JP2007284392A - Anti-pcb monoclonal antibody that can be used for determining mixed sample of a plurality of pcb homologs of various numbers of chlorine atom, hybridoma that produces the antibody and method for determining pcb using the antibody - Google Patents

Anti-pcb monoclonal antibody that can be used for determining mixed sample of a plurality of pcb homologs of various numbers of chlorine atom, hybridoma that produces the antibody and method for determining pcb using the antibody Download PDF

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JP2007284392A
JP2007284392A JP2006114732A JP2006114732A JP2007284392A JP 2007284392 A JP2007284392 A JP 2007284392A JP 2006114732 A JP2006114732 A JP 2006114732A JP 2006114732 A JP2006114732 A JP 2006114732A JP 2007284392 A JP2007284392 A JP 2007284392A
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pcb
monoclonal antibody
antibody
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JP5041725B2 (en
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Yoko Takagi
陽子 高木
Hirohisa Kitagawa
博久 北川
Chiwa Kataoka
千和 片岡
Naoya Omura
直也 大村
Kazuhiro Sasaki
和裕 佐々木
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Central Research Institute of Electric Power Industry
Kyoto Electronics Manufacturing Co Ltd
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Kyoto Electronics Manufacturing Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-PCB monoclonal antibody having cross reactivity to a plurality of PCB homologs of various numbers of chlorine atom and having high affinity to PCB homologs of 3 to 6 chlorine atoms among all PCB homologs of 1 to 10 chlorine atoms, a method for producing it and a method for determining PCB using the anti-PCB monoclonal antibody. <P>SOLUTION: The method for producing the anti-PCB monoclonal antibody is characterized by using, as a hapten, a mixture of isomers of biphenyl derivatives represented by general formula (I), wherein n indicates an integer of 1 to 10; X and Y indicate each an integer of 0 to 2 respectively; and the sum of X and Y indicates an integer of 1 to 4. The anti-PCB monoclonal antibody is characterized by being produced by the above method and having cross reactivity to two or more kinds of PCB homologs having a different number of chlorine atom from each other. The method for determining PCB is characterized by using the above anti-PCB monoclonal antibody. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、環境中、工業製品中、及び生物試料中などに含まれるＰＣＢｓ（ポリ塩化ビフェニル類）の測定に用いられる抗ＰＣＢモノクローナル抗体、及びその製造方法、並びに前記抗ＰＣＢモノクローナル抗体を用いたＰＣＢ測定方法に関し、特に、塩素数の異なる二種以上のＰＣＢ同族体に対して交差反応性を有し、１〜１０塩素化ＰＣＢ全同族体のうち、３〜６塩素化ＰＣＢ同族体に高い親和性を有する抗ＰＣＢモノクローナル抗体及びその製造方法、並びに前記抗ＰＣＢモノクローナル抗体を用いたＰＣＢ測定方法に関する。   The present invention uses an anti-PCB monoclonal antibody used for measurement of PCBs (polychlorinated biphenyls) contained in the environment, industrial products, biological samples, and the like, a method for producing the same, and the anti-PCB monoclonal antibody. With respect to the PCB measurement method, in particular, it has cross-reactivity with respect to two or more kinds of PCB homologues having different chlorine numbers, and is high in 3-6 chlorinated PCB homologues among 1-10 chlorinated PCB all homologues. The present invention relates to an anti-PCB monoclonal antibody having affinity, a production method thereof, and a PCB measurement method using the anti-PCB monoclonal antibody.

化学物質による環境汚染問題は、健康問題や社会生活に大きな不安を与えると共に産業活動等にも影響を与えるため、早急に解決すべき重大な課題である。特に、人体や環境への有害性が確認されたＰＣＢｓ（ポリ塩化ビフェニル類、以下、単に「ＰＣＢ」と表すことがある）は過去、コンデンサやトランスの絶縁油、熱媒体、機械油等に多用されるなど地球規模で環境汚染が拡大しており、難分解性であることからその残留性や生物濃縮が問題となっている。
ＰＣＢｓについては、１９７４年に「化学物質の審査及び製造に関する法律」により製造、輸入、及び新たな使用について原則的に禁止措置が取られ、更に、２００１年に「ポリ塩化ビフェニル廃棄物の適正な処理の推進に関する特別措置法(ＰＣＢ特措法)」が施行され、２０１６年までに全てのＰＣＢ廃棄物の適性処理が法的に義務づけられた。これにより、ＰＣＢｓ廃棄物の処理過程でのモニタリングや、処理後のＰＣＢ濃度の測定が重要な課題となっている。
また、ＰＣＢｓを含まないとされていたトランス中の代替絶縁油においても、残留していた微量ＰＣＢｓの混入が明らかになった事より、大量の代替絶縁油やトランス本体のＰＣＢｓ濃度の迅速な分析も急務となっている。 The problem of environmental pollution caused by chemical substances is a serious problem that should be solved as soon as possible because it causes great concerns about health problems and social life, and also affects industrial activities. In particular, PCBs that have been confirmed to be harmful to the human body and the environment (polychlorinated biphenyls, hereinafter may be simply referred to as “PCB”) have been widely used in the past as insulation oil, heat medium, and machine oil for capacitors and transformers. Environmental pollution is expanding on a global scale, and its persistence and bioconcentration are problematic because it is difficult to decompose.
As for PCBs, in 1974 the “Law on the Examination and Production of Chemical Substances” was prohibited in principle for production, import, and new use. In 2001, “Appropriate disposal of polychlorinated biphenyl waste” The Special Measures Law on the Promotion of Disposal (PCB Special Measures Law) was enforced, and by 2016, all PCB wastes were legally mandated. Accordingly, monitoring in the process of processing PCBs waste and measurement of PCB concentration after processing are important issues.
In addition, in the alternative insulating oil in the transformer that was supposed not to contain PCBs, it was revealed that the trace amount of PCBs remaining remained, so that a rapid analysis of the PCBs concentration in a large amount of alternative insulating oil and transformer body Is also an urgent need.

従来のＰＣＢの測定としては、例えば、公定法として、高分解能ガスクロマトグラフィー／マススペクトロメトリー（ＨＲＧＣ／ＨＲＭＳ）や、電子捕獲検出器付きガスクロマトグラフ法（ＧＣ−ＥＣＤ）が用いられて来た。しかしながら、これらの方法は設備投資や分析消耗品等にコストが嵩み、また、煩雑なクリーンアップ操作や分析者の技術習熟等の必要性から、データ取得や解析に多大な時間を要する等の問題があり、分析業務が特定の分析機関に限定される等、大量の試料を処理する方法としては限界があった。
これに対し、特異的抗体を利用する免疫学測定法は、迅速かつ簡便な測定方法として知られ、近年、環境分析の分野にも応用されつつある。 For the measurement of conventional PCBs, for example, high resolution gas chromatography / mass spectrometry (HRGC / HRMS) and gas chromatography with electron capture detector (GC-ECD) have been used as official methods. However, these methods are costly for capital investment, analysis consumables, etc., and require a lot of time for data acquisition and analysis due to the need for complicated clean-up operations and analysts' technical skill. There is a problem, and there is a limit as a method for processing a large amount of samples, such as analysis work being limited to a specific analysis organization.
On the other hand, an immunological measurement method using a specific antibody is known as a rapid and simple measurement method, and has recently been applied to the field of environmental analysis.

ＰＣＢ類似物質であるダイオキシン類の迅速分析法としても、レセプターバインディングアッセイや免疫測定法が注目されており、特に、免疫測定法では抗体が単一の測定対象物に対し特異的に結合するという性質を利用し、ダイオキシン類の毒性等量と相関を有する特定の同族体量を測定する事により、多数の同族体が存在するダイオキシン類の毒性等量総和（ダイオキシン量）を迅速、簡便に求める方法が開発されている。   Receptor binding assays and immunoassays are also attracting attention as rapid analysis methods for dioxins that are PCB-analogous substances. In particular, immunoassays have the property that antibodies specifically bind to a single analyte. A method to quickly and easily determine the sum of the toxic equivalents (dioxin content) of dioxins containing a large number of congeners by measuring the amount of a specific homologue that correlates with the toxic equivalents of dioxins. Has been developed.

さらに、ＰＣＢは水に難溶性であるため、分析するための試料を調製するにあたり、両親媒性溶媒存在下にＰＣＢを溶解させる必要があるため、前記免疫学的測定法に用いられる抗ＰＣＢ抗体としては、試料中に存在する両親媒性溶媒に対する耐性が求められる。これに対し、前記免疫学的測定法に用いられる抗ＰＣＢ抗体としては、例えば、５０（ｖ／ｖ）％有機溶媒水溶液中でも抗原認識特性が実質的に低下しない抗モノクローナル抗体（特許文献１参照）等が提案されている。   Furthermore, since PCB is poorly soluble in water, it is necessary to dissolve PCB in the presence of an amphiphilic solvent when preparing a sample for analysis. Therefore, the anti-PCB antibody used in the immunoassay As for, the tolerance with respect to the amphiphilic solvent which exists in a sample is calculated | required. On the other hand, as an anti-PCB antibody used in the immunological assay, for example, an anti-monoclonal antibody whose antigen recognition property does not substantially decrease even in a 50 (v / v)% organic solvent aqueous solution (see Patent Document 1). Etc. have been proposed.

このように、抗体を用いる免疫学的測定法では、交差反応性が低く、測定対象物に対し高特異性という抗体本来の性質を利用し、特定の物質を測定する事に重点が置かれて来た。
このような考えに基づき、多数の異性体が存在するＰＣＢにおいては、交差反応性が制御された特異性の高い抗ＰＣＢ抗体が求められており、例えば、コプラナーＰＣＢの一つである3,3’,5,5’-四塩化ビフェニル（ＰＣＢ８０）に対して特異性を持つ抗体（特許文献２参照）等が提案されている。 In this way, immunoassays using antibodies focus on measuring specific substances by utilizing the intrinsic nature of antibodies, which have low cross-reactivity and high specificity for the analyte. I came.
Based on such an idea, a PCB having a large number of isomers is required to have a highly specific anti-PCB antibody with controlled cross-reactivity, for example, 3,3 which is one of coplanar PCBs. An antibody having specificity for ', 5,5'-bichlorinated biphenyl (PCB80) (see Patent Document 2) has been proposed.

一方、ＰＣＢ分析では、ダイオキシン類分析における「毒性等量」という考え方が無いため、免疫測定法においても、多数のＰＣＢ同族体について可能な限り広く検出し、測定できる事が重要である。しかしながら、特異性の高い単一の抗体を利用した測定系においては、同族体を網羅することが難しく、公定法による分析値との乖離、フォールスネガティブ（偽陰性の判定や見落とし）の危険性、さらに再現性の低さ等が懸念される。   On the other hand, in PCB analysis, since there is no concept of “toxic equivalent” in dioxin analysis, it is important that many PCB congeners can be detected and measured as widely as possible even in immunoassays. However, in a measurement system using a single antibody with high specificity, it is difficult to cover homologues, deviation from analysis values by official methods, risk of false negative (false negative judgment or oversight), Furthermore, there are concerns about low reproducibility.

そこで、２種類の特異性の異なる抗体、あるいは測定系を混合し、その結果得られる抗体の特異性を利用して絶縁油中のＰＣＢ濃度を分析する方法が提案されている（非特許文献１参照）。また、ダイオキシン類、ＰＣＢ類を検出する低分子物質検出用器具において、毒性を推定する場合と総量を推定する場合に応じて標的物質の異性体を選択し、該標的物質の抗体を用いることが開示されている（特許文献３参照）。
他方、異性体間において交差反応性が認められる複数種類の抗体を用いて、ダイオキシン類を異性体レベルで検出する方法が提案されている（特許文献４参照）。 Therefore, a method has been proposed in which two types of antibodies having different specificities or measurement systems are mixed, and the PCB concentration in the insulating oil is analyzed using the specificity of the resulting antibody (Non-patent Document 1). reference). Further, in a low molecular weight substance detection device for detecting dioxins and PCBs, it is possible to select an isomer of a target substance according to a case of estimating toxicity and a case of estimating a total amount, and use an antibody of the target substance It is disclosed (see Patent Document 3).
On the other hand, a method of detecting dioxins at the isomer level using a plurality of types of antibodies in which cross-reactivity is recognized between isomers has been proposed (see Patent Document 4).

しかしながら、特許文献３及び４に記載された抗ＰＣＢ抗体を用いた免疫学的測定方法における測定対象は、コプラナーＰＣＢであり、そこで用いられる抗体は、異なる塩素数群の同族体混合物（例えば、鐘淵化学工業製のカネクロール、及び三菱モンサント（現三菱化学）製のアロクロールなどに由来するＰＣＢ）を、高感度に定量するための抗体としては満足なものではない。   However, the measurement target in the immunoassay method using the anti-PCB antibody described in Patent Documents 3 and 4 is a coplanar PCB, and the antibody used therein is a homologous mixture of different chlorine groups (for example, bell It is not satisfactory as an antibody for quantitatively quantifying Kanechlor manufactured by Sakai Chemical Industry and PCB derived from Arrochlor manufactured by Mitsubishi Monsanto (currently Mitsubishi Chemical) with high sensitivity.

特開２０００−１９１６９９号公報JP 2000-191699 A 特開２００５−２４７８２２号公報JP 2005-247822 A 特開２００４−１３８５５０号公報JP 2004-138550 A 特開２００２−２２８６６０号公報JP 2002-228660 A 25th International Symposium on Halogenated Environmental Organic Pollutant and POPs,2005,ID238725th International Symposium on Halogenated Environmental Organic Pollutant and POPs, 2005, ID2387

本発明は、従来における前記問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、３塩化ビフェニル、４塩化ビフェニル、５塩化ビフェニル、及び６塩化ビフェニル等の塩素数の異なる複数のＰＣＢ同族体の混合物、具体的には、油性試料中に含まれるカネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、ＫＣ−６００：鐘淵化学工業社製）、及びアロクロール（Ａｒｏｃｌｏｒ．１２４２、Ａｒｏｃｌｏｒ．１２４８、Ａｒｏｃｌｏｒ．１２５４、Ａｒｏｃｌｏｒ．１２６０：三菱モンサント（現三菱化学）製）中に存在比の高い複数のＰＣＢ同族体に交差反応性を有し、１〜１０塩素化ＰＣＢ全同族体のうち、３〜６塩素化ＰＣＢ同族体混合物に高い親和性を有する抗ＰＣＢモノクローナル抗体、及びその製造方法、並びに前記抗ＰＣＢモノクローナル抗体を用いたＰＣＢ測定方法を提供することを目的とする。   An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention is a mixture of a plurality of PCB homologues having different chlorine numbers, such as biphenyl trichloride, tetrachloride biphenyl, pentachloride biphenyl, and hexachloride biphenyl, specifically, Kanechlor ( KC-300, KC-400, KC-500, KC-600: manufactured by Kaneka Chemical Co., Ltd.), and Arochlor (Arochlor. 1242, Arolor. 1248, Arolor. 1254, Arolor. 1260: Mitsubishi Monsanto (current Mitsubishi Chemical) Made of) a plurality of PCB homologues having a high abundance ratio, and among 1-10 chlorinated PCB all homologues, anti-PCB having a high affinity for 3-6 chlorinated PCB homologue mixtures Provided are a monoclonal antibody, a production method thereof, and a PCB measurement method using the anti-PCB monoclonal antibody. For the purpose of theft.

前記課題を解決するため、本発明者らが鋭意検討を重ねた結果、下記一般式（Ｉ）で表されるビフェニル誘導体の異性体混合物をハプテンとし、該ハプテンをキャリアータンパク質に結合した化合物を用いることにより、塩素数の異なる二種以上のＰＣＢ同族体に交差反応性を有し、かつ親和性の高い抗ＰＣＢモノクローナル抗体が得られることを見出し、本発明を完成するに至った。   As a result of extensive studies by the present inventors to solve the above problems, a compound in which an isomer mixture of biphenyl derivatives represented by the following general formula (I) is used as a hapten and the hapten is bound to a carrier protein is used. Thus, it was found that an anti-PCB monoclonal antibody having cross-reactivity and high affinity to two or more kinds of PCB homologues having different chlorine numbers was obtained, and the present invention was completed.

ただし、前記一般式（Ｉ）中、ｎは１〜１０の整数を表し、Ｘ及びＹはそれぞれ０〜２の整数を表し、ＸとＹとの和は１〜４の整数を表す。 However, in said general formula (I), n represents the integer of 1-10, X and Y represent the integer of 0-2 respectively, and the sum of X and Y represents the integer of 1-4.

本発明は、本発明者による前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ 下記一般式（Ｉ）で表されるビフェニル誘導体の異性体混合物を、ハプテンとして用いることを特徴とする抗ＰＣＢモノクローナル抗体の製造方法である。
ただし、前記一般式（Ｉ）中、ｎは１〜１０の整数を表し、Ｘ及びＹはそれぞれ０〜２の整数を表し、ＸとＹとの和は１〜４の整数を表す。
＜２＞ 前記一般式（Ｉ）で表されるビフェニル誘導体から得られた下記一般式（II）で表される化合物を免疫原として動物を免疫し、免疫した前記動物の抗体産生細胞を回収し、該抗体産生細胞とミエローマ細胞とを融合して得たハイブリドーマを培養することを含む前記＜１＞に記載の抗ＰＣＢモノクローナル抗体の製造方法である。
ただし、前記一般式（II）中、ｎは１〜１０の整数を表し、Ｘ及びＹはそれぞれ０〜２の整数を表し、ＸとＹとの和は１〜４であり、Ｚはタンパク質を表す。
＜３＞ 前記一般式（II）中、Ｚが、酵素、蛍光色素、放射性同位元素、磁性体、色素封入ポリマー、希土類、補酵素、金コロイド、及び金原子クラスターのいずれかの標識物質により標識されている前記＜２＞に記載の抗ＰＣＢモノクローナル抗体の製造方法である。
＜４＞ 前記一般式（Ｉ）及び前記一般式（II）のいずれかで表される化合物を用いて、ハイブリドーマ細胞株をスクリーニングすることを含む前記＜１＞から＜２＞のいずれかに記載の抗ＰＣＢモノクローナル抗体の製造方法である。 This invention is based on the said knowledge by this inventor, and as a means for solving the said subject, it is as follows. That is,
<1> A method for producing an anti-PCB monoclonal antibody, wherein an isomer mixture of a biphenyl derivative represented by the following general formula (I) is used as a hapten.
However, in said general formula (I), n represents the integer of 1-10, X and Y represent the integer of 0-2 respectively, and the sum of X and Y represents the integer of 1-4.
<2> An animal is immunized with a compound represented by the following general formula (II) obtained from the biphenyl derivative represented by the general formula (I) as an immunogen, and antibody-producing cells of the immunized animal are collected. The method for producing an anti-PCB monoclonal antibody according to <1>, further comprising culturing a hybridoma obtained by fusing the antibody-producing cells and myeloma cells.
However, in said general formula (II), n represents the integer of 1-10, X and Y represent the integer of 0-2 respectively, the sum of X and Y is 1-4, Z is protein. To express.
<3> In the general formula (II), Z is labeled with any labeling substance of enzyme, fluorescent dye, radioisotope, magnetic substance, dye-encapsulated polymer, rare earth, coenzyme, gold colloid, and gold atom cluster. The method for producing an anti-PCB monoclonal antibody according to <2>.
<4> The method according to any one of <1> to <2>, further comprising screening a hybridoma cell line using the compound represented by any one of the general formula (I) and the general formula (II). This is a method for producing an anti-PCB monoclonal antibody.

＜５＞ 前記＜１＞から＜４＞のいずれかに記載の抗ＰＣＢモノクローナル抗体の製造方法により製造され、塩素数の異なる二種以上のＰＣＢ同族体に交差反応性を有することを特徴とする抗ＰＣＢモノクローナル抗体である。
＜６＞ １〜１０塩素化ＰＣＢ全同族体のうち、１〜２塩素化ＰＣＢ同族体及び７〜１０塩素化同族体に対する親和性よりも、３〜６塩素化ＰＣＢ同族体混合物に対する親和性が高い前記＜５＞に記載の抗ＰＣＢモノクローナル抗体である。 <5> Produced by the method for producing an anti-PCB monoclonal antibody according to any one of <1> to <4>, wherein two or more kinds of PCB homologs having different chlorine numbers have cross-reactivity. Anti-PCB monoclonal antibody.
<6> Among all 1-10 chlorinated PCB homologues, the affinity for 3-6 chlorinated PCB homologue mixture is greater than the affinity for 1-2 chlorinated PCB homologue and 7-10 chlorinated homologue. The anti-PCB monoclonal antibody according to <5>, which is high.

＜７＞ 前記＜５＞から＜６＞のいずれかに記載の抗ＰＣＢモノクローナル抗体を産生することを特徴とするハイブリドーマである。
＜８＞ 受託番号がＦＥＲＭ Ｐ−２０８４５である前記＜７＞に記載のハイブリドーマである。 <7> A hybridoma characterized by producing the anti-PCB monoclonal antibody according to any one of <5> to <6>.
<8> The hybridoma according to <7>, wherein the accession number is FERM P-20845.

＜９＞ 被測定試料に、前記＜５＞から＜６＞のいずれかに記載の抗ＰＣＢモノクローナル抗体を添加し、
（１）前記抗ＰＣＢモノクローナル抗体と結合した前記被測定試料中のＰＣＢ、及び
（２）前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢモノクローナル抗体
のいずれかを検出する方法を含むことを特徴とするＰＣＢ測定方法である。
＜１０＞ 被測定試料に、前記＜５＞から＜６＞のいずれかに記載の抗ＰＣＢモノクローナル抗体、及び前記一般式（Ｉ）及び前記一般式（II）のいずれかで表される化合物をＰＣＢ競合物質として添加し、前記抗ＰＣＢモノクローナル抗体と結合した記競合物質を検出することを含むことを特徴とするＰＣＢ測定方法である。
＜１１＞ 被測定試料に、前記＜５＞から＜６＞のいずれかに記載の抗ＰＣＢモノクローナル抗体を添加し、前記一般式（II）で表される化合物をＰＣＢ競合物質として固定化した担体を用い、前記抗ＰＣＢモノクローナル抗体と前記被測定試料中のＰＣＢとを競争させ、前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢモノクローナル抗体を検出することを含むことを特徴とするＰＣＢ測定方法である。 <9> To the sample to be measured, add the anti-PCB monoclonal antibody according to any one of <5> to <6>,
(1) including a method of detecting any of PCB in the sample to be measured bound to the anti-PCB monoclonal antibody and (2) the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured. Is a PCB measurement method characterized by
<10> An anti-PCB monoclonal antibody according to any one of <5> to <6> and a compound represented by any one of the general formula (I) and the general formula (II) It is a method for measuring PCB, comprising detecting a competitive substance added as a PCB competitor and bound to the anti-PCB monoclonal antibody.
<11> A carrier obtained by adding the anti-PCB monoclonal antibody according to any one of <5> to <6> to a sample to be measured and immobilizing the compound represented by the general formula (II) as a PCB competitor And the competition between the anti-PCB monoclonal antibody and the PCB in the sample to be measured, and detecting the anti-PCB monoclonal antibody not bound to the PCB in the sample to be measured. This is a measurement method.

本発明によると、塩素数の異なる複数のＰＣＢ同族体の混合物、具体的には、油性試料中に含まれるカネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、ＫＣ−６００：鐘淵化学工業社製）、及びアロクロール（Ａｒｏｃｌｏｒ．１２４２、Ａｒｏｃｌｏｒ．１２４８、Ａｒｏｃｌｏｒ．１２５４、Ａｒｏｃｌｏｒ．１２６０：三菱モンサント（現三菱化学）製）中に存在比の高い複数のＰＣＢ同族体に交差反応性を有し、１〜１０塩素化ＰＣＢ全同族体のうち、３〜６塩素化ＰＣＢ同族体に高い親和性を有する抗ＰＣＢモノクローナル抗体、及びその製造方法、並びに前記抗ＰＣＢモノクローナル抗体を用いたＰＣＢ測定方法を提供することができる。   According to the present invention, a mixture of a plurality of PCB homologues having different chlorine numbers, specifically, Kanechlor (KC-300, KC-400, KC-500, KC-600: Kaneka Chemical) contained in an oily sample. ), And Arochlor (Arochlor.1242, Arochlor.1248, Arochlor.1254, Arochlor.1260: Mitsubishi Monsanto (currently Mitsubishi Chemical)) have high reactivity in PCB homologs. An anti-PCB monoclonal antibody having high affinity for 3-6 chlorinated PCB homologs among all 1-10 chlorinated PCB homologs, a method for producing the same, and a PCB measurement method using the anti-PCB monoclonal antibodies Can be provided.

（抗ＰＣＢモノクローナル抗体）
＜抗ＰＣＢモノクローナル抗体の製造方法＞
本発明の抗ＰＣＢモノクローナル抗体は、下記一般式（Ｉ）で表されるビフェニル誘導体の異性体混合物からなるハプテンを用い、本発明の抗ＰＣＢモノクローナル抗体の製造方法により製造される。 (Anti-PCB monoclonal antibody)
<Method for producing anti-PCB monoclonal antibody>
The anti-PCB monoclonal antibody of the present invention is produced by the method for producing an anti-PCB monoclonal antibody of the present invention using a hapten composed of an isomer mixture of biphenyl derivatives represented by the following general formula (I).

ただし、前記一般式（Ｉ）中、ｎは１〜１０の整数を表し、Ｘ及びＹはそれぞれ０〜２の整数を表し、ＸとＹとの和は１〜４の整数を表す。   However, in said general formula (I), n represents the integer of 1-10, X and Y represent the integer of 0-2 respectively, and the sum of X and Y represents the integer of 1-4.

なお、前記一般式（Ｉ）で表されるビフェニル誘導体の異性体混合物における異性体としては、例えば、３塩化ビフェニル及び四塩化ビフェニルのいずれかであることが好ましく、例えば、下記一般式（III）及び（IV）のいずれかで表される化合物などが挙げられる。
The isomer in the isomer mixture of the biphenyl derivative represented by the general formula (I) is preferably, for example, any one of biphenyl trichloride and tetraphenyl bichloride. For example, the following general formula (III) And a compound represented by any one of (IV) and the like.

これらの異性体の混合割合としては、３−フェニルフェノールに対する塩素化剤の添加量及び塩素原子の配向性から、前記一般式（III）の化合物が主要異性体と考えられる。   Regarding the mixing ratio of these isomers, the compound of the above general formula (III) is considered to be the main isomer from the amount of chlorinating agent added to 3-phenylphenol and the orientation of chlorine atoms.

前記一般式（III）で表されるビフェニル誘導体は、例えば、以下の反応式に従い製造することができる。   The biphenyl derivative represented by the general formula (III) can be produced, for example, according to the following reaction formula.

ただし、前記反応式中、Ｒは、低級アルキル基で直鎖状、分岐鎖状で炭素原子数１〜４の整数を表し、ｎは１〜１０の整数を表し、Ｘ及びＹはそれぞれ０〜２の整数を表し、ＸとＹとの和は１〜４の整数を表す。 In the above reaction formula, R represents a lower alkyl group that is linear or branched, and represents an integer of 1 to 4 carbon atoms, n represents an integer of 1 to 10, and X and Y are each 0 to 2 represents an integer, and the sum of X and Y represents an integer of 1 to 4.

前記一般式（Ｉ）で表される化合物は、単独では抗原性を持たないため、抗ＰＣＢモノクローナル抗体を作製するために、下記一般式（II）で表される化合物（以下、「コンジュゲート」ということがある）を調製し、これを免疫原（抗原）として用い、前記抗ＰＣＢモノクローナル抗体を作製することが好ましい。   Since the compound represented by the general formula (I) does not have antigenicity by itself, a compound represented by the following general formula (II) (hereinafter, “conjugate”) is used to produce an anti-PCB monoclonal antibody. It is preferable to prepare the anti-PCB monoclonal antibody using this as an immunogen (antigen).

ただし、前記一般式（II）中、ｎは１〜１０の整数を表し、Ｘ及びＹはそれぞれ０〜２の整数を表し、ＸとＹとの和は１〜４であり、Ｚはタンパク質を表す。   However, in said general formula (II), n represents the integer of 1-10, X and Y represent the integer of 0-2 respectively, the sum of X and Y is 1-4, Z is protein. To express.

前記一般式（II）中、Ｚで表されるタンパク質（「キャリアータンパク質」ということがある）としては、免疫原性を有し、かつ前記一般式（Ｉ）で表される化合物とスペーサー部を介して連結可能である限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、スカシガイ由来ヘモシアニン（ＫＬＨ）、卵白アルブミン（ＯＶＡ）、ウシ血清アルブミン（ＢＳＡ）、ヒト血清アルブミン、ウサギ血清アルブミン、ヤギ血清アルブミン、ウシガンマグロブリン、ミオグロビン、Ｍｕｌｔｉｐｌｅ ａｎｔｉｇｅｎ ｐｅｐｔｉｄｅ（ＭＡＰ）等が挙げられる。   In the general formula (II), the protein represented by Z (sometimes referred to as “carrier protein”) has an immunogenicity and a compound represented by the general formula (I) and a spacer part. As long as it can be linked via a hydride, can be appropriately selected according to the purpose. For example, mussel hemocyanin (KLH), ovalbumin (OVA), bovine serum albumin (BSA), human serum albumin, Examples include rabbit serum albumin, goat serum albumin, bovine gamma globulin, myoglobin, and multiple antigen peptide (MAP).

前記一般式（II）で表される化合物（コンジュゲート）の調製方法としては、特に制限はなく、公知の合成方法により調製することができ、例えば、下記反応式に示すように、前記一般式（Ｉ）で表されるビフェニル誘導体のカルボキシル基を、Ｎ−ヒドロキシスクシンイミドなどの縮合反応により活性エステル誘導体に変換し、次いで、該活性エステル誘導体とキャリアータンパク質のアミノ基とを常法に従って結合させる方法が挙げられる。   The method for preparing the compound (conjugate) represented by the general formula (II) is not particularly limited and can be prepared by a known synthesis method. For example, as shown in the following reaction formula, the general formula A method in which the carboxyl group of the biphenyl derivative represented by (I) is converted into an active ester derivative by a condensation reaction such as N-hydroxysuccinimide, and then the active ester derivative and the amino group of the carrier protein are bonded according to a conventional method. Is mentioned.

得られた前記一般式（II）で表される化合物（コンジュゲート）は、ゲルクロマトグラフィー等の公知の方法により反応液を生理的リン酸緩衝液などの中性溶液に置換すると共に精製することが好ましい。   The obtained compound (conjugate) represented by the general formula (II) is purified by substituting the reaction solution with a neutral solution such as a physiological phosphate buffer by a known method such as gel chromatography. Is preferred.

前記一般式（II）中、Ｚで表されるタンパク質は、標識物質により標識されていてもよい。前記標識物質としては、例えば、酵素、蛍光色素、放射性同位元素、磁性体、色素封入ポリマー、希土類、希土類、補酵素、金コロイド、及び金原子クラスター等の公知の標識物質から適宜選択することができる。   In the general formula (II), the protein represented by Z may be labeled with a labeling substance. The labeling substance may be appropriately selected from known labeling substances such as enzymes, fluorescent dyes, radioisotopes, magnetic substances, dye-encapsulated polymers, rare earths, rare earths, coenzymes, gold colloids, and gold atom clusters. it can.

前記抗ＰＣＢモノクローナル抗体は、例えば、前記一般式（II）で表される化合物を免疫原とし、該免疫原とアジュバントとを乳化させて得た乳化液を用いて動物を免疫し、免疫した前記動物の脾臓細胞を回収し、該脾臓細胞とミエローマ細胞とを融合して得たハイブリドーマを培養することにより製造される。
モノクローナル抗体の製造方法としては、例えば、ケーラーとミルシュタインの方法（Kohler G, Milstein C, Nature, 256,495-7(1975)）等に準じて行うことが出来る。 The anti-PCB monoclonal antibody, for example, immunized an animal using an emulsion obtained by emulsifying the immunogen and an adjuvant using the compound represented by the general formula (II) as an immunogen, It is produced by collecting spleen cells of an animal and culturing a hybridoma obtained by fusing the spleen cells and myeloma cells.
The monoclonal antibody can be produced according to, for example, the method of Kohler and Milstein (Kohler G, Milstein C, Nature, 256, 495-7 (1975)).

免疫する前記動物としては、マウス、ラット、ウサギ、ヤギ等の哺乳動物が好ましい。
前記動物の免疫方法としては、特に制限はなく、公知の方法から適宜選択することができ、例えば、前記免疫原とアジュバントとを乳化させて得た乳化液を、皮下、静脈内、腹腔内に注射等により投与する方法が挙げられる。
前記免疫原は、例えば、マウスの場合、０．０１〜０．５ｇ／ｂｏｄｙを、２〜３週間程度の間隔で３〜１５回投与することが好ましい。
前記アジュバントとしては、特に制限はなく、目的に応じて適宜選択することができるが、例えば、フロイントの完全アジュバント、不完全アジュバント、ＢＣＧ、トレハロースダイマイコレート（ＴＤＭ）、リポ多糖（ＬＰＳ）、ミョウバンアジュバント、シリカアジュバント等が挙げられるが、抗体の誘導能、動物への負荷等の関係から、最適なものを選択して使用することが好ましい。 As the animal to be immunized, mammals such as mice, rats, rabbits and goats are preferable.
The animal immunization method is not particularly limited and can be appropriately selected from known methods. For example, an emulsion obtained by emulsifying the immunogen and an adjuvant is subcutaneously, intravenously or intraperitoneally. The method of administering by injection etc. is mentioned.
For example, in the case of mice, the immunogen is preferably administered in an amount of 0.01 to 0.5 g / body 3 to 15 times at intervals of about 2 to 3 weeks.
The adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose. For example, Freund's complete adjuvant, incomplete adjuvant, BCG, trehalose dimycolate (TDM), lipopolysaccharide (LPS), alum Adjuvants, silica adjuvants, and the like can be mentioned, but it is preferable to select and use the most suitable one in view of the inducing ability of the antibody, the load on the animal, and the like.

前記免疫原の最後の投与から数日後に、免疫した前記動物から抗体産生細胞を回収する。前記抗体産生細胞としては、例えば、脾臓、リンパ節由来Ｂ細胞等が挙げられる。
前記抗体産生細胞をミエローマ細胞と融合し、その後所望の抗体を産生する融合細胞をスクリーニングすることにより、前記ハイブリドーマが得られる。 Several days after the last administration of the immunogen, antibody-producing cells are collected from the immunized animal. Examples of the antibody-producing cells include spleen and lymph node-derived B cells.
The hybridoma can be obtained by fusing the antibody-producing cells with myeloma cells and then screening for fused cells that produce the desired antibody.

前記ミエローマ細胞は、特に限定されず、公知のものを使用できる。マウス、ラット、ヒト由来の細胞株等から選択することができ、例えば、マウス由来細胞であれば、Ｐ３−Ｘ６３／Ａｇ８、Ｘ６３−Ａｇ８．６５３、ＮＳ１／１．Ａｇ ４ １,Ｓｐ２１０−Ａｇ１４、 ＦＯ、ＮＳＯ／Ｕ、ＭＰＣ−１１、ＭＰＣ１１−Ｘ４５−ＧＴＧ １．７、Ｓ１９４／５ＸＸＯ・Ｂｕｌ等、ラット由来細胞であれば、Ｒ２１０．ＲＣＹ３、Ｙ３−Ａｇ・１．２．３、ＩＲ９８３Ｆ、４Ｂ２１０等が挙げられる。
前記抗体産生細胞とミエローマ細胞との融合方法としては、センダイウイルス法、ポリエチレングリコール法、プロトプラスト法等、当業者に公知の方法で行えばよく、特に限定されないが、ポリエチレングリコール法で融合させる方法が細胞毒性も比較的少なく、融合操作も簡単であるという理由から好ましい。 The myeloma cells are not particularly limited, and known ones can be used. For example, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.1 can be selected from mouse, rat, and human-derived cell lines. If it is a rat-derived cell such as Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, S194 / 5XXO · Bul, R210. RCY3, Y3-Ag · 1.2.3, IR983F, 4B210 and the like.
Methods for fusing the antibody-producing cells and myeloma cells may be performed by methods known to those skilled in the art, such as Sendai virus method, polyethylene glycol method, protoplast method, and the like. It is preferable because it has relatively little cytotoxicity and the fusion operation is simple.

前記所望の抗体を産生する融合細胞をスクリーニングする方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ラジオイムノアッセイ（ＲＩＡ）、エンザイムイムノアッセイ（ＥＩＡ、ＥＬＩＳＡ）、フルオロイムノアッセイ（ＦＩＡ）などが挙げられる。
例えば、前記一般式（II）で表されるコンジュゲートを固相化し、前記融合細胞（ハイブリドーマ）の培養上清を反応させる事より、該培養上清の抗体価を測定し、抗体価が高いウエルを抗ＰＣＢ抗体産生ウエルとして選択することができる。さらに、抗体価測定時にＰＣＢ代替化合物（例えば、前記一般式（Ｉ）で表されるビフェニル誘導体）やＰＣＢｓ等を共存させる事より、これら共存物質に対し反応性の高いウエルを選択し、限界希釈法にて目的の抗ＰＣＢ抗体産生ハイブリドーマのモノクローン化を行うことができる。
また、前記一般式（II）で表されるコンジュゲート中、Ｚで表されるタンパク質を標識物質により標識し、これを抗ＰＣＢ抗体産生ウエルの抗体価を測定時に共存させ、直接競合反応を行う方法によって抗ＰＣＢ抗体産生ハイブリドーマをスクリーニングすることもできる。 The method for screening the fused cells producing the desired antibody is not particularly limited and may be appropriately selected according to the purpose. For example, radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), fluoroimmunoassay (FIA).
For example, by immobilizing the conjugate represented by the general formula (II) and reacting the culture supernatant of the fused cell (hybridoma), the antibody titer of the culture supernatant is measured, and the antibody titer is high. Wells can be selected as anti-PCB antibody producing wells. Furthermore, by coexisting PCB substitute compounds (for example, biphenyl derivatives represented by the above general formula (I)) and PCBs at the time of antibody titer measurement, wells that are highly reactive with these coexisting substances are selected, and limiting dilution is performed. The desired anti-PCB antibody-producing hybridoma can be monocloned by the method.
Further, in the conjugate represented by the general formula (II), the protein represented by Z is labeled with a labeling substance, and this is allowed to coexist at the time of measurement of the antibody titer of the anti-PCB antibody production well to perform a direct competitive reaction. An anti-PCB antibody-producing hybridoma can also be screened by the method.

測定対象のＰＣＢは水に難溶であるため、両親媒性溶媒に溶解させて測定に供せられる。このため、前記抗ＰＣＢ抗体は、試料中に存在する前記両親媒性溶媒に対する耐性を有することが好ましい。よって、前記抗ＰＣＢ抗体産生ハイブリドーマのスクリーニングも前記両親媒性溶媒存在下で行うことが好ましい。
前記両親媒性溶液としては、ＤＭＳＯ、メタノール、エタノール等が挙げられる。
前記スクリーニングに用いられる前記両親媒性溶液の濃度としては、５０質量％以下が好ましく、３０質量％以下がより好ましく、５〜２０質量％が特に好ましい。 Since PCB to be measured is hardly soluble in water, it is dissolved in an amphiphilic solvent and used for measurement. For this reason, it is preferable that the said anti- PCB antibody has tolerance with respect to the said amphiphilic solvent which exists in a sample. Therefore, it is preferable that the anti-PCB antibody-producing hybridoma is also screened in the presence of the amphiphilic solvent.
Examples of the amphiphilic solution include DMSO, methanol, ethanol and the like.
The concentration of the amphiphilic solution used for the screening is preferably 50% by mass or less, more preferably 30% by mass or less, and particularly preferably 5 to 20% by mass.

前記抗ＰＣＢ抗体産生ハイブリドーマのスクリーニングに用いられるセンサとしては、ＳＰＲ（表面プラズモン）センサ、ＱＣＭ（水晶振動子）センサ、光学的センサ、及び電気化学的センサ等のトランスデューサーのセンサ官能部へ、前記一般式（Ｉ）で表されるビフェニル誘導体、又は前記一般式（II）で表されるビフェニル誘導体とキャリアータンパク質とのコンジュゲートを固定化したものが挙げられる。
また、予め前記融合細胞の培養上清にＰＣＢ等を添加して反応させたものを、前記センサ官能部に接触させてスクリーニングを行うこともできる。この場合、前記官能部に結合した抗体量を評価することにより、種々のトランスデューサーでのＰＣＢ測定への適用が可能な、高親和性を有する抗ＰＣＢ抗体産生ハイブリドーマを選抜することができる。 As a sensor used for screening of the anti-PCB antibody-producing hybridoma, the sensor functional part of a transducer such as an SPR (surface plasmon) sensor, a QCM (quartz crystal) sensor, an optical sensor, and an electrochemical sensor, Examples include a biphenyl derivative represented by the general formula (I) or a conjugate of a biphenyl derivative represented by the general formula (II) and a carrier protein.
Screening can also be carried out by bringing PCBs or the like added to the culture supernatant of the fusion cells and reacting them in advance with the sensor functional part. In this case, an anti-PCB antibody-producing hybridoma having high affinity that can be applied to PCB measurement using various transducers can be selected by evaluating the amount of the antibody bound to the functional part.

前記抗ＰＣＢ抗体産生ハイブリドーマとしては、ＰＣＢを認識する本発明の抗ＰＣＢモノクローナル抗体を産生する限り、特に限定されないが、上記の方法により作製され、樹立されたものが好ましく、具体的にはＰＣＢ−Ｋ２Ａ−０株が好ましい。
前記ＰＣＢ−Ｋ２Ａ−０株は、下記一般式（II）に示す化合物を用いて作製されたハイブリドーマであり、独立行政法人産業技術総合研究所 特許生物寄託センターに平成１８年３月１６日付で、受託番号ＦＥＲＭ Ｐ−２０８４５として寄託されている。 The anti-PCB antibody-producing hybridoma is not particularly limited as long as it produces the anti-PCB monoclonal antibody of the present invention that recognizes PCB, but is preferably prepared and established by the above method. Specifically, PCB- The K2A-0 strain is preferred.
The PCB-K2A-0 strain is a hybridoma produced by using a compound represented by the following general formula (II), and is dated March 16, 2006 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. Deposited under the deposit number FERM P-20845.

前記抗ＰＣＢモノクローナル抗体は、例えば、前記抗ＰＣＢ抗体産生ハイブリドーマ細胞株を、予めプリスタン（２，６，１０，１４−テトラメチルペンタデカン）を投与したマウスの腹腔内に移植して培養し、１０〜１４日後に抗ＰＣＢモノクローナル抗体を含む腹水を回収し、得られた腹水から得ることができる。
また、前記ハイブリドーマを、培地（例えば、１０％ウシ胎児血清を含むＤＭＥＭ等）を用いて培養し、得られた培養液の上清を、抗ＰＣＢモノクローナル抗体溶液とすることができる。
前記腹水、又はウシ胎児血清不含培地より得た前記抗ＰＣＢ抗体産生ハイブリドーマ細胞株培養液上清から、前記抗ＰＣＢモノクローナル抗体を精製する方法としては、特に制限はなく、目的に応じて公知の方法から適宜選択することができ、例えば、限外ろ過、塩析、イオン交換クロマトグラフィー、ゲルろ過クロマトグラフィー、アフィニティクロマトグラフィー等により精製することができる。 The anti-PCB monoclonal antibody is cultured, for example, by transplanting the anti-PCB antibody-producing hybridoma cell line into the abdominal cavity of a mouse previously administered with pristane (2,6,10,14-tetramethylpentadecane), After 14 days, ascites containing the anti-PCB monoclonal antibody can be collected and obtained from the obtained ascites.
In addition, the hybridoma can be cultured using a medium (for example, DMEM containing 10% fetal bovine serum), and the supernatant of the obtained culture solution can be used as an anti-PCB monoclonal antibody solution.
The method for purifying the anti-PCB monoclonal antibody from the culture supernatant of the anti-PCB antibody-producing hybridoma cell line obtained from the ascites or fetal bovine serum-free medium is not particularly limited and is known according to the purpose. It can select suitably from the method, For example, it can refine | purify by ultrafiltration, salting-out, ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc.

＜抗ＰＣＢモノクローナル抗体の評価＞
本発明の抗ＰＣＢモノクローナル抗体は、前記本発明の抗ＰＣＢモノクローナル抗体の製造方法により製造され、塩素数の異なる二種以上のＰＣＢ同族体に交差反応性を有する。また、前記抗ＰＣＢモノクローナル抗体は、１〜１０塩素化ＰＣＢ全同族体のうち、３〜６塩素化ＰＣＢ同族体に対する親和性が高いことが好ましい。 <Evaluation of anti-PCB monoclonal antibody>
The anti-PCB monoclonal antibody of the present invention is produced by the above-described method for producing an anti-PCB monoclonal antibody of the present invention, and has cross-reactivity with two or more kinds of PCB homologs having different chlorine numbers. Moreover, it is preferable that the said anti- PCB monoclonal antibody has high affinity with respect to 3-6 chlorinated PCB homologs among 1-10 chlorinated PCB all homologues.

前記抗ＰＣＢモノクローナル抗体の親和性は、例えば、前記抗ＰＣＢモノクローナル抗体のＰＣＢとの結合を５０％阻害する抗原濃度（ＩＣ５０値）により評価することができるため、前記交差反応性は、例えば、塩素数の異なる二種以上のＰＣＢ同族体について、それぞれのＩＣ５０値を求め、それらの比を求めることにより評価することができる。 The affinity of the anti-PCB monoclonal antibody can be evaluated by, for example, the antigen concentration (IC 50 value) that inhibits the binding of the anti-PCB monoclonal antibody to PCB by 50%. Two or more kinds of PCB homologues having different chlorine numbers can be evaluated by obtaining respective IC 50 values and obtaining their ratios.

前記ＩＣ５０値の算出方法としては、特に制限はなく、目的に応じて適宜選択することができるが、例えば、蛍光光度計による測定データを、下記数式（１）で表される近似式に当てはめて求めることにより解析することができる。 The method for calculating the IC 50 value is not particularly limited and may be appropriately selected depending on the intended purpose. For example, measurement data obtained by a fluorometer is applied to an approximate expression represented by the following formula (1). It is possible to analyze by obtaining.

前記数式（１）中、ｙは、抗原を加えなかったときの蛍光強度の値を１００％とした時の相対的な蛍光強度を表し、ｘは、抗原濃度を表し、Ｐ１とＰ２は、近似のパラメータを表す。 In the formula (1), y represents relative fluorescence intensity when the value of fluorescence intensity when no antigen is added is 100%, x represents antigen concentration, and P1 and P2 are approximate. Represents the parameters.

測定データは、抗原を加えなかったときの蛍光強度の値を１００％として相対値に変換した後、グラフ作成ソフトウェア（例えば、Ｏｒｉｇｉｎ ｖｅｒｓｉｏｎ ６．０（ＯｒｉｇｉｎＬａｂ製）等）を用いて最適なＰ１及びＰ２を決定する。
このようにして得られた近似式を、前記抗体と前記抗原との結合曲線とし、ｙ＝５０％となるときのｘの値(＝Ｐ１)をＩＣ５０値とする。
なお、ＩＣ５０値に対して抗体濃度が十分に小さい時（例えば、１０分の１以下の時）、ＩＣ５０値と平衡解離定数（Ｋｄ）がほぼ一致することが知られている。 The measurement data is converted into a relative value with the fluorescence intensity value when no antigen is added as 100%, and then the optimum P1 and P1 are obtained using graphing software (for example, Origin version 6.0 (manufactured by OriginLab)). P2 is determined.
The approximate expression obtained in this way is the binding curve between the antibody and the antigen, and the value of x (= P1) when y = 50% is the IC 50 value.
It is known that when the antibody concentration is sufficiently small with respect to the IC 50 value (for example, 1/10 or less), the IC 50 value and the equilibrium dissociation constant (Kd) are almost the same.

前記抗ＰＣＢモノクローナル抗体の塩素数の異なるＰＣＢに対する特異性は、例えば、カネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、ＫＣ−６００）を被測定試料として用い、評価することができる
前記カネクロールは、それぞれ下記表１に示す比率で異なる塩素数のＰＣＢを含有しているため、例えば、本発明の抗ＰＣＢモノクローナル抗体は、塩素数の異なる二種以上のＰＣＢ同族体に交差反応性を有し、１〜１０塩素化ＰＣＢ全同族体のうち、３〜６塩素化ＰＣＢ同族体に高い親和性を有するため、ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、及びＫＣ−６００のいずれに対して最も高い特異性を示す。 The specificity of the anti-PCB monoclonal antibody for PCBs having different chlorine numbers can be evaluated using, for example, Kanechlor (KC-300, KC-400, KC-500, KC-600) as a sample to be measured. Since Kanechlor contains PCBs having different chlorine numbers in the ratios shown in Table 1 below, for example, the anti-PCB monoclonal antibody of the present invention is cross-reactive with two or more kinds of PCB homologs having different chlorine numbers. Among all the homologues of 1-10 chlorinated PCBs, it has a high affinity for 3-6 chlorinated PCB homologues, so any of KC-300, KC-400, KC-500, and KC-600 Shows the highest specificity for.

（出典：高菅卓三ら、「各種クリーンアップ法とＨＲＧＣ／ＨＲＭＳを用いたポリ塩化ビフェニル（ＰＣＢｓ）の全異性体詳細分析方法」，環境化学，Ｖｏｌ．５，Ｎｏ．３，ｐｐ．６４７−６７５，１９９５） (Source: Takuzo Takatsuki et al., “Detailed analysis method of all isomers of polychlorinated biphenyls (PCBs) using various cleanup methods and HRGC / HRMS”, Environmental Chemistry, Vol. 5, No. 3, pp. 647- 675, 1995)

（ＰＣＢ測定方法）
本発明のＰＣＢ測定方法は、本発明の抗ＰＣＢモノクローナル抗体を用いる方法であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記抗ＰＣＢモノクローナル抗体を用い、試料中のＰＣＢを免疫学的に検出、定量する方法、具体的には、ラジオイムノアッセイ（ＲＩＡ）、エンザイムイムノアッセイ（ＥＩＡ、ＥＬＩＳＡ）、フルオロイムノアッセイ（ＦＩＡ）、免疫クロマトグラフィー、蛍光免疫測定法（ＦＩＡ）、化学発光酵素免疫測定法（ＣＬＥＩＡ）、免疫比濁法（ＴＩＡ）、ラテックス免疫比濁法（ＬＴＩＡ）、及び結合平行除外法等が挙げられる。 (PCB measurement method)
The PCB measurement method of the present invention is not particularly limited as long as it is a method using the anti-PCB monoclonal antibody of the present invention, and can be appropriately selected according to the purpose. For example, using the anti-PCB monoclonal antibody, For immunological detection and quantification of PCBs, specifically, radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), fluoroimmunoassay (FIA), immunochromatography, fluorescence immunoassay (FIA), Examples include chemiluminescent enzyme immunoassay (CLEIA), immunoturbidimetry (TIA), latex immunoturbidimetry (LTIA), and binding parallel exclusion method.

前記ＰＣＢ測定方法としては、競合法（間接競合法あるいは直接競合法）、結合平衡除外法等が挙げられ、具体的には、下記（Ｉ）〜（III）の方法が挙げられる。
（Ｉ）前記被測定試料に、本発明の抗ＰＣＢモノクローナル抗体を添加し、
（１）前記抗ＰＣＢモノクローナル抗体と結合した前記被測定試料中のＰＣＢ、及び
（２）前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢモノクローナル抗体
のいずれかを検出する方法を含むＰＣＢ測定方法。
（II）前記被測定試料に、本発明の抗ＰＣＢモノクローナル抗体、及び前記一般式（Ｉ）及び前記一般式（II）のいずれかで表される化合物をＰＣＢ競合物質として添加し、前記抗ＰＣＢモノクローナル抗体と結合した前記競合物質を検出することを含むＰＣＢ測定方法。
（III）前記被測定試料に、本発明の抗ＰＣＢモノクローナル抗体を添加し、前記一般式（II）で表される化合物をＰＣＢ競合物質として固定化した担体を用い、前記抗ＰＣＢモノクローナル抗体と前記被測定試料中のＰＣＢとを競争させ、前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢモノクローナル抗体を検出することを含むＰＣＢ測定方法。 Examples of the PCB measurement method include a competition method (indirect competition method or direct competition method), a binding equilibrium exclusion method, and the like, and specifically include the following methods (I) to (III).
(I) The anti-PCB monoclonal antibody of the present invention is added to the sample to be measured,
(1) PCB including a method for detecting any of PCB in the sample to be measured bound to the anti-PCB monoclonal antibody and (2) the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured Measuring method.
(II) The anti-PCB monoclonal antibody of the present invention and the compound represented by any one of the general formula (I) and the general formula (II) are added as a PCB competitor to the sample to be measured, and the anti-PCB A method for measuring PCB comprising detecting the competitor substance bound to a monoclonal antibody.
(III) The carrier to which the anti-PCB monoclonal antibody of the present invention is added to the sample to be measured and the compound represented by the general formula (II) is immobilized as a PCB competitor is used, and the anti-PCB monoclonal antibody and the A PCB measurement method comprising: competing with a PCB in a sample to be measured and detecting the anti-PCB monoclonal antibody not bound to the PCB in the sample to be measured.

以下、前記間接競合法及び前記結合平衡除外法の一例を説明する。
前記間接競合法及び前記結合平衡除外法において、担体に固定化する抗原（以下、「固定化抗原」という）としては、例えば、前記一般式（Ｉ）で表されるビフェニル誘導体を含む化合物、及び前記一般式（II）で表される化合物等が挙げられる。
前記間接競合法及び前記結合平衡除外法は、前記固定化抗原を前記担体上に固定化し、前記担体の前記固定化抗原が結合していない部分をブロッキング剤でブロッキングする。次いで、該担体に被測定試料と前記抗ＰＣＢモノクローナル抗体とを加え、前記固定化抗原と前記被測定試料中のＰＣＢとを前記抗ＰＣＢモノクローナル抗体に対して前記間接競合法においては競合反応させ、前記結合平衡除外法においては結合平衡除外化反応させる。
前記固定化担体と結合しなかった前記抗ＰＣＢモノクローナル抗体を洗浄除去した後、二次抗体として標識した抗体を加え、前記固定化抗原と結合した抗ＰＣＢモノクローナル抗体と結合させ、洗浄を行った後、前記標識の発色又は発光を測定する。また、二次抗体を使用せず、前記抗ＰＣＢモノクローナル抗体を直接標識して用いても良い。得られた吸光度や光強度について、試料を添加しない場合の値に対する減少率を求め、これを阻害率とし、既知の濃度のＰＣＢを添加したときの阻害率からあらかじめ求めておいた検量線を用い、前記試料中のＰＣＢ濃度を算出することができる。 Hereinafter, an example of the indirect competition method and the binding equilibrium exclusion method will be described.
In the indirect competition method and the binding equilibrium exclusion method, as an antigen to be immobilized on a carrier (hereinafter referred to as “immobilized antigen”), for example, a compound containing a biphenyl derivative represented by the general formula (I), and The compound etc. which are represented with the said general formula (II) are mentioned.
In the indirect competition method and the binding equilibrium exclusion method, the immobilized antigen is immobilized on the carrier, and a portion of the carrier to which the immobilized antigen is not bound is blocked with a blocking agent. Next, the sample to be measured and the anti-PCB monoclonal antibody are added to the carrier, and the immobilized antigen and PCB in the sample to be measured are subjected to a competitive reaction with the anti-PCB monoclonal antibody in the indirect competition method, In the binding equilibrium exclusion method, a binding equilibrium exclusion reaction is performed.
After washing and removing the anti-PCB monoclonal antibody that did not bind to the immobilized carrier, an antibody labeled as a secondary antibody was added, and the anti-PCB monoclonal antibody bound to the immobilized antigen was bound and washed. The color development or luminescence of the label is measured. Further, the anti-PCB monoclonal antibody may be directly labeled without using a secondary antibody. For the absorbance and light intensity obtained, the rate of decrease relative to the value when no sample was added was determined, and this was used as the inhibition rate, using a calibration curve obtained in advance from the inhibition rate when PCB of a known concentration was added. The PCB concentration in the sample can be calculated.

前記標識としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、酵素としてペルオキシダーゼを用い、基質に過酸化水素、発色剤にｏ−フェニレンジアミンやテトラメチルベンジジンを使用する場合、及び酵素としてアルカリフォスファターゼを用い、基質にｐ−ニトロフェニルリン酸を使用する場合には、発色を吸光度で測定することができる。一方、基質の反応生成物が蛍光である場合や、蛍光物質を用いて標識されている場合には、蛍光光度計等を用いて蛍光を測定することができる。また、化学発光物質を用いる場合には、発光を化学発光測定器等により測定することができ、金コロイドを用いる場合には、透過光度等を透過光量測定器等により測定することができる。   The label is not particularly limited and may be appropriately selected according to the purpose. For example, when peroxidase is used as an enzyme, hydrogen peroxide is used as a substrate, and o-phenylenediamine or tetramethylbenzidine is used as a color former. In addition, when alkaline phosphatase is used as an enzyme and p-nitrophenyl phosphate is used as a substrate, color development can be measured by absorbance. On the other hand, when the reaction product of the substrate is fluorescent or is labeled with a fluorescent substance, the fluorescence can be measured using a fluorometer or the like. When a chemiluminescent substance is used, the luminescence can be measured with a chemiluminescence measuring instrument or the like, and when using a gold colloid, the transmitted light intensity or the like can be measured with a transmitted light quantity measuring instrument or the like.

前記被測定試料としては、例えば、被験物質中のＰＣＢを、ジメチルスルホキシド（ＤＭＳＯ）やアルコール系溶媒等の極性溶媒で液−液抽出してなる試料、及び前記被験物質中のＰＣＢを極性溶媒で液−液抽出し、抽出された前記ＰＣＢを含む前記極性溶媒を吸着剤で固相抽出し、前記吸着剤に吸着した成分を所望の溶媒に転溶させてなる試料、並びに前記被験物質にノルマルヘキサン、シクロヘキサン、トルエン等の低極性溶媒を加え、前記被験物質中のＰＣＢを親水性溶媒で抽出してなる試料などが挙げられる。
前記液−液抽出において、前記被験物質と前記極性溶媒との混合比（質量比）としては、例えば、（前記被験物質）：（前記極性溶媒）＝１：１〜：１：１０が好ましい。 Examples of the sample to be measured include a sample obtained by liquid-liquid extraction of PCB in a test substance with a polar solvent such as dimethyl sulfoxide (DMSO) or an alcohol solvent, and PCB in the test substance with a polar solvent. Liquid-liquid extraction, the sample obtained by solid-phase extraction of the polar solvent containing the extracted PCB with an adsorbent, and the components adsorbed on the adsorbent transferred to a desired solvent, and the test substance in normal Examples include a sample obtained by adding a low-polarity solvent such as hexane, cyclohexane, and toluene and extracting PCB in the test substance with a hydrophilic solvent.
In the liquid-liquid extraction, the mixing ratio (mass ratio) between the test substance and the polar solvent is preferably, for example, (the test substance) :( the polar solvent) = 1: 1 to 1:10.

前記被験物質としては、少なくともＰＣＢを含む可能性がある物質（ＰＣＢ汚染物）であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、絶縁油、絶縁油以外の油（例えば、植物油、動物油、合成油、鉱物油等）、工場排水、土壌、排ガス、動物の血液や体液、絶縁油の処理作業等において使用した部材等の廃棄物などが挙げられる。これらの中でも、絶縁油が好適に挙げられる。
前記絶縁油としては、脱塩素化処理等のＰＣＢ分解処理を行った後の絶縁油も好適に挙げられる。 The test substance is not particularly limited as long as it is a substance (PCB contaminant) that may contain at least PCB, and can be appropriately selected according to the purpose. For example, insulating oil, oil other than insulating oil (For example, vegetable oils, animal oils, synthetic oils, mineral oils, etc.), factory wastewater, soil, exhaust gas, animal blood and body fluids, wastes such as members used in the treatment of insulating oils, and the like. Among these, insulating oil is preferably exemplified.
As said insulating oil, the insulating oil after performing PCB decomposition processes, such as a dechlorination process, is also mentioned suitably.

前記被測定試料中の極性溶媒の濃度としては、前記抗ＰＣＢモノクローナル抗体のＰＣＢに対する前記親和性が著しく劣化しない限り、特に制限はなく、例えば、１〜２０質量％であることが好ましく、１０〜２０質量％であることがより好ましい。
なお、本発明の抗ＰＣＢモノクローナル抗体は、２０質量％の極性溶媒を含む被測定試料溶液中において、ＰＣＢｓに対し７０〜９０％の親和性を有する。 The concentration of the polar solvent in the sample to be measured is not particularly limited as long as the affinity of the anti-PCB monoclonal antibody for PCB is not significantly deteriorated. For example, it is preferably 1 to 20% by mass, More preferably, it is 20 mass%.
The anti-PCB monoclonal antibody of the present invention has an affinity of 70 to 90% for PCBs in a sample solution to be measured containing 20% by mass of a polar solvent.

本発明の抗ＰＣＢモノクローナル抗体は、カネクロールやアロクロールに含まれる複数の塩素が置換されたＰＣＢに対し親和性を有するため、環境試料中、工業製品中、生物試料中に含まれるＰＣＢの迅速かつ再現性の良い分析が可能となり、前記抗ＰＣＢモノクローナル抗体を用いたＰＣＢ分析方法は、ＰＣＢｓの迅速なモニタリングや分析技術として好適である。
また、ＳＰＲ（表面プラズモン）センサ、ＱＣＭ（水晶振動子）センサ、光学的センサ、および電気化学的センサ等のトランスデューサーを使用した系において、センサ官能部へ、本発明の抗ＰＣＢモノクローナル抗体、又は前記ビフェニル誘導体等を固定化して、免疫センサとして使用することができる。
さらに、本発明の抗ＰＣＢモノクローナル抗体を固定化した担体は、ＰＣＢの効率的濃縮や除去に適用することができる。 The anti-PCB monoclonal antibody of the present invention has an affinity for PCBs in which a plurality of chlorines contained in Kanechlor and Aroclor are substituted, so that PCBs contained in environmental samples, industrial products, and biological samples can be rapidly and Analysis with good reproducibility becomes possible, and the PCB analysis method using the anti-PCB monoclonal antibody is suitable as a rapid monitoring and analysis technique for PCBs.
Further, in a system using transducers such as an SPR (surface plasmon) sensor, a QCM (quartz crystal) sensor, an optical sensor, and an electrochemical sensor, the anti-PCB monoclonal antibody of the present invention or The biphenyl derivative or the like can be immobilized and used as an immunosensor.
Furthermore, the carrier on which the anti-PCB monoclonal antibody of the present invention is immobilized can be applied to the efficient concentration and removal of PCB.

以下、本発明の実施例について説明するが、本発明はこの実施例に何ら限定されるものではなく、本技術分野において行われるこれらに対する通常の改変及び修飾を含むものとする。   EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples, and includes ordinary alterations and modifications to these performed in the technical field.

（実施例１）
（１）ビフェニル誘導体の異性体混合物、及びコンジュゲートの調製
下記反応式に従い、３−フェニルフェノールに、スペーサー分子を導入したハプテンとしてのビフェニル誘導体（下記反応式中、化合物３）を調製し、さらにキャリアータンパク質と結合させてなるコンジュゲート（下記反応式中、化合物５）以下の反応式に従って合成した。 Example 1
(1) Preparation of isomer mixture of biphenyl derivative and conjugate According to the following reaction formula, a biphenyl derivative (compound 3 in the following reaction formula) as a hapten obtained by introducing a spacer molecule into 3-phenylphenol was prepared. A conjugate formed by binding to a carrier protein (compound 5 in the following reaction formula) was synthesized according to the following reaction formula.

３−フェニルフェノール５１１ｍｇ(３．０ｍｍｏｌ)に、トリフルオロ酢酸４ｍＬを加え、氷冷下、用事調製したＮ−クロロモルホリン１．２０ｇ(９．９０ｍｍｏｌ)を滴下した。同温にて３０分間撹拌した後、室温まで昇温し、さらに１時間撹拌した。その後、蒸留水を加え、水酸化ナトリウム水溶液で反応液を中和した。有機層をジクロロメタンで抽出し、有機層を飽和食塩水で洗浄後、溶媒を減圧下に留去した。残渣をシリカゲルカラムクロマトグラフィーで精製(ジクロロメタン：ヘキサン＝１:１０で溶出)し、塩素化異性体混合物として前記反応式中の化合物１を６１９ｍｇ得た。   To 511 mg (3.0 mmol) of 3-phenylphenol, 4 mL of trifluoroacetic acid was added, and 1.20 g (9.90 mmol) of N-chloromorpholine prepared for use under ice cooling was added dropwise. After stirring at the same temperature for 30 minutes, the temperature was raised to room temperature and further stirred for 1 hour. Thereafter, distilled water was added, and the reaction solution was neutralized with an aqueous sodium hydroxide solution. The organic layer was extracted with dichloromethane, and the organic layer was washed with saturated brine, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluted with dichloromethane: hexane = 1: 10) to obtain 619 mg of Compound 1 in the above reaction formula as a chlorinated isomer mixture.

前記化合物１ ５１５ｍｇを無水Ｎ，Ｎ−ジメチルホルムアミドに溶解させ、炭酸カリウム３９０ｍｇ(２．８２ｍｍｏｌ)、次いで６−ブロモへキサン酸エチルエステル６３０ｍｇ(２．８２ｍｍｏｌ)を加え、６０℃に加温し、１７時間撹拌した。反応後、室温まで放冷した後、蒸留水を加え、有機層をジクロロメタンで抽出し、飽和食塩水で洗浄後、溶媒を減圧下に留去し、塩素化異性体混合物として前記反応式中の化合物２を得た。前記化合物２は、これ以上精製せずに次の反応に用いた。   515 mg of the compound 1 was dissolved in anhydrous N, N-dimethylformamide, 390 mg (2.82 mmol) of potassium carbonate and then 630 mg (2.82 mmol) of 6-bromohexanoic acid ethyl ester were added, and the mixture was heated to 60 ° C., Stir for 17 hours. After the reaction, the mixture was allowed to cool to room temperature, distilled water was added, the organic layer was extracted with dichloromethane, washed with saturated brine, and the solvent was distilled off under reduced pressure to give a chlorinated isomer mixture in the above reaction formula. Compound 2 was obtained. Compound 2 was used in the next reaction without further purification.

前記化合物２を、ジオキサン１０ｍＬ、メタノール１２．５ｍＬに溶解させ、１０Ｎ水酸化ナトリウム水溶液２．５ｍＬを加え、室温にて２時間撹拌した。反応後、６Ｎ塩酸で反応液を弱酸性にし、有機層をジクロロメタンで抽出した。飽和食塩水で洗浄した後、溶媒を減圧下に留去した。残渣をシリカゲルカラムクロマトグラフィーで精製(ジクロロメタンで溶出)し、塩素化異性体混合物として前記反応式中の化合物３を４８４ｍｇ得た。   The compound 2 was dissolved in 10 mL of dioxane and 12.5 mL of methanol, 2.5 mL of 10N aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for 2 hours. After the reaction, the reaction solution was made weakly acidic with 6N hydrochloric acid, and the organic layer was extracted with dichloromethane. After washing with saturated brine, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluted with dichloromethane) to obtain 484 mg of compound 3 in the above reaction formula as a chlorinated isomer mixture.

次に、前記化合物３ ３８４ｍｇ、及びＮ-ヒドロキシスクシンイミド１３７ｍｇ(１．１９ｍｍｏｌ)を、無水Ｎ，Ｎ−ジメチルホルムアミド２ｍＬに溶解した。この反応液にトリエチルアミン２０７μＬ(１．４９ｍｍｏｌ)、１−エチル−３−(３−ジメチルアミノプロピル)−カルボジイミド塩酸塩２８５ｍｇ(１．４９ｍｍｏｌ)を加え、室温で１４時間撹拌した。反応後、蒸留水を加え、有機層をジクロロメタンで抽出した。有機層を飽和食塩水で洗浄した後、溶媒を減圧下に留去した。残渣をシリカゲルカラムクロマトグラフィーで精製(ジクロロメタンで溶出)し、塩素化異性体混合物として、前記反応式中の化合物４を３６１ｍｇ得た。   Next, 384 mg of the compound 3 and 137 mg (1.19 mmol) of N-hydroxysuccinimide were dissolved in 2 mL of anhydrous N, N-dimethylformamide. To this reaction solution, 207 μL (1.49 mmol) of triethylamine and 285 mg (1.49 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride were added and stirred at room temperature for 14 hours. After the reaction, distilled water was added and the organic layer was extracted with dichloromethane. The organic layer was washed with saturated brine, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluted with dichloromethane) to obtain 361 mg of Compound 4 in the reaction formula as a chlorinated isomer mixture.

前記化合物４を、ジメチルスルホキシド(ＤＭＳＯ)に溶解し、５０ｍＭリン酸緩衝液（ｐＨ８．０）に溶解したＢＳＡ溶液、又はＫＬＨ溶液に氷冷撹拌下に滴下し、室温にて一晩撹拌した。反応液をＰＢＳ(−)で平衡化したセファデックスカラムに添加し、同緩衝液で展開して、キャリアータンパク質を分離し、コンジュゲート（前記反応式中の化合物５）を精製した。   The compound 4 was dissolved in dimethyl sulfoxide (DMSO) and added dropwise to a BSA solution or KLH solution dissolved in 50 mM phosphate buffer (pH 8.0) under ice-cooling and stirring at room temperature overnight. The reaction solution was added to a Sephadex column equilibrated with PBS (−) and developed with the same buffer to separate the carrier protein, and the conjugate (compound 5 in the above reaction formula) was purified.

（２）マウスの免疫
得られたコンジュゲートを免疫原として、マウスを免疫した。
前記化合物５と、アジュバントＲＡＳ Ｒ−７００(ＲＩＢＩ社)とを１：１容量に混合して十分に乳化させ、得られた乳化液をＢＡＬＢ／ｃマウス（７〜８週齢、雌）の腹腔内に２００μＬ投与する事によりマウスを免疫感作した。
追加免疫は、約２〜３週間間隔で行い、追加免疫から１週間経過後に尾静脈より採血し、ＥＬＩＳＡ法により血中抗体価を測定して抗体価の推移を観察した。 (2) Immunization of mice Mice were immunized using the obtained conjugate as an immunogen.
The compound 5 and adjuvant RAS R-700 (RIBI) were mixed in a 1: 1 volume and sufficiently emulsified, and the resulting emulsion was used for the abdominal cavity of BALB / c mice (7-8 weeks old, female). The mice were immunized by administration of 200 μL.
Booster immunization was performed at intervals of about 2 to 3 weeks, and blood was collected from the tail vein 1 week after booster immunization, and the antibody titer in blood was measured by ELISA to observe the change in antibody titer.

（３）ハイブリドーマの作製
血中に免疫原に対する高い抗体産生が認められたマウス尾静脈内に、さらに免疫原を投与して最終免疫を行った。最終免疫から３日後に、免疫した前記マウスより脾臓を摘出し、脾臓細胞を調製し、対数増殖期にあるミローマ細胞（Ｐ３−Ｘ６３−Ａｇ８．６５３あるいはＳｐ２／Ｏ）と脾臓細胞を１：５になるように混合し、ポリエチレングリコール（ＰＥＧ）法にて細胞融合を行った。融合後の細胞を、ＨＡＴ（ヒポキサンチン、アミノプテリンおよびチミジン）添加の１０％ＦＣＳ含有ＲＰＭＩ１６４０培地中に懸濁させ脾臓細胞数で１〜２×１０5細胞／ウエルになるように９６ウエル培養プレートに播種し、３７℃、５％ＣＯ2下で培養した。 (3) Preparation of hybridoma Final immunization was carried out by further administering the immunogen into the tail vein of mice in which high antibody production against the immunogen was observed in the blood. Three days after the final immunization, the spleen is removed from the immunized mouse, spleen cells are prepared, and myeloma cells (P3-X63-Ag8.653 or Sp2 / O) in the logarithmic growth phase and spleen cells are 1: 5. And cell fusion was performed by the polyethylene glycol (PEG) method. The cells after the fusion are suspended in RPMI 1640 medium containing 10% FCS supplemented with HAT (hypoxanthine, aminopterin and thymidine), and 96-well culture plate so that the number of spleen cells is 1-2 × 10 5 cells / well. And cultured at 37 ° C. under 5% CO 2.

（４）抗体産生ハイブリドーマのスクリーニングおよびクローニング
細胞融合より７〜１０日後に、クローンの増殖が見られたウエルの培養上清を用いてＥＬＩＳＡ法にて抗体価を測定した。 (4) Screening and cloning of antibody-producing hybridomas 7 to 10 days after cell fusion, antibody titers were measured by ELISA using the culture supernatant of wells in which clone growth was observed.

（５）抗体産生ハイブリドーマのスクリーング（抗体価の測定）
マイクロタイタープレート（ＣＯＲＮＩＮＧ社製）の各ウエルに、０．２５〜１μｇ／ｍＬの各免疫原のＰＢＳ(−)希釈溶液（５０μＬ）を加え、室温で１時間静置して固相化した。また、前記コンジュゲート中のキャリアータンパク質に対する抗体産生クローンを除外する為に、ＢＳＡ、又はＫＬＨを固定化したウエルを対照として用いた。
０．０５％Ｔｗｅｅｎ ２０を含むＰＢＳ(−)（洗浄液）３００μＬで各ウエルを３回洗浄した後、ブロックエース粉末（雪印社製、ＵＫ−Ｂ８０）４ｇを１００ｍＬの精製水にて溶解させたのち精製水で４倍希釈して調製した溶液（３００μＬ）をウエルに添加し、室温で２時間静置してブロッキングを行った。ウエルを洗浄液（３００μＬ）で３回洗浄後、２５μＬの２０％ＤＭＳＯ、０．０１％Ｔｉｔｏｎ−Ｘ１００、及び１次抗体を含む培養上清（２５μＬ）を各ウエルに添加して混和した後、室温条件下で１０〜３０分間静置して抗原抗体反応を行わせた。
次いで、洗浄液（３００μＬ）で各ウエルを３回洗浄した後、固相化した各免疫原に結合した１次抗体を検出するために、精製水で１０倍希釈したブロックエース溶液にて３０００倍希釈に調製した西洋ワサビペルオキシダーゼ標識抗マウスＩｇＧ（γ鎖認識）（ＫＰＬ社製）を２次抗体溶液として加えて（５０μＬ）、室温で１時間反応させた。ウエルを洗浄液３００μＬで同様に３回洗浄した後、３，３'，５，５'−テトラメチルベンジジン（ＴＭＢ）溶液（ＫＰＬ社製）を各ウエルに加え、室温条件下にて発色反応を行った。
５〜１５分間後に１Ｍリン酸５０μＬを加えて反応停止し、マイクロプレートリーダー（マルチスキャンＭＳ、Ｌａｂｓｙｓｔｅｍｓ社製）にて直ちに５４０ｎｍの吸光度を測定した。
各免疫原を固定化したウエルと対照ウエルとの吸光度差を、前記免疫原に対する抗体の結合活性とした。 (5) Screening of antibody-producing hybridoma (measurement of antibody titer)
To each well of a microtiter plate (CORNING), 0.25 to 1 μg / mL of each immunogen in PBS (−) diluted solution (50 μL) was added and allowed to stand at room temperature for 1 hour to be immobilized. Further, in order to exclude antibody-producing clones against the carrier protein in the conjugate, wells immobilized with BSA or KLH were used as controls.
Each well was washed three times with 300 μL of PBS (−) (washing solution) containing 0.05% Tween 20 and 4 g of Block Ace powder (Yuki-Ink, UK-B80) was dissolved in 100 mL of purified water. A solution prepared by diluting 4 times with purified water (300 μL) was added to the wells and allowed to stand at room temperature for 2 hours for blocking. After the wells were washed three times with a washing solution (300 μL), 25 μL of 20% DMSO, 0.01% Titon-X100, and a culture supernatant (25 μL) containing the primary antibody were added to each well, mixed, and then mixed at room temperature. The antigen-antibody reaction was allowed to stand for 10 to 30 minutes under the conditions.
Next, each well was washed three times with a washing solution (300 μL), and then diluted 3000 times with a Block Ace solution diluted 10 times with purified water in order to detect the primary antibody bound to each immobilized immunogen. Horseradish peroxidase-labeled anti-mouse IgG (γ chain recognition) (manufactured by KPL) was added as a secondary antibody solution (50 μL) and reacted at room temperature for 1 hour. After the wells were washed three times with 300 μL of the washing solution in the same manner, 3,3 ′, 5,5′-tetramethylbenzidine (TMB) solution (manufactured by KPL) was added to each well, and a color reaction was performed at room temperature. It was.
After 5 to 15 minutes, 50 μL of 1M phosphoric acid was added to stop the reaction, and the absorbance at 540 nm was immediately measured with a microplate reader (Multiscan MS, manufactured by Labsystems).
The difference in absorbance between the well in which each immunogen was immobilized and the control well was defined as the binding activity of the antibody to the immunogen.

（６）間接競合法による特異抗体産生クローンの選抜
前記（５）に従い、マイクロタイタープレートの各ウエルに各免疫原を固相化し、洗浄ののち精製水で４倍希釈したブロックエース溶液３００μＬをウエルに添加し、室温で２時間静置してブロッキングを行った。ウエルを洗浄液３００μＬで３回洗浄した。
次いで、いずれも溶媒終濃度が０．０１％Ｔｉｔｏｎ−Ｘ１００含有２０％ＤＭＳＯとなるように調製した(i)各免疫原、(ii)個々のＰＣＢ同族体、(iii)４種類のカネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、ＫＣ−６００）、(iv)４種類のカネクロールの等量混合物（ＫＣ−Ｍｉｘ）、及び（ｖ）対照（２０％ＤＭＳＯ及び０．０１％Ｔｉｔｏｎ−Ｘ１００）を用意し、各々２５μＬを、免疫原固相化プレートの各ウエルに加えた。
次に、ハイブドーマ培養上清（２５μＬ）ウエル内に添加して混和した後、室温条件下で１０〜３０分間静置して抗原抗体反応を行わせた。
洗浄液３００μＬで各ウエルを３回洗浄した後、固相化免疫原に結合した１次抗体を検出するために精製水で１０倍希釈したブロックエースにて３０００倍希釈に調製した西洋ワサビペルオキシダーゼ標識抗マウスＩｇＧ（γ鎖認識）を２次抗体溶液として５０μＬ加え、室温条件下で１時間反応させた。
ウエルを洗浄液３００μＬで同様に３回洗浄したのち、ＴＭＢ溶液を各ウエルに加えて室温で発色反応を行った。５〜１５分間後に１Ｍリン酸５０μＬを加えて反応停止し、マイクロプレートリーダー（マルチスキャンＭＳ、Ｌａｂｓｙｓｔｅｍｓ社製）にて直ちに４５０ｎｍの吸光度を測定した。対照ウエルの吸光度値（コントロールＯＤ；Ｂ０）に対するＰＣＢ、カネクロール等共存下のウエル吸光度値(Ｂ)の割合（Ｂ／Ｂ０）を算出し、共存物質に対する抗体の反応性を判断した。ＰＣＢに対する結合活性が高く、かつ継続的に高い抗体産生が確認されたウエルのハイブリドーマを選抜し、限界希釈法によるクローニングに供した。 (6) Selection of specific antibody-producing clones by indirect competition method According to (5) above, each immunogen is solid-phased in each well of a microtiter plate, and after washing, 300 μL of a block ace solution diluted 4-fold with purified water is added to the well. And allowed to stand at room temperature for 2 hours for blocking. The wells were washed 3 times with 300 μL of washing solution.
Subsequently, all were prepared so that the final concentration of the solvent was 20% DMSO containing 0.01% Titon-X100. (I) Each immunogen, (ii) Individual PCB congeners, (iii) 4 types of Kanechlor ( Kiv-300, KC-400, KC-500, KC-600), (iv) an equal mixture of four types of kanechlor (KC-Mix), and (v) a control (20% DMSO and 0.01% Titon) -X100) and 25 μL of each was added to each well of the immunogen-immobilized plate.
Next, after adding and mixing in a hybridoma culture supernatant (25 microliters) well, it left still for 10 to 30 minutes under room temperature conditions, and performed the antigen antibody reaction.
Each well was washed 3 times with 300 μL of washing solution, and then a horseradish peroxidase-labeled anti-antigen labeled with 3000 fold diluted with Block Ace diluted 10 times with purified water to detect the primary antibody bound to the immobilized immunogen. 50 μL of mouse IgG (γ chain recognition) was added as a secondary antibody solution and reacted at room temperature for 1 hour.
After the wells were washed three times with 300 μL of the washing solution in the same manner, a TMB solution was added to each well and a color reaction was performed at room temperature. After 5 to 15 minutes, 50 μL of 1M phosphoric acid was added to stop the reaction, and the absorbance at 450 nm was immediately measured with a microplate reader (Multiscan MS, manufactured by Labsystems). The ratio (B / B0) of the well absorbance value (B) in the coexistence of PCB, kanechlor, etc. to the absorbance value of the control well (control OD; B0) was calculated, and the reactivity of the antibody to the coexisting substance was judged. Hybridomas in wells that had high binding activity to PCB and were confirmed to have high antibody production continuously were selected and subjected to cloning by limiting dilution.

（７）トランスデューサーへの適応性可能性を有するクローンの選抜
前記（６）にて選抜した前記ハイブリドーマのうち、１回以上クローニングを実施したハイブリドーマを、更に蛍光分析に基づくイムノセンサ（ＤＸＳ−６００）を用いて選抜を行った。
拡大培養したクローン培養上清を、予め５％ＤＭＳＯ，０．１ｗ／ｖ％ＢＳＡ含有ＰＢＳ(−)の溶媒中でＣｙ５標識抗マウスＩｇＧ（Ｊａｃｋｓｏｎ社製）と室温条件下で２時間以上反応させたのち、４〜８００倍の範囲に希釈し、トランスデューサーに供した。
センサ官能部に固定化した前記化合物５に対し、Ｃｙ５で標識された抗体を含むハイブリドーマ培養上清を０．７５ｍＬ／分の流速で約０．５ｍＬ通過させた。
非特異的に結合した抗体や夾雑物質を５％ＤＭＳＯ，０．１ｗ／ｖ％ＢＳＡ含有ＰＢＳ(−)溶液にて洗浄後、得られたＣｙ５由来の蛍光強度をセンサ官能部に結合した抗体量として検出した。
さらに、ＰＣＢ共存下にクローン培養上清をＣｙ５標識抗マウスＩｇＧと反応させた後、同様に前記センサ官能部へ送液した場合に検出される蛍光強度を基に、ＰＣＢへの高親和性を示す抗体産生クローンを取得した。 (7) Selection of clones having adaptability to transducers Among the hybridomas selected in (6) above, hybridomas that have been cloned one or more times are further subjected to immunosensor (DXS-600) based on fluorescence analysis. ) Was used for selection.
The expanded culture supernatant of the clone was reacted with Cy5-labeled anti-mouse IgG (Jackson) for 2 hours or more in advance in a solvent of PBS (−) containing 5% DMSO and 0.1 w / v% BSA in advance. After that, it was diluted to a range of 4 to 800 times and used for a transducer.
About 0.5 mL of the hybridoma culture supernatant containing the antibody labeled with Cy5 was passed through the compound 5 immobilized on the sensor functional part at a flow rate of 0.75 mL / min.
Amount of antibody that binds non-specifically bound antibody or contaminants with 5% DMSO, 0.1 w / v% BSA-containing PBS (-) solution, and then binds Cy5-derived fluorescence intensity to the sensor functional part Detected as.
Furthermore, after reacting the clonal culture supernatant with Cy5-labeled anti-mouse IgG in the presence of PCB, the high affinity for PCB is obtained based on the fluorescence intensity detected when the solution is similarly sent to the sensor functional part. The indicated antibody producing clones were obtained.

以上の方法により、ＰＣＢに高い反応性を示す抗ＰＣＢモノクローナル抗体を産生するハイブリドーマを樹立した。該ハイブリドーマＰＣＢ−Ｋ２Ａ−０は、平成１８年３月１６日に、独立行政法人産業技術総合研究所 特許生物寄託センターに寄託され、受託番号としてＦＥＲＭ Ｐ−２０８４５を取得している。   By the above method, a hybridoma producing an anti-PCB monoclonal antibody having high reactivity with PCB was established. The hybridoma PCB-K2A-0 was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on March 16, 2006, and has acquired FERM P-20845 as a deposit number.

（８）モノクローナル抗体の大量調製と精製
８週齢を超えるＢＡＬＢ／ｃマウスの腹腔内へ、プリスタン［２，６，１０，１４−テトラメチルペンタデカン（和光純薬）］（０．５ｍＬ）を投与し、２〜３週間飼育した。予め、対数増殖期に維持しておいた抗ＰＣＢモノクローナル抗体産生ハイブリドーマＰＣＢ−Ｋ２Ａ−０の培養液を回収し、遠心分離にて培養上清を除いたのちＦＣＳ不含のＲＰＭＩ１６４０にて沈査を１×１０６細胞／０．５ｍＬになるよう細胞液を調製した。
この細胞液をプリスタン前投与のＢＡＬＢ／ｃマウス腹腔中に移植し、１０〜１４日後に腹腔内に漏出した腹水を腹部より２５Ｇのシリンジを装着した注射器で回収した。
採取した腹水を、ポアサイズ０．２２μｍ径のフィルターを用いてろ過後、得られたろ液をプロテインＧ−セファロースカラム（Ａｍｅｒｓｈａｍ Ｂｉｏｎｓｉｅｎｓ社製）を用いたアフィニティクロマトグラフィーによって精製し、前記抗ＰＣＢモノクローナル抗体を得た。 (8) Large-scale preparation and purification of monoclonal antibody Pristan [2,6,10,14-tetramethylpentadecane (Wako Pure Chemicals)] (0.5 mL) was administered into the peritoneal cavity of BALB / c mice over 8 weeks of age. And raised for 2-3 weeks. The culture solution of the anti-PCB monoclonal antibody-producing hybridoma PCB-K2A-0 previously maintained in the logarithmic growth phase was collected, the culture supernatant was removed by centrifugation, and then the precipitate was examined with RPMI1640 without FCS. A cell solution was prepared so as to be × 10 6 cells / 0.5 mL.
This cell solution was transplanted into the abdominal cavity of BALB / c mice pre-administered with pristane, and ascites leaked into the abdominal cavity after 10 to 14 days was collected from the abdomen with a syringe equipped with a 25G syringe.
The collected ascites was filtered using a filter having a pore size of 0.22 μm, and the obtained filtrate was purified by affinity chromatography using a protein G-Sepharose column (manufactured by Amersham Biosciences), and the anti-PCB monoclonal antibody was purified. Obtained.

＜抗ＰＣＢモノクローナル抗体の特性評価＞
（１）アイソタイプ分析
固相化抗原として下記構造式（II）で表される化合物を用い、マウスタイパーキット（ＢＩＯ−ＲＡＤ社製）を用いて前記抗ＰＣＢモノクローナル抗体のアイソタイプ分析を行った。
この結果、前記抗ＰＣＢモノクローナル抗体産生ハイブリドーマＰＣＢ−Ｋ２Ａ−０によって生産された前記抗ＰＣＢモノクローナル抗体のＨ鎖はＩｇＧ１、Ｌ鎖はκであった。 <Characteristic evaluation of anti-PCB monoclonal antibody>
(1) Isotype analysis The compound represented by the following structural formula (II) was used as a solid-phased antigen, and the anti-PCB monoclonal antibody was subjected to isotype analysis using a mouse typer kit (manufactured by BIO-RAD).
As a result, the H chain of the anti-PCB monoclonal antibody produced by the anti-PCB monoclonal antibody-producing hybridoma PCB-K2A-0 was IgG1, and the L chain was κ.

（２）間接競合ＥＬＩＳＡ法による標準曲線の作成
前記抗ＰＣＢモノクローナル抗体を用い、カネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、ＫＣ−６００、及びこれらの混合溶液）による阻害実験を実施した。
マイクロタイタープレートに前記一般式（II）で表される化合物を室温条件下にて１時間固相化後、洗浄液３００μＬで３回洗浄し、精製水で原液を４倍希釈して調製したブロックエース溶液３００μＬをウエルに添加し、室温条件下にて２時間ブロッキングを行った。
ウエルを３回洗浄後、測定試料としてＫＣ−３００、ＫＣ−４００、ＫＣ−５００、及びＫＣ−６００（ＧＬサイエンス）並びにこれらの混合溶液を、１００ｎｇ／ｍＬから３倍階段希釈を行い、種々の濃度のＫＣ溶液（溶媒：２０％ＤＭＳＯ、０．０１％Ｔｉｔｏｎ−Ｘ１００溶液）を調製した。
各々、前記抗ＰＣＢモノクローナル抗体（ＰＣＢ−Ｋ２Ａ−１）２５μＬと共にウエルに添加して混和し、室温条件下にて１０〜３０分間静置して抗原抗体反応を行った。
再びウエルを洗浄後、１０倍希釈ブロックエースにて３０００倍希釈に調製した西洋ワサビペルオキシダーゼ標識抗マウスＩｇＧ（γ鎖認識）を５０μＬ加え、室温条件下にて１時間反応させた。ウエルを洗浄したのち、ＴＭＢ溶液を加え、室温条件下にて発色反応を行い、５〜１５分間後に１Ｍリン酸５０μＬを加えて反応停止して、マイクロプレートリーダーにて直ちに４５０ｎｍの吸光度を測定した。
溶媒（２０％ＤＭＳＯ，０．０１％Ｔｉｔｏｎ−Ｘ１００溶液）のみを添加したウエルの吸光度をコントロールＯＤ（Ｂ０）とし、Ｂ０に対するカネクロール存在下のウエル吸光度値(Ｂ)のＢ／Ｂ０を算出した値を添加した試料濃度対してプロットし、典型的シグモイド曲線を得た。結果を図１に示す。 (2) Preparation of standard curve by indirect competitive ELISA method Using the anti-PCB monoclonal antibody, inhibition experiments with Kanechlor (KC-300, KC-400, KC-500, KC-600, and mixed solutions thereof) were performed. did.
Block Ace prepared by immobilizing the compound represented by the general formula (II) on a microtiter plate for 1 hour at room temperature, washing with 300 μL of a washing solution three times, and diluting the stock solution four times with purified water. 300 μL of the solution was added to the well, and blocking was performed for 2 hours at room temperature.
After washing the wells three times, KC-300, KC-400, KC-500, and KC-600 (GL Science) as a measurement sample and a mixed solution thereof were subjected to 3-fold serial dilution from 100 ng / mL to various samples. A concentration KC solution (solvent: 20% DMSO, 0.01% Titon-X100 solution) was prepared.
Each was added to and mixed with 25 μL of the anti-PCB monoclonal antibody (PCB-K2A-1) in a well, and allowed to stand at room temperature for 10 to 30 minutes to carry out an antigen-antibody reaction.
After washing the wells again, 50 μL of horseradish peroxidase-labeled anti-mouse IgG (γ chain recognition) prepared to be diluted 3000 times with 10-fold diluted Block Ace was added and reacted at room temperature for 1 hour. After washing the wells, a TMB solution was added and a color reaction was carried out at room temperature. After 5 to 15 minutes, 50 μL of 1M phosphoric acid was added to stop the reaction, and the absorbance at 450 nm was immediately measured with a microplate reader. .
The absorbance of the well to which only the solvent (20% DMSO, 0.01% Titon-X100 solution) was added was taken as the control OD (B0), and B / B0 of the well absorbance value (B) in the presence of Kanechlor relative to B0 was calculated. Values were plotted against added sample concentration to obtain a typical sigmoid curve. The results are shown in FIG.

該抗体は評価に使用した４種類のカネクロールのうち、ＫＣ−４００に最も強い反応性を示し、次いで、ＫＣ−３００、ＫＣ−５００、ＫＣ−６００の順で反応性を示した。反応性に差異はあったものの、いずれのカネクロールに対してもｐｐｂレベルの感度を有し、本発明の抗ＰＣＢモノクローナル抗体は、塩素数の異なる二種以上のＰＣＢ同族体に交差反応性を示すとともに、１〜１０塩素化ＰＣＢ全同族体のうち、存在比の高い３〜６塩素化ＰＣＢ同族体に高い親和性を有し、高い検出感度を示すことがわかった。また、ＥＬＩＳＡ測定系においてもＰＣＢ含有試料への適用性が示唆された。結果を表２に示す。   The antibody showed the strongest reactivity to KC-400 among the four types of canechlor used in the evaluation, and then showed the reactivity in the order of KC-300, KC-500, and KC-600. Although there is a difference in reactivity, it has ppb level sensitivity to any kanechlor, and the anti-PCB monoclonal antibody of the present invention exhibits cross-reactivity to two or more kinds of PCB homologs having different chlorine numbers. In addition, it was found that among all the homologues of 1 to 10 chlorinated PCBs, it has a high affinity for 3 to 6 chlorinated PCB analogues having a high abundance ratio and exhibits high detection sensitivity. In addition, applicability to PCB-containing samples was also suggested in the ELISA measurement system. The results are shown in Table 2.

（実施例２）
実施例１で得た抗ＰＣＢモノクローナル抗体について、蛍光分析に基づくイムノセンサ（京都電子工業社製、ＤＸＳ−６００、又はＳａｐｉｄｙｎｅ社製、ＥｎｄｏＢｉｏｓｅｎｓｏｒTM）を用いて親和性、交差反応性を評価した。 (Example 2)
The anti-PCB monoclonal antibody obtained in Example 1 was evaluated for affinity and cross-reactivity using an immunosensor based on fluorescence analysis (Kyoto Electronics Industry Co., Ltd., DXS-600, or Sapidyne Co., Ltd., EndoBiosensor ™ ).

N−ヒドロキシスクシンイミジルエステル基が導入されたアガロースビーズ（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅ社製）に、前記化合物５（ビフェニル誘導体とキャリアータンパク質とのコンジュゲート）を共有結合にて結合させ、ＢＳＡ溶液でブロッキングを行い、抗体結合用担体とした。
実施例１で得られた抗ＰＣＢモノクローナル抗体ＰＣＢ−Ｋ２Ａ−１を１ｍｇ／ｍＬの濃度に調製した後、ＣｙＴＭ５ｍＡｂ Ｌａｂｅｌｌｉｎｇ ｋｉｔ（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅ社製）によりＣｙ５を導入し、Ｃｙ５標識抗ＰＣＢモノクローナル抗体とした。
終濃度０．８ｎＭの前記Ｃｙ５標識抗ＰＣＢモノクローナル抗体を、終濃度０．０１〜５０ｎｇ／ｍＬになるように調製したカネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、ＫＣ−６００）（ＧＬサイエンス社製）の溶液とそれぞれ反応させたのち、直ちに、抗体結合担体へ流速０．７５ｍＬ／分で作用させた。
次いで、５％ＤＭＳＯ，０．１ｗ／ｖ％ＢＳＡ含有ＰＢＳ(−)を流速０．７５ｍＬ／分で通過させ、担体への非特異的結合を排除したのち蛍光強度を測定した。カネクロールを添加しない抗体溶液の示す蛍光量をコントロール（Ｂ０）値とし、Ｂ０値に対するカネクロール添加により低下した蛍光量(Ｂ)の割合（Ｂ／Ｂ０）を、カネクロール濃度に対しプロットしたところ、いずれのカネクロールに対しても、検出下限値(ＩＣ８５)は約５０〜６０ｐｐｔを示し、極めて高感度な抗体である事が確認された。
また、ＫＣ−４００に対する反応性が高い傾向が観察されたものの、ＫＣ−３００、ＫＣ４００、ＫＣ−５００、及びＫＣ−６００の阻害曲線はほぼ一致したことから、前記抗ＰＣＢモノクローナル抗体を用いることにより、４種類のカネクロールに対し、同程度の反応性を有するＰＣＢ測定系を構築できることがわかった。結果を図２に示す。 Compound 5 (conjugate of biphenyl derivative and carrier protein) is covalently bound to agarose beads (manufactured by Amersham Bioscience) into which an N-hydroxysuccinimidyl ester group has been introduced, and blocking is performed with a BSA solution. An antibody-binding carrier was obtained.
After preparing the anti-PCB monoclonal antibody PCB-K2A-1 obtained in Example 1 to a concentration of 1 mg / mL, Cy5 was introduced by Cy ™ 5 mAb Labeling kit (manufactured by Amersham Bioscience), and Cy5-labeled anti-PCB monoclonal antibody It was.
Kanechlor (KC-300, KC-400, KC-500, KC-600) prepared by adjusting the Cy5-labeled anti-PCB monoclonal antibody having a final concentration of 0.8 nM to a final concentration of 0.01 to 50 ng / mL ( Each was reacted with a solution (manufactured by GL Science) and immediately allowed to act on the antibody-binding carrier at a flow rate of 0.75 mL / min.
Subsequently, PBS (−) containing 5% DMSO and 0.1 w / v% BSA was passed at a flow rate of 0.75 mL / min to eliminate non-specific binding to the carrier, and then the fluorescence intensity was measured. When the amount of fluorescence exhibited by the antibody solution not added with canechlor was taken as the control (B0) value, the ratio (B / B0) of the amount of fluorescence (B) decreased by the addition of canechlor to the B0 value was plotted against the canechlor concentration. For any canechlor, the lower limit of detection (IC 85 ) was about 50-60 ppt, confirming that the antibody was extremely sensitive.
In addition, although a tendency toward high reactivity with KC-400 was observed, the inhibition curves of KC-300, KC400, KC-500, and KC-600 almost coincided, so that the anti-PCB monoclonal antibody was used. It was found that a PCB measurement system having the same degree of reactivity can be constructed for four types of kanechlor. The results are shown in FIG.

また、前記蛍光光度計により評価した抗ＰＣＢモノクローナル抗体ＰＣＢ−Ｋ２Ａ−１のカネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、及びＫＣ−６００）の交差反応性を表３に示す。   Table 3 shows the cross-reactivity of the anti-PCB monoclonal antibody PCB-K2A-1 kanechlor (KC-300, KC-400, KC-500, and KC-600) evaluated by the fluorometer.

本発明の抗ＰＣＢモノクローナル抗体は、極性溶媒存在下においてＰＣＢとの結合性が低下することなく、塩素数の異なる複数のＰＣＢ同族体の混合物、具体的には、油性試料中に含まれるカネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、ＫＣ−６００：鐘淵化学工業社製)、及びアロクロール（Ａｒｏｃｌｏｒ．１２４２、Ａｒｏｃｌｏｒ．１２４８、Ａｒｏｃｌｏｒ．１２５４、Ａｒｏｃｌｏｒ．１２６０：三菱モンサント（現三菱化学）製）中に存在比の高い複数のＰＣＢ同族体に交差反応性を有し、１〜１０塩素化ＰＣＢ全同族体のうち、存在比の高い３〜６塩素化ＰＣＢ同族体に高い親和性を有するため、組成が未知の絶縁油等、ＰＣＢを含有する可能性のある検査対象を、最小限の誤差で高感度に検出、定量することができる。
本発明の抗ＰＣＢモノクローナル抗体を用いたＰＣＢ測定方法は、廃棄物処理後のＰＣＢ残留濃度、環境中のＰＣＢ濃度等の検査に好適に使用することができる。 The anti-PCB monoclonal antibody of the present invention is a mixture of a plurality of PCB homologues having different chlorine numbers, specifically, Kanechlor contained in an oily sample, without reducing the binding property to PCB in the presence of a polar solvent. (KC-300, KC-400, KC-500, KC-600: Kaneka Chemical Industry Co., Ltd.), and Arochlor (Arochlor. 1242, Arochlor. 1248, Arolor. 1254, Arolor. 1260: Mitsubishi Monsanto (current Mitsubishi Chemical) ) Made in), a cross-reactivity to a plurality of PCB homologues with high abundance ratio, among 1-10 chlorinated PCB all homologues, high affinity to 3-6 chlorinated PCB homologues with high abundance ratio Therefore, it is possible to detect and quantify with high sensitivity the inspection object that may contain PCB such as insulating oil whose composition is unknown. Can do.
The PCB measurement method using the anti-PCB monoclonal antibody of the present invention can be suitably used for inspection of PCB residual concentration after waste treatment, PCB concentration in the environment, and the like.

図１は、実施例１においてＥＬＩＳＡ法により評価した、抗ＰＣＢモノクローナル抗体ＰＣＢ−Ｋ２Ａ−１によるカネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、及びＫＣ−６００）の阻害曲線(ｎ＝３)である。図１中、「○」はＫＣ−３００、「●」はＫＣ−４００、「△」はＫＣ−５００、及び「□」はＫＣ−６００の結果をそれぞれ示す。FIG. 1 shows an inhibition curve (n == canechlor (KC-300, KC-400, KC-500, and KC-600) by anti-PCB monoclonal antibody PCB-K2A-1 evaluated by ELISA in Example 1. 3). In FIG. 1, “◯” indicates the results of KC-300, “●” indicates the results of KC-400, “Δ” indicates the results of KC-500, and “□” indicates the results of KC-600. 図２は、実施例２において蛍光光度計により評価した、抗ＰＣＢモノクローナル抗体ＰＣＢ−Ｋ２Ａ−１によるカネクロール（ＫＣ−３００、ＫＣ−４００、ＫＣ−５００、及びＫＣ−６００）の阻害曲線(ｎ＝２)である。図２中、「○」はＫＣ−３００、「●」はＫＣ−４００、「△」はＫＣ−５００、及び「□」はＫＣ−６００の結果をそれぞれ示す。FIG. 2 shows the inhibition curve (n) of canechlor (KC-300, KC-400, KC-500, and KC-600) by the anti-PCB monoclonal antibody PCB-K2A-1 evaluated by a fluorometer in Example 2. = 2). In FIG. 2, “◯” indicates the results of KC-300, “●” indicates the results of KC-400, “Δ” indicates the results of KC-500, and “□” indicates the results of KC-600.

Claims (10)

下記一般式（Ｉ）で表されるビフェニル誘導体の異性体混合物を、ハプテンとして用いることを特徴とする抗ＰＣＢモノクローナル抗体の製造方法。
ただし、前記一般式（Ｉ）中、ｎは１〜１０の整数を表し、Ｘ及びＹはそれぞれ０〜２の整数を表し、ＸとＹとの和は１〜４の整数を表す。 The manufacturing method of the anti- PCB monoclonal antibody characterized by using the isomer mixture of the biphenyl derivative represented by the following general formula (I) as a hapten.
However, in said general formula (I), n represents the integer of 1-10, X and Y represent the integer of 0-2 respectively, and the sum of X and Y represents the integer of 1-4. 前記一般式（Ｉ）で表されるビフェニル誘導体から得られた下記一般式（II）で表される化合物を免疫原として動物を免疫し、免疫した前記動物の抗体産生細胞を回収し、該抗体産生細胞とミエローマ細胞とを融合して得たハイブリドーマを培養することを含む請求項１に記載の抗ＰＣＢモノクローナル抗体の製造方法。
ただし、前記一般式（II）中、ｎは１〜１０の整数を表し、Ｘ及びＹはそれぞれ０〜２の整数を表し、ＸとＹとの和は１〜４であり、Ｚはタンパク質を表す。 An animal is immunized with a compound represented by the following general formula (II) obtained from the biphenyl derivative represented by the general formula (I) as an immunogen, and antibody-producing cells of the immunized animal are collected, and the antibody The method for producing an anti-PCB monoclonal antibody according to claim 1, comprising culturing a hybridoma obtained by fusing the production cell and myeloma cell.
However, in said general formula (II), n represents the integer of 1-10, X and Y represent the integer of 0-2 respectively, the sum of X and Y is 1-4, Z is protein. To express. 前記一般式（Ｉ）及び前記一般式（II）のいずれかで表される化合物を用いて、ハイブリドーマ細胞株をスクリーニングすることを含む請求項１から２のいずれかに記載の抗ＰＣＢモノクローナル抗体の製造方法。   The anti-PCB monoclonal antibody according to any one of claims 1 to 2, comprising screening a hybridoma cell line using the compound represented by any one of the general formula (I) and the general formula (II). Production method. 請求項１から３のいずれかに記載の抗ＰＣＢモノクローナル抗体の製造方法により製造され、塩素数の異なる二種以上のＰＣＢ同族体に交差反応性を有することを特徴とする抗ＰＣＢモノクローナル抗体。   An anti-PCB monoclonal antibody produced by the method for producing an anti-PCB monoclonal antibody according to any one of claims 1 to 3, wherein the anti-PCB monoclonal antibody has cross-reactivity with two or more kinds of PCB congeners having different chlorine numbers. １〜１０塩素化ＰＣＢ全同族体のうち、１〜２塩素化ＰＣＢ同族体及び７〜１０塩素化同族体に対する親和性よりも、３〜６塩素化ＰＣＢ同族体に対する親和性が高い請求項４に記載の抗ＰＣＢモノクローナル抗体。   5. The affinity for 3-6 chlorinated PCB congeners is higher than the affinity for 1-2 chlorinated PCB congeners and 7-10 chlorinated congeners among all 1-10 chlorinated PCB congeners. An anti-PCB monoclonal antibody described in 1. 請求項４から５のいずれかに記載の抗ＰＣＢモノクローナル抗体を産生することを特徴とするハイブリドーマ。   A hybridoma that produces the anti-PCB monoclonal antibody according to any one of claims 4 to 5. 受託番号がＦＥＲＭ Ｐ−２０８４５である請求項６に記載のハイブリドーマ。   The hybridoma according to claim 6, wherein the accession number is FERM P-20845. 被測定試料に、請求項４から５のいずれかに記載の抗ＰＣＢモノクローナル抗体を添加し、
（１）前記抗ＰＣＢモノクローナル抗体と結合した前記被測定試料中のＰＣＢ、及び
（２）前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢモノクローナル抗体
のいずれかを検出する方法を含むことを特徴とするＰＣＢ測定方法。 An anti-PCB monoclonal antibody according to any one of claims 4 to 5 is added to a sample to be measured,
(1) including a method of detecting any of PCB in the sample to be measured bound to the anti-PCB monoclonal antibody and (2) the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured. PCB measuring method characterized by this. 被測定試料に、請求項４から５のいずれかに記載の抗ＰＣＢモノクローナル抗体、及び前記一般式（Ｉ）及び前記一般式（II）のいずれかで表される化合物をＰＣＢ競合物質として添加し、前記抗ＰＣＢモノクローナル抗体と結合した前記競合物質を検出することを含むことを特徴とするＰＣＢ測定方法。   The anti-PCB monoclonal antibody according to any one of claims 4 to 5 and a compound represented by any one of the general formula (I) and the general formula (II) are added as a PCB competitor to a sample to be measured. And a method for measuring PCB, comprising detecting the competitor substance bound to the anti-PCB monoclonal antibody. 被測定試料に、請求項４から５のいずれかに記載の抗ＰＣＢモノクローナル抗体を添加し、前記一般式（II）で表される化合物をＰＣＢ競合物質として固定化した担体を用い、前記抗ＰＣＢモノクローナル抗体と前記被測定試料中のＰＣＢとを競争させ、前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢモノクローナル抗体を検出することを含むことを特徴とするＰＣＢ測定方法。
An anti-PCB monoclonal antibody according to any one of claims 4 to 5 is added to a sample to be measured, and a carrier obtained by immobilizing the compound represented by the general formula (II) as a PCB competitor is used. A method for measuring PCB, comprising: competing a monoclonal antibody with PCB in the sample to be measured, and detecting the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured.
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