WO2005105846A1 - Antibody specifically recognizing resin plasticizer compound - Google Patents

Abstract

An antibody which specifically recognizes a resin plasticizer compound, characterized by specifically recognizing DEHP in a solid-phase state; and a process for producing the antibody. Also provided are a method of immunologically analyzing a resin plasticizer compound with the antibody; a concentration method; and a kit therefor.

Description

 Antibodies that specifically recognize resin plasticizer compounds

 Technical field

 The present invention relates to an antibody for immunological analysis that specifically recognizes a resin plasticizer compound, a method for producing the antibody, and use thereof.

 Background art

 In recent years, environmental pollution by endocrine disrupting substances (also called “environmental hormones”) has become a problem, and endocrine disrupting substances and their degradation products in the environment are measured and analyzed, and the results are used for environmental conservation. There is a need for immediate use. Various methods for measuring and analyzing endocrine disrupting substances have been known in the past, and at present, a method using a gas chromatograph-one mass spectrometer (GC-MS) is mainly used. However, this method does not provide the sensitivity required for quantification of very small amounts of endocrine disrupting substances that do not exist, and requires high-concentration enrichment by extraction with a solvent, etc. In addition, it is a very expensive device and requires proficiency in operation, and it takes a long time to concentrate, extract, and detect each sample (several weeks for some substances such as dioxin).

 As a means for solving the above problem, an enzyme immunoassay (hereinafter sometimes abbreviated as ELISA) may be mentioned. Quantitative kits for various other environmental pollutants by the ELISA method have been reported (for example, WO94 / 125336), and these analytical kits composed of enzymes and antibodies are not available. It has the advantage that measurement is completed quickly and usually in a matter of hours. The operation is also very simple. In addition, the use of antibodies, particularly monoclonal antibodies, allows very specific quantification (see, for example, WO99 / 43979).

On the other hand, typical endocrine disruptors include alkylphenol ethoxylates, alkylphenols, resin components, black phenols, resin plasticizers, and the like. Among them, as resin plasticizer compounds, dibutyl phthalate (hereinafter abbreviated as DBP) and butylpentyl phthalate (hereinafter abbreviated as BBP) And di (2-ethylhexyl) phthalate (hereinafter may be abbreviated as DEHP). Antibodies that recognize DBP and BBP have been known so far (see WO99 / 43799). An antibody recognizing D EHP (DH-150) has also been reported (Goda Y. et al .; “Development of the ELIS As for Detection of Endocrine Dispersers J, Fifth International Symposium on Environmental Biotechnology (ISEB2000). ), Program / Abstracts, p. 119), and the conventional antibody that recognizes DEHP (DH-150) could measure DEHP only by the indirect competition method in which the antigen was immobilized. It has been desired to produce an antibody that can specifically recognize DEHP even in such a state and can measure DEHP by a direct competition method.

 Disclosure of the invention

 The present inventors have conducted intensive studies in order to establish a detection and measurement method by a direct competition method among immunoassay methods for resin plasticizer compounds, particularly DEHP, and particularly for the ELISA method. Then, by using a specific haptenized resin plasticizer compound for immunization, we succeeded in producing an antibody that specifically recognizes DEHP even in the solid-phase state.

 That is, the present invention is as follows.

 (1) An antibody that specifically recognizes a resin plasticizer compound, and specifically recognizes DEHP in a solid-phase state.

 (2) The antibody according to the above (1), wherein the concentration of DEHP is 50 mg / L or less when B / Bo of DEHP is 50% in a solid phase.

(3) The resin plasticizer compound is represented by the formula (I):

[Wherein, R ¹ is O-phenylene or tetramethylene, R ² and R ³ are the same or different, and each is H, linear or branched alkyl having 1 to 20 carbon atoms, benzyl, Or cyclohexyl), the antibody according to the above (1).

(4) The antibody according to (1), which is a monoclonal antibody.

 (5) The monoclonal antibody according to (4), which is 2F4A4Y.

 (6) A hybridoma that produces the monoclonal antibody described in (4) above.

 (7) The hybrid dorma according to (6), which is 2F4A4y (FERM BP-08601).

 (8) The antibody according to (1), which is a polyclonal antibody.

(9) The method for producing the monoclonal antibody according to the above (4), comprising the following steps: (a) Formula (II) _:

[Wherein, R ⁴ and R ⁵ are the same or different and each is a linear or branched alkyl group having 5 to 20 carbon atoms which may be substituted with a carboxyl group, or one of them is (C ₂ When H ₄ 0) _n COC ₂ H ₄ COOH (n represents an integer of 1 to 20), the other is a linear or branched alkyl group having 5 to 20 carbon atoms, and R ⁶ is H or Fusing myeloma cells with B cells sensitized with a compound represented by the following formula:

 (b) a step of recovering a monoclonal antibody from the obtained antibody-producing hybridoma. (10) The method according to (9), wherein the fusion is performed by an electric pulse method.

 (11) Equation (II):

[Wherein, R ⁴ and R ⁵ are the same or different and are each a linear or branched alkyl group having 5 to 20 carbon atoms which may be substituted with a carboxyl group, or — When (C ₂ H ₄₀ ) _n COC ₂ H ₄ COOH (n is an integer of 1 to 20), the other is a linear or branched alkyl group having 5 to ²⁰ carbon atoms , ¹⁶ means a group having an HLB value of 5 to 16 according to the Griffin method of 11 or the whole molecule.] B cells sensitized with the compound represented by The method for producing a hybridoma according to the above (6), comprising:

 (12) The method for producing a polyclonal antibody according to the above (8), comprising the following steps:

(a) Formula (II):

[Wherein, R ⁴ and R ⁵ are the same or different and each is a straight-chain or branched-chain alkyl group having 5 to 20 carbon atoms which may be substituted with a carboxyl group; when _{2 H 4 0) n COC 2} H 4 COOH (n is an integer of 1 to 20), the other a linear or branched alkyl group having 5 to 20 carbon atoms, R ⁶ is H also Means a group having an HLB value of 5 to 16 according to the Dariffin method for the whole molecule]. Immunizing a mammal with a compound represented by the formula:

 (b) a step of isolating a polyclonal antibody from the immunized mammal;

 (13) An immunological analysis method for a resin plasticizer compound, comprising using the antibody according to (1).

 (14) After reacting the antibody according to (1) immobilized on the carrier surface with the specimen and the resin plasticizer compound labeled with the labeling agent, the antibody is retained or retained on the carrier. (13) The method according to (13) above, wherein the activity of the labeling agent that is not performed is measured.

(15) A kit for immunological analysis of resin plasticizer compounds, comprising the antibody according to (1) above.

 (16) A method for concentrating a resin plasticizer compound, comprising using the antibody according to (1).

(17) Concentration of resin plasticizer compounds containing the antibody according to (1) above Kit

 Brief Description of Drawings

 Figure 1 shows the reactivity of the seven selected antibodies to DEHP by the direct competition method.

 FIG. 2 shows the cross-reactivity of the two selected antibodies against various resin plasticizer compounds.

 FIG. 3 shows the measurement sensitivities of the indirect competitive method of DH-150 and the direct competitive method of 2F4A4γ when DEHP is measured.

 Figure 4 shows the measurement sensitivity of the direct competition method between DF-34 and 2F444γ when DΒ is used as the measurement target.

 Detailed description of the invention

 Hereinafter, the present invention will be described in detail.

 The present invention provides an antibody that specifically recognizes a resin plasticizer compound and specifically recognizes DEHP in a solid phase.

 `` Antibody specifically recognizing DEHP in the immobilized state '' means, for example, in the immobilized state, the DEHP concentration when BZB O for DEHP is 50% is 1 OnigZL or less, preferably 5 mgZL or less, Preferably, the antibody is 3 mgZL or less.

 Further, the antibody of the present invention can specifically recognize DEHP even in a non-solid state (for example, a state of being dissolved in a solvent).

In the present invention, the resin plasticizer compound is not particularly limited.

[Wherein, R ¹ is O-phenylene or tetramethylene, R ² and R ³ are the same or different, and each is H, straight-chain or branched-chain alkyl having 1 to 20 carbon atoms, benzyl. Or hexyl).

 In the formula (I), the number of carbon atoms in the alkyl is not particularly limited, but is usually 1 to 20, and preferably 7 to 12.

 The alkyl includes, for example, methyl, ethyl, propyl, isopropyl, pentinole, sec-butyl, t-pentinole, pentynole, isopentinole, t-pentinole, hexyl, t-hexinole, heptinole, t-heptyl, octyl , T-octyl, noel, t-nonyl, dodecyl, t-decyl and the like, but are not limited thereto.

 Specifically, examples of resin plasticizer compounds include dibutyl phthalate (DBP), butylbenzyl phthalate (BBP), dipentyl phthalate (DPnP), dihexyl phthalate (DHP), and phthalic acid. To di (2-ethylhexyl) (DEHP), getyl phthalate (DEP), dipropyl phthalate (DPrP), diisononyl phthalate (DI NP), diisododecyl phthalate (DI DP), dicyclo phthalate Xyl (DCHP) and getylhexyl adipate (DEHA). Preferred resin plasticizer compounds include DBP, BBP, DPnP, DHP, DEHP, DEP, DPrP, DCHP and DEHA. More preferred resin plasticizer compounds include DBP, BBP, DPnP, DHP and DEHP.

Examples of the antibody include natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAb), recombinant antibodies produced using gene recombination techniques (eg, single-chain Fv fragments (scFv), bispecific-chimeric scFV-scFv ), Tandem scFV ^ scfv2, bispecific- (scFv) 2, disulfide-linked scFv, disulfide-stabilized Fv fragments (dsFv) diabod, single-chain diabody (scDb), bivalent diabody _N bispecific diabody, knob-into—hole stabilized diabody, disulfide-stabilized diabody triabody ^ tetrabody ^ trispecific triabody _N CL-dimerized scFv, CHl-CL-dimerized scFv, CH3-dimerized scFv, knob— into— hole CH3-dimerized scFv, CH3-dimerized biva 丄 ent diabody, Fc— dimerized scFv, Fab-scFv fusions ^ Ig-scFv fusions ^ leucine-zipper stabilized scFv dimers ^ helix-stabilized scFv dimers, 4 helix- bundle stabilized scFv tetramers, streptavidin-scFv, intrabody _N Fab 'fragment s F (ab fragment s Fv fragments (Fv), Kimef antibody, Human antibodies, single-chain antibodies, etc.) and human antibodies that can be produced using human antibody-producing transgenic animals, etc., but are not limited thereto. The antibody is preferably a monoclonal antibody or a polyclonal antibody, more preferably a monoclonal antibody. Suitable examples of such monoclonal antibodies, 2 F4A4 _Y like that shown in the 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 aforementioned antibody, and specifically, for example, F (ab ') ₂ , Fab, Fab, Fv ^ variable fragment of antibody), sFv, dsFv (disulfide stabilized Fv) ), DAb (single domain antibody), (Exp. Op in. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996), antibody fragments produced by Fab expression library, etc. Is done.

 The present invention also provides a hybridoma producing the above antibody and a method for producing the same. Preferred examples of the hybridoma include 2F4B7D6F6 and 2F4 44γ shown in Examples below. Among them, 2F4A4γ is superior in sensitivity to 2F4B7D6F6, and has particularly high sensitivity to medium to high concentrations of DΕΗΡ. Hypri-Dorma 2F4-4γ was deposited with the Patent Organism Depositary at the National Institute of Advanced Industrial Science and Technology at the National Institute of Advanced Industrial Science and Technology at Tsukuba East, Ibaraki, Japan, on January 28, 2004. (Accession number: FERM BP-086 01).

 The monoclonal antibody of the present invention can be produced by fusing B cells sensitized with a haptenized resin plasticizer compound with myeloma cells and recovering the monoclonal antibody from the resulting antibody-producing hybridoma. Can be. The present invention provides a method for producing the monoclonal antibody.

In sensitizing B cells, for example, haptenized resin plasticizer compounds and A complex with the rear protein is used.

The haptenized resin plasticizer compound is not particularly limited, but may be, for example, a compound represented by the formula (II):

[Wherein, R ⁴ and R ⁵ are the same or different, and each is a linear or branched alkyl group having 5 to 20 carbon atoms which may be substituted with a carbonyl group. When (C ₂ H ₄₀ ) _n COC ₂ H ₄ COOH (n represents an integer of 1 to 20), the other is a linear or branched alkyl group having 5 to 20 carbon atoms, and ¹⁶ is 11 or a group having an HLB value of 5 to 16 according to the Griffin method of the whole molecule].

When R ⁴ and R ⁵ are an alkyl group optionally substituted with a carboxyl group, preferably R ⁴ and R ⁵ are the same group. In this case, R ⁴ and R ⁵ are preferably an alkyl group substituted by a hydroxyl group.

R ⁶ is H or a group having an HLB value of 5 to 16 according to the Griffin method of the whole molecule. The HLB value is represented by hydrophilic group molecular weight / total molecular weight X 100/5, and examples of such a group include, for example, CH ₂ OOCH ₂ COCH ₂ COOH and CH ₂ OOCH ₂ CH ₂ CO (CH ₂ CH ₂ 0) ₆ OOCCH ₂ CH ₂ CO OH. Preferably, R ⁶ is H.

 In the formula (II), the number of carbon atoms in the alkyl is not particularly limited, but is usually 5 to 20, and preferably 7 to 12.

Examples of the alkyl include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, pentyl, isopentyl, t-pentyl, hexyl, t-hexinole, heptyl, t-heptyl, octinole, and t-octinole. , Nonyl, _t -noryl, dodecyl, t-dodecyl and the like, but are not limited thereto. , Specifically, as the haptenized resin plasticizer compound,

A compound represented by the following formula (III) (hereinafter referred to as DEHP-1):

A compound represented by the following formula (IV) (hereinafter, referred to as DEHP-7):

Compound represented by the following formula (V) (hereinafter, referred to as DEHP-14)

A compound represented by the following formula (VI) (hereinafter, referred to as DEHP-15)

-COOCH ₂ CH H ₂ ) ₈ COOH

 (VI)

COOCH ₂ CH (CH ₂ ) ₈ COOH

C ₂ H ₅

And the like. Preferred is DEHP-7 represented by the formula (IV) or DEHP-14 represented by the formula (V).

The above-mentioned haptenized resin plasticizer compound can be easily produced by a person skilled in the art from a known compound by a production method described in Examples below. The complex in which the haptenized resin plasticizer compound and the carrier protein are bound together can be obtained, for example, by reacting the compound of the formula (II) with N-hydroxysuccinimide to obtain an activated ester. It can be obtained by reacting an ester with a carrier protein.

 Examples of the carrier-protein include various carrier proteins that have been widely used in the art, such as KLH (keyhole limpet hemocyannin), BSA (bovine serum albumin), and OVA (ovalbumin).

 The sensitization of B cells is usually carried out by inoculating an animal with the complex of the haptenized resin plasticizer compound and the carrier protein and immunizing the animal. As animals to be inoculated, for example, goats, sheep, rats, mice, guinea pigs, and chickens are used, and mice and rats are preferably used.

 The method of inoculation may be a commonly used method, for example, 1 to 1 time for mice.

1 0 0 mu _§, preferably 5 0~1 0 0 μ _§ equal volumes with milk I fire (0. 1 ml) complete adjuvant or RIBI ™ adjuvant Pando system saline Contact Yopi Freund's, They are taken subcutaneously in the back and abdomen or intraperitoneally 3 to 6 times every 2 to 3 weeks.

 From these immunized animals, for example, mice, select individuals with high antibody titers, 3 to 5 days after the final immunization, collect the spleen or lymph nodes by a conventional method, and use the haptenized resin plasticizer compounds contained in them. The B cells sensitized in step 1 are fused with myeoma cells.

 In the present invention, the sensitized B cells used for cell fusion are obtained by aseptically removing a spleen or a lymph node from an immunized animal such as a mouse in accordance with a conventional method, washing, crushing, and It can be obtained as cells or lymph node cells. Splenocytes are preferred because they are rich in B cells and other immune response cells.

The myeloma cells (bone marrow B heavy cells) used in the present invention are not particularly limited, and include, for example, conventionally known NS-1, P3U1, and Sp2 / θ PAI. PAI is also preferred because of its high fusion efficiency. Myeloma cells may be those that have been previously subcultured according to a method commonly used in the art. Examples of the cell fusion method used in the present invention include a method using a conventionally known Sendai virus, a method using polyethylene glycol (PEG) as a fusion promoter, an electric pulse (PEF: pulsed electric field) method, a laser radiation method ( laser radiation) can be used without particular limitation. Among these, the cell fusion method using PEG is a method that is widely used because it is relatively simple.However, an a / red substance produced by oxidizing PEG has strong cytotoxicity. It has been shown to adversely affect the development of Hypri-Doma. In addition, in such a fusion method, when performing cell fusion, not only between immunized cells and myeoma cells, but also between immunized cells and

Non-specific cell fusion also occurs between Z or myeloid cells, which has the disadvantage that much effort and time is required to obtain the desired monoclonal antibody. Therefore, in the present invention, the immunized cells and the myeloma cells can be selectively fused without using a cytotoxic substance among the above-mentioned cell fusion methods, and can be compared with the cell fusion method using PEG. Cell fusion is preferably performed by the PEF method, which can produce a hybridoma producing an antigen-specific monoclonal antibody of interest at a 10- to 20-fold higher efficiency.

In the PEF method, the first complex, in which avidin is bound to immunized cells via an antigenic substance, and the second complex, in which biotin is bound to myeloma cells, have a strong relationship between biotin and avidin. Utilizing affinity, a complex of immunized cell-antigen substance-avidin-biotin-myeloma cell is produced using biotin / avidin cross-linking, and cell fusion is performed by applying a high-voltage square wave pulse to this complex. Is the way. The most significant feature of the PEF method is that immunized cells can be selected as antigens in advance via antigen receptors on the cells, so that by using an avidin-bound antigen substance, only immunized cells can be selectively used. (Lo, MMS et al .: Nature 310, 792-794 (1984), Masahiro Tomita et al .: Biochim. Biophy. Acta. 1055, 199-2 06 (1990) ), Masahiro Tomita et al .: J. Immunol. Methods. 251, 31-43 (2001), Tsong, T.Y. Masahiro Tomita: Methods Enzymol. 220, 238-246 (1993), Masahiro Tomita: Protein Nucleic Acid Enzyme 45, 600-606 (2000)).

 Specifically, it may be performed as follows according to a conventionally known procedure. The following is an example of a case where a conjugate of the compound represented by the above formula (II) (referred to as hapten compound (II)) and carrier monoprotein is used as an antigenic substance and splenocytes are used as B cells. . In the case of other thickening substances, it can be carried out in the same manner as described below.

 (1) In a suitable solvent (such as DMSO), a carboxyl-containing hapten compound (II) prepared in advance was added to N-hydroxysuccinimide and 1-ethyl-3-1- (3-dimethylaminopropyl). Carestimid hydrochloride is added to calo to make estenolate. Avidin is added thereto to prepare a conjugate in which the hapten compound (II) and avidin are bound.

 (2) The above conjugate is mixed with the suspension of the immunized cells, and after stirring, the precipitate obtained by centrifugation is mixed with a suspension containing an appropriate antibiotic (for example, RPMI 1640 containing kanamycin sulfate). After washing, suspend in the suspension. In this way, a first complex in which avidin is bound to the immunized cell via the antigenic substance is formed.

 (3) Wash the myeloma cells that have been subcultured in advance by an ordinary method, and suspend them in an appropriate suspension (for example, PBS) to prepare a suspension containing the myeloma cells. Suspend biotin (N-hydroxysuccinimide-biotin) in an appropriate solvent (eg, DMF) to prepare a suspension containing biotin. These are mixed, rotated in a 5% carbon dioxide incubator at 37 ° C, centrifuged, and the resulting precipitate is washed with the above suspension (RPI 1640 containing kanamycin sulfate) and then washed. Suspend in liquid. In this way, a second complex in which biotin is bound to myeloma cells is formed.

(4) The suspension containing the first complex prepared in (2) and the suspension containing the second complex prepared in (3) are mixed. Mix proportion of spleen containing immunized cells It is preferable to carry out the cell suspension and the myeloma cells at a ratio of 10: 1 to: L: 2, more preferably 1: 1. The liquid obtained by mixing is centrifuged, and the obtained precipitate is suspended in the above suspension liquid. After centrifugation, leave for an appropriate time, and rotate. After rotation, the precipitate obtained by centrifugation is mixed with an isotonic sucrose buffer (0.25 M sucrose + 2 mM sodium dihydrogen phosphate / disodium hydrogen phosphate (pH 7.2) + 0.1 mM magnesium chloride + 0.1 mM calcium chloride). In this way, a complex of immunized cell, hapten compound (II), avidin, biotin, and myeloma cell utilizing the strong affinity between biotin and avidin is formed.

 (5) The suspension containing the immunized cell-hapten compound (II) -avidin-biotin-myeloma cell complex obtained in (4) above is placed on a platinum-prepared plate in a volume of 0.5 mL to 1 mL. In addition, an electric palace is loaded on this. The load of the electric pulse is, for example, using a commercially available cell fusion device such as electro square porat or T820 (manufactured by BTX), ECM830 (manufactured by BTX), ECM2001 (manufactured by BTX), or the like. Apply a square wave pulse. The loading conditions are usually 2 kVZcm (10 times / sec x 4 times) or 3 kV / cm (10 times / fsec x 4 times), and 2 kVZcm (10; isec X 4 Times) is preferred. As a result, the cross-linked immunized cells and myeloma cells are selectively fused to produce a hybridoma.

 The hybridoma produced by the method of the present invention is cultured according to a conventional method so as to be subjected to screening.

After adding, for example, RPMI 1640 complete medium (90% RPMI 1 640 + 10% FCS + 100 ^ g / mL kanamycin sulfate + 2 mM L-glutamine + 5ΟβΜβ-mercaptoethanol) to the above hybridoma, I do. Culturing is usually performed on a 96-well or 48-well microphone plate. For example, as described above, the supernatant is subjected to complete HAT-containing RPMI 1640 medium (100 / M hypoxanthine + 0. 4 μΜ aminopterin + 16 μΜ After exchanging the medium for 5 to 2 weeks (containing 0.1 ml / we) for 1 to 5 weeks, the supernatant was replaced with RPMI 1640 complete medium containing HT (containing 100 iM hypoxanthine + 16Μ thymidine). The medium may be changed for 5 to 2 weeks (for example, 0.1 mL / we are changed by 11), and thereafter, the medium may be changed with RPMI 1640 complete medium.

 A variety of methods can be used to screen for the desired hybridomas, including; if performed by ELISA (enzyme-linked immunosorbent assay), fe? I can do that. Hybridomas that have a positive antibody activity by the above ELISA assay may be cloned by a method usually used in the art, for example, a limiting dilution method. The antibody titer of the cloned hybridoma supernatant is measured by the above-described method, and a hybridoma that produces a stable and high-titer antibody is selected to obtain the desired monoclonal antibody-producing hybridoma. it can.

Subsequently, the monoclonal antibody is recovered from the obtained antibody-producing hybridoma. Production (production), isolation and purification of monoclonal antibodies by hybridomas can be performed by methods known per se. Specific examples of antibody production and purification methods are described in, for example, "Enzymimnoassy", pp. 46-71, pp. 85-110, and salting out (Na ₂ SO ₄ , ( NH _{4) 2} S0 _4), ion exchanger (DEAE, QAE, CM / cellulose ^ Sephadex, Sepharose, etc. Servacel), hydrophobic chromatography (L- Fueyuruara two Lou Sepharose etc.), gel filtration (Sephadex G-200, Bio -Ge 1 p-300, etc.), electrophoresis (zone electrophoresis with agarose gel, isoelectric focusing, isokinetic electrophoresis, etc.), ultracentrifugation (sucrose density gradient centrifugation), affinity chromatography Immobilization "(such as Protein-A Sepharose ^ Protein-A superose).

When the antibody is a polyclonal antibody, a mammal is immunized with a haptenized resin plasticizer compound in the same manner as a monoclonal antibody. The immunization is usually performed by inoculating a conjugate of the above-mentioned haptenized resin plasticizer compound and protein into a mammal together with itself or a carrier, a diluent or the like. The inoculation method and the like are the same as the production method of the monoclonal antibody. 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 immunoglobulin separation and purification method as in the above-described isolation of the monoclonal antibody.

 In addition, a gene encoding the monoclonal antibody is cloned from the hybridoma producing the monoclonal antibody, and the gene is used. Can be manufactured.

 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). , Cloning method of antibody gene from hybridoma cell line (Hybridoma Cell Line), preparation method of antibody gene library (Antibody Gene Libraries), production of recombinant antibody from gene library A selection (Recombinant Antibodies From Gene Libraries) method, an antibody gene engineering (Antibody Engineering) method, and the like are described, and a recombinant antibody can be produced by these methods.

Further, chimeric antibodies are described in, for example, “Experimental Medicine (Extra Extra Number), Vol. 1.6, No. 10, 1988”, and Japanese Patent Publication No. 3-73280, and humanized antibodies are described in, for example, No. 506458, JP-A-62-296890 and the like, and human antibodies are described in, for example, `` Nature Genetics, Vol. 15, p. 146-156, 1997 '', `` Nature Genetics, Vol. 7, p. 13- 21, 1994 ", Japanese Translation of PCT International Publication No. 4-504365, International Application Publication W094 / 25585," Nikkei Science, June, pages 40-50, 19995 ", rNature, Vol. 368, p. 856-859, 1994 ", and Japanese Patent Publication No. 6-500233. The antibody of the present invention can be used as a reagent for quantitatively measuring a target resin plasticizer compound, or can be used for concentrating the target resin plasticizer compound by immobilizing it on various carriers. It can be used for the production of affinity columns.

 Examples of the carrier for immobilizing the antibody of the present invention include microplates (eg, 96-well microplate, 24-well microplate, 192-well microplate, and 38-well microplate). , Test tubes (eg, glass test tubes, plastic test tubes), glass particles, polystyrene particles, modified polystyrene particles, polyvinylidene / vinyl particles, latex (eg, polystyrene.latex), nitrosenorelose membrane, cyanogen bromide-activated filter paper, DBM-activated filter paper, granular solid phase (eg, Sepharose, Sephadettas, Agarose, Senollose, Cefacryl etc.), iron-containing polycarbonate membrane, magnet-containing beads and the like. In order to support the monoclonal antibody obtained in the present invention on a carrier, a method known per se, "Enzymim Noassy", pages 268-296, "Affinity Chromatography Handbook" (Amersham Pharmacia) It can be supported by the method described in Biotech Co., Ltd. (issued on February 20, 1998).

 In the immunological analysis method of the present invention, a specimen (for example, a water or soil sample containing a resin plasticizer compound, a decomposition product thereof or a mixture thereof) and a resin plasticizer labeled with a known amount are used. A so-called direct competition method is used in which the amount of a compound, its degradation product or a mixture thereof is quantitatively determined by a competitive reaction for binding to the antibody of the present invention. In the direct competition method, a certain amount of antibody held on a carrier is added to a sample solution containing an unknown antigen, and then a certain amount of antigen labeled with a labeling agent is added. The activity of the labeling agent retained on the carrier or the labeling agent not retained on the carrier is measured. It is desirable to add the antibody and the labeled antigen almost simultaneously.

 The direct competition method is generally simpler to operate and can be measured in a relatively shorter time than the sandwich method.

Labeling agents for labeling antigens include radioisotopes (hereinafter abbreviated as RI), enzymes, Enzyme substrates, fluorescent substances, biotin and the like can be mentioned. The binding between these antigens and the labeling agent is performed by the maleimide method (Journal of Biochem., J. Biochem., 79, 233 (1976)), the active-dani biotin method (Journal of American Chemical's Society (J. Am. Chem. Soc.) _S 100, 3585 (1978)) is used.

 Specifically, in order to carry out a specific immunological analysis measurement method by a competition method for a resin plasticizer compound, a test sample containing an unknown amount of the resin plasticizer compound is physically and physically tested by a known conventional method. Alternatively, a solid phase chemically bound to the antibody is added and reacted. At the same time, a certain amount of the resin plasticizer compound labeled with the labeling agent is added and reacted. Next, the solid phase is usually washed well, and the activity of the labeling agent bound to the solid phase is measured. If the labeling agent is RI, measure with a well counter or a liquid scintillation counter. If the labeling agent is an enzyme, add the substrate and allow to stand. Measure the enzyme activity using a colorimetric, colorimetric, or fluorescent method. Whether the labeling agent is a fluorescent substance or a luminescent substance, the measurement is performed according to a known method.

 Using the antibody of the present invention, the conventional anti-DEHP antibody could only be measured by the indirect competition method in which the antigen was immobilized, whereas the direct competition method in which the antibody was immobilized (antibody immobilization method). Of DEHP is possible.

In the method for concentrating a resin plasticizer compound of the present invention, a large amount of a sample is passed through an immunoadsorbent force ram or mixed with immunoadsorbent particles to utilize an antigen-antibody reaction. The resin adsorbent compound is captured by the immunoadsorbent, then the pH is changed (lower to 2.5 to 3 or higher to 1.5), and the ionic strength is changed (1 MNaCl, etc.). ), change of polarity (10% Jiokisan, 50% E Ji glycol, 3M chaotropic salt _{(S CN-, CC l 3 COO-} , I) etc.), the addition of protein denaturant (8M urea, etc. 6M hydrochloric acid guanidine) Elution by a known method such as electrophoresis or dissociation by electrophoresis makes it possible to concentrate resinous plasticizer compounds having few immunological contaminants at a high magnification of thousands to tens of thousands of times.

By the concentration method, a resin plasticizer compound that is present only in an extremely small amount in the environment, Compared with conventional concentration methods such as solvent extraction method and solid phase extraction method, it is possible to obtain a concentrated liquid that can be concentrated at a much higher magnification and that has a low content of contaminants and the like that interfere with quantification. Can be.

 In addition, the antibody of the present invention is combined with, for example, an enzyme agent for ELISA, a buffer, or the like, to provide a kit for immunological analysis of a resin plasticizer compound, a decomposition product thereof, or a mixture thereof, or an immunological analysis kit. It can be a kit for concentration, and such a kit is also included in the scope of the present invention.

 Example

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

Example 1

 1. Hapten synthesis.

 (1) Synthesis of DEHP-1

 After 10 g of 8-bromooctanoic acid was dissolved in 30 OmL of tetrahydrofuran (THF), 20 mL of diphenylaminoaminomethane was added thereto, and the mixture was reacted at room temperature for one hour. The next day, 2 OmL of diphenylaminomethane was further added, and the mixture was further reacted at room temperature. After concentration under reduced pressure, the reaction product was dissolved in hexane-ethyl acetate (9: 1) and crudely purified by silica gel column.

This crude product (12 Og), phthalic acid (2.42 _§ ) and 1,8-diazabicyclo [5,4,0] -7-pandenecene (DBU) were refluxed under heating in 6 OmL of benzene. The next day, 2.22 g of DBU was added, the mixture was heated and refluxed roughly for 6 hours, cooled to room temperature, added with water and chloroform, and washed with water. After drying over sodium sulfate, the concentrate under reduced pressure was applied to a silica gel column (300 g) and eluted with ethyl hexane monoacetate (9: 1) followed by the same (2: 1) to obtain 8.2 g of phthalate ester Was.

4.1 g of this phthalate was dissolved in 10 OmL of THF, and 0.4 g of 10% Pd / C (50% water-containing product) was added thereto. After injecting H ₂ (0.3 mL / min, 5 hours), 1.2 g of 10% Pd / C (50% water-containing product) was added. In addition, inject H ₂ (0. After 5 mL, 2 hours), remove the catalyst, apply the concentrated solution under reduced pressure to an ODS column (Da osp-120 30ι50 μιη, 3 X 50 cm), elute with 75% methanol, concentrate under reduced pressure, III):

(DEHP-1) was obtained as a pale yellow oil.

 The target structure was confirmed by 1H-NMR, and the HPLC purity was 99% or more. (HPLC conditions: column: Daiso SP-120-5-ODS-BP 4.6 thigh ID x 150 thigh, mobile phase: MeOH: ¾0 = 3: 1 TFA 0.1%; 1.0 mL / min, detection UV254 nm, temperature: room temperature)

 (2) Synthesis of DEHP-3

 To a mixture of 25 g of 4-methylphthalic acid, 42 g of DBU and 50 OmL of dry toluene was added 50 mL of 2-ethylhexylpromide, and the mixture was stirred under reflux for 3.5 hours. After confirming the disappearance of the starting material by TLC (chloroform-methanol di- 4: 1), the mixture was ice-cooled, the precipitated crystals were separated by filtration, and the filtrate was concentrated under reduced pressure to obtain 61 g of a colorless oil. This was purified by silica gel column chromatography (solvent: n-hexane monoethyl acetate = 20: 1) to obtain 37.5 g of a crude product.

 A mixture of 37.5 g of the crude product, 19.8 g of NBS, 1.6 g of AIBN, and 1.6 L of carbon tetrachloride was stirred under reflux for 1 hour. The reaction was checked by TLC (n-hexane monoacetate = 9: 1), cooled to room temperature, insolubles were filtered off, and the filtrate was concentrated under reduced pressure to obtain a brown oil (60 g). This was purified by silica gel column chromatography (solvent: n-hexane monoacetate = 20: 1) to obtain a pale yellow oil (2%).

28 g of the obtained pale yellow oil was dissolved in 224 mL of pyridine, and the mixture was stirred at 105 ° C for 1.5 hours. After confirming the disappearance of the raw materials by TLC (n-hexane monoacetate = 9: 1), the mixture was concentrated under reduced pressure to obtain 32.8 g of a brown oil. This was dissolved in 150 mL of ethanol, 6.3 g of 4-nilolo-N, N-dimethylaniline was added, and 43 mL of 1N sodium hydroxide was added dropwise at -20 ° C to -10 ° C over 10 minutes. Stirred at −10 ° C. for 30 minutes. TLC (n-hexane monoethyl acetate = 9: 1) After checking the reaction with, water and ethyl acetate were added, and the mixture was partitioned. The oil layer was washed with 1N hydrochloric acid and saturated brine in that order, dried over sodium sulfate and concentrated under reduced pressure to obtain 27 g of a brown oil. This was purified by silica gel gel chromatography (solvent: n-hexane monoethyl acetate = 91) to obtain 10.5 g of a crude product.

 A solution consisting of 11.7 g of the crude product obtained above and 55 mL of ethanol was added dropwise to a solution consisting of 530 mg of sodium borohydride and 55 mL of ethanol over a period of 10 minutes at 15 ° C at 6 ° C under a nitrogen stream. Stirred at room temperature for 10 minutes. After confirming the disappearance of the starting materials by TLC (n-hexane monoacetate = 91), 12 mL of 1N hydrochloric acid was added dropwise over 10 minutes under ice cooling. This mixture was concentrated under reduced pressure, ethyl acetate and saturated saline were added thereto, and the mixture was separated, washed with saturated saline, dried over sodium sulfate, and concentrated under reduced pressure to obtain 11.5 g of a pale yellow oil. This was purified by silica gel column chromatography (solvent: n-hexane monoethyl acetate = 31) to obtain 7.2 g of a crude product.

 A mixture consisting of 3.0 g of the obtained crude product and 866 mg of diglycolic anhydride was stirred at 100 ° C. for 6 hours under a nitrogen stream. After confirming the disappearance of the raw material by TLC (cloth form-methanol = 91), cool to room temperature, and purify by silica gel column chromatography (cloth form → cloth form-methanol = 91). Formula (VII):

The hapten DEHP represented by the following formula was obtained.

 (3) Synthesis of DEHP-6

12.6 g of 4-methynolephthalenoic acid and 25 ml of DBU21.5 mL of bromo-2-ethynolehexane were dissolved in 30 OmL of toluene and stirred at 80 ° C. for 2.5 hours. 30 OmL of water was added to the mixture, liquids were separated, and the organic layer was washed with 15 OmL of saturated saline. Then, the reaction product was dried over sodium sulfate and concentrated under reduced pressure to obtain 22.5 _{g of} a reaction product. The residue was purified by silica gel chromatography (solvent: ethyl hexane monoacetate = 10: 1) to obtain 11.4 g of a crude product.

 110 g of the crude product, 5.3 g of NBS (N-bromosuccineimide), and 0.4 g of AIBN (Azobisisobutyronitrile) were dissolved in 120 mL of tetrachloromethane carbon and heated under reflux for 4 hours. After allowing to cool, it was washed three times with 10 OmL of water, dried over sodium sulfate, and concentrated under reduced pressure. As a result, 12.8 g of a mixture in which the target substance and the starting material were present in a ratio of about 1: 1 (by TLC) were obtained. The mixture was applied to a silica gel short column (solvent: n-hexane monoacetate = 20: 1) to remove the highly polar part, and rebrominated with NBS 2.1 g and AI BN 0.1 g under the above conditions. . The reaction product was purified by a silica gel column to obtain 27.5 g of a crude product as a yellow oil.

 27.5 g of the obtained crude product was added to 5 OmL of pyridine, and the mixture was stirred at 100 ° (1 hour at 3). The mixture was concentrated under reduced pressure to obtain 8.8 g of a black-brown tar-like reactant, which was purified. Used for the next step without.

 8.8 g of the crude reaction product and 3.2 g of p-ditrosodimethylaniline were dissolved in 40 mL of ethanol, and after cooling with ice, 15.5 mL of IN NaOH solution was added dropwise at 5 ° C or lower. The mixture was further stirred for 20 minutes under ice cooling. After adding 15 OmL of water to the mixture, the mixture was extracted three times with 5 OmL of ethyl acetate.

 The obtained mixture of the target substance and ethyl acetate was added dropwise to 80 mL of a 1N HCl aqueous solution at room temperature. The mixture was vigorously stirred at room temperature for 30 minutes, separated, and washed twice with saturated saline. Thereafter, the mixture was concentrated under reduced pressure, and the highly polar part was removed with a silica gel short column (solvent: hexane-ethyl acetate = 10: 1) to obtain 32.8 g of a crude product. The mixture was further subjected to pressure ram in silica gel (silica gel 1 kg, solvent: hexane monoethyl acetate = 10: 1) to obtain 41.8 g of a crude product.

After the crude product 4 was added Na BH ₄ 73 mg, ethanol 8 m L, 4 a mixture of mono-methyl phthalate 1. 6 g of ethanol 8mL added, followed by stirring for 30 minutes under ice-cooling. The mixture was charged with 1.92 mL of IN HC1 and concentrated under reduced pressure. Then, water and ethyl acetate were added, stirred, and separated. The organic layer was washed with saturated saline, Washed with sodium hydrogen sulfate. The brown oil obtained by concentration under reduced pressure was applied to a silica gel column (solvent: ethyl hexane monoacetate = 6: 1 to 2: 1) to give 51.5 g of a crude product as a pale brown oil. Was.

 51.5 g of the crude product and 0.36 g of succinic anhydride were mixed and stirred at 110 ° C for 17 hours. After allowing to cool, the mixture was applied to a silica gel column (solvent: hexane monoacetate = 2: 1) to obtain 1.37 g of a crude product 6 (containing about 10 mol% of a crude product 5). Crude product 6 0.52 g, Hexaethylene glycol mono THP ether 0.

47 g, was ice cooled CH ₂ C 1 ₂ 5mL, was added WSCD0. 2 g, the DMAP 0. 03 g, sequentially, and stirred overnight at room temperature. Thereafter, CH ₂ C 1 ₂ was added 10 mL to the mixture, 11 ^ hydrochloric 101111 ^, saturated aqueous sodium bicarbonate solution 20 mL, washed successively with saturated brine, and concentrated under reduced pressure and dried with anhydrous sodium sulfate. The reaction product was applied to a silica gel column (solvent: hexane monoethyl acetate = 1: 3 → ethyl acetate), and the crude product 7 was subjected to 0.

Obtained as 5 g of a colorless oil.

 70.48 g of the crude product was dissolved in 2 OmL of 1,4-dioxane, and 1.0 mL of 1 N hydrochloric acid was added thereto. The mixture was stirred at room temperature for 1.5 hours. The mixture was poured into a saturated aqueous sodium hydrogen carbonate solution (80 mL), and extracted with ethyl acetate. Then, it was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The reaction product was applied to a silica gel column (solvent: ethyl acetate → ethyl acetate-methanol = 10: 1) to obtain 80.32 g of a crude product as a colorless oil.

80.32 g of the crude product was mixed with 0.08 g of succinic anhydride, and the mixture was stirred at 110 ° C for 8 hours. After cooling, the mixture was dissolved in a mixture of diisopropyl ether-hexane (1: 1) and washed with water four times. Since the mixture was easily emulsified and had poor liquid separation properties, a saturated saline solution was appropriately added to accelerate the liquid separation. The washing water was extracted with diisopropyl ether, and the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The reaction product is applied to a silica gel column (solvent: ethyl acetate-2-propanol = 10: 1) to obtain the following formula (VIII): CH2CH3

HOOCCH2GH ₂ GOO (OH2CH ₂ C) ₆ OCCH ₂ CH2COOCH ₂ --COOCH ₂ CH (CH ₂ ) ₃ CH ₃

 (in)

-COOCH ₂ (H (GH ₂ ) ₃ CH ₃

 CH2CH3

0.32 g of a hapten (DEHP-6) represented by the following formula was obtained. (HPLC purity 91.4%) (4) Synthesis of DEHP-7

 Hexaethylene glycol (28.2 g) was dissolved in DMF (30 OmL), 4.0 g of NaH (60% dilution in mineral oil) was added under ice cooling, and the mixture was stirred for 15 minutes under ice cooling. Further, 15.2 g of benzyl chloride was added dropwise at 10 ° C for about 5 minutes. After stirring at room temperature for 1.5 hours, the mixture was filtered, and the filtrate was concentrated under reduced pressure. Further purification was performed using a silica gel column (200 g silica, ethyl acetate) to obtain 20 g of hexaethylene glycol monobenzyl ether as a colorless liquid.

A mixture consisting of 0.76 g of phthalic anhydride and 1.86 g of hexaethylene glycol / monobenzene / reatel was stirred at 110 ° C. for 17 hours. Without purification, 3 OmL of toluene, 0.9 mL of 1-bromo-1-ethylhexane and 0.7 g of DBUO were added to the entire amount of the obtained reaction product. Mixture 110. After stirring at C for 4 hours, 30 mL of water was added. After liquid separation, the mixture was washed with saturated saline and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the reaction product was dissolved in hexane monoethyl acetate (1: 1) and purified by silica gel column to obtain 12.45 g of a crude product as a colorless oil. 12.45 g of this crude product was dissolved in 4 OmL of THF, and 0.4 g of 10% Pd / C (50% water-containing product) was added. After blowing H ₂ (3 OmL / min, 4 hours), the catalyst was removed by filtration. To the reaction product, 1 OmL of purified water and 0.1 g of Pd black were added, and after blowing in H ₂ (S OmLZ, 4 hours), TLC confirmed that considerable raw materials had been consumed. The reaction was filtered, THF was distilled off under reduced pressure, and extracted with ethyl acetate. After further drying over anhydrous sodium sulfate, concentration under reduced pressure 1.8 g were obtained as a colorless oil.

 166.6 mg of succinic anhydride was added to 863.9 mg of the obtained crude product, and the mixture was stirred at 100 ° C for 6 hours. Further, 109.6 mg of succinic anhydride was added, and the mixture was stirred at 110 ° C for a while. The reaction product was dissolved in ethyl 2-propanol acetate (9: 1) and purified by a silica gel column to obtain 0.51 g of a crude product as a colorless oil. The same purification was repeated to obtain 0.45 g of a crude product as a colorless oil, which was then dissolved in diisopropyl ether and washed five times with water. Since liquid separation was difficult, saturated saline was appropriately added on the way. After drying over anhydrous sodium sulfate and concentration under reduced pressure, the following formula (IV)

0.41 g of a hapten (DEHP-7) represented by the formula was obtained as a colorless oil. (HP. 94.7% purity)

 (5) Synthesis of DEHP-10

 1 OmL of dry pyridine was ice-cooled, 0.5 g of phthalic acid chloride was added dropwise, and the mixture was stirred at room temperature at room temperature. The mixture was concentrated to remove pyridine to give 7.66 g of a yellow oil. This was subjected to silica gel column chromatography (silica gel 150 g, solvent: chloroform-methanol = 20: 1) to obtain 2.2 g of a colorless oil. The crude product was further subjected to ODS column chromatography (solvent: 30% acetonitrile) to obtain 1138 mg of a crude product as an anhydrous oil.

10 Omg of succinic anhydride was added to 135 Omg of the crude product, and the mixture was stirred at 90 ° C overnight. After returning the mixture to room temperature, the mixture is subjected to silica gel column chromatography (silica gel 30 g, solvent: chloroform-methanol = 10: 1) to obtain the following formula (IX): -COO (C ₂ H ₄ 0) ₆ COC ₂ H ₄ COOH

(IX)

COO (C ₂ H ₄ 0) ₆ COC ₂ H ₄ COOH

14 Omg of the hapten (DEHP-10) represented by was obtained as a colorless oil. (H PLC purity 92.9%)

 (6) Synthesis of DEHP-14

1.0 g of 11-promoundecanoic acid was added to 1 mL of dry THF, and 1 OmL of Ph ₂ CH ₂ N ₂ was further added to the mixture, followed by stirring at room temperature. Thereafter, the product was purified by silica gel column chromatography (developing solvent: dichloromethane-methane-η-hexane monoacetate == 4: 1). N-Hexane was added to the purified product to remove insolubles. The obtained reaction product was further purified by silica gel column chromatography (silica gel 60 g, eluent: n-hexane monoethyl acetate = 10: 1) to obtain 1.1.9 g of a crude product as a yellow oil. Was.

 Purified product 11.9 g, 193 mg of phthalic acid, 353 mg of 1,8-diazabicyclo (5,4,0) —7-pandecene (DBU) were added to 6 mL of toluene, and the mixture was stirred at 80 ° C for 3.5 hours. . The mixture was returned to room temperature, added with water and black-mouthed form, stirred, and then separated. The extract was washed with saturated brine, dried over sodium sulfate and concentrated. The obtained reaction product was eluted with silica gel gel ram chromatograph (silica gel 50 g, developing solvent: n-hexane monoethyl acetate = 20: 1 → 1: 1) to obtain 20.6 g of a crude product. Was obtained as a colorless oil.

20.6 g of the crude product was added to 8 mL of THF, and a catalyst amount of 10% Pd / C (50% water content) was added. After blowing H ₂ (3 OmL / min, 3 hours), the catalyst was filtered and concentrated. The reaction product was applied to silica gel column chromatography (silica gel 10 g, developing solvent: hexane monoacetate = 20: 1 → 1: 1), and the following formula (V):

269 g of the hapten (DEHP-14) represented by the formula was obtained as white crystals. (HPL C purity 98.8%)

 (7) Synthesis of DEHP-15

 2. 1 g of 60% sodium hydroxide was dispersed in 50 mL of N, N-dimethylformamide, cooled to 10 ° C, 10 g of getyl ethylmalonate was added dropwise, and the mixture was stirred for 35 minutes. This was added dropwise to a solution in which 14.5 g of 1,8-dibromooctane was dissolved in 5 OmL of N, N-dimethylformamide and cooled to 110 ° C in advance. After the completion of the dropwise addition, the mixture was returned to room temperature and stirred for 1.5 hours. Pour into 500 mL of ice water and add ethyl acetate

Extracted with 20 OmL. The extract was washed three times with 40 mL of water, dried over sodium sulfate, and concentrated under reduced pressure to obtain 20 g of a pale yellow oil. This was purified by silica gel column chromatography (silica gel 200 g, developing solvent: hexane monoacetate = 1: 0 → 20: 1) to obtain a crude product 1 as 10 g of a colorless oil.

 To 20.3 g of the crude product, 4 OmL of 48% hydrogen bromide was added, and the mixture was refluxed at 120 for 34 hours. After returning to room temperature, 30 OmL of ice water and 30 OmL of black hole form were added, and the mixture was separated. The organic layer was washed twice with 30 OmL of saturated saline, dried over sodium sulfate, and concentrated under reduced pressure to obtain 19.6 g of a yellow oil. This was purified by silica gel column chromatography (silica gel 200 g, eluent: ethyl hexane monoacetate = 50: 1 → 1: 1) to obtain 23.7 g of a crude product as a colorless oil. In addition, 14.7 g of the starting material ester was recovered.

 23.7 g of the resulting crude product was heated at 170 ° C for 2.5 hours. The mixture was returned to room temperature, and purified by silica gel column chromatography (silica gel 60 g, eluent: hexane monoethyl acetate = 10: 1) to obtain 33.36 g of a crude product as a pale yellow oil.

583 lng of the polan 'dimethylsulfide complex was dissolved in 1 OmL of THF, cooled with ice, and 2 mL of the crude product 32.36 g of ZTHF obtained was added dropwise. The mixture was heated to 80 ° C with an oil path and stirred for 2 hours. The temperature was returned to room temperature, and water (1 mL) and potassium carbonate were added in that order. Extract with 20 mL of methyl tert-butyl ether and 20 mL of saturated saline. Washed twice. After drying over sodium sulfate, the reaction mixture was concentrated under reduced pressure and a yellow oil was obtained. 2. lg was purified and purified by silica gel column chromatography (silica gel 50 g, developing solvent: hexane / monoethyl acetate = 10: 1). 41.8 g was obtained as a pale yellow oil.

 42.1 g of the crude product and 0.39 g of sodium cyanide were dissolved in 2 mL of dimethyl sulfoxide, and the mixture was stirred at 100 ° C for 2 hours. After returning to room temperature, 2 OmL of water was added, and the mixture was extracted with 2 OmL of methyl tert-butyl ether. Washed twice with 20 mL, once with 20 mL of 1N hydrochloric acid, and three times with 20 mL of saturated saline. After drying over sodium sulfate, the mixture was concentrated under reduced pressure to obtain 2.O g of a pale yellow oil. This was purified by silica gel column chromatography (silica gel 50 g, developing solvent: chromatoform) to obtain 51.54 g of a crude product as a slightly yellow oil.

51.88 g of the crude product obtained and 2.49 g of potassium hydroxide were dissolved in 3 mL of ethanol and 3 OmL of water, followed by stirring at 80 ° C. for 1 day. After removing the ethanol under reduced pressure and adding 1 N hydrochloric acid to make it acidic, the mixture was extracted with 10 mL of ethyl acetate. The extract was washed three times with 10 mL of saturated brine, dried over sodium sulfate, and concentrated under reduced pressure to obtain 2.0 _{g of} a reaction product as yellow oil.

 Dissolve 2.0 g of the obtained reaction product, 1.5 g of benzyl bromide, 1.4 g of 1,8-diazabicyclo mouth (5,4,0) -7-pandencene in 2 mL of toluene, and add 5 g at 80 ° C. Stir for 5 hours. After returning to room temperature, 0.3 g of benzyl bromide and 0.26 g of 1,8-diazabicyclo (5,4,0) -7-dandencene were added, and the mixture was stirred at 80 ° C. for 1 hour. After returning to room temperature, 50 mL of water and 5 OmL of chloroform were added for extraction. The extract was washed three times with 50 mL of saturated saline, dried over sodium sulfate, and concentrated under reduced pressure to obtain 3.2 g of a yellow oil. This was purified by silica gel column chromatography (silica gel 70 g, developing solvent: ethyl hexane monoacetate = 5: 1) to obtain 61.38 g of a crude product as a yellow oil.

6 1.38 g of crude product and 0.35 g of phthalic acid were dissolved in 15 mL of methylene chloride, and under ice-cooling, 0.1 g of 4-dimethylaminopyridine and 10.8 g of EDCHC were added. ice The mixture was stirred under cooling. After the reagent was dissolved, the mixture was returned to room temperature and stirred for 17 hours. Further, 10.4 g of EDO HC and 5 mL of methylene chloride were added, and the mixture was stirred for 3.5 hours. Add 1 OmL of methylene salt, wash twice with 1 ^ 1 hydrochloric acid 10111, twice with 10 mL of saturated sodium bicarbonate, twice with 1 OmL of saturated saline, dry over sodium sulfate, and concentrate under reduced pressure to a yellow oil. 1.6 g was obtained. This was purified by silica gel column chromatography (silica gel 25 g, eluent: hexane monoacetate = 10: 1) to obtain 70.86 g of a crude product as a colorless oil.

 70.86 g of the crude product was dissolved in 1 OmL of tetrahydrofuran, a catalytic amount of palladium Z carbon was added, and hydrogen gas was blown in for 7.5 hours. The catalyst was removed by filtration, and concentrated under reduced pressure to obtain 1.0 g of an oil. This was purified by silica gel column chromatography (silica gel 15 g, developing solvent: hexane monoacetate = 2: 1) to obtain the following formula (VI):

 258 mg of hapten DEHP-15 represented by the following formula was obtained as a colorless oil. (HPL C purity 98.4%)

 2. Confirmation of antibody titer

 (1) Preparation of antigens for immunogens and Atsusei

The various haptens synthesized in 1 above, 35 μιηο1, 7-soluble carbodiimide 42 μιηο1 and Ν-hydroxysuccinimide 42 / imol, were reacted once in 0.5 mL of dimethyl sulfoxide to prepare an activated ester. Then dissolved in bovine serum albumin (BSA) or Oboaru Pumin (0VA) a 0.13M sodium bicarbonate (NaHCO ₃₎ solution LML, after addition of the activated ester 120, or 50 teeth, are reacted with over晚4 ° C Was. Unreacted reagents are removed by dialysis against dalbecco's phosphate buffer (PBS), and hapten-BSA is Hapten-OVA was cryopreserved as an antigen for Atsey.

 (2) Mouse antiserum atsay method

 (I) Indirect competition method (antigen immobilization method)

Assi antigens prepared in (1) to (hapten -OVA) diluted to 0. lM aHC0 ₃ 7 with a solution (pH9.8) 10 g / mL, plated in 96-well plate at 50μ L / well, 4 ° C The antigen was adsorbed on the plate by allowing the plate to stand overnight. Next, the antigen was recovered, 350 L / well of 1% gelatin diluted with PBS was added, and the mixture was allowed to stand at 4 ° C for 24 hours, and blocking was performed. After leaving still at 37 ° C for 2 hours, the plate was washed three times with PBST (0.05 ° / oTween20 in PBS), and the primary antibody (serum diluted with PBST) was added at 50 / xL / well to 37 ° C, 1 hour. Allowed to react for hours. Then, after washing 3 times with PBST, add 50 L / well of secondary antibody [goat anti-mouse IgG (H + L) conjugated with HRP (horseradish peroxidase)] diluted 10,000 times with PBST. It was made to react at ° C for 1 hour. Finally, after 5 washes with PBST, coloring agents [0. 1M sodium citrate buffer (pH5.2 ) + o- phenyl ene diamine (lmg / ml) + 0.02% H 2 0 2] was added 100 L / well The color was developed at 37 ° (for 10 minutes, 1 MH ₂ SO ₄ was added at 50 Ai L / well, and the reaction was stopped. This was measured with 0D _49tam using a microplate reader.

 (II) Direct competition method (antibody immobilization method)

 (I 1-1) Preparation of antibody-immobilized plate

A goat anti-mouse IgG antibody (ICN / Cappel Code No. 55479) dissolved in 25 mM Bis-Tris-HCl (pH 5.6-5.7) (Dojin Code No. 345-04741) at 5 / zg / mL lOO ^ L / well was dispensed into a 96-well 1 microplate (Coster EIA / RIA plate strip8 Code No.2581), allowed to stand at 4 ° C for 1 hour, and then washed twice with 300 µL of PBST. 200 / iL / well of Ploque Ace (Yukishira Milk Products Co., Ltd., Tokyo) diluted 4 times with PBS was added, left at 4 ° C for 1 hour, and then washed twice with 300 μL of PBST. Add 100 μL / well of anti-DEHP monoclonal antibody dissolved in PBS + 0.1% BSA to a concentration of 0.075 μg / mL, and incubate at 4 ° C. 200 μL / well of the above blocking solution was added, and the plate was allowed to stand at 4 ° C. The entire volume was aspirated with an aspirator, and the dehydrated and dried solid-phased plate was sealed in an aluminum bag and vacuum dried. Degas and seal by machine, 2 ~ 8C Stored in the refrigerator.

 (I 1 -2) Antibody titer measurement

 DEHP (hapten) -HRP (50 mM phosphate buffer (pH7.10) dissolved in 10% methanol aqueous solution and 100 μL of DEHP or other plasticizer and prepared in (5)-(II) of Example 2 described later 100 / iL was mixed in a 96-well microplate (Nunc 167008). Next, 100 mixed solutions were added to the antibody-immobilized plate prepared in the above (II-1) and reacted at room temperature for 60 minutes. Unreacted substances are washed and removed three times with PBST 300 ^ L / well, and then TMB /, ° -Oxita ', -S', substrate kit (Nippon Pio'Rad, Tokyo, # 172-1066: the following coloring substrate) Was added at 100 μL / well. After the reaction at room temperature for 30 minutes, 1 N phosphoric acid was added at 10 μl / well to stop the color reaction, and the absorbance at a wavelength of 450 nm was measured using a plate reader.

 (3) Immunity

The immunogen prepared in (1) was dissolved in PBS to a concentration of 500 μg / mL, and immunized with BALB / c mice (SPF specification, female, 4 weeks old). For the first immunization, mix the antigen solution lOO ^ L ^ O.Smg / mL) with an equal volume of RIBI adjuvant (RIBI MPL + TDM EMULSION) (purchased from Corixa) per mouse to make 200 L. An oil-in-water emulsion was prepared by vortexing for ^2-3 minutes. This was injected intraperitoneally into mice. The second immunization was performed 14 days after the first immunization, and the antigen solution was mixed with an equal amount of RIBI adjuvant (100 し /0.311¾/), and the preparation was performed in the same manner as in the first immunization. In addition, the immunization interval was 2 weeks in all cases.

 Three days after the third immunization, blood was collected from the orbital vein of the mouse, and after bleeding, it was centrifuged at 10,000 rpm (8, 200 G) for 10 minutes to obtain serum. The antibody titer of the obtained serum was measured by the method described in 2- (2), and immunization was repeated every two weeks to measure the antibody titer.

 (4) Antibody titer

Table 1 shows the antibody titers of mice immunized with immunogens prepared from various haptens in this manner. table 1

 Note) OD of antiserum diluted 1000 times

 + +:> 1.5, +: 0.5-1.5, 1: 0.5

 Thus, by using the hapten, an antibody (antiserum) recognizing a desired resin plasticizer compound was obtained. Example 2

 1. Obtaining anti-DEHP monoclonal antibody

 (1) Preparation of immunogen, antigen for Atsusei, antigen for cell fusion

The hapten (DEHP-7) synthesized in Example 1 (35 mol), 7-soluble carbodiimide (42 mol _s) N-hydroxysuccinic acid imide (42 μτΆθΙ) was reacted in 0.5 mL of dimethyl sulfoxide to give a 晚 reaction. An activated ester was made. Then bovine serum albumin (BSA), was dissolved Opoarupumin the (OVA) or streptavidin § avidin 5 mg to 0. 1 3 M bicarbonate sodium © beam (NaHCO ₃₎ solution 1 m L, the activated ester 1 2 0, After adding 50 or 2 L, the reaction was carried out at −4 ° C. Unreacted reagents are removed by analysis with Dulbecco's phosphate buffer (PBS). saved.

 (2) Mouse antiserum method

(1) Assi antigens prepared in (hapten - OVA) to 0. lMNaHC0 ₃ solution (. PH 9 8) with diluted to 10 μ g / mL, seeded by 50 μ L / well in 96-well plates, 4 ° The antigen was adsorbed to the plate by allowing to stand overnight at C. Next, collect the antigen and dilute with PBS. The diluted 1% gelatin was added at 350 iL / well, and allowed to stand at 4 ° C for 24 hours to perform blocking. After leaving still at 37 ° C for 2 hours, the plate was washed three times with PBST (0.05 Tween20 in PBS), and the primary antibody (serum diluted with PBST) was added at 50 / zL / well and reacted at 37 ° C for 1 hour. I let it. Subsequently, after washing three times with PBST, add a secondary antibody [goat anti-mouse IgG (H + L) conjugated with HRP (horseradish peroxidase)] diluted 10,000 times with PBST ¾ ■ 50 L / well, and add It was made to react at ° C for 1 hour. Finally, after 5 washes with PBST, coloring agents [0. 1M sodium citrate buffer (pH5.2 ) + o- phenylene diamine (lmg / ml) + 0.02% H 2 0 2] in addition 100 / zL / well of 37 ° and developed C, 10 min, then quenched by the addition of _{1 M ¾S0 4 50 / z L} / well. Using a microplate reader

Was measured.

 (3) Immunity

 The immunogen prepared in (1) was dissolved in PBS to a concentration of 500 g / mL, and immunized with BALB / c mice (SPF specification, female, 4 weeks old). For the first immunization, mix 100 g / ml (0.5 mg / ml) of antigen solution with an equal volume of RIBI adjuvant (RIBI MPL + TDM EMULSION) (purchased from Corixa) per mouse to make 200 μL. An oil-in-water emulsion was prepared by vortexing for 2-3 minutes. This was injected intraperitoneally into mice. The second immunization was performed 14 days after the first immunization. The antigen solution was mixed with 100 parts (0.3 mg / mL) of RIBI adjuvant, and the preparation was performed in the same manner as in the first immunization. In addition, the immunization interval was 2 weeks in all cases.

 Three days after the third immunization, blood was collected from the orbital vein of the mouse, and after bleeding, it was centrifuged at 10,000 rpm (8, 200 G) for 10 minutes to obtain serum. The antibody titer of the obtained serum is measured by the method described in (1) (2) .If the antibody titer is sufficiently high, the last immunization is performed two weeks after the third immunization, and three days later, the mouse spleen cells are removed. Used for cell fusion. If the antibody titer was not sufficient, immunization every two weeks was repeated once or twice again, and after a sufficient increase in the antibody titer was confirmed, cell fusion was performed.

 (4) Cell fusion by electric pulse method (PEF method)

 (I) Preparation of splenocytes-DEHP-7-streptavidin complex

The spleen was removed from the mouse with an increased antibody titer obtained in (3) according to a standard method, and sulfated. A 2.5 mL spleen cell suspension was prepared in RPMI1640 with force namycin. On the other hand, (1) was prepared in DEHP- 7- streptavidin § avidin complex (lm _g / mL) 20 The mu iota was添Ka卩sulfate kanamycin-containing RPMI1640 2. 5 mL, spleen cell suspension prepared above The solution was mixed with ^2.5 mL. After reacting with a rotator at 4 ° C for 2 hours, the precipitate was suspended in 10 mL of RPMI1640 containing kanamycin sulfate after centrifugation (800GX5min). After the same centrifugation operation again, the precipitate was suspended in 5 mL of RPMI1640 containing kanamycin sulfate to prepare a spleen cell-DEHP-7-streptavidin complex.

 (II) Preparation of biotin-myeloma cell complex

Myeloma cells (PAI) cultured in RPMI1640 complete medium (three T-150 culture flasks) were collected, washed with 40 mL of PBS, centrifuged, and then suspended in 5 mL of PBS. Meanwhile, 30 μL of N-hydroxysuccimidimbiotin (lmg / 30 / zL in DMF) was dissolved in 5 mL of PBS and mixed with 5 mL of the previously prepared myeloma cell suspension (37 ° C, 5 C0 ₂ rotation in incubator for 30 minutes). After centrifugation, the plate was washed with 50 mL of RPMI1640 containing kanamycin sulfate, then centrifuged again, and suspended in 5 mL of RPMI1640 with kanamycin sulfate.

 (I I I) Cell fusion by PEF

(4) Each of the suspensions prepared in (1) and (II) was mixed so that spleen cells-DEHP-7-streptavidin and piotin-myeloma cells became 1: 1. This was centrifuged at 200 G for 10 min, and the precipitate was suspended in RPMI1640 containing 1 mL of kanamycin sulfate. After centrifugation at 50G for 1-2 minutes, the mixture was left in a clean bench for 30 minutes. Thereafter, further 30min, after 5% C0 ₂ incubator tapping make a rotation, 200G, and centrifuged at LOmin, isotonic sucrose puffer of 2 mL (0. 25M sucrose + 2 mM sodium dihydrogen phosphate Z hydrogen phosphate Suspended in Ninadium (pH 7.2) +0. ImM magnesium chloride +0. ImM calcium chloride. Add 0.5-1.0 mL each of this to a platinum-prepared plate, and use a cell fusion device (electro square porator T820 or ECM 201, manufactured by ΒΤΧ) to obtain 2 kV / cm (10 ^ secX4times). Electrofusion (PEF fusion) was performed under the conditions of 3 kV / cm (10 μsecX4times). The PEF-fused cells were suspended in 20 mL of RPMI1640 complete medium prepared in advance, allowed to stand for 30 min, and then dispensed to a 96-well microplate at 0.2 mL / well. And cultured at 37 ° C, 5% C0 ₂ incubator was carried out medium添Ka卩and media exchange by HAT medium by a conventional method.

 (5) Screening and cloning of Hypri-Doma

 (I) Preparation of Atsushi plate

Anti-mouse IgAGM

Diluted with (Goat IgG fraction to mouse immunoglobulins ( IgG, I gA, IgM) cappel made, part number 55461) with 5 mu g / mL to become as _{0. 1Μ NaHC0 3 (pH9. 8} ), 96well microplates (coaster 1: 2592) was added with 50 μL / well. After standing overnight at 4 ° C, the cells were collected, 350 µL / well of 1% gelatin diluted with PBS was added, and the mixture was incubated at 37 ° C for 2 hours to perform blocking.

 Preparation of (II) antigen-enzyme complex (DEHP-7-HRP)

 20 μmol of the hapten (DEHP-7) prepared in Example 1, 24 μmol of water-soluble carbodiimide, and 24 mol of N-hydroxysuccinimide were allowed to react in ImL of dimethyl sulfoxide to prepare an activated ester. Next, 3.3 mg of horseradish peroxidase (HRP) was dissolved in 0.1 mL of a 0.13 M sodium bicarbonate (NaHCO) solution, and 2 L of activated histamine was added, followed by reaction at 4 ° C overnight. Unreacted reagent was removed by ultrafiltration to obtain DEHP-7-HRP. It was stored refrigerated with HRP-3.3 mg / mL together with the prepared DEHP-7-HRP preservative.

 (I I I) Atsushi Method

(5)-After washing the assay plate prepared in (I) three times with PBST, the culture supernatant was added at 50 L / well each and incubated at 37 ° C for 1 hour. After washing three times with PBST, separate the enzyme-enzyme complex (DEHP-7-HRP) diluted 3,000-fold with PBS and DEHP dissolved in 20% Me0H separately in a mixing microplate (Nunc: 167008). Equal volumes were mixed, and each mixture was added to the antibody-immobilized plate at 50 μL / well, and incubated at 37 ° C for 1 hour. Finally, after washing ⁵ times with PBST, 100 μL / well of a coloring agent [0.1 M sodium citrate buffer (pH 5.2) + o-phenylene diamine (lmg / ml) + 0.02% H ₂ O ₂ ] 37 at a time. C, 10 After incubation for 1 minute to develop color, 1M H ₂ SO ₄ was added at 50 / zL / well to stop the reaction. Use a microplate reader to set this to 0D ₄₉ . _The measurement was performed in _nB , and the inhibition rate by DEHP was measured. .

 (IV) High Pridoma Selection, Crawling

 (5)-In the (III) Atsushi, cloning was performed according to a conventional method for the hybridomas having a high DEHP inhibition rate, and seven anti-DEHP antibody-producing hybridomas were selected and obtained.

 (6) Obtaining purified antibodies

 Purified antibodies were obtained from the 7 hybridomas described above by protein G affinity chromatography from 45-50% saturated ammonium sulfate fraction of the culture supernatant or mouse ascites according to a conventional method.

2. Evaluation of purified antibody

 (1) Atsushi by direct competition method

 (I) Preparation of antibody-immobilized plate

 A goat anti-mouse IgG antibody (ICNZCappel Code No. 55479) dissolved in 25 mM Bis-Tris-HC1 (pH 5.6-5.7) (Dojin Code No. 345-04741) at 5 / xg / mL / 100 / iL / The wells were dispensed into 96-well 11 microplates (Coster EIA / RIA plate strip8 Code No. 2581), allowed to stand at 4 ° C for one minute, and then washed twice with 300 μL of PBST. Block Ace (Snow Brand Milk Products Co., Ltd., Tokyo) diluted 4 times with PBS was added with SOO L / well, and allowed to stand at 4 ° C for 10 minutes, and then washed twice with 300 L of PBST. PBS + 0.1 ° /. An anti-DEHP monoclonal antibody dissolved in BSA at a concentration of 0.075 μg / mL was added at 100 / wellL / well, and the plate was allowed to stand at 4 ° C for 1 hour, followed by washing twice with PBST300; Add 200 μL / well of the blocking solution described above, allow to stand at 4 for a while, aspirate the entire volume with an aspirator, seal the dehydrated and dried solid phase plate in an aluminum bag, and degas with a vacuum dryer.ぴ Seal and store in a refrigerator at 2-8 ° C.

 (I I) E L I S A Atsushi

100 μL of DEHP or other plasticizer dissolved in 10% or 20% methanol aqueous solution And DEHP-7-HRP (diluted in 50 mM phosphate buffer (pH 7.5)) 100 / xL prepared in 1- (5)-(II) were mixed in a 96-well microplate (Nunc 167008). Next, the mixed solution IOOL was added to the antibody-immobilized plate prepared in 2- (1)-(1), and reacted at room temperature for 60 minutes. The unreacted substances were washed and removed three times with PBST 300 / zL / well. A substrate kit (Nippon Bio-Rad, Tokyo, # 172-1066: coloring substrate) was added at 100 ZL / well. After the reaction at room temperature for 30 minutes, 1 N phosphoric acid was added at 100 L / wel 1 to stop the color development reaction, and the absorbance at a wavelength of 450 nm was measured using a plate reader.

 (2) Inhibition test using DEHP

 For the above 7 antibodies, a DEHP standard curve was prepared by a direct competition method. The DEHP standard solution was prepared using 10% methanol. The results are shown in Figure 1. Of the above 7 antibodies, 2F4B7D6F6, which was the most sensitive at low concentration (DEHP: 0.1 mg / L), and 2F4A4Y, which was most sensitive at medium to high concentration (DEHP: 0.5 to 1 mg / L) Selected as a promising antibody.

 (3) Promising 2 Optimization of Atsey conditions for antibodies

 Under the above assay conditions (the concentration of the antigen-enzyme complex was changed as appropriate), the methanol concentration (10, 20, 40, 60%) for dissolving DEHP was optimized. The sensitivity of both antibodies decreased at DEHP lmg / L at a methanol concentration of 40% or more. From this, the methanol concentration for dissolving DEHP was set to 20%. '

 (4) Cross-reactivity with other plasticizers

 Assuming that the methanol concentration of the sample was 20%, the cross-reactivity with other plasticizers was examined. Figure 2 shows the results when the reactivity with DEHP is 100%. Both antibodies reacted extensively with ester phthalate adipates, especially with phthalates with 4-6 alkyl chains (DBP, BBP, DPnP and DHP).

 (5) Sensitivity comparison with conventional antibodies

When DHEP and DBP measurement sensitivities were compared under optimal conditions for 2F4A4 _Y and 2F4B7D6F6, 2F4A4y was slightly superior in sensitivity. Therefore, the study was conducted using 2F4A4 _Y below. Fig. 3 shows the results of comparison with DH-150, a previously acquired anti-DEHP antibody, when DEHP was measured. 2F4A4 y, which has a sensitivity equivalent to 0.2 mg / L, has a wider quantifiable range, and DH-150 could only be measured by the indirect competition method in which the antigen was immobilized, whereas 2F4A4 y _{In the case of Y} , it was possible to measure by the direct competition method with immobilized antibody. Fig. 4 shows the results of comparison with DF-34, which has already been acquired, when DBP is measured. In terms of sensitivity, DF-34 was 0.25 mg / L, while 2F4A4y was 0.02 to 0.05 mg / L, about 5 to 10 times higher sensitivity.

 Industrial applicability

 As is clear from the above description, conventionally, DEHP could only be measured by the indirect competition method in which the antigen was immobilized, whereas the antibody of the present invention specifically recognized DEHP in the immobilized state. It is possible to measure DEHP by the direct competition method. By using such an antibody, it is possible to provide a method of immunoassay and concentration of a resin plasticizer compound, which is significantly more useful than conventional methods.

Claims

The scope of the claims

1. An antibody that specifically recognizes a resin plasticizer compound, and specifically recognizes DEHP in a solid-phase state.

2. The antibody according to claim 1, wherein in a solid phase state, the DEHP concentration when BZBo for DEHP is 50% is 1 Omg / L or less.

3. When the resin plasticizer compound has the formula (I)

[Wherein, R ¹ is O-phenylene or tetramethylene, R ² and R ³ are the same or different, and are each H, linear or branched alkyl having 1 to 20 carbon atoms, benzyl or [Which means cyclohexyl]]. 4. The antibody according to claim 1, which is a monoclonal antibody.

5. The monoclonal antibody according to claim 4, which has 2F4Α4γ.

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

7. The hybrid dorma according to claim 6, which is 7.2 F4Α4γ (FERMΒ—08601). The antibody according to claim 1, which is a polyclonal antibody,

9. The method for producing the monoclonal antibody according to claim 4, comprising the following steps: (a) Formula (II):

[Wherein R ⁴ and R ⁵ are the same or different and each is a linear or branched alkyl group having 5 to 20 carbon atoms which may be substituted with a carboxyl group, or — C ₂ H ₄ 0) _n COC ₂ H ₄ COOH

In addition, ^ is a straight-chain or branched-chain alkyl group having 5 to 20 carbon atoms, and the length ⁶ is 11 or the HLB value of the whole molecule according to the Dariffin method is 5 A B-cell sensitized with a compound represented by the formula: and a myeloma cell;

(b) The step of recovering a monoclonal antibody from the obtained antibody-producing hybridoma, 10. The method according to claim 9, wherein the fusion is performed by an electric pulse method. 11. Formula (II):

[Wherein, R ⁴ and R ⁵ are the same or different and each is a linear or branched alkyl group having 5 to 20 carbon atoms which may be substituted with a carboxyl group, or one of them is (C ₂ H ₄ 〇) _n COC ₂ H ₄ COOH (n is an integer of 1 to 20), and the other is a linear or branched alkyl group having 5 to 20 carbon atoms, and R ⁶ is H or And a myeloma cell sensitized with the compound represented by the formula (1), wherein the HLB value of the whole molecule according to the Griffin method is 5 to 16. 7. The method for producing a hybrid dorma according to 6.

12. The method for producing a polyclonal antibody according to claim 8, comprising the following steps: (a) Formula (II):

[Wherein, R ⁴ and R ⁵ are the same or different and each is a linear or branched alkyl group having 5 to 20 carbon atoms which may be substituted with a carbonyl group; When C ₂ H ₄ O) _n COC ₂ H ₄ COOH (n represents an integer of 1 to 20), the other is a linear or branched alkyl group having 5 to 20 carbon atoms, and R ⁶ is H Or a group having an HLB value of 5 to 16 according to the Griffin method of the whole molecule].

 (b) a step of isolating a polyclonal antibody from the immunized mammal;

13. An immunological analysis method for a resin plasticizer compound, comprising using the antibody according to claim 1. 14. After reacting the antibody according to claim 1 immobilized on a carrier surface, a sample, and a resin plasticizer compound labeled with a labeling agent, the antibody is retained on the carrier or not retained 14. The method according to claim 13, wherein the activity of the labeling agent is measured.

1 5. An immunological analysis kit for a resin plasticizer compound, comprising the antibody according to claim 1 as a component.

16. A method for concentrating a resin plasticizer compound, comprising using the antibody according to claim 1. 17. A kit for concentrating a resin plasticizer compound comprising the antibody according to claim 1 as a component. Four

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