JP2006321766A - Haptene compound of fipronyl, antibody, hybridoma and its measuring means, and measuring kit or measuring method - Google Patents
Haptene compound of fipronyl, antibody, hybridoma and its measuring means, and measuring kit or measuring method Download PDFInfo
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- JP2006321766A JP2006321766A JP2005147693A JP2005147693A JP2006321766A JP 2006321766 A JP2006321766 A JP 2006321766A JP 2005147693 A JP2005147693 A JP 2005147693A JP 2005147693 A JP2005147693 A JP 2005147693A JP 2006321766 A JP2006321766 A JP 2006321766A
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- fipronil
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
Description
本発明は、フィプロニルのハプテン化合物、抗体、抗体を産出するハイブリドーマおよびその測定手段、測定キットまたは測定方法に関する。 The present invention relates to a fipronil hapten compound, an antibody, a hybridoma that produces the antibody, and a measurement means, measurement kit, or measurement method thereof.
フィプロニル（化学名：５−アミノ−１−（２，６−ジクロロ−４−（トリフルオロメチル）フェニル）−４−（（トリフルオロメチル）スルフィニル）−１Ｈ−ピラゾール−３−カルボニトリル）は、以下の式（２）： Fipronil (chemical name: 5-amino-1- (2,6-dichloro-4- (trifluoromethyl) phenyl) -4-((trifluoromethyl) sulfinyl) -1H-pyrazole-3-carbonitrile) is The following formula (2):
殺虫剤として、ウンカ・ヨコバイ類，アブラムシ類，コナジラミ類，カメムシ類，コナカイガラムシ類，ツノロウムシ類等の半翅目害虫，シンクイムシ類，チャノホソガ，キンモンホソガ，ギンモンハモグリガ，モモハモグリガ，ミカンハモグリガ等の鱗翅目害虫，更には鞘翅目害虫や直翅目害虫などの幅広い害虫に優れた薬効を示し、浸透移行や残効性を有する。 Pesticides such as leafhoppers, leafhoppers, aphids, stink bugs, stink bugs, leafworms, hornworms, etc. as insecticides It exhibits excellent medicinal effects on a wide range of pests, such as Coleoptera and Coleoptera pests, and has penetration and residual effects.
近年、土壌、水、大気等の環境中での残留農薬や、最近特に増加してきた輸入農産物の農薬等の残留に大きな社会的関心が寄せられている。フィプロニルについては、食品衛生法の残留農薬基準値が、例えば、米（０．０１ｐｐｍ）、とうもろこし（０．０２ｐｐｍ）、その他の穀類（０．０１ｐｐｍ）、ばれいしょ（０．０１ｐｐｍ）、てんさい（０．０１ｐｐｍ）、さとうきび（０．０１ｐｐｍ）、はくさい（０．１ｐｐｍ）、キャベツ（０．０５ｐｐｍ）、カリフラワー（０．０５ｐｐｍ）、ブロッコリー（０．０５ｐｐｍ）、その他あぶらな科野菜（０．０５ｐｐｍ）、マッシュルーム（０．０２ｐｐｍ）、バナナ（０．０１ｐｐｍ）、ひまわりの種（０．０１ｐｐｍ）、なたね（０．０１ｐｐｍ）と定められている。従って、環境や食品に関する安全確保のためには、上記の農作物に含有されるフィプロニルの量を迅速かつ正確に測定することが必要である。 In recent years, there has been a great social interest in the residue of pesticide residues in the environment such as soil, water, and the atmosphere, and recently the increase in agricultural chemicals of imported agricultural products. For fipronil, the standard values for residual agricultural chemicals in the Food Sanitation Law are, for example, rice (0.01 ppm), corn (0.02 ppm), other cereals (0.01 ppm), potato (0.01 ppm), and sugar beet (0. 01ppm), sugar cane (0.01ppm), Japanese cabbage (0.1ppm), cabbage (0.05ppm), cauliflower (0.05ppm), broccoli (0.05ppm), other oilseed vegetables (0.05ppm), mushrooms (0.02 ppm), banana (0.01 ppm), sunflower seed (0.01 ppm), rapeseed (0.01 ppm). Therefore, in order to ensure safety related to the environment and food, it is necessary to quickly and accurately measure the amount of fipronil contained in the above-mentioned crops.
また、一方、フィプロニルは木材などの建築材の害虫として知られるシロアリなどに対しても優れた薬効を示すことから、シロアリ駆除剤などとして多く使用されている。こうした用途においては、その薬効の維持あるいは保守の観点から、建築物などの基盤となる土壌などに残存するフィプロニルの測定が重要となる。 On the other hand, fipronil is also widely used as a termite control agent because it exhibits excellent medicinal properties against termites known as pests of building materials such as wood. In such applications, it is important to measure fipronil remaining in soil that is the basis of buildings and the like from the viewpoint of maintaining or maintaining its medicinal properties.
従来、例えば農作物中のフィプロニルの残留量は、米、果実、野菜、いも類等から抽出し、精製した後、高速液体クロマトグラフィー（ＨＰＬＣ）により分析されてきた。即ち、試料をアセトニトリルで抽出し、多孔性ケイソウ土カラムクロマトグラフィー、シリカゲルカラムクロマトグラフィーで精製後、ＨＰＬＣで測定する方法が採用されている。 Conventionally, for example, the residual amount of fipronil in agricultural products has been extracted from rice, fruits, vegetables, potatoes, etc., purified, and then analyzed by high performance liquid chromatography (HPLC). That is, a method is adopted in which a sample is extracted with acetonitrile, purified by porous diatomaceous earth column chromatography or silica gel column chromatography, and then measured by HPLC.
一方、免疫学的測定法は、抗原抗体反応を利用して抗原の測定を行うもので、測定精度が優れているばかりでなく、迅速、簡便かつ経済的な測定法である。従来、免疫学的測定法は、臨床診断の分野で患者の病態解析法の一つとして大きな役割を担ってきたが、環境負荷化学物質の測定への適用が進んでいる。 On the other hand, an immunological measurement method measures an antigen using an antigen-antibody reaction, and is not only excellent in measurement accuracy but also a quick, simple and economical measurement method. Conventionally, immunological measurement methods have played a major role as one of patient pathological analysis methods in the field of clinical diagnosis, but their application to the measurement of environmentally hazardous chemical substances is progressing.
しかしながら、上記ＨＰＬＣで測定する方法は、試料の調製が煩雑で多大の手順と時間を必要とし、分析に熟練を要すること、並びに、測定装置や設備等に高額の費用を必要とする等の問題点がある。フィプロニルやの測定は短時間で膨大な数の試料の分析結果を出す必要があり、精度面だけでなく、簡便性、迅速性及び経済性をも具備した新規測定方法が要求されてきている。特に、シロアリ駆除剤としての薬効の維持および保守においては個別の建物の基盤の土壌を測定することが必要となることから、測定方法の簡便さとともに、簡易な測定キットの要請が強まっている。 However, the method of measuring by the above-mentioned HPLC is a problem that sample preparation is complicated, requires a lot of procedures and time, requires skill in analysis, and requires high costs for measuring devices and equipment. There is a point. The measurement of fipronil needs to produce an analysis result of a huge number of samples in a short time, and there is a demand for a new measurement method that is not only accurate but also simple, quick, and economical. In particular, the maintenance and maintenance of the medicinal effect as a termite control agent requires the measurement of soil on the foundations of individual buildings. Therefore, there is an increasing demand for a simple measurement kit as well as a simple measurement method.
また、免疫学的測定法においても、フィプロニルに対する抗体の作製、それを用いた測定方法についての報告はなく、実用化の要請が強まっている。 In immunological measurement methods, there is no report on production of antibodies against fipronil and measurement methods using the same, and there is an increasing demand for practical use.
本発明の目的は、フィプロニルに対して高感度、かつ選択性の高い抗体を作製するためのハプテン化合物、フィプロニルに対する抗体、ならびに当該抗体を用いた高感度かつ定量性に優れたフィプロニルの免疫学的測定手段、測定キットおよび免疫学的測定方法を提供することにある。 An object of the present invention is to provide a hapten compound for producing an antibody with high sensitivity and selectivity to fipronil, an antibody against fipronil, and immunological analysis of fipronil with high sensitivity and excellent quantification using the antibody. It is to provide a measurement means, a measurement kit, and an immunological measurement method.
本発明者らは、前記課題を解決すべく鋭意検討を重ねた結果、以下に示すフィプロニルのハプテン化合物、抗体、および測定手段、測定キットまたは測定方法により前記目的を達成することができることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that the object can be achieved by the following haptenic compound of fipronil, antibody, and measurement means, measurement kit or measurement method, The present invention has been completed.
すなわち、本発明の対象となる化合物は、下記式（１）： That is, the compound which is the subject of the present invention is represented by the following formula (1):
で表わされる構造を有する。
It has the structure represented by these.
前記式（１）で表される化合物において、Ａが硫黄原子、Ｌがカルボキシル基、ｎが２であることが好ましい。 In the compound represented by the formula (1), it is preferable that A is a sulfur atom, L is a carboxyl group, and n is 2.
本発明者は、上記化合物１が、フィプロニルのハプテンとして好適に用いることが可能であることを案出したもので、高感度、かつ選択性の高い抗体を作製するが可能であり、当該抗体を用いた高感度かつ定量性に優れたフィプロニルの免疫学的測定手段および免疫学的測定方法を提供することができる。
The present inventor has devised that the
本発明は、上記化合物をハプテンとし、当該ハプテンと高分子化合物との複合体を抗原として用いることにより得られるフィプロニルに対する抗体またはそのフラグメントである。 The present invention is an antibody against fipronil or a fragment thereof obtained by using the above compound as a hapten and using a complex of the hapten and a polymer compound as an antigen.
本発明においては、上記ハプテンと高分子化合物との複合体を抗原として用いることにより、動物においてフィプロニルに対する免疫応答を良好に惹起することができ、特異的かつ高感度な抗フィプロニル抗体を得ることができる。また、本発明にはＦａｂフラグメントやＦ（ａｂ’）２フラグメントなどのように抗原結合性を有する抗体の一部も包含される。 In the present invention, by using the complex of the hapten and the polymer compound as an antigen, an immune response against fipronil can be successfully induced in animals, and a specific and highly sensitive anti-fipronil antibody can be obtained. it can. The present invention also includes a part of an antibody having antigen-binding properties such as Fab fragment and F (ab ') 2 fragment.
本発明においては、前記抗体がモノクローナル抗体またはそのフラグメントであることが好ましい。 In the present invention, the antibody is preferably a monoclonal antibody or a fragment thereof.
一般に抗体には、ポリクローナル抗体またはモノクローナル抗体が包含され、これら抗体の中でも、本発明ではフィプロニルに対してモノクローナル抗体が高感度であり、しかも選択性が高いことを見出した。 In general, the antibody includes a polyclonal antibody or a monoclonal antibody, and among these antibodies, the present invention has found that the monoclonal antibody is highly sensitive to fipronil and has high selectivity.
本発明は、上記抗体を産生するハイブリドーマである。 The present invention is a hybridoma that produces the antibody.
本発明に係るハイブリドーマによって、前記モノクローナル抗体を安定して産生することができるとともに、当該ハイブリドーマを培養することにより、大量のモノクローナル抗体を製造することができる。 The above-mentioned monoclonal antibody can be stably produced by the hybridoma according to the present invention, and a large amount of monoclonal antibody can be produced by culturing the hybridoma.
具体的には、本発明においては、前記ハイブリドーマが[ＦＰＮ−１Ｅ９−２５]であることが好ましい。 Specifically, in the present invention, the hybridoma is preferably [FPN-1E9-25].
こうしたハイブリドーマによって、フィプロニルに対する高感度かつ選択性が高い抗体を安定的に産出することができることを見出したもので、フィプロニルの免疫学的測定に好適に用いることができる。 It has been found that such a hybridoma can stably produce a highly sensitive and highly selective antibody to fipronil, and can be suitably used for immunoassay of fipronil.
本発明は、上記いずれかの抗体またはそのフラグメントを含んでなるフィプロニルの測定手段である。 The present invention is a means for measuring fipronil comprising any one of the above-described antibodies or fragments thereof.
本発明の測定手段は、本発明のモノクローナル抗体を含むことにより、フィプロニルの免疫学的測定方法に好適に用いられ、フィプロニルを特異的、高感度および簡便に測定することのできる手段を提供することができる。本発明の測定手段が前記式（１）とは異なる構造を有する化合物を含有する固相化抗原をさらに含む場合、より高感度に測定することができる手段を提供することもできる。 By including the monoclonal antibody of the present invention, the measurement means of the present invention is suitably used for the immunological measurement method of fipronil, and provides a means capable of measuring fipronil specifically, highly sensitively and easily. Can do. When the measurement means of the present invention further comprises a solid-phased antigen containing a compound having a structure different from the formula (1), a means capable of measuring with higher sensitivity can be provided.
本発明は、上記いずれかの抗体またはそのフラグメントを含んでなるフィプロニルの測定キットである。 The present invention is a kit for measuring fipronil comprising any of the above-described antibodies or fragments thereof.
上記測定手段のうち、測定キットとした場合には、特に現場でのフィプロニルの免疫学的測定作業などに好適に用いられ、フィプロニルを特異的、高感度および簡便に測定することのできる手段を提供することができる。 Among the above measurement means, when a measurement kit is used, it is particularly suitable for on-site immunological measurement of fipronil, and provides a means for measuring fipronil specifically, highly sensitively and easily. can do.
本発明は、上記いずれかの抗体またはそのフラグメントを含んでなるフィプロニルの測定方法である。 The present invention is a method for measuring fipronil comprising any of the above-described antibodies or fragments thereof.
本発明のモノクローナル抗体または測定手段を用いることにより感度、特異性および操作の簡便性にすぐれた効果を奏する測定方法が可能である。 By using the monoclonal antibody or the measuring means of the present invention, a measuring method having an effect excellent in sensitivity, specificity and ease of operation is possible.
本発明の化合物は、フィプロニルのハプテンとして好適に用いられるものである。当該ハプテン化合物と高分子化合物との複合体を抗原として用いることにより、動物においてフィプロニルに対する免疫応答を良好に惹起することができ、特異的かつ高感度な抗フィプロニルおよび抗抗体を得ることができる。前記式（１）で表される化合物において、Ｌはカルボキシル基、ｎは２の場合、フィプロニルのハプテンとして特に優れた効果を奏する。 The compound of the present invention is suitably used as a hapten for fipronil. By using the complex of the hapten compound and the polymer compound as an antigen, an immune response against fipronil can be successfully induced in animals, and specific and highly sensitive anti-fipronil and anti-antibody can be obtained. In the compound represented by the formula (1), when L is a carboxyl group and n is 2, a particularly excellent effect as a hapten of fipronil is obtained.
本発明の抗体がモノクローナル抗体である場合、フィプロニルに対して高感度であり、しかも他の類似化合物に対する交差反応性が認められず、フィプロニルを特異的に検出することができる。本発明のハイブリドーマは、前記モノクローナル抗体を安定して産生することができ、当該ハイブリドーマを培養または由来の動物の腹腔内に投与し腹水を作らせることにより、大量のモノクローナル抗体を製造することができる。本発明の測定手段は、本発明のモノクローナル抗体を含むことにより、フィプロニルの免疫学的測定方法に好適に用いられ、フィプロニルを特異的、高感度および簡便に測定することができる。さらに、測定キットとした場合には、特に現場でのフィプロニルの免疫学的測定作業などに好適に用いることができる。本発明のフィプロニルの測定方法は、本発明のモノクローナル抗体または測定手段を用いることにより感度、特異性および操作の簡便性にすぐれた効果を奏する。 When the antibody of the present invention is a monoclonal antibody, it is highly sensitive to fipronil, and cross-reactivity to other similar compounds is not observed, so that fipronil can be specifically detected. The hybridoma of the present invention can stably produce the monoclonal antibody, and a large amount of monoclonal antibody can be produced by administering the hybridoma into the abdominal cavity of a cultured or derived animal to produce ascites. . By including the monoclonal antibody of the present invention, the measurement means of the present invention is suitably used for a fipronil immunoassay method, and fipronil can be specifically, highly sensitively and easily measured. Furthermore, when it is set as a measurement kit, it can be used suitably especially for the immunological measurement operation | work etc. of the fipronil on the spot. The method for measuring fipronil of the present invention exhibits excellent effects in sensitivity, specificity, and ease of operation by using the monoclonal antibody or measurement means of the present invention.
本発明は、下記式（１）： The present invention provides the following formula (1):
上記式（１）で表される化合物において、Ａ−（ＣＨ２）ｎ−Ｌはフィプロニル構造の一部分に導入したスペーサーアームを表し、ｎは１〜１０の整数である。フィプロニルと結合対象の高分子化合物との間に適度なスペースを有するためには、ｎは２〜５が好ましい。 In the compound represented by the above formula (1), A- (CH 2 ) nL represents a spacer arm introduced into a part of the fipronil structure, and n is an integer of 1 to 10. In order to have an appropriate space between fipronil and the polymer compound to be bound, n is preferably 2 to 5.
上記式（１）で表される化合物において、Ｌが対象高分子と共有結合することにより、複合体を形成する。 In the compound represented by the above formula (1), L is covalently bonded to the target polymer to form a complex.
ハプテン化合物として用いる上記式（１）で表される化合物の製造は、公知の合成方法により行うことができ、特に限定されるものではないが、例えば式（１）で表される化合物のＡが硫黄でＬがカルボキシル基のものについては、下記反応式： The production of the compound represented by the above formula (1) used as the hapten compound can be carried out by a known synthesis method and is not particularly limited. For example, the compound A represented by the formula (1) For sulfur and L being a carboxyl group, the following reaction formula:
具体的には、前記反応式において、
（１）工程１
出発原料として５−アミノ−４−トリフルオロメチルスルホニル−１−（２，６−ジクロロ−４−トリフルオロメチルフェニル）ピラゾール−３−カルボニトリル（１）を用い、酸化剤と反応させて５−アミノ−４−トリフルオロメチルスルホニル−１−（２，６−ジクロロ−４−トリフルオロメチルフェニル）ピラゾール−３−カルボニトリル（２）を得る。
Specifically, in the reaction formula,
(1)
5-Amino-4-trifluoromethylsulfonyl-1- (2,6-dichloro-4-trifluoromethylphenyl) pyrazole-3-carbonitrile (1) is used as a starting material and reacted with an oxidizing agent to give 5- Amino-4-trifluoromethylsulfonyl-1- (2,6-dichloro-4-trifluoromethylphenyl) pyrazole-3-carbonitrile (2) is obtained.
この反応で酸化剤としては特に限定されないが、例えば過酸化水素、過酢酸、過安息香酸、メタクロロ過安息香酸などの過酸類、オゾン、過マンガン酸カリウム、クロム酸などを使用することができる。また触媒としてタングステンやパナジウムを用いることも出来る。溶媒としては、例えばメタノール、エタノールなどのアルコール類、ベンゼン、トルエンなどの芳香族炭化水素類、ジエチルエーテル、ジプロピルエーテル、テトラヒドロフランなどのエーテル類、アセトン、メチルエチルケトンなどのケトン類、アセトニトリル、プロピオニトリルなどのニトリル類、ジメチルホルムアミド、ジメチルアセトアミドなどの酸アミド類、ジメチルスルホキシドなどのスルホキシド類、酢酸などの酸、水およびそれらの混合溶媒などを挙げることができる。反応温度は通常室温から溶媒の沸点の温度で、３０分から１０時間程度行う。 Although it does not specifically limit as an oxidizing agent by this reaction, For example, hydrogen peroxide, peracetic acid, perbenzoic acid, peracids, such as a metachloro perbenzoic acid, ozone, potassium permanganate, chromic acid, etc. can be used. Further, tungsten or panadium can also be used as a catalyst. Examples of the solvent include alcohols such as methanol and ethanol, aromatic hydrocarbons such as benzene and toluene, ethers such as diethyl ether, dipropyl ether, and tetrahydrofuran, ketones such as acetone and methyl ethyl ketone, acetonitrile, and propionitrile. Nitriles such as, amides such as dimethylformamide and dimethylacetamide, sulfoxides such as dimethylsulfoxide, acids such as acetic acid, water, and mixed solvents thereof. The reaction temperature is usually from room temperature to the boiling point of the solvent and is carried out for about 30 minutes to 10 hours.
（２）工程２
得られた化合物を塩基の存在下、3-メルカプトアルキルカルボン酸と反応させてω−［５−アミノ−３−シアノ−１−（２，６−ジクロロ−４−トリフルオロメチルフェニル）ピラゾール−４−イルチオ］アルキルカルボン酸（３）を得る。
本反応で用いられる塩基、溶媒については工程１と同様のものを用いることができる。反応温度は通常０℃から溶媒の沸点の温度で、反応時間は３０分から１０時間程度行う。
上記各工程における詳細な合成方法は、実施例に記載する。
(2) Step 2
The obtained compound is reacted with 3-mercaptoalkylcarboxylic acid in the presence of a base to give ω- [5-amino-3-cyano-1- (2,6-dichloro-4-trifluoromethylphenyl) pyrazole-4. -Ilthio] alkylcarboxylic acid (3) is obtained.
As the base and solvent used in this reaction, those similar to those in
Detailed synthesis methods in the above steps are described in Examples.
このようにして得られたフィプロニルのハプテン化合物は、牛血清アルブミン（ＢＳＡ）、ウサギ血清アルブミン（ＲＳＡ）、オボアルブミン（ＯＶＡ）、スカシ貝ヘモシアニン（ＫＬＨ）、チログロブリン（ＴＧ）、免疫グロブリン等の高分子化合物（タンパク質）との複合体を形成させた後、抗原として用いる。 The fipronil hapten compound thus obtained includes bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), mussel hemocyanin (KLH), thyroglobulin (TG), immunoglobulin and the like. After forming a complex with a polymer compound (protein), it is used as an antigen.
複合体の形成方法は、公知の方法により行うことができ、特に限定されるものではない。例えば、混合酸無水物法または活性エステル法等によって、フィプロニルのハプテン化合物のカルボキシ基と前記高分子化合物の官能基とを反応させて、複合体を形成することができる。 The formation method of a composite_body | complex can be performed by a well-known method, and is not specifically limited. For example, a complex can be formed by reacting the carboxy group of the hapten compound of fipronil with the functional group of the polymer compound by a mixed acid anhydride method or an active ester method.
本発明は、上記ハプテン化合物と高分子化合物との複合体を抗原として用いることにより得られるフィプロニルに対する抗体を提供する。当該抗体は、フィプロニルに対する特異性を有する抗体である。 The present invention provides an antibody against fipronil obtained by using a complex of the hapten compound and the polymer compound as an antigen. The antibody is an antibody having specificity for fipronil.
抗体には、一般に免疫したウサギやヤギなどから血液を採取後その中に含まれる抗体を分離・精製するいわゆるポリクローナル抗体や、抗体産生能を持つクローン化ハイブリドーマの分泌する抗体を分離・精製するいわゆるモノクローナル抗体がある。本発明においては、ポリクローナル抗体またはモノクローナル抗体が抱合される。モノクローナル抗体が有する高感度性および高選択性、さらには、複数のモノクローナル抗体の組み合わせによる複数成分との分離可能な反応性など適用の汎用性の広さから、特にモノクローナル抗体が好ましい。 In general, antibodies are collected from blood from immunized rabbits or goats, and so-called polyclonal antibodies that separate and purify antibodies contained therein, and so-called polyclonal antibodies that secrete cloned hybridomas capable of producing antibodies are separated and purified. There are monoclonal antibodies. In the present invention, a polyclonal antibody or a monoclonal antibody is conjugated. Monoclonal antibodies are particularly preferred because of the high versatility of application, such as high sensitivity and high selectivity possessed by monoclonal antibodies, and the ability to be separated from a plurality of components by a combination of a plurality of monoclonal antibodies.
前記モノクローナル抗体は、フィプロニルに対する特異性と他の物質に対する交差反応性とを明確にするため、下記のようなＩＣ５０値を有することが好ましい。ここでＩＣ５０値とは、間接競合ＥＬＩＳＡまたは直接競合ＥＬＩＳＡにより標準阻害曲線を求めて、５０％阻害を示す検体の濃度をいう。 The monoclonal antibody preferably has the following IC50 value in order to clarify the specificity for fipronil and the cross-reactivity for other substances. Here, the IC50 value refers to the concentration of a sample exhibiting 50% inhibition when a standard inhibition curve is obtained by indirect competitive ELISA or direct competitive ELISA.
すなわち、本抗体は、フィプロニルを測定対象とする場合は、直接競合ＥＬＩＳＡによるフィプロニルに対するＩＣ５０が５００ｎｇ／ｍＬ以下であることが好ましく、５０ｎｇ／ｍＬ以下がより好ましい。 That is, when fipronil is used as the measurement target, this antibody preferably has an IC50 for fipronil by direct competition ELISA of 500 ng / mL or less, more preferably 50 ng / mL or less.
前記抗体は、通常の製造方法に従って製造することができる（Current Protocol in Molecular Biology、Chapter 11.12〜11.13（2000））。具体的には、本発明の抗体がポリクローナル抗体の場合には、常法に従ってフィプロニルのハプテン化合物と高分子化合物との複合体を形成させた後、当該複合体を家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。一方、モノクローナル抗体の場合には、前記複合体を常法に従ってマウス等の非ヒト動物に免疫し、得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞をスクリーニングし、モノクローナル抗体産生ハイブリドーマを培養することにより得ることができる（Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4 〜11.11 ）。 The antibody can be produced according to a usual production method (Current Protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, a complex of a fipronil hapten compound and a polymer compound is formed according to a conventional method, and then the complex is applied to a non-human animal such as a rabbit. Immunization can be obtained from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, the complex is immunized to a non-human animal such as a mouse according to a conventional method, and a hybridoma cell prepared by cell fusion of the obtained spleen cell and myeloma cell is screened. It can be obtained by culturing an antibody-producing hybridoma (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4 to 11.11).
抗体の調製は、限外ろ過、硫安分画、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、アフィニティークロマトグラフィーなどの濃縮・精製法を適宜組み合わせて行うことができる。 The antibody can be prepared by appropriately combining concentration and purification methods such as ultrafiltration, ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography, and affinity chromatography.
また、本発明は、前記モノクローナル抗体を産生するハイブリドーマを提供する。以下、マウスでのハイブリドーマの作製方法についてより詳細に説明する。 The present invention also provides a hybridoma that produces the monoclonal antibody. Hereinafter, a method for producing a hybridoma using a mouse will be described in more detail.
前記のように調製した抗原を２ｍｇ／ｍＬ程度になるように生理的リン酸緩衝液に溶解し、アジュバントと等量混合した後、ＢａＬｂ／ｃマウスの腹腔内に投与する。その後、約２週間毎に追加免疫する。尾血管から採取した血液の血清中の抗体力価が高くなった前記マウスの脾臓を摘出し、無血清ＤＭＥＭ培地（ダルベッコ改変イーグル培地）中で、組織片等を取り除いた後に新しい培地中に移し、脾細胞を完全に培地中に浮遊させる。遠心、上清除去を数回繰り返し、細胞を洗った後、マウスのミエローマ細胞（Ｐ３Ｘ６３Ａｇ８．６５３）と細胞数の比５：１〜１０：１（脾細胞：ミエローマ）で混合する。細胞を沈殿させ上清を取り除いたあと、攪拌しながら５０％ポリエチレングリコール（分子量１５００）をゆっくり加え細胞融合を行う。細胞融合後、遠心分離によって集めた細胞に、細胞数が５×１０５個／ｍＬになるようにＨＡＴ培地を加えて懸濁し、細胞懸濁液を９６穴プラスチックプレートに２５０μＬ／ウェルの量で分注して、３７℃、５％炭酸ガス、加湿条件下のインキュベーター中で培養する。１週間後、ウェル中の培地の半量をＨＡＴ培地で置換して、１０日から１４日間培養する。培養液中の抗体の活性をＥＬＩＳＡで調べ、目的とする抗体を産生しているウェルの細胞について、限界希釈法によりハイブリドーマのクローニングを行う。クローニングにより、抗フィプロニルおよび抗抗体を産生している安定なハイブリドーマ株を得る。 The antigen prepared as described above is dissolved in a physiological phosphate buffer so as to be about 2 mg / mL, mixed with an equal amount of adjuvant, and then administered intraperitoneally to BaLb / c mice. Thereafter, booster immunization is carried out about every 2 weeks. The spleen of the mouse in which the antibody titer in the serum of blood collected from the tail blood vessel has been increased is removed, removed in a serum-free DMEM medium (Dulbecco's modified Eagle medium), and transferred to a new medium. The spleen cells are completely suspended in the medium. Centrifugation and supernatant removal are repeated several times to wash the cells, and then mixed with mouse myeloma cells (P3X63Ag8.653) at a cell number ratio of 5: 1 to 10: 1 (spleen cells: myeloma). After the cells are precipitated and the supernatant is removed, cell fusion is performed by slowly adding 50% polyethylene glycol (molecular weight 1500) while stirring. After cell fusion, the cells collected by centrifugation are suspended by adding HAT medium so that the number of cells is 5 × 10 5 cells / mL, and the cell suspension is added to a 96-well plastic plate in an amount of 250 μL / well. Dispense and incubate in an incubator at 37 ° C., 5% carbon dioxide, humidified conditions. One week later, half of the medium in the well is replaced with HAT medium and cultured for 10 to 14 days. The activity of the antibody in the culture solution is examined by ELISA, and the hybridoma is cloned by limiting dilution for the cells of the well producing the target antibody. Cloning yields a stable hybridoma strain producing anti-fipronil and anti-antibody.
本発明では、前記方法によりハイブリドーマを作製しＦＰＮ−１Ｅ９−２５について、寄託番号ＦＥＲＭ ＡＰ−２０３８４の下、２００５年２月２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した。 In the present invention, a hybridoma was prepared by the above-described method, and FPN-1E9-25 was deposited at the National Institute of Advanced Industrial Science and Technology (AIST) on February 2, 2005 under the deposit number FERM AP-20384. .
本発明のハイブリドーマは、培地（例えば、１０％牛胎児血清を含むＤＭＥＭ）を用いて培養し、その培養液の遠心上清をモノクローナル抗体溶液とすることができる。また、本ハイブリドーマを由来する動物の腹腔に注入することにより、腹水を生成させ、得られた腹水をモノクローナル抗体溶液とすることができる。これらの抗体溶液は、さらに上述のように精製・濃縮することができる。 The hybridoma of the present invention can be cultured using a medium (for example, DMEM containing 10% fetal bovine serum), and the centrifuged supernatant of the culture solution can be used as a monoclonal antibody solution. In addition, ascites can be generated by injecting the hybridoma into the abdominal cavity of an animal from which the hybridoma is derived, and the obtained ascites can be used as a monoclonal antibody solution. These antibody solutions can be further purified and concentrated as described above.
また、本発明においては、フィプロニルに特異的に結合する抗体を含むことにより、フィプロニルを簡便に測定することができ、測定手段としての測定キットおよび後述するフィプロニルの測定方法に好適に使用することができる。前記キットは、さらに、測定法に応じて、標識された二次抗体もしくは標識されたフィプロニルのハプテン化合物、緩衝液、検出試薬および／またはフィプロニルの標準溶液等を含む。好ましいキットは、ＥＬＩＳＡ法に用いられうるものであり、下記直接競合ＥＬＩＳＡ法に用いる場合、固相化されたフィプロニルに対する抗体、固相化抗体を保持する担体、酵素標識された抗原および検出試薬などを含む。 In addition, in the present invention, fipronil can be easily measured by including an antibody that specifically binds to fipronil, and can be suitably used for a measurement kit as a measurement means and a method for measuring fipronil described later. it can. The kit further contains a labeled secondary antibody or a labeled fipronil hapten compound, a buffer, a detection reagent, and / or a standard solution of fipronil, depending on the measurement method. Preferred kits are those that can be used in the ELISA method. When used in the direct competitive ELISA method described below, an antibody against immobilized fipronil, a carrier holding the immobilized antibody, an enzyme-labeled antigen, a detection reagent, etc. including.
さらに、本発明は、前記抗体または測定手段を用いることを特徴とするフィプロニルの測定方法に関する。測定方法としては、通常の抗原−抗体反応を利用する方法であれば特に制限されず、放射性同位元素免疫測定法（ＲＩＡ）、酵素免疫測定法（ＥＬＩＳＡ）、蛍光もしくは発光測定法、凝集法、イムノブロット法、イムノクロマト法等(Meth. Enzymol., 92, 147-523 (1983), Antibodies Vol.II IRL Press Oxford (1989))が挙げられるが、感度や簡便性等の点からＥＬＩＳＡが好ましい。ＥＬＩＳＡに用いる酵素としては、ペルオキシダーゼ、アルカリホスファターゼ、β−ガラクトシダーゼ、ルシフェラーゼ等が挙げられる。 Furthermore, the present invention relates to a method for measuring fipronil, which comprises using the antibody or the measuring means. The measurement method is not particularly limited as long as it uses a normal antigen-antibody reaction. Radioisotope immunoassay (RIA), enzyme immunoassay (ELISA), fluorescence or luminescence assay, aggregation method, Examples include immunoblotting, immunochromatography and the like (Meth. Enzymol., 92, 147-523 (1983), Antibodies Vol. II IRL Press Oxford (1989)), and ELISA is preferred from the viewpoint of sensitivity and simplicity. Examples of the enzyme used for ELISA include peroxidase, alkaline phosphatase, β-galactosidase, luciferase and the like.
ＥＬＩＳＡによる測定法は、間接競合ＥＬＩＳＡまたは直接競合ＥＬＩＳＡなどが挙げられる。例えば以下に述べるような本発明のモノクローナル抗体を用いた直接競合ＥＬＩＳＡによって行うことができる。 Examples of the measurement method by ELISA include indirect competitive ELISA and direct competitive ELISA. For example, it can be performed by direct competition ELISA using the monoclonal antibody of the present invention as described below.
（１）本発明のモノクローナル抗体を、担体に固相化する
用いる担体は、９６穴、４８穴、１９２穴等のマイクロタイタープレートが好ましい。固相化は、例えば、固相化用抗体を含む緩衝液を担体上に載せ、インキュベーションすればよい。緩衝液中の抗体の濃度は、通常０．０１μｇ／ｍＬから１００μｇ／ｍＬ程度である。緩衝液としては、検出手段に応じて公知のものを使用することができる。
(1) Immobilizing the monoclonal antibody of the present invention on a carrier The carrier used is preferably a microtiter plate having 96 holes, 48 holes, 192 holes or the like. For immobilization, for example, a buffer containing the immobilization antibody may be placed on a carrier and incubated. The concentration of the antibody in the buffer is usually about 0.01 μg / mL to 100 μg / mL. As the buffer solution, a known solution can be used according to the detection means.
（２）担体の固相表面へのタンパク質の非特異的吸着を防止するため、固相化用抗体が吸着していない固相表面部分を、抗体と無関係なタンパク質等によりブロッキングする
ブロッキング剤としては、ＢＳＡもしくはスキムミルク溶液、または市販のブロックエース（大日本製薬社製）等を使用することができる。ブロッキングは、前記ブロッキング剤を担体に添加し、例えば、約４℃で一晩インキュベーションした後、洗浄液で洗浄することにより行われる。洗浄液としては特に制限はないが、前記（１）と同じ緩衝液を使用することができる。
(2) In order to prevent non-specific adsorption of proteins to the solid phase surface of the carrier, the solid phase surface portion to which the antibody for solid phase immobilization is not adsorbed is blocked with a protein unrelated to the antibody. , BSA or skim milk solution, or commercially available Block Ace (Dainippon Pharmaceutical Co., Ltd.) can be used. Blocking is performed by adding the blocking agent to the carrier and, for example, incubating overnight at about 4 ° C. and then washing with a washing solution. Although there is no restriction | limiting in particular as a washing | cleaning liquid, The same buffer solution as said (1) can be used.
（３）各種濃度のフィプロニルを含む試料に、フィプロニルのハプテン化合物と酵素を結合させた酵素結合ハプテンを加えた混合物を調製する
酵素結合ハプテンの調製は、フィプロニルのハプテン化合物を酵素に結合する方法であれば特に制限なく、いかなる方法で行ってもよい。
(3) Prepare a mixture of fipronil hapten compound and enzyme-bound hapten, which is obtained by binding fipronil hapten compound and enzyme. Preparation of enzyme-bound hapten is a method of binding fipronil hapten compound to enzyme. Any method may be used without particular limitation.
（４）工程（３）の混合物を工程（２）で得られた抗体固相化担体と反応させる
フィプロニルと酵素結合ハプテンとの競合阻害反応により、これらと固相化担体との複合体が生成する。反応は例えば、約２５℃で約１時間行う。反応終了後、緩衝液で担体を洗浄し、固相化抗体と結合しなかった酵素結合ハプテンを除去する。
固相化抗体−酵素結合ハプテン複合体の量を測定することにより、予め作成した検量線から試料中のフィプロニルの量を決定する。
(4) The mixture of step (3) is reacted with the antibody-immobilized carrier obtained in step (2). By the competitive inhibition reaction of fipronil and enzyme-bound hapten, a complex of these and the immobilized carrier is produced. To do. The reaction is carried out, for example, at about 25 ° C. for about 1 hour. After completion of the reaction, the carrier is washed with a buffer to remove the enzyme-bound hapten that did not bind to the immobilized antibody.
By measuring the amount of the immobilized antibody-enzyme linked hapten complex, the amount of fipronil in the sample is determined from a calibration curve prepared in advance.
（５）担体に結合した標識酵素と反応する発色基質溶液を加え、吸光度を測定することによって検量線からフィプロニルの量を算出することができる
標識酵素としてペルオキシダーゼを使用する場合には、例えば、過酸化水素と、３，３’，５，５’−テトラメチルベンジジンまたはｏ−フェニレンジアミンを含む発色基質溶液を使用することができる。通常、発色基質溶液を加えて室温で約１０分程度反応させた後、硫酸を加えることにより酵素反応を停止させる。３，３’，５，５’−テトラメチルベンジジンを使用する場合、４５０ｎｍの吸光度を測定する。ｏ−フェニレンジアミンを使用する場合、４９２ｎｍの吸光度を測定する。なお、バックグランド値を補正するため、６３０ｎｍの吸光度も同時に測定することが望ましい。
(5) The amount of fipronil can be calculated from the calibration curve by adding a chromogenic substrate solution that reacts with the labeled enzyme bound to the carrier and measuring the absorbance. When peroxidase is used as the labeled enzyme, for example, excess A chromogenic substrate solution containing hydrogen oxide and 3,3 ′, 5,5′-tetramethylbenzidine or o-phenylenediamine can be used. Usually, after adding a chromogenic substrate solution and reacting at room temperature for about 10 minutes, the enzyme reaction is stopped by adding sulfuric acid. When 3,3 ′, 5,5′-tetramethylbenzidine is used, the absorbance at 450 nm is measured. When o-phenylenediamine is used, the absorbance at 492 nm is measured. In order to correct the background value, it is desirable to simultaneously measure the absorbance at 630 nm.
標識酵素としてアルカリホスファターゼを使用する場合には、例えばｐ−ニトロフェニルリン酸を基質として発色させ、ＮａＯＨ溶液を加えて酵素反応を止め、４１５ｎｍでの吸光度を測定する方法があげられる。 In the case of using alkaline phosphatase as a labeling enzyme, for example, a method of coloring with p-nitrophenyl phosphate as a substrate, adding an NaOH solution to stop the enzyme reaction, and measuring the absorbance at 415 nm can be mentioned.
フィプロニルを添加しない反応溶液の吸光度に対して、フィプロニルを添加して抗体と反応させた溶液の吸光度の減少率を阻害率として計算する。既知の濃度のフィプロニルを添加した反応液の阻害率により予め作成しておいた検量線を用いて、試料中のフィプロニルの濃度の濃度を算出することができる。 For the absorbance of the reaction solution to which no fipronil is added, the decrease rate of the absorbance of the solution reacted with the antibody by adding fipronil is calculated as the inhibition rate. The concentration of fipronil in the sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of the reaction solution to which fipronil at a known concentration is added.
別の態様としてフィプロニルの測定は以下のような手順により間接競合ＥＬＩＳＡによって行うことができる。 In another embodiment, fipronil can be measured by indirect competitive ELISA according to the following procedure.
（１）固相化抗原を担体に固相化する。
用いる担体は、通常のＥＬＩＳＡに用いる担体であれば特に制限されないが、９６穴等のマイクロタイタープレートが好ましい。固相化は、例えば、固相化用抗原を含む緩衝液を担体上に載せ、インキュベーションすればよい。緩衝液中の抗原の濃度は、通常０.０１μｇ／ｍＬから１００μｇ／ｍＬ程度である。緩衝液としては、検出手段に応じて公知のものを使用することができる。
(1) An immobilized antigen is immobilized on a carrier.
The carrier to be used is not particularly limited as long as it is a carrier used in ordinary ELISA, but a microtiter plate having 96 holes or the like is preferable. For immobilization, for example, a buffer containing the immobilization antigen may be placed on a carrier and incubated. The concentration of the antigen in the buffer is usually about 0.01 μg / mL to 100 μg / mL. As the buffer solution, a known solution can be used according to the detection means.
（２）担体の固相表面へのタンパク質の非特異的吸着を防止するため、固相化用抗原が吸着していない固相表面部分を、抗原と無関係なタンパク質等によりブロッキングする。ブロッキング剤としては、ＢＳＡもしくはスキムミルク溶液、または市販のブロックエース（大日本製薬社製）等を使用することができる。ブロッキングは、前記ブロッキング剤を担体に添加し、例えば、約４℃で一晩インキュベーションした後、洗浄液で洗浄することにより行われる。洗浄液としては特に制限はないが、前記（１）と同じ緩衝液を使用することができる。 (2) In order to prevent nonspecific adsorption of the protein to the solid phase surface of the carrier, the solid phase surface portion where the antigen for immobilization is not adsorbed is blocked with a protein unrelated to the antigen. As the blocking agent, BSA or skim milk solution, or commercially available Block Ace (Dainippon Pharmaceutical Co., Ltd.) can be used. Blocking is performed by adding the blocking agent to the carrier and, for example, incubating overnight at about 4 ° C. and then washing with a washing solution. Although there is no restriction | limiting in particular as a washing | cleaning liquid, The same buffer solution as said (1) can be used.
（３）前記（１）および（２）で処理された固相表面に各種濃度のフィプロニルを含む試料および本発明のモノクローナル抗体溶液を加え、該抗体を前記固相化抗原およびフィプロニルに競合的に反応させて、固相化抗原−抗体複合体およびフィプロニルに対する抗体複合体を生成させる。
反応は、通常室温、１〜２時間程度で行うことができる。
フィプロニルは、水に不溶性であるため、反応溶液中には各種有機溶媒を含有することができる。前記有機溶媒としては、フィプロニルを溶解させ、かつ抗原−抗体反応を阻害しない範囲で有機溶媒およびその含有量を選択すればよい。具体的には、メタノール、ジメチルスルホキシドなどがあげられ、含有量は、５〜５０重量％程度である。
(3) Samples containing various concentrations of fipronil and the monoclonal antibody solution of the present invention are added to the solid phase surface treated in (1) and (2) above, and the antibody is competitive with the immobilized antigen and fipronil. Reacting to produce an immobilized antigen-antibody complex and an antibody complex against fipronil.
The reaction can usually be performed at room temperature for about 1 to 2 hours.
Since fipronil is insoluble in water, the reaction solution can contain various organic solvents. What is necessary is just to select an organic solvent and its content as the said organic solvent in the range which dissolves fipronil and does not inhibit an antigen-antibody reaction. Specific examples include methanol and dimethyl sulfoxide, and the content is about 5 to 50% by weight.
（４）固相化抗原−抗体複合体の量を測定することにより、予め作成した検量線から試料中のフィプロニルの量を決定することができる。
固相化抗原−抗体複合体の量は、酵素標識した二次抗体（マウス抗体を認識する抗体）を添加して測定することができる。例えばフィプロニルに対する抗体としてマウスモノクローナル抗体を用いる場合、酵素標識（例えば、ペルオキシダーゼまたはアルカリホスファターゼ等）した抗マウス−ヤギ抗体を用いて、担体に結合したフィプロニルに対する抗体と反応させるのが望ましい。反応は、前記（３）と同様の条件下で行えばよい。反応後、緩衝液で洗浄する。
(4) By measuring the amount of the immobilized antigen-antibody complex, the amount of fipronil in the sample can be determined from a calibration curve prepared in advance.
The amount of the immobilized antigen-antibody complex can be measured by adding an enzyme-labeled secondary antibody (an antibody that recognizes a mouse antibody). For example, when a mouse monoclonal antibody is used as an antibody against fipronil, it is desirable to react with an antibody against fipronil bound to a carrier using an enzyme-labeled (eg, peroxidase or alkaline phosphatase) anti-mouse-goat antibody. The reaction may be performed under the same conditions as in (3) above. After the reaction, wash with buffer.
（５）担体に結合した二次抗体の標識酵素と反応する発色基質溶液を加え、酵素結合ハプテンの酵素に反応する発色基質溶液を前述の直接競合阻害ＥＬＩＳＡ法と同様に加え、吸光度を測定することにより検量線からフィプロニルの量を算出することができる。 (5) Add a chromogenic substrate solution that reacts with the labeling enzyme of the secondary antibody bound to the carrier, add a chromogenic substrate solution that reacts with the enzyme of the enzyme-bound hapten in the same manner as the direct competitive inhibition ELISA method described above, and measure the absorbance. Thus, the amount of fipronil can be calculated from the calibration curve.
前記本発明の測定方法においては、測定対象物に応じた前処理をして試料とした後、前記直接競合ＥＬＩＳＡの工程（３）または間接競合ＥＬＩＳＡに供することができる。例えば 測定対象物が土壌または食品の場合、フィプロニルが抽出できる全ての方法を用いることができる。抽出物は、メタノールに転溶させて緩衝液で希釈後、測定試料にする。簡便法として、メタノールで抽出し緩衝液で希釈したものをそのまま試料とすることも可能である。具体的には、例えば試料５ｇに、メタノール５ｍＬを加えて２０分間振とうする。室温で３０分間静置した後、抽出物の上層２ｍＬを２５００ｒｐｍで１０分間遠心分離する。上清を蒸留水で１：４に希釈し、これを前記工程（３）に供する。 In the measurement method of the present invention, the sample can be pretreated according to the measurement object to obtain a sample, and then subjected to the step (3) of the direct competitive ELISA or the indirect competitive ELISA. For example, when the measurement object is soil or food, all methods capable of extracting fipronil can be used. The extract is dissolved in methanol, diluted with a buffer solution, and used as a measurement sample. As a simple method, a sample extracted with methanol and diluted with a buffer can be used as it is. Specifically, for example, 5 mL of methanol is added to 5 g of a sample and shaken for 20 minutes. After standing at room temperature for 30 minutes, 2 mL of the upper layer of the extract is centrifuged at 2500 rpm for 10 minutes. The supernatant is diluted 1: 4 with distilled water and subjected to step (3).
また、測定対象物が環境水の場合、抽出工程を必要とせず、直接測定試料とすることが可能である。 Further, when the measurement object is environmental water, an extraction step is not required, and it can be directly used as a measurement sample.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の技術的範囲を限定するためのものではない。当業者は本明細書の記載に基づいて容易に本発明に修飾、変更を加えることができ、それらは本発明の技術的範囲に含まれる。 EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.
＜実施例１＞
（１）５−アミノ−１−（２，６−ジクロロ−４−（トリフルオロメチル）フェニル）−４−（（トリフルオロメチル）スルホニル）−１Ｈ−ピラゾール−３−カルボニトリルの合成
５−アミノ−１−（２，６−ジクロロ−４−（トリフルオロメチル）フェニル）−４−（（トリフルオロメチル）スルフィニル）−１Ｈ−ピラゾール−３−カルボニトリル０．１７ｇ（０．４ｍｍｏｌ）、タングステン酸ナトリウム二水和物０．０１ｇ（０．０３ｍｍｏｌ）、３０％過酸化水素水０．０７ｇ（０．６ｍｍｏｌ）を酢酸１ｍＬ、に溶かし、５５℃で６時間反応させた。反応液を冷却後、水１０ｍＬを加え、酢酸エチル１０ｍＬで２回抽出した。酢酸エチル層を水で洗った後、無水硫酸マグネシウムで脱水、ろ過、濃縮し、淡黄色結晶の５−アミノ−１−（２，６−ジクロロ−４−（トリフルオロメチル）フェニル）−４−（（トリフルオロメチル）スルホニル）−１Ｈ−ピラゾール−３−カルボニトリルを０．２ｇ得た。
<Example 1>
(1) Synthesis of 5-amino-1- (2,6-dichloro-4- (trifluoromethyl) phenyl) -4-((trifluoromethyl) sulfonyl) -1H-pyrazole-3-carbonitrile -1- (2,6-dichloro-4- (trifluoromethyl) phenyl) -4-((trifluoromethyl) sulfinyl) -1H-pyrazole-3-carbonitrile 0.17 g (0.4 mmol), tungstic acid Sodium dihydrate 0.01 g (0.03 mmol) and 30% aqueous hydrogen peroxide 0.07 g (0.6 mmol) were dissolved in 1 mL of acetic acid and reacted at 55 ° C. for 6 hours. After cooling the reaction solution, 10 mL of water was added, and the mixture was extracted twice with 10 mL of ethyl acetate. The ethyl acetate layer was washed with water, dehydrated with anhydrous magnesium sulfate, filtered and concentrated to give pale yellow crystals of 5-amino-1- (2,6-dichloro-4- (trifluoromethyl) phenyl) -4- 0.2 g of ((trifluoromethyl) sulfonyl) -1H-pyrazole-3-carbonitrile was obtained.
（２）３−［５−アミノ−３−シアノ−１−（２，６−ジクロロ−４−トリフルオロメチルフェニル）−１Ｈ−ピラゾール−４−イルチオ］プロピオン酸の合成
５−アミノ−１−（２，６−ジクロロ−４−（トリフルオロメチル）フェニル）−４−（（トリフルオロメチル）スルホニル）−１Ｈ−ピラゾール−３−カルボニトリル０．１５ｇ（０．３３ｍｍｏｌ）をエタノール４ｍＬに溶かし、３−メルカプトプロピオン酸０．０４ｇ（０．３６ｍｍｏｌ）と炭酸カリウム０．１ｇ（０．６９ｍｍｏｌ）を加え、７０℃で１時間加熱し、その後４時間還流させた。反応液の溶媒を留去後、水を加え、１Ｎ塩酸でｐＨ３にし、酢酸エチルで２回抽出した。酢酸エチル層を無水硫酸マグネシウムで脱水後、ろ過、濃縮し、シリカゲルカラム（ヘキサン：酢酸エチル＝７０：３０）で精製して、淡黄色結晶の３−［５−アミノ−３−シアノ−１−（２，６−ジクロロ−４−トリフルオロメチルフェニル）−１Ｈ−ピラゾール−４−イルチオ］プロピオン酸０．１２ｇ（収率８５．３％）を得た。
(2) Synthesis of 3- [5-amino-3-cyano-1- (2,6-dichloro-4-trifluoromethylphenyl) -1H-pyrazol-4-ylthio] propionic acid 5-amino-1- ( 2,5-dichloro-4- (trifluoromethyl) phenyl) -4-((trifluoromethyl) sulfonyl) -1H-pyrazole-3-carbonitrile 0.15 g (0.33 mmol) was dissolved in 4 mL of ethanol, 3 -Mercaptopropionic acid 0.04g (0.36mmol) and potassium carbonate 0.1g (0.69mmol) were added, and it heated at 70 degreeC for 1 hour, Then, it was made to recirculate | reflux for 4 hours. After the solvent of the reaction solution was distilled off, water was added, pH was adjusted to 3 with 1N hydrochloric acid, and extracted twice with ethyl acetate. The ethyl acetate layer was dehydrated with anhydrous magnesium sulfate, filtered, concentrated, and purified with a silica gel column (hexane: ethyl acetate = 70: 30) to give 3- [5-amino-3-cyano-1- 0.12 g (yield 85.3%) of (2,6-dichloro-4-trifluoromethylphenyl) -1H-pyrazol-4-ylthio] propionic acid was obtained.
１Ｈ ＮＭＲ （ＤＭＳＯ−ｄ６） δ ２．５６（２Ｈ，ｍ，ＣＨ２），３．２９（２Ｈ，ｍ，ＣＨ２），７．８５（２Ｈ，ｓ，ＮＨ２），７．９０（１Ｈ，ｓ，ＣＨ２），８．０１（１Ｈ，ｓ，ＣＨ），１２．４（１Ｈ，ｓ，ＣＯＯＨ） 1 H NMR (DMSO-d 6 ) δ 2.56 (2H, m, CH 2 ), 3.29 (2H, m, CH 2 ), 7.85 (2H, s, NH 2 ), 7.90 ( 1H, s, CH 2), 8.01 (1H, s, CH), 12.4 (1H, s, COOH)
＜実施例２＞（免疫原の調製）
免疫原としてウシ血清アルブミン（ＢＳＡ）と本発明フィプロニルハプテンとの結合体を、活性エステル法を用いて作製した。
<Example 2> (Preparation of immunogen)
A conjugate of bovine serum albumin (BSA) and the fipronil hapten of the present invention as an immunogen was prepared using the active ester method.
実施例１で製造した３−［５−アミノ−３−シアノ−１−（２，６−ジクロロ−４−トリフルオロメチルフェニル）−１Ｈ−ピラゾール−４−イルチオ］プロピオン酸（フィプロニルハプテン）２．１ｍｇ、Ｎ−ヒドロキシスクシンイミド１．２ｍｇおよび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１．９ｍｇを、Ｎ，Ｎ−ジメチルホルムアミド２００μＬに溶解し、この溶液を２５℃の暗所に１．５時間放置しフィプロニルハプテン溶液とした。 3- [5-Amino-3-cyano-1- (2,6-dichloro-4-trifluoromethylphenyl) -1H-pyrazol-4-ylthio] propionic acid (fipronyl hapten) prepared in Example 1 1 mg, 1.2 mg of N-hydroxysuccinimide and 1.9 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride were dissolved in 200 μL of N, N-dimethylformamide, The fipronil hapten solution was left for 1.5 hours.
別途、０．１Ｍホウ酸緩衝液（ｐＨ８．０）１ｍＬにＢＳＡを１０ｍｇ加え、一晩攪拌することにより、ＢＳＡ溶液を得た。 Separately, 10 mg of BSA was added to 1 mL of 0.1 M borate buffer (pH 8.0) and stirred overnight to obtain a BSA solution.
このＢＳＡ溶液に、先に調製したフィプロニルハプテン溶液を徐々に滴下し、暗所にて室温で１．５時間攪拌した。反応終了後、４℃で２日間生理的リン酸緩衝液（ＰＢＳ、１０ｍＭリン酸緩衝液、１５０ｍＭ ＮａＣｌ、ｐＨ７．０）に対して透析した後、−４０℃で貯蔵した。このようにして得られたフィプロニルハプテンとＢＳＡとの結合体を免疫原として使用した。 The previously prepared fipronil hapten solution was gradually added dropwise to this BSA solution and stirred at room temperature in the dark for 1.5 hours. After completion of the reaction, the mixture was dialyzed against physiological phosphate buffer (PBS, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0) at 4 ° C. for 2 days, and stored at −40 ° C. The conjugate of fipronil hapten and BSA thus obtained was used as an immunogen.
＜実施例３＞（固相化抗原の調製）
固相化抗原としてうさぎ血清アルブミン（ＲＳＡ）とフィプロニルハプテンとの結合体を、活性エステル法を用いて作製した。
<Example 3> (Preparation of immobilized antigen)
A conjugate of rabbit serum albumin (RSA) and fipronil hapten as a solid-phased antigen was prepared using the active ester method.
フィプロニルハプテン２．１ｍｇ、Ｎ−ヒドロキシスクシンイミド１．２ｍｇおよび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１．９ｍｇを、Ｎ，Ｎ−ジメチルホルムアミド２００μＬに溶解し、この溶液を２５℃の暗所に１．５時間放置しフィプロニルハプテン溶液とした。 2.1 mg of fipronil hapten, 1.2 mg of N-hydroxysuccinimide and 1.9 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride were dissolved in 200 μL of N, N-dimethylformamide. A fipronil hapten solution was left in a dark place at 25 ° C for 1.5 hours.
別途、０．１Ｍホウ酸緩衝液（ｐＨ８．０）１ｍＬにＲＳＡを１０ｍｇ加え、一晩攪拌することにより、ＲＳＡ溶液を得た。 Separately, 10 mg of RSA was added to 1 mL of 0.1 M borate buffer (pH 8.0) and stirred overnight to obtain an RSA solution.
このＲＳＡ溶液に、先に調製したフィプロニルハプテン溶液を徐々に滴下し、暗所にて室温で１．５時間攪拌した。反応終了後、４℃で一晩生理的リン酸緩衝液（ＰＢＳ、１０ｍＭリン酸緩衝液、１５０ｍＭ ＮａＣｌ、ｐＨ７．０）に対して透析した後、３０℃で貯蔵した。このようにして得られたフィプロニルハプテンとＲＳＡとの結合体を固相化抗原として使用した。 The fipronil hapten solution prepared previously was gradually added dropwise to this RSA solution, and the mixture was stirred at room temperature in the dark for 1.5 hours. After completion of the reaction, the mixture was dialyzed against physiological phosphate buffer (PBS, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0) overnight at 4 ° C., and stored at 30 ° C. The conjugate of fipronil hapten and RSA thus obtained was used as the immobilized antigen.
＜実施例４＞（モノクローナル抗体産生ハイブリドーマの作製）
実施例２で調製した免疫源を２ｍｇ／ｍＬとなるようにＰＢＳに溶解し、これに等量の完全アジュバント（商品名：フロイント完全アジュバント；ＦＣＡ）を等量混合しエマルジョン化し、その１００μＬを６〜７週齢のメスのＢＡＬＢ／Ｃマウスに腹腔投与した。これと同様の手順で、不完全アジュバント（商品名：フロイント不完全アジュバント；ＦＩＣＡ）を等量混合した０．５ｍｇ／ｍＬの免疫原１００μＬを２週間毎に追加免疫した。４回の免疫後、眼底から採血し、血清中の抗体力価を間接競合法で確認した。十分に力価が高くなったことを確認し、１週間後に最終免疫として免疫原１０μｇ／１００μＬ・ＰＢＳを尾静脈から投与した。その３日後に当該マウスから脾臓を摘出し細胞融合に供した。
<Example 4> (Production of monoclonal antibody-producing hybridoma)
The immunogen prepared in Example 2 was dissolved in PBS to 2 mg / mL, and an equal amount of complete adjuvant (trade name: Freund's complete adjuvant; FCA) was mixed in an equal amount to make an emulsion. It was administered intraperitoneally to 7-week-old female BALB / C mice. In the same procedure, booster immunization was performed every 2 weeks with 100 μL of 0.5 mg / mL immunogen mixed with an equal amount of incomplete adjuvant (trade name: Freund's incomplete adjuvant; FICA). After four immunizations, blood was collected from the fundus and the antibody titer in the serum was confirmed by the indirect competition method. After confirming that the titer was sufficiently high, an
摘出した脾臓を無血清ＤＭＥＭ培地（ダルベッコ改変イーグル培地）中で余分な組織片を切除したあと、脾臓から完全に細胞を取り出し、培地中に浮遊させた。浮遊している大きな組織片を沈降させるために５分間静置、細胞浮遊液を遠沈管に集め、１５００ｒｐｍで遠心し、上清を吸引除去して、新しい無血清ＤＭＥＭを添加して細胞を浮遊させた。この操作を２回繰り返した。 Excess tissue fragments were excised from the excised spleen in serum-free DMEM medium (Dulbecco's modified Eagle medium), and then the cells were completely removed from the spleen and suspended in the medium. Allow to settle for 5 minutes to settle large floating tissue pieces. Collect the cell suspension in a centrifuge tube, centrifuge at 1500 rpm, aspirate and remove the supernatant, and add new serum-free DMEM to float the cells. I let you. This operation was repeated twice.
あらかじめ培養してあったミエローマ細胞（Ｐ３Ｘ６３Ａｇ８．６５３）を回収し、遠沈、上清除去、無血清ＤＭＥＭ培地で再浮遊を２回繰り返した。 The myeloma cells (P3X63Ag8.653) that had been cultured in advance were collected, centrifuged, removed from the supernatant, and resuspended twice in serum-free DMEM medium.
それぞれの細胞数を計数して脾細胞とミエローマ細胞との比率が１０：１〜７．５：１になるように混合し、１５００ｒｐｍで５分間遠心して，上清を吸引除去した。 Each cell number was counted, mixed so that the ratio of spleen cells to myeloma cells was 10: 1 to 7.5: 1, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed by suction.
遠沈管を激しく攪拌しながら５０％ポリエチレングリコール（分子量１５００）溶液２ｍＬを約６０秒かけて添加した。次いで約１０ｍＬの無血清ＤＭＥＭを攪拌しながら３〜４分かけて添加した。 While vigorously stirring the centrifuge tube, 2 mL of a 50% polyethylene glycol (molecular weight 1500) solution was added over about 60 seconds. About 10 mL of serum-free DMEM was then added over 3-4 minutes with stirring.
遠沈管を１０００ｒｐｍ，５分で遠心して上清を完全に吸引除去し、脾細胞が２．５×１０６個／ｍＬになるようにＨＴ培地（ヒポキサンチン、チミジン、１０％牛胎児血清入ＤＭＥＭ培地）に浮遊させ、９６穴培養プレートに１００μＬ／ウェル分注し、３７℃、８％炭酸ガス、加湿条件下で培養を開始した。 The centrifuge tube is centrifuged at 1000 rpm for 5 minutes to completely remove the supernatant, and HT medium (Hypoxanthine, thymidine, 10% fetal bovine serum-containing DMEM is added so that the number of splenocytes becomes 2.5 × 10 6 cells / mL. The suspension was suspended in a medium), dispensed at 100 μL / well into a 96-well culture plate, and the culture was started under conditions of 37 ° C., 8% carbon dioxide and humidification.
翌日に約４０μＬ／ウェルのＨＡＴ培地（ヒポキサンチン、チミジン、アミノプテリン、１０％牛胎児血清入ＤＭＥＭ培地）を添加し、ミエローマ細胞が死滅し、ハイブリドーマ細胞のコロニーが形成されるまで観察を続け、以後は細胞の状態を見ながらＨＴ培地を添加した。 On the next day, add about 40 μL / well of HAT medium (hypoxanthine, thymidine, aminopterin, 10% fetal bovine serum-containing DMEM medium), and continue observation until myeloma cells die and hybridoma cell colonies form, Thereafter, HT medium was added while observing the state of the cells.
培養開始から１０日後に培養液を採取し、間接競合阻害法でフィプロニルに対する抗体を産生しているウェルを選別し、９６ウェル、４８ウェル、２４ウェルと順次培養スケールを上げた。 Ten days after the start of the culture, the culture medium was collected, and wells producing antibodies against fipronil were selected by the indirect competitive inhibition method, and the culture scale was sequentially increased to 96, 48, and 24 wells.
２４ウェルの段階で限界希釈法によるクローニングを行ない、フィプロニルに対するモノクローナル抗体産生ハイブリドーマ株 [ＦＰＮ−１Ｅ９−２５]を得た。 Cloning by the limiting dilution method was performed at the stage of 24 wells to obtain a hybridoma strain [FPN-1E9-25] producing a monoclonal antibody against fipronil.
＜実施例５＞（モノクローナル抗体の作製）
実施例４で得られたハイブリドーマ株を１０％牛胎児血清入りＤＭＥＭで培養し、約２×１０６個の細胞をＢａＬｂ／ｃ メスＲｅｔｉｒｅ マウスの腹腔内に注射し、腹水液を採取した。得られた腹水はプロテインＧ カラムによりＩｇＧ精製を行った。
<Example 5> (Preparation of monoclonal antibody)
The hybridoma strain obtained in Example 4 was cultured in DMEM containing 10% fetal bovine serum, about 2 × 10 6 cells were injected into the peritoneal cavity of BaLb / c female Retire mice, and ascites fluid was collected. The obtained ascites was subjected to IgG purification using a protein G column.
＜実施例６＞（フィプロニルハプテンと西洋ワサビペルオキシダーゼ（ＨＲＰ）との結合体の調製）
直接競合ＥＬＩＳＡにおいてトレーサーとして用いるため、活性エステル法によりフィプロニルハプテンと西洋ワサビペルオキシダーゼとの結合体を調製した。
<Example 6> (Preparation of conjugate of fipronil hapten and horseradish peroxidase (HRP))
A conjugate of fipronil hapten and horseradish peroxidase was prepared by the active ester method for use as a tracer in a direct competition ELISA.
フィプロニルハプテン２．１ｍｇ、Ｎ−ヒドロキシスクシンイミド１．２ｍｇおよび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１．９ｍｇを、Ｎ，Ｎ−ジメチルホルムアミド２００μＬに溶解し、この溶液を２５℃の暗所に１．５時間放置しフィプロニルハプテン溶液とした。 2.1 mg of fipronil hapten, 1.2 mg of N-hydroxysuccinimide and 1.9 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride were dissolved in 200 μL of N, N-dimethylformamide. A fipronil hapten solution was left in a dark place at 25 ° C for 1.5 hours.
別途、０．１Ｍホウ酸緩衝液（ｐＨ８．０）１ｍＬにＨＲＰを１０ｍｇ加え、一晩攪拌することにより、ＨＲＰ溶液を得た。 Separately, 10 mg of HRP was added to 1 mL of 0.1 M borate buffer (pH 8.0) and stirred overnight to obtain an HRP solution.
このＨＲＰ溶液に、先に調製したフィプロニルハプテン溶液を徐々に滴下し、暗所にて室温で１．５時間攪拌した。反応終了後、４℃で一晩生理的リン酸緩衝液（ＰＢＳ、１０ｍＭリン酸緩衝液、１５０ｍＭ ＮａＣｌ、ｐＨ７．０）に対して透析した後、３０℃で貯蔵した。ゲル濾過で精製を行い、濃度の測定はＤＣプロテインアッセイで行った。 The previously prepared fipronil hapten solution was gradually added dropwise to this HRP solution and stirred at room temperature in the dark for 1.5 hours. After completion of the reaction, the mixture was dialyzed against physiological phosphate buffer (PBS, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0) overnight at 4 ° C., and stored at 30 ° C. Purification was performed by gel filtration, and the concentration was measured by a DC protein assay.
＜実施例７＞（直接競合ＥＬＩＳＡ法によるフィプロニルの測定）
（１）抗マウスＩｇＧヤギ抗体をＰＢＳ緩衝液（１０ｍＭ ＮａＰＢ、１５０ｍＭ ＮａＣｌ）で１０μｇ／ｍＬに希釈し、９６穴マイクロプレートに１００μｇ／ウェルづつ分注し、４℃で一晩放置することにより固相化した。次に液を吸引除去後、ＰＢＳ緩衝液（０．４％ＢＳＡ、１０ｍＭ ＮａＰＢ、１５０ｍＭ ＮａＣｌ、ｐＨ７．０）を３００μｇ／ウェル分注し、４℃で一晩静置することによりブロッキングを行った後、ブロッキング液を吸引除去した。
<Example 7> (Measurement of fipronil by direct competitive ELISA method)
(1) Anti-mouse IgG goat antibody is diluted to 10 μg / mL with PBS buffer (10 mM NaPB, 150 mM NaCl), dispensed at 100 μg / well into a 96-well microplate, and allowed to stand overnight at 4 ° C. Phased. Next, after removing the solution by suction, blocking was performed by dispensing 300 μg / well of PBS buffer (0.4% BSA, 10 mM NaPB, 150 mM NaCl, pH 7.0) and allowing to stand at 4 ° C. overnight. Thereafter, the blocking solution was removed by suction.
（２）実施例５で得られたモノクローナル抗体ＦＰＮ−１Ｅ９−２５をＰＢＳ緩衝液（０．２％ＢＳＡ、１０ｍＭリン酸緩衝液、１５０ｍＭ ＮａＣｌ、ｐＨ７．０）で２．０μｇ／ｍＬに希釈し、（１）で作製した抗マウスＩｇＧヤギ抗体固相化プレートに１００μｇ／ウェルづつ分注した、２０℃で１時間静置した後、ＰＢＳで洗浄し抗体固相化プレートとした。 (2) The monoclonal antibody FPN-1E9-25 obtained in Example 5 was diluted to 2.0 μg / mL with PBS buffer (0.2% BSA, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0). The anti-mouse IgG goat antibody immobilized plate prepared in (1) was dispensed at 100 μg / well, allowed to stand at 20 ° C. for 1 hour, and then washed with PBS to obtain an antibody immobilized plate.
（３）ＨＲＰ（西洋ワサビペルオキシダーゼ）とフィプロニルハプテンの結合体をＰＢＳ緩衝液（０．４％ＢＳＡ、１０ｍＭリン酸緩衝液、１５０ｍＭ ＮａＣｌ、ｐＨ７．０）で０．２５μｇ／ｍＬに希釈しＨＲＰ希釈液とした。 (3) HRP dilution by diluting a conjugate of HRP (horseradish peroxidase) and fipronil hapten to 0.25 μg / mL with PBS buffer (0.4% BSA, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0) Liquid.
（４）上記ＨＲＰ希釈液と１０％メタノール溶液に溶解したフィプロニルの標準溶液（０．１、１．０、３．０、５．０、１０、３０、１００および１０００μｇ／ｍＬ）を等量混合し、その混合液の１００μｇ／ウェルを（２）で得られたＦＰＮ−１Ｅ９−２５抗体固相化プレートに加え、２０℃で１時間静置した後、ＰＢＳで洗浄した。 (4) Equal volume mixing of the above HRP diluted solution and fipronil standard solution (0.1, 1.0, 3.0, 5.0, 10, 30, 100 and 1000 μg / mL) dissolved in 10% methanol solution Then, 100 μg / well of the mixture was added to the FPN-1E9-25 antibody-immobilized plate obtained in (2), allowed to stand at 20 ° C. for 1 hour, and then washed with PBS.
（５）ＨＲＰ基質溶液（１００μｇ／ｍＬの３，３’，５，５’−テトラメチルベンチジンおよび０．００６％過酸化水素を添加した０．１Ｍ酢酸ナトリウム緩衝液（ｐＨ５．５））１００μＬをウェルに加え、２５℃で１０分間インキュベーションした後、１Ｎ硫酸１００μＬをウェルに加えて酵素反応を止め、４５０ｎｍの吸光度をマイクロプレートリーダーで測定した。 (5) HRP substrate solution (100 μg / mL of 3,3 ′, 5,5′-tetramethylbenzidine and 0.1 M sodium acetate buffer (pH 5.5) supplemented with 0.006% hydrogen peroxide) 100 μL Was added to the well and incubated at 25 ° C. for 10 minutes. Then, 100 μL of 1N sulfuric acid was added to the well to stop the enzyme reaction, and the absorbance at 450 nm was measured with a microplate reader.
モノクローナル抗体ＦＰＮ−１Ｅ９−２５溶液についての直接競合阻害法によるフィプロニルの標準阻害曲線を図１に示す。モノクローナル抗体ＦＰＮ−１Ｅ９−２５溶液を用いた直接競合ＥＬＩＳＡ法によるフィプロニルの測定可能な範囲は４．５ｎｇ／ｍＬ〜８０ｎｇ／ｍＬであり、５０％阻害を示す値（ＩＣ５０値）は１６．３ｎｇ／ｍＬであった。 A standard inhibition curve of fipronil by the direct competitive inhibition method for the monoclonal antibody FPN-1E9-25 solution is shown in FIG. The measurable range of fipronil by the direct competitive ELISA method using the monoclonal antibody FPN-1E9-25 solution is 4.5 ng / mL to 80 ng / mL, and the value indicating 50% inhibition (IC50 value) is 16.3 ng / mL.
以上の結果から、本発明のフィプロニル誘導体を用いて得られるモノクローナル抗体を使用する直接競合ＥＬＩＳＡ法により、フィプロニル分析の大幅な簡略化および測定時間の短縮が可能となり、多数の検体を迅速、簡便且つ低コストで測定できることがわかる。 From the above results, the direct competitive ELISA method using the monoclonal antibody obtained by using the fipronil derivative of the present invention can greatly simplify the fipronil analysis and shorten the measurement time, and can quickly and easily measure a large number of samples. It turns out that it can measure at low cost.
＜実施例８＞（抗体のメタノール耐性）
抗体ＦＰＮ−１Ｅ９−２５のメタノール耐性を調べた。実施例７の（４）において、１０％メタノールに対して各設定濃度のメタノール溶液（１，５，１０，１５，２０，３０，４０％）に溶解したフィプロニル標準液（３．２５，７．５，１５，３０，６０ｎｇ/ｍＬ）のＩＣ５０値を求めた。
<Example 8> (Methanol tolerance of antibody)
The methanol resistance of the antibody FPN-1E9-25 was examined. In Example 7 (4), fipronil standard solution (3.25,7.6) dissolved in methanol solution (1, 5, 10, 15, 20, 30, 40%) of each set concentration with respect to 10% methanol. IC50 values of 5, 15, 30, 60 ng / mL) were determined.
その結果を図２に示す。フィプロニル抗体ＦＰＮ−１Ｅ９−２５は３０％メタノール中でもＩＣ５０値が１５ｎｇ／ｍＬと高い反応性を示し、メタノールに対し高い耐性を有していた。農作物中あるいは土壌中に残留しているフィプロニルを抽出する際、メタノールは一般的に使用される非常に優れた溶媒であり、メタノールに対し高い耐性を有することは残留フィプロニル測定用の抗体としてＦＰＮ−１Ｅ９−２５は極めて有用であることを示している。 The result is shown in FIG. The fipronil antibody FPN-1E9-25 showed a high reactivity with an IC50 value of 15 ng / mL even in 30% methanol, and had high resistance to methanol. When extracting fipronil remaining in crops or soil, methanol is a very good solvent that is generally used, and having high resistance to methanol is FPN- as an antibody for measuring residual fipronil. 1E9-25 has been shown to be very useful.
Claims (9)
で表わされる構造を有する化合物。 Following formula (1):
A compound having a structure represented by:
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| CN107505421A (en) * | 2017-09-25 | 2017-12-22 | 重庆市农业科学院 | The rapid extraction and purification method of ethiprole and its metabolite residue in a kind of birds, beasts and eggs |
| CN109696548A (en) * | 2017-10-23 | 2019-04-30 | 北京维德维康生物技术有限公司 | A kind of kit and its preparation method and application detecting Fipronil |
| CN111060690A (en) * | 2019-10-08 | 2020-04-24 | 杭州佰昕科技有限公司 | Time-resolved fluorescence immunoassay kit for detecting olaquindox and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH11147878A (en) * | 1997-11-19 | 1999-06-02 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of iprodione and metabolite of iprodione, antibody and determination |
| JP2001139552A (en) * | 1999-11-11 | 2001-05-22 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Haptenic compound and antibody to acetamiprid and method for assaying |
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| JPH11147878A (en) * | 1997-11-19 | 1999-06-02 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of iprodione and metabolite of iprodione, antibody and determination |
| JP2001139552A (en) * | 1999-11-11 | 2001-05-22 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Haptenic compound and antibody to acetamiprid and method for assaying |
Cited By (3)
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|---|---|---|---|---|
| CN107505421A (en) * | 2017-09-25 | 2017-12-22 | 重庆市农业科学院 | The rapid extraction and purification method of ethiprole and its metabolite residue in a kind of birds, beasts and eggs |
| CN109696548A (en) * | 2017-10-23 | 2019-04-30 | 北京维德维康生物技术有限公司 | A kind of kit and its preparation method and application detecting Fipronil |
| CN111060690A (en) * | 2019-10-08 | 2020-04-24 | 杭州佰昕科技有限公司 | Time-resolved fluorescence immunoassay kit for detecting olaquindox and application thereof |
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