) of 60% NaH (in oil), the mixture was stirred for 4 minutes under ice cooling. It was added dissolved in _{DMF 5mL Ph CH 2 B r 1.} 73 g (10. lmmo l), and stirred for 1 hour under ice-cooling. The reaction solution was poured into ice water (80 mL), extracted with ethyl acetate (50 mL × 2), and washed with 2 OmL of water. After drying over Na ₂ SO ₄ , the filtrate was concentrated under reduced pressure to obtain 3.52 g of crude 2-formyl-14-phenol-O-benzyl ether (step 2).

The crude 2-formyl one 4-Bruno loose phenol O- benzyl ether 3. 52 g was dissolved in ethanol _{5 OmL, Na BH 4 0. 2} g of (5. 3 mm o 1) was added Caro, 30 minutes at room temperature After stirring, the mixture was concentrated under reduced pressure. After adding 5 OmL of water and 50 mL of ethyl acetate to the residue, separating, washing with saturated saline, drying over Na ₂ SO ₄ and concentration under reduced pressure, the residue was subjected to a silica gel column (100 g of silica, hexane : Ethyl acetate 10: 1 elution) to give 2-phenoxy-1-5-nonylbenzyl alcohol. 2.15 g (6.3 mmo 1) of coal was obtained (total yield of step 3, steps 2 and 3 was 63%).

 2-phenoxy-5-nonylbenzyl alcohol 2.15 g (6.3 mmo 1) and 0.75 g (6.58 mmo 1) of dtaltaric anhydride were stirred at 100 ° C overnight, then allowed to cool to room temperature. Purification with silica gel column (100 g of silica, elution with hexane: ethyl acetate 10: 1) yielded 1.85 g (4.07 mmol) of mono (2-phenoxy-1-5-norbenzyl) daltalate ester ) Was obtained (Step 4, yield 65%). Dissolve 1.85 g (4.07 mmo 1) of dartano mono (2-phenoxy-1-5-nonylbenzyl) esterol in 5 OmL of ethyl acetate and add 0.1 g of 10% Pd / C (50% water-containing product). Then, it was 3 o'clock under 7k blowing. 0.2 g of 10% Pd / C (50% water-containing product) was added to ®S solution, and the square was filtered off for 2.5 hours while blowing 7 elements. Anti

After drying J¾ night with Na ₂ SO ₄ , purification was carried out using a silica gel column (100 g of silica, elution with hexane: ethyl acetate 1: 1), and the formula (18):

0.6 g (1.65 mmo 1) of the compound (AP-3) represented by the formula was obtained (step 5, yield: 40%).

 (1) Manufacturing of —3 AP-6

4-Noni Bruno reflex Hainaut Honoré 12. 5 g of (56. 7 mmo 1) was dissolved in CH ₂ C 1 ₂ 6 OmL, and cooled to a 30 ° C. Was added dropwise _{T i C l 4 12. 5mL (} 113. 7 mm o 1), was added dropwise _{C l 2 CHOCH 3 5mL (57.} 2 mm o 1). The cooling bath was removed and the reaction was allowed to come to room temperature (about 30 minutes). After introducing the reaction mixture into ice water 10 OML, and the mixture is separated, and the aqueous layer was extracted with CH ₂ C 1 _2, the combined organic layers were washed with saturated brine. After drying over Na ₂ SO ₄ , the mixture was concentrated under reduced pressure to obtain a brown oil. Purification was performed using a silica gel column (hexane: ethyl acetate, elution 100: 0, 10: 1) to obtain 11.7 g (47.1 mmo 1) of a yellow oil (step 1, yield: 83%).

Dissolve 11.7 g (47.lmmol) of yellow oil in 100 mL of DMF, then add ice Under cold i one P r NE t 24. 2mL (141. 3mmo l), C 1 CH 2 OCH 3 4. added 6mL (61. 2mmo 1), and stirred at room temperature for 1 hour. After stirring at 75 ° C for 1 hour, the mixture was returned to room temperature, poured into 15 OmL of ice water, and extracted with ethyl acetate. After drying over N a ₂ S0 _4, and concentrated under reduced pressure to give a yellow oil 14. O _g (step 2).

Na BH ₄ 0. 9 g of (23. 9 mm o 1) was dissolved in ethanol 60 mL, was added dropwise the yellow oil 14. 0 g dissolved in Etanoru 60 m L under ice-cooling, 30 under ice-cooling For a minute. After concentration under reduced pressure, water and ethyl acetate were added to the residue to separate the layers, and the organic layer was washed with saturated saline. After drying over Na ₂ SO ₄ , the filtrate was concentrated under reduced pressure to obtain 13.8 g of a yellow oil (Step 3).

After dissolving the yellow oil 7. 0 g in CH ₂ C 1 ₂ 175mL, it was added dropwise PPh ₃ 7. 48 g and _{CB r 4 l l. 84 g} , was stirred for 30 minutes at room temperature. The reaction solution was poured into saturated aqueous sodium bicarbonate solution (10 OmL), separated, and the organic layer was washed with saturated saline. After drying over Na ₂ SO ₄ , the mixture was concentrated under reduced pressure, and ethyl acetate / hexane was added to the residue to remove a precipitate. After concentration under reduced pressure, the residue was purified by a silica gel column (hexane: ethyl acetate eluted 4: 1) to obtain 7. Og (19.6 mmo 1) of a yellow oil (Step 4, Steps 2 to 4). The total yield is 82%).

Dissolve HO (CH ₂ CH ₂ O) ₆ H 7.5 g (26.6 mm o 1) in 25 mL of DMF and then, under ice cooling, 0.53 g of NaH (60%, in oil) 0.53 g (13.2 mmo 1) Was added and stirred for 30 minutes. 4.7 g (13.2 mmo 1) of the yellow oil dissolved in 25 mL of DMF was added dropwise, stirred for 30 minutes, returned to room temperature, and stirred for 3 hours. The reaction solution was poured into 150 mL of ice water, extracted with IPE, and washed with saturated saline. After drying over Na ₂ SO ₄ , the mixture was concentrated under reduced pressure to obtain a brown oil. Purification by silica gel column (elution with ethyl acetate and methanol) gave 3.6 g (6.4 mmo 1) of a yellow oil (Step 5, yield 48%).

 To 1.0 g (1.79 mmo 1) of the yellow oil was added 18 Omg (1.79 mmo 1) of succinic anhydride, and the mixture was stirred at 100 ° C under reduced pressure to obtain 1.35 g of a brown oil (Step 6). ).

Dissolve 20% Omg (0.3 Ommo 1) in 10% OPA After adding 5 mL of HC 1 / MeOHO, the mixture was covered for 10 minutes. Overnight, 250 mg of brown oil was obtained. Applying the difficulty to the gel ram (black mouth form: methanol = 50: 1 elution), the formula (19):

(AP-6) 17 Omg (0.28 mmo 1) was obtained as a dark-colored oil (Step 7, 91% yield).

 (1) Manufacturing of 4 AP-8

After mixing 1.1 g (5.0 mm o 1) of noninolephene and 0.5 g (5. Ommo 1) of conodic anhydride, 100. For 3 hours to obtain 1.6 g of a colorless oil. Silica gel column (silica gel 32 g, n-hexane: ethyl acetate = 20: 1, 1: 1 elution) After purification, formula (20):

(AP-8) 85 Omg (2.65 mmo 1) was obtained as a colorless oil (yield 53%).

 (2) Production of conjugate of hapten and BSA or KLH

 Hapten (AP-1, AP-3, AP-6, AP-8) produced in (1) above 60

^ mo1,7-soluble carbodiimide 72 μmol, N_hydroxysuccinic imide 72 mol1 were allowed to stand overnight in 1 mL of dimethyl sulfoxide to produce an activated ester. Next, 10 mg of serum albumin (BSA, manufactured by Sigma, # A-7906) or squash shell hemocyanin (KLH, CALBIOCHEM difficult, # 374805) is added.

13 || ^ Sodium acid (NaHCO ₃ )

After admitting 67 L, overnight 4. ¾ with C. Dialysis against Danolec-Becolin thigh stock solution (PBS) to remove the remaining difficulties, and the conjugate of hapten and BSA or KLH

^^). The immunogen was stored ^; ^. (3) Immunization

 The immunogen produced in (2) above was dissolved in PBS so as to have a concentration of 500 / igZmL, and mixed with an equal amount of Freund's adjuvant or Libya adjuvant system to prepare an emulsion. The emulsion was subcutaneously administered to a BALBZC mouse (female) at a dose of 100 gZ mouse, and the immunity was stimulated every two weeks. After 4 to 6 immunizations, the serum antibody titer of 4-nourfenol was measured by a competition method. The results are shown in Table 1.

 (Note *: Indicates the antibody titer of the serum diluted 1000 times. ++:> 2.0, +: 0.5 ~ 2.0, 1: <0.5 (Each number indicates absorbance.))

The immunogen was administered intravenously (20 ^ g / O. 2mL PBSZ mouse) to the individual with the highest serum antibody titer (AP-6 as hapten and BSA as protein). Final immunity).

 (4) Cell fusion

The spleen was removed from the mouse that had been subjected to the final immunization in (3) above (3 days after the final immunization), Splenic erectile spores were collected. Mouse myeloma cells and spleen cells were cultured at a ratio of 1:10 under polyethylene glycol (average amount: 4000), and cell fusion was performed. The fusion cells (hypridoma) were suspended in HAT medium and cultured in a carbon dioxide incubator (37 ° C, 5% CO ₂ ), locked in a 96-well 11 microplate.

 (5) screening of hybridomas

 (5) One 1 Atsushi plate creation

A goat anti-mouse IgAGM ^ -body (C appel, # 5546 1) was dissolved in PBS at a concentration of mL, and the solution was added to a microplate (100 / i L / we 11). In 4 ° C - after晚^ & is allowed, then with PBS containing ^{0. 05 0 / oTwe en 20 (} T-PBS) (300 μ L / we 1 1 twice), 1% was «in PBS Block Ace (Snow Brand Milk Products Co., Ltd.) was added at {200 μL we 11}. Four. After over-pressing at C, the plates were stored at 4 ° C until use.

 (5) — 2 Production of ^ body with hapten and HRP

The hapten (AP-1, AP-3, AP-6, AP-8) produced in (1) above was treated with 20 mo 1, water-soluble carbodiimide 24 μmo 1, and N-hydroxysuccinimide 24 mo 1 as dimethino. The activated ester was made by reconstitution in 1 mL of resnorreoxide. Next, 10 mg of horseradish peroxidase (HRP) is dissolved in 3 mL of 0.13 sodium salt (ΝaHCO ₃ ) sickle, 62.5 L of the activated ester is added, and the mixture is allowed to cool at −4 ° C. Was. Unremoved fiber was removed by P-jump filtration to obtain a conjugate of hapten and HRP (hereinafter referred to as enzyme complex). The enzyme complex was prepared at a concentration of 3.3 mg / mL and stored refrigerated.

 (5) -3 Primary screening

In the microplate showing the cells in (4) above, apply 100 μL of the culture medium of we11, which had cell proliferative potential, to the assay plate prepared in (5) -1 above. Returned to PBS (300 ^ LZwe 11 twice). After 1 hour at room temperature ^^? ¾ »83 (3 times with 300 L / wel 1), 5000 times with T-PBS,« Micro-play the ^]! Enzyme complex ^: produced in (5) 1-2 above Garnished with tomato. After one hour of HiS at room temperature, the solution was washed with T-PBS (3 times with 300 L / we 11), and the 液 ^ solution was injected (100 LZ _we 11). After 30 minutes at room temperature, HJ¾f Tei stop solution was urged (lOO LZwe 11), and Wei ^ was stopped. The absorbance at a wavelength of 450 nm was measured, and the cells in we11 over the absorbent fiber 1 were transferred to a 24 we11 microplate.

 (5) -4 Secondary screening (NP inhibition test)

In the 24we11 microplate to which the cells were transferred in step (5) above, sufficient cell proliferation was obtained. The culture medium of we11 was added to the Atsushi plate prepared in (5) -1 above (before use) The cells were washed with PBS or T-PBS (2 times with 300 iLZwe 11). After 1 hour at room temperature, return with T-PBS (3 times with 300 μL / we 11), fermentation complex (8000 in T-PBS) and 1 Omg / L phenol (hereinafter referred to as NP) (10% methanol +1 _^0/0 DMSO, dissolved in super night) or 10%: \ ^ 011 + 1% 0: mixed solution of \ ^ 0裔夜(MM) (1: 1) ^ / をAfter 1 ®S at room temperature, the mixture was ¾fed with T-PBS (3 times with 300 μL / we 11), and the substrate was added (100 L / we 11). After SJ for 30 minutes at room temperature, the stop solution was purified (100 L / we 11) and stopped. The absorption at a wavelength of 450 nm was measured.

 (5) 1-5 Secondary screening

 In the above (5) 1-4, sufficient cell proliferation was observed for cells whose absorbance decreased by 50% or more due to ¾E of NP (10 mg / L) compared to the control. Using the same method, the reciprocity was examined by the same male as in (5) -4 above. Two kinds of noninolephenol ethoxylates (NP1EO, NP10EO) and noninolephenoki, g-sodium acid (NP1EC) were used as exchangeable substances. A standard curve was prepared for each of the NP (control) and the three kinds of cross-linked substances, the concentration at which the inhibition rate was 50% (hereinafter referred to as I C50) was determined, and the cross-reactivity was determined by the following equation.

-Exchange rate (%) = (IC 50 for NP) / (IC 50 for replacement) X 100 The specificity of the NP was high, and the other exchanges were almost identical to the other substances. Cloying was performed according to a conventional method to obtain cells, and the following hybridomas were obtained.

 (6) Screening results

 Table 2 shows the results of Screening. The ス ク リ ー ニ ン グ -215 antibody selected by the above screening; ^ had low production of ^^ '! "With foreign substances other than Ν 、 and high specificity to NP. Table 2 Cross-reactive fiber of AP-215 antibody

 Paternal reactant Manufacturer Code No. Cross-reactivity (%)

 NP Kanto Chemical 28 640-96 100

 NP1EO Hayashi Junyaku 52377 2.4

 NP10EO Tokyo Chemical P0707 4.0

 NP1EC Hayashi Jun 52 393 0.6

(7) Purification of monoclonal antibody

 The cell culture supernatant fractionated with mouse BIzK or 45-50% saturated ammonium sulfate by a conventional method was subjected to protein G affinity chromatography to purify the antibody.

Interaction test

As in Difficult Example 1 (5) -15, the interaction of the antibody AP-215 with an alkylphenol-like compound and other fibrous substances was determined. As shown in Table 3, AP—2 1

5 Antibody was very specific for the alkylphenol compound. Table 3 Cross-reactivity of AP-215 antibody

 Cross-reactivity rate Cross-reactant manufacturer Code No.

 (%) Alkyl phenol (AP)

 4-Nonylphenol Kanto Chemical 28640-96 100

4-octylphenol * -L Science 1021-33045 814

4-Pentylphenol Wako Pure Chemicals 019-11022 86

 Nonylphenol xylate

 (NPnEO)

 n = l Hayashi Junyaku 52377 4.0 n = 2 Tokyo Kasei P0704 3.5 n = 5 Tokyo Kasei P0705 7.0 n = 7.5 Tokyo Kasei P0706 6.0 n = 10 Tokyo Kasei P0707 5.9 Octylphenol ethoxylate

 (OPnEO)

 n = 10 Tokyo Chemical P0873 18

 Nonylphenoxycarboxylic acid

 (PnEC)

 n = l Hayashi Junyaku 52393 0.91 n = 2 Hayashi Junyaku 52394 1.1 n = 3 Hayashi Junyaku 52395 2.1

Row 3

Manufacture of ELISA kit for measurement of alkylphenol compounds. (1) Preparation of “Immobilized plate”

 Goat anti-mouse IgG antibody (ICN / C appe 1 company Cod) dissolved in 25mM B is—Tris—HCl (pH 5.6-5.7) (Dojin Code No. 345-04741) eNo. 55479) into a plate for immobilization (Coster EIA / RIA latestrip 8 Code eNo. 2581) (5 zg / l 00 / z 1

/ we ll), 4. After standing at room temperature for 1 B in C, the mixture was twice played with 300 L of »solution (T-PBS). Purokingu liquid (10 g B loc kAc e (Snow stamping L industry) + 500 ^ 1 Slur off 72 N (Nippon Environmental Chemicals) +0.584 g NaC 1 (Wakorin Cod eNo. 191-01665) +10 g Suc rose (Wako Co de No. 196-00015) +25 ml 20 OmM Tris—HC 1 (pH 7.5), make up to 100 OmL with (200 ^ L / we 11), and 4. C was used twice with 300 μL of static fiber and washing solution (T-PBS). Next, Du 1 becco, s PBS (I) (including Wako Fuji Code No. 041-20211, including 0.05% slough off 72 N + 0.1% BSA (Sigma Code No. A—7638)) The anti-alkyl phenol antibody (AP-783) dissolved in the solution was excited (0.075 μg / 100 1 / we 11), allowed to stand at 4 ° C for a while, and then 300 L of liquid (T-PBS) was added. Pressed twice. After adding the blocking solution 200 1 / we 11 and leaving it at 4 ° C for a while, aspirate the whole volume with an aspirator, enclose the dried solid phase plate in an aluminum bag, and use a vacuum-dried fiber. It was degassed, sealed, and stored in cold shoulder at 2-8 ° C.

 (2) `` Preparation of AP draft

Undiluted solution 1 (1000 mg AP / L): 4-Noninolephenol standard (Kanto Chemical Co. No. 28640-96) was accurately weighed at 10 Omg with respect to 100% content, and 100 ml The flask was made up with At methanol.

Preparation of stock solution 2 (1 OmgAP / L) [20% methanol + 10% DMSO]: Weigh accurately 2 ml of stock solution, 38 mL of methanol, 20 mL of DMSO and Sluroff 72N100 with a hole pipet into a 200 ml volumetric flask. Ab, made up with distilled water.

 Preparation of 20% methanol + 10% DMSO venom (containing 0.05N suloff 72N): 200 mL of methanol, 10 OmL of DMSO and 500 iL of suloff 72N were put into a 1000 ml volumetric flask with Ab and distilled water.

Preparing the target night (0 g AP / L): 20% methanol + 10% DMS θ¾> Preparing the target night (50 / gAP / L): Stock solution 2 1 ml of undiluted solution into a 200m 1 volumetric flask, Al, 20% Methanol + 10% DMS 0 Preparation of standard solution (20 gAP / L): 24 ml of undiluted solution was placed in a 20-ml volumetric flask, and the volume was increased with 20% methanol + 10% DMS O-sickle.

Preparation of standard solution (1000 / igAP / L): 20 ml of undiluted solution was made up in a 200 ml 1 volumetric flask with AtK 20% methanol + 10% DMS 0 overnight.

Preparation of Shimoe night (5000 gAP / L): Undiluted solution (2 10 Oml) was made up into 20 Oml Mesflasco with AK 20% methanol / le + 10% DMSO intense night.

1.5 ml of each of the above standard solutions was stored in a refrigerator at AK 2 to 8 ° C. in five Mighty-pial tubes.

 (3) Preparation of `` listening complex powder ''

27 mg of water-soluble carbodiimide (WS C Dojindo Chemical Co. No. 348-03631 for peptide synthesis) and 16 mg of N-hydroxysuccinimide (NHS I Wako Fiber Code No. 089-04032) for dimethinores Norreoxide (D MSO Wako «Special grade) Diluted to 0.5 ml, mixed with 61.4 mg of the noptene (AP-6) produced in Example 1 above, 0.5 ml of DMS O and 0.5 ml of wisteria, Allowed ®S overnight at room temperature. The obtained SJi ^ night 12.5 µl and 10 ml of peroxidase (Code No. 814393 for POD Roche EIA) dissolved in ₃ ml of 1 l% NaHC03 were HJ¾ed overnight at 4 ° C with stirring. The resulting solution was filtered through a molecular weight filter having a molecular weight cut-off of 30,000, and the final volume was adjusted to 3 ml with PBS containing 0.05% suloff 72N to obtain a yeast enzyme complex. After dispensing 200 L each, the mixture was mixed to obtain an antigen-enzyme complex (this powder was obtained.

 (4) Preparation of ¾ »element complex

_{Na 2 HPO 4 - 12H 2 0} 13. 26 g, NaH 2 PO 4 · 2Η 2 0 2. 02g, N aC l 14. 61 §, Suraofu 72 200 1, f volume angle军後distilled water 1 L, The 8 ml was dispensed into an appropriate fiber, closed, and stored cold at 2 to 8 ° C.

 (5) Preparation of “6x solution”

Dissolve 20ml Tween-20 in 200ml distilled water, 12 bags of Dulbecco's PBS (one), 0.3ml of Sluroff 72N, 10% Tween-20 30 After dissolving 1 ml in 1 L of distilled water, 5 Oml was dispensed into an appropriate container, capped, and stored at 2-8 ° C. [W].

 (6) «of" ^ substrate super night-A "«

 3, · 3, 5, 5, 5 Tetramethylbenzidine (TMBZ for Dojindo chemistry test, Cod eNo. 346-04030 1) 1 Omg to dimethylformamide (DMF Wakomitsu difficult grade Code No. 045-029 16) Dissolved in 1 ml, dispensed 250 μL each into observation vessels, capped and stored at 2-8 ° C cold.

 (7) Duck Substrate Nada-B

Ur ea hy drogenperoxide (Sigma Co de No. U —1 753) '350 mg was adjusted to pH 5.0 with citric acid (citrate ^^ hydrate Wakoto's reagent Code No. 035—03495). It prepared 4 Omm phosphate _{(N a 2 ΗΡ0 4 · 1} 2Η 2 0 Wako Lin flame grade Co d eNo. 1 96- 02835) to 1 L with buffer! After dispensing 15 mL each, capping was performed and stored in a refrigerator at 2 to 8 ° C.

 (8) Duck stop solution "

 1N phosphoric acid 赚. (Kendetology Code No. 328 14-08) was dispensed into appropriate containers in 15 ml increments, capped, and stored in cold water at 2-8 ° C.

Determination of alkynolephenolized ^) with ELISA kit for the determination of alkylphenol compounds (preparation of standard curve)

 (1) Preparation of NPTP

 NP standard solution (0, 50, 200, 1000, 5000 μg / L 20% methanol + 10% DMSO † night) is # ^ using methanol and distilled water, and NP standard water (0, 5, 20) , 100, 500 μg / L in 10% methanol and 1% DMS O).

(2) Preparation of nitrogen complex

Add 7 mL of the complex solution to “Thigh enzyme complex powder” and dissolve. »Preparation of parent compound surgery. .

 (3) Preparation of “mixture”

"NP target, night" (The deviation is also 10% methanol + 1% DMS

100 iL / we 11 and the iodine compound arts were mixed on a “mixing microplate”.

 In the case of using, as in "NP night", mix the mixture with "¾enzyme complex ^ ¾".

 (4) ^)

 The “mixture” prepared in the above (3) was dispensed (100 L / we 11) to an “anti-AP monoclonal antibody-immobilized plate” and allowed to stand at room temperature for 60 minutes.

 (5) Preparation of “赚 Solution”

 Antibody During the time, the “sixfold concentrated lysate” and distilled water were mixed at a ratio of 1: 5 to prepare a “wash solution”.

 (6) Removal of precious items

 The anti-source antibody SJ¾ night was discarded, and the inside of the well 1 was washed three times with “wash solution” (300 L / we 11).

 (7) Color development ^ ^ Stop

 After adding a test (10 L / we 11) and letting it run at room temperature for 30 minutes ^: Then, the "color stop solution" was recommended (100 L / well) and stopped.

 (8) Colorimetric and density calculation

 The absorbance at a wavelength of 450 nm was measured using a plate reader, and the AP concentration in the sample was calculated from the standard curve.

 (9) Result

The generated standard curve is shown in FIG. In the figure, NPitit is plotted on the X-axis, and the ratio (inhibition rate, B / B0%) of absorption at each concentration of nourphenol to 0 μg ZL is plotted on the Y-axis. This result, quantifiable range Roh Elf enol. 2 to 200 mu GZL away and ideas we were _D

Male example 5

Measurement of phenolic phenolation ^^ in seawater sump nore (comparison with the measured value by GC-MS / MS)

 In the same manner as in Example 4, 34 Tokyo Bay Seawater Samples (50 OmL) that had been subjected to the pretreatment shown in Fig. 2 (refer to the National Fiber Research Institute Special Research Report (S-46), 2002, etc.) The alkylphenol compound concentration was measured. At the same time, the concentration of alkynolephenol compounds (sum of nonylphenol and otachi / refenol) in the same sample was measured by GC-MS / MS, and the measured values were compared. The results are shown in Figure 3.

In FIG. 3, the Y-axis shows measured values by the ELISA method, and the X-axis shows measured values by GC-MSZMS. As shown in FIG. 3, the correlation between the value measured by the ELISA method and the value measured by GC-MS / MS was high (Y = 0.88 ΟΧ + 6.6: R ² = 0.95). Industrial applicability

 By using the antibody of the present invention in a measurement method such as ELISA, even in the presence of an alkylphenol ethoxylate compound, an alkylphenol compound can be specifically measured without being affected by the compound. . In addition, when the antibody is used, even in the presence of an alkylphenol ethoxylate compound, the alkylphenol compound can be specifically concentrated without being affected.

Claims

The scope of the claims

1. An antibody that specifically recognizes alkyl phenol compounds and does not recognize alkyl phenol ethoxylate compounds.

 2. When the molar concentration of the alkylphenol compound required to inhibit the binding to the alkylphenol phenol compound by 50% is set to 1, the alkylphenol ethoxylate compound required to inhibit the binding by 50% is 1. The antibody according to claim 1, wherein the molar concentration of the antibody is 5 or more.

 3. An alkylphenol compound is represented by the formula (1):

H)

[Wherein I ¹ , R ² and R ³ are the same or different and each represent H or alkyl], and the alkylphenol ethoxylate compound is represented by the formula (2):

R ⁵ R ⁶

[Wherein R ⁴ , R ⁵ and R ⁶ are the same or different and are each H or alkyl, and R ⁷ is (OC ₂ H ₄ ) _m OH or (OC ₂ H ₄ ) _m COOH (wherein m represents an integer of 1 to 70).] The antibody according to claim 1, wherein

 4. The antibody according to any one of claims 1 to 3, which is a monoclonal antibody.

5. The antibody of claim 4, which is AP-215.

 6. A hybridoma that produces the monoclonal antibody according to claim 4.

7. The method according to claim 6, which is AP-215 (FERM BP—08567). Hypri dorma.

 8. The method for producing an antibody according to claim 1, comprising the following steps: (a) immunizing a mammal with a conjugate of an alkylphenol compound and a protein;

 (b) a step of isolating an antibody that specifically recognizes an alkylphenol compound and does not recognize an alkylphenoloxylate compound.

9. The conjugate of the alkylphenol compound and the protein is represented by the formula (3): R ² R ³

Z

[Wherein RR ² and R ³ are the same or different and are each H or alkyl, L is a spacer, Z is a protein residue] or a compound represented by the formula (4) :

[Wherein, RR ² and R ³ are the same or different and are each H or alkyl, L is a spacer, and Z is a protein residue]. 8. The production method according to 8.

 10. The method according to claim 9, wherein the spacer is a divalent group containing alkylene or a divalent group derived from a hydrophilic polymer.

 11. The production method according to claim 10, wherein the hydrophilic polymer is polyoxyalkylene dalicol.

1 2. The spacer is given by equation (5): One X— Y— 0C0 (CH ₂ ) _n C0NH— (5)

Wherein X is a bond or alkylene, Y is a bond or one (z) _p — (wherein, Z is a repeating unit of a hydrophilic polymer, and p is an integer of 1 to 20), n represents an integer of 1 to 10], or a divalent group represented by the following formula (6):

— X——Y——C0NH—— (6)

[Wherein, X represents a bond or alkylene, Y represents a bond or one (z) p— (wherein, Z represents a repeating unit of a hydrophilic polymer, and p represents an integer of 1 to 20)] 11. The method according to claim 10, which is a divalent group represented by the formula:

 13. A method for measuring an alkylphenol compound, comprising using the antibody according to claim 1.

 14. A kit for measuring an alkylphenol compound, comprising the antibody according to claim 1.

 15. A method for concentrating an alkylphenol compound, comprising using the antibody according to claim 1.

 16. A kit for concentrating an alkyl phenol / Reich mixture, comprising the antibody according to claim 1.