JP2004323508A - Anti-heavy metal monoclonal antibody and method of using the same - Google Patents
Anti-heavy metal monoclonal antibody and method of using the same Download PDFInfo
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- JP2004323508A JP2004323508A JP2004083097A JP2004083097A JP2004323508A JP 2004323508 A JP2004323508 A JP 2004323508A JP 2004083097 A JP2004083097 A JP 2004083097A JP 2004083097 A JP2004083097 A JP 2004083097A JP 2004323508 A JP2004323508 A JP 2004323508A
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Abstract
【課題】 環境汚染物質としてカドミウムおよび／または水銀を免疫学的に検出・定量するためモノクローナル抗体およびこれを用いる免疫学的方法を提供する。
【解決手段】 カドミウム錯体または水銀錯体をタンパク質などのキャリアと結合させ、抗原として用いてモノクローナル抗体を作製し、得られたモノクローナル抗体を用いてカドミウムおよび／または水銀を錯体として検出・定量する。PROBLEM TO BE SOLVED: To provide a monoclonal antibody for immunologically detecting and quantifying cadmium and / or mercury as an environmental pollutant and an immunological method using the same.
SOLUTION: A cadmium complex or a mercury complex is bound to a carrier such as a protein, and a monoclonal antibody is prepared using the antigen as an antigen. Cadmium and / or mercury is detected and quantified as a complex using the obtained monoclonal antibody.
Description
本発明は、重金属としてカドミウムまたはカドミウムと水銀を特異的に認識するモノクローナル抗体、並びにその使用方法に関する。詳細には、カドミウム錯体またはカドミウム錯体と水銀錯体を特異的に認識しうるモノクローナル抗体、および該抗体を用いるカドミウムおよび／または水銀を定性的または定量的に検出する方法に関する。 The present invention relates to a monoclonal antibody that specifically recognizes cadmium or cadmium and mercury as heavy metals, and a method of using the same. More specifically, the present invention relates to a monoclonal antibody capable of specifically recognizing a cadmium complex or a cadmium complex and a mercury complex, and a method for qualitatively or quantitatively detecting cadmium and / or mercury using the antibody.
近年、環境保全などの社会的な環境意識や健康に対する影響への関心の高まりから、産業や生活に伴う様々な場面における環境汚染物質の排出・蓄積の動向が注視されている。環境汚染物質の中でも重金属類、特にカドミウムや水銀は過去にその毒性による重篤な問題を起こしていることもあり、公的機関により飲料水や地下水における水質基準、土壌における環境基準、環境への排出基準が設けられている。このため、これらの基準を満たしているかどうかを種々の検査対象についてカドミウムや水銀の測定が行われているが、通常はガスクロマトグラフィーや質量分析法などが用いられる。しかし、これらの方法は高価な測定機器が必要であり、また測定も測定機器を設置した施設で行う必要がある。このため、環境汚染物質としてのカドミウムや水銀を現場で簡易的に測定する手段が求められていた。 2. Description of the Related Art In recent years, attention has been paid to trends in the emission and accumulation of environmental pollutants in various scenes associated with industry and daily life due to an increase in social environmental consciousness such as environmental protection and an interest in health. Among environmental pollutants, heavy metals, especially cadmium and mercury, have caused serious problems due to their toxicity in the past, and water quality standards for drinking water and groundwater, environmental standards for soil, environmental Emission standards are established. For this reason, cadmium and mercury are measured for various test objects to determine whether or not these standards are satisfied. Usually, gas chromatography or mass spectrometry is used. However, these methods require expensive measurement equipment, and the measurement also needs to be performed at a facility where the measurement equipment is installed. For this reason, means for simply measuring cadmium and mercury as environmental pollutants on site has been required.
一方、免疫反応を利用した測定法としては、ラジオイムノアッセイ（ＲＩＡ）、蛍光イムノアッセイ（ＦＩＡ）、免疫発光測定法、エンザイムイムノアッセイ（ＥＩＡまたはＥＬＩＳＡ）などの方法が知られている。また、微量物質の測定法としては、間接蛍光抗体法、競合アッセイ法の他に蛍光センサーを利用する方法（非特許文献１）などがあり、その応用範囲は広い。近年、環境汚染物質の測定にも免疫学的測定方法が適用されており、環境汚染物質として内分泌攪乱物質または環境ホルモンを簡易かつ高感度に測定する方法が報告されている（特許文献１）。しかし、測定すべき環境汚染物質がカドミウムなどの金属元素やそのイオンの場合、一般的にはそれ自体が抗原性を持たないため、カドミウムおよびそのイオンを抗原として用いること不可能であると考えられており、カドミウムなどの重金属を特異的に認識する抗体は知られていない。 On the other hand, methods such as radioimmunoassay (RIA), fluorescence immunoassay (FIA), immunoluminescence measurement, and enzyme immunoassay (EIA or ELISA) are known as measurement methods utilizing an immune reaction. As a method for measuring a trace substance, there are a method using a fluorescent sensor in addition to an indirect fluorescent antibody method and a competitive assay method (Non-Patent Document 1), and the application range is wide. In recent years, immunological measurement methods have also been applied to the measurement of environmental pollutants, and a method for simply and highly sensitively measuring endocrine disrupters or environmental hormones as environmental pollutants has been reported (Patent Document 1). However, if the environmental pollutant to be measured is a metal element such as cadmium or its ions, it is generally considered impossible to use cadmium and its ions as antigens, since they themselves do not have antigenicity. And no antibody that specifically recognizes heavy metals such as cadmium is known.
従って、本発明の課題は、環境汚染物質としての重金属であるカドミウムや水銀を免疫学的に検出・定量しうる方法およびこれに用いるカドミウムを特異的に認識するモノクローナル抗体を提供することである。 Therefore, an object of the present invention is to provide a method capable of immunologically detecting and quantifying cadmium and mercury, which are heavy metals as environmental pollutants, and a monoclonal antibody specifically recognizing cadmium used in the method.
そこで、本発明者らは、抗カドミウム抗体、抗水銀抗体としてこれらの錯体を特異的に認識しうる抗体は作成可能であると考えて鋭意研究を行ったところ、それ自体では抗原としては小さすぎるが該錯体とタンパク質を結合して高分子量化することによりカドミウムや水銀を特異的に認識するモノクローナル抗体を作成しうることを見出した。 Therefore, the present inventors have conducted intensive studies on the assumption that an antibody capable of specifically recognizing these complexes as an anti-cadmium antibody and an anti-mercury antibody can be prepared, and as such, it is too small as an antigen. Found that a monoclonal antibody that specifically recognizes cadmium or mercury can be prepared by binding the complex to a protein to increase the molecular weight.
従って、本発明は、
［１］重金属としてカドミウムまたはカドミウムと水銀を特異的に認識するモノクローナル抗体；
［２］錯体を形成した重金属を特異的に認識する前記［１］記載のモノクローナル抗体；
［３］受託番号ＦＥＲＭ Ｐ−１９２４０として寄託されたハイブリドーマにより産生される前記［１］または［２］記載のモノクローナル抗体；
［４］受託番号ＦＥＲＭ Ｐ−１９７０３として寄託されたハイブリドーマにより産生される前記［１］または［２］記載のモノクローナル抗体；
［５］前記［１］〜［４］のいずれかに記載のモノクローナル抗体を産生するハイブリドーマ；
［６］試料中のカドミウムおよび／または水銀を定性的または定量的に測定する免疫学的方法において、前記［１］〜［４］のいずれかに記載のモノクローナル抗体を用いることを特徴とする方法；並びに
［７］前記［１］〜［４］のいずれかに記載のモノクローナル抗体を含む、試料中のカドミウムおよび／または水銀を定性的または定量的に測定するためのキット；
に関する。
Therefore, the present invention
[1] a monoclonal antibody that specifically recognizes cadmium or cadmium and mercury as heavy metals;
[2] the monoclonal antibody of [1], which specifically recognizes a complexed heavy metal;
[3] the monoclonal antibody of [1] or [2], which is produced by a hybridoma deposited under accession number FERM P-19240;
[4] the monoclonal antibody of [1] or [2], which is produced by a hybridoma deposited under accession number FERM P-19703;
[5] a hybridoma that produces the monoclonal antibody according to any of [1] to [4];
[6] An immunological method for qualitatively or quantitatively measuring cadmium and / or mercury in a sample, wherein the monoclonal antibody according to any of the above [1] to [4] is used. And [7] a kit for qualitatively or quantitatively measuring cadmium and / or mercury in a sample, comprising the monoclonal antibody according to any of the above [1] to [4];
About.
本発明のモノクローナル抗体は、重金属としてカドミウムおよび／または水銀の法的規準（例えば環境基準、水質基準など）以下の検出限界を有し、これら重金属について環境基準濃度の測定が可能であることが好ましい。具体的には、抗カドミウム抗体は、その検出限界が排水基準の１００ｐｐｂ以下、好ましくは環境基準の１０ｐｐｂ以下であることが好ましい。抗水銀抗体は、検出限界が環境基準である０.５ｐｐｂ以下であることが好ましい。 The monoclonal antibody of the present invention has a detection limit below the legal standards of cadmium and / or mercury as heavy metals (for example, environmental standards, water quality standards, and the like), and it is preferable that the concentrations of these heavy metals can be measured based on the environmental standards. . Specifically, the detection limit of the anti-cadmium antibody is preferably 100 ppb or less based on a wastewater standard, and more preferably 10 ppb or less based on an environmental standard. The anti-mercury antibody preferably has a detection limit of 0.5 ppb or less, which is an environmental standard.
また、本発明の抗カドミウムモノクローナル抗体は、正確な測定を与えるためにカルシウム、マグネシウム、銅、鉄、ニッケル、鉛、亜鉛などのその他の金属とはほとんどまたは全く交差反応せず、カドミウム以外の重金属との親和性はカドミウムの１０％以下、好ましくは１％以下である。 In addition, the anti-cadmium monoclonal antibody of the present invention has little or no cross-reactivity with other metals such as calcium, magnesium, copper, iron, nickel, lead, and zinc, and provides heavy metals other than cadmium to provide accurate measurements. Has an affinity for cadmium of 10% or less, preferably 1% or less.
また、本発明の抗水銀モノクローナル抗体は、同様にカルシウム、マグネシウム、銅、鉄、ニッケル、鉛、亜鉛などのその他の金属とはほとんどまたは全く交差反応せず、カドミウムを除くその他の重金属との親和性は１０％以下、好ましくは１％以下である。また、この抗水銀モノクロナール抗体は、同時にカドミウムとも反応し、水銀を除くその他の金属即ちカルシウム、マグネシウム、銅、鉄、ニッケル、鉛、亜鉛などのその他の金属とは区別して検出することができる。 Similarly, the anti-mercury monoclonal antibody of the present invention has little or no cross-reactivity with other metals such as calcium, magnesium, copper, iron, nickel, lead, and zinc, and has an affinity for other heavy metals except cadmium. The property is 10% or less, preferably 1% or less. In addition, this anti-mercury monoclonal antibody simultaneously reacts with cadmium and can be detected separately from other metals except mercury, that is, calcium, magnesium, copper, iron, nickel, lead, and zinc. .
本発明の抗カドミウムモノクローナル抗体としては、カドミウムを特異的に認識するモノクローナル抗体、特に錯体を形成したカドミウムを特異的に認識するモノクローナル抗体であればいずれの抗体でもよいが、例えばＳｏ２５Ａ１、Ｓｏ２１Ｄ５、Ｓｏ２６Ｇ８が挙げられる。これらのモノクローナル抗体はカドミウムやその他の金属に対してほとんど同じ特性を示すため、即ち主にカドミウムと反応してカルシウム、マグネシウム、銅、鉄、水銀、ニッケル、鉛、亜鉛などのその他の金属とはほとんどまたは全く交差反応しないという特性を示すため、同一の親細胞に由来するハイブリドーマから産生されるものと考えられる。なお、モノクローナル抗体Ｓｏ２６Ｇ８を産生するハイブリドーマは、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１５年２月２７日付けで受託番号ＦＥＲＭ Ｐ−１９２４０として寄託されている。 The anti-cadmium monoclonal antibody of the present invention may be any monoclonal antibody specifically recognizing cadmium, in particular, any monoclonal antibody specifically recognizing complexed cadmium, such as So25A1, So21D5, and So26G8. Is mentioned. These monoclonal antibodies show almost the same properties for cadmium and other metals, i.e., reacting mainly with cadmium to separate other metals such as calcium, magnesium, copper, iron, mercury, nickel, lead and zinc. It exhibits little or no cross-reactivity and is considered to be produced from hybridomas derived from the same parental cell. The hybridoma producing the monoclonal antibody So26G8 has been deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the accession number FERM P-19240 on February 27, 2003.
また、本発明の抗水銀抗体としては、水銀を特異的に認識するモノクローナル抗体、特に錯体を形成した水銀を特異的に認識するモノクローナル抗体であればいずれの抗体でもよいが、例えばモノクローナル抗体Ｎｘ２２Ｃ３が挙げられる。モノクローナル抗体Ｎｘ２２Ｃ３を産生するハイブリドーマは、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１６年２月２６日付けで受託番号ＦＥＲＭ Ｐ−１９７０３として寄託されている。 The anti-mercury antibody of the present invention may be any monoclonal antibody that specifically recognizes mercury, particularly any antibody that specifically recognizes complexed mercury. For example, monoclonal antibody Nx22C3 may be used. No. The hybridoma producing the monoclonal antibody Nx22C3 has been deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the accession number FERM P-19703 on February 26, 2004.
本発明のモノクローナル抗体を用いるカドミウムおよび／または水銀を定性的または定量的に測定する免疫学的方法において、カドミウムと水銀はキレート剤に配位させ、形成された錯体を本発明のモノクローナル抗体により検出・測定する。故に、本発明は、（ｉ）試験試料にキレート剤を添加して錯体を形成させ、（ii）該錯体を特異的に認識する抗体を用いる免疫学的手法によりカドミウムおよび／または水銀を定性的または定量的に測定する方法に関する。本発明のモノクローナル抗体は、前述のようにカドミウムや水銀に対して親和性が高く、且つ他の金属との交差反応性が低いため、試験試料中のカドミウムや水銀をより正確に測定することができる。 In the immunological method for qualitatively or quantitatively measuring cadmium and / or mercury using the monoclonal antibody of the present invention, cadmium and mercury are coordinated with a chelating agent, and the formed complex is detected by the monoclonal antibody of the present invention. ·Measure. Therefore, the present invention provides a method for qualitatively converting cadmium and / or mercury by an immunological technique using (i) a chelating agent added to a test sample to form a complex, and (ii) an antibody that specifically recognizes the complex. Or, it relates to a method of quantitatively measuring. As described above, the monoclonal antibody of the present invention has a high affinity for cadmium and mercury and a low cross-reactivity with other metals, so that cadmium and mercury in a test sample can be measured more accurately. it can.
その中でも、本発明のモノクローナル抗体Ｎｘ２２Ｃ３は、水銀とカドミウムの双方に対して非常に高い親和性・特異性を有している。そこで、試験試料にあらかじめＳＨ基含有物質（例えば、メルカプトエタノールやグルタチオンなどのＳＨ基を有する化合物、あるいは牛血清アルブミンなどシステイン残基を含むタンパク質）を添加して、そのＳＨ基と水銀を共有結合させる。次いで、ＳＨ基含有物質で前処理した試料と前処理しない試料についてＮｘ２２Ｃ３抗体を用いて測定することにより、試験試料中に水銀およびカドミウムが存在するか否かを区別して判定し、かつ２つの測定値から水銀とカドミウムの各々の濃度を算出することができる。従って、本発明は、試験試料にＳＨ基含有物質を添加して前処理すること、およびその前処理した試料と前処理しない試料について、モノクローナル抗体Ｎｘ２２Ｃ３を用いて金属イオン濃度を測定し、その測定結果から試験試料中に水銀またはカドミウムの存在・不存在を判定し、かつ水銀とカドミウムの各々について同時に濃度を測定する方法に関する。 Among them, the monoclonal antibody Nx22C3 of the present invention has very high affinity and specificity for both mercury and cadmium. Therefore, an SH group-containing substance (for example, a compound having an SH group such as mercaptoethanol or glutathione, or a protein containing a cysteine residue such as bovine serum albumin) is added to the test sample in advance, and the SH group and the mercury are covalently bonded. Let it. Then, by using an Nx22C3 antibody to measure a sample pretreated with an SH group-containing substance and a sample not pretreated, it is determined whether mercury and cadmium are present in the test sample, and two determinations are made. From the values, the respective concentrations of mercury and cadmium can be calculated. Accordingly, the present invention provides a method for pre-treating a test sample by adding an SH group-containing substance, and measuring the metal ion concentration of the pre-treated sample and the untreated sample using the monoclonal antibody Nx22C3, The present invention relates to a method for judging the presence / absence of mercury or cadmium in a test sample from a result and simultaneously measuring the concentrations of mercury and cadmium.
本発明において用いる免疫学的手法としては、本発明のモノクローナル抗体を用いればいずれでも良いが、例えば免疫クロマトグラフィー、ラジオイムノアッセイ（ＲＩＡ）、蛍光イムノアッセイ（ＦＩＡ）、免疫発光測定法、エンザイムイムノアッセイ（ＥＩＡまたはＥＬＩＳＡ）、ＣＬＥＩＡ（化学発光酵素免疫測定法）、免疫比濁法（ＴＩＡ）、ラテックス免疫比濁法（ＬＴＩＡ）、蛍光センサー法などの方法が挙げられる。 As the immunological technique used in the present invention, any method may be used as long as the monoclonal antibody of the present invention is used. For example, immunochromatography, radioimmunoassay (RIA), fluorescence immunoassay (FIA), immunoluminescence assay, enzyme immunoassay (EIA) Or ELISA), CLEIA (chemiluminescent enzyme immunoassay), immunoturbidimetry (TIA), latex immunoturbidimetry (LTIA), a fluorescence sensor method, and the like.
本発明のカドミウムおよび／または水銀を定性的または定量的に測定するためのキットは、本発明の抗カドミウムあるいは抗水銀のモノクローナル抗体のみから構成されていてもよいが、他の試薬例えばキレート剤、キレート剤−タンパク質複合体、ポジティブコントロール試料、ネガティブコントロール試料などを包含してもよい。また、モノクローナル抗体、キレート剤−タンパク質複合体のいずれか、または両方が標識されていてもよい。さらに、本発明のキットは、モノクローナル抗体を含め必要な試薬がフィルターなどに吸着されている免疫クロマトグラフィー装置（例えば、試験紙）の形態でもよい。尚、免疫クロマトグラフィー装置には、モノクローナル抗体あるいはキレート剤−タンパク質複合体のいずれか一方が固定され、他方が流動可能に供給されれば良い。 The kit for qualitatively or quantitatively measuring cadmium and / or mercury of the present invention may be composed of only the anti-cadmium or anti-mercury monoclonal antibody of the present invention, but other reagents such as a chelating agent, A chelating agent-protein complex, a positive control sample, a negative control sample and the like may be included. In addition, one or both of the monoclonal antibody and the chelator-protein complex may be labeled. Further, the kit of the present invention may be in the form of an immunochromatography apparatus (for example, test paper) in which necessary reagents including monoclonal antibodies are adsorbed on a filter or the like. It is sufficient that either the monoclonal antibody or the chelating agent-protein complex is immobilized on the immunochromatography apparatus and the other is supplied in a flowable manner.
本発明によれば、環境汚染物質として注目されている重金属のカドミウムや水銀を特異的に認識するモノクローナル抗体およびこれを用いるカドミウムおよび／または水銀を検出・定量するための免疫学的方法、該方法に用いるキット（装置）が提供される。本発明のモノクローナル抗体はカドミウムおよび／または水銀に対して高い親和性・特異性を有するため微量のこれら重金属を検出することができ、環境への排出基準のみならず、高い検出感度が要求される水質基準や環境基準に対する判定にも用いることができる。また、この方法は免疫学的手法を用いるため、環境調査の現場でカドミウムおよび／または水銀を迅速且つ簡便に測定できる。 According to the present invention, a monoclonal antibody that specifically recognizes cadmium or mercury, a heavy metal that is attracting attention as an environmental pollutant, and an immunological method for detecting and quantifying cadmium and / or mercury using the monoclonal antibody, and the method A kit (apparatus) for use in is provided. Since the monoclonal antibody of the present invention has a high affinity and specificity for cadmium and / or mercury, it can detect trace amounts of these heavy metals, and is required to have high detection sensitivity as well as an emission standard for the environment. It can also be used to determine water quality standards and environmental standards. In addition, since this method uses an immunological technique, cadmium and / or mercury can be measured quickly and easily at the site of an environmental investigation.
＜抗原＞
本発明では、カドミウムイオン、水銀イオン単独では抗原性を持たないため、抗カドミウム抗体および抗水銀抗体を作成するためこれら重金属をキレート剤に配位させ、形成された金属錯体を抗原として用いた。キレート剤としては、カドミウムや水銀を配位しうるものであれば任意のキレート剤を用いることができるが、例えばエチレンジアミンテトラ酢酸（ＥＤＴＡ）、ジエチレントリアミンペンタ酢酸（ＤＴＰＡ）、テトラエチレントリアミン（ＴＥＴ）、エチレンジアミン（ＥＤＡ）、ジエチレントリアミン（ＤＥＴＡ）、クエン酸、シュウ酸、クラウンエーテル、ニトリロテトラ酢酸、エデト酸二ナトリウム、エデト酸ナトリウム、エデト酸三ナトリウム、ペニシラミン、ペンテテートカルシウム三ナトリウム、ペンテト酸、スクシメルおよびエデト酸トリエンチンを挙げることができるが、好ましくはＥＤＴＡである。
<Antigen>
In the present invention, since cadmium ions and mercury ions alone do not have antigenicity, these heavy metals are coordinated to a chelating agent to prepare anti-cadmium antibodies and anti-mercury antibodies, and the formed metal complexes are used as antigens. As the chelating agent, any chelating agent can be used as long as it can coordinate cadmium or mercury. For example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), tetraethylenetriamine (TET), Ethylenediamine (EDA), diethylenetriamine (DETA), citric acid, oxalic acid, crown ether, nitrilotetraacetic acid, disodium edetate, sodium edetate, trisodium edetate, penicillamine, trisodium pentetate calcium, pentetate, succimer and Trientine edetate can be mentioned, but EDTA is preferred.
また、カドミウム錯体、例えばカドミウムとＥＤＴＡの錯体（以後、Ｃｄ−ＥＤＴＡと略記する）、水銀錯体、例えば水銀とＥＤＴＡの錯体（以後、Ｈｇ−ＥＤＴＡと略記する）では免疫応答を誘導するには分子として小さすぎるため、キャリアとなる高分子量物質に結合させ、これを抗原または免疫源として用いる。キャリアとして用いることができる高分子量物質の例としては多糖類、タンパク質などが挙げられるが、タンパク質が好ましい。アルブミン、オバルブミン、ヘモシアニン、グロブリン、ゼラチン、コラーゲンなどが挙げられるが、これらに限定されない。 In addition, a cadmium complex such as a complex of cadmium and EDTA (hereinafter abbreviated as Cd-EDTA) and a mercury complex such as a complex of mercury and EDTA (hereinafter abbreviated as Hg-EDTA) require a molecule to induce an immune response. Is too small to bind to a high molecular weight substance as a carrier, and this is used as an antigen or an immunogen. Examples of high molecular weight substances that can be used as carriers include polysaccharides and proteins, but proteins are preferred. Examples include, but are not limited to, albumin, ovalbumin, hemocyanin, globulin, gelatin, collagen and the like.
これら金属錯体とタンパク質の複合体を作成するには、タンパク質と結合しうる官能基を有するキレート剤または該官能基を導入したキレート剤を用いるか、あるいはリンカーを介してタンパク質とキレート剤を結合させることができる。そのようなキレート剤は市販されており、例えばイソチオシアノベンジル−ＥＤＴＡ（同仁化学）が挙げられる。複合体の形成は常法により行うことができる。 In order to form a complex of the metal complex and the protein, a chelating agent having a functional group capable of binding to the protein or a chelating agent having the functional group introduced therein is used, or the protein and the chelating agent are bound via a linker. be able to. Such chelating agents are commercially available and include, for example, isothiocyanobenzyl-EDTA (Dojindo). The formation of the complex can be performed by a conventional method.
＜抗重金属モノクローナル抗体の作製＞
マウス免疫、ハイブリドーマの作製およびその培養などの一連のモノクローナル抗体の作製は、常法に従って、例えばモノクローナル抗体作製マニュアル、多田ら著、学際企画発行、１９９５年（ISBN 4-906514-19-7）を参照して適宜行うことができ、免疫するマウスの系統、脾臓細胞と融合させるミエローマのなども特に限定されない。
<Preparation of anti-heavy metal monoclonal antibody>
The production of a series of monoclonal antibodies, such as mouse immunization, production of hybridomas, and culture thereof, is carried out according to a conventional method. For example, a manual for producing monoclonal antibodies, published by Tada et al. The strain can be appropriately performed with reference to the mouse strain, and the strain of the mouse to be immunized, the myeloma to be fused with spleen cells, and the like are not particularly limited.
＜抗体生産細胞のスクリーニング＞
抗体生産細胞のスクリーニングには一般的にはＥＬＩＳＡ法を用いるが、より少ない抗体量でスクリーニングが可能であるため、蛍光センサー法（特許文献１）を用いるのが好ましい。この方法は、一次スクリーニングおよび二次スクリーニングからなり、一次スクリーニングは担体に固定化した抗原（カドミウム錯体−タンパク質複合体または水銀錯体−タンパク質複合体）に抗体を含有するハイブリドーマ培養上清を添加し、固定化抗原に結合した抗体を蛍光標識した二次抗体（抗マウスＩｇＧ抗体）を用いて蛍光センサーで検出する。二次スクリーニングでは、培養上清に過剰量の金属錯体を添加して平衡化した後、錯体と結合しなかった抗体の固定化抗原との結合を一次スクリーニングと同様に測定する。一次スクリーニングと二次スクリーニングの差から所望の金属錯体と特異的に結合する抗体を特定する。
<Screening of antibody-producing cells>
In general, ELISA is used to screen antibody-producing cells. However, since screening can be performed with a smaller amount of antibody, it is preferable to use a fluorescent sensor method (Patent Document 1). This method comprises a primary screening and a secondary screening. In the primary screening, a hybridoma culture supernatant containing an antibody is added to an antigen (cadmium complex-protein complex or mercury complex-protein complex) immobilized on a carrier, The antibody bound to the immobilized antigen is detected by a fluorescent sensor using a fluorescent-labeled secondary antibody (anti-mouse IgG antibody). In the secondary screening, an excess amount of the metal complex is added to the culture supernatant to equilibrate, and then the binding of the antibody not bound to the complex to the immobilized antigen is measured in the same manner as in the primary screening. An antibody that specifically binds to a desired metal complex is specified based on the difference between the primary screening and the secondary screening.
＜モノクローナル抗体の産生および精製＞
特定したクローンを培養し、単離したコロニーは徐々に培地量を増やしながら継代する。得られた培地は、脱塩カラムを用いて培地自体に含まれる若干の重金属を除去するのが好ましい。さらに、培地中に他のタンパク質の影響を除くために、抗体を精製する方法、例えばプロテインＡアフィニティーカラムクロマトグラフィーにより抗体を精製するのが好ましい。
<Production and purification of monoclonal antibody>
The specified clone is cultured, and the isolated colonies are subcultured while gradually increasing the amount of medium. It is preferable to remove some heavy metals contained in the medium itself using a desalting column from the obtained medium. Furthermore, in order to eliminate the influence of other proteins in the medium, it is preferable to purify the antibody by a method for purifying the antibody, for example, protein A affinity column chromatography.
＜モノクローナル抗体の評価＞
得られたモノクローナル抗体は、カドミウム錯体、水銀錯体と他の金属の錯体との親和性を比較することによりその抗体の抗原に対する親和性および特異性を評価する。親和性は、抗体の抗原との結合を５０％阻害する抗原濃度（ＩＣ５０）により評価する。そして、特異性は、カドミウムや水銀に対するＩＣ５０と他の金属に対するＩＣ５０を比較することにより評価する。ＩＣ５０はいずれの方法で算出してもよいが、測定結果をソフトウェア（例えばOrigin Version 6.0など）により解析して求めてもよい。例えば、蛍光センサー法などで得られた測定値は、抗原を加えなかったときの測定値を１００％として相対値に変換した後、抗体と抗原の結合曲線の近似式：
ｙ＝９９／（１＋（ｘ／Ｐ１）Ｐ２）＋０.５
［式中、ｘは抗体量であり、ｙは抗体と抗原の結合量（％）であり、Ｐ１およびＰ２は近似のパラメーターである］に導入する。また、Ｐ１およびＰ２はソフトフェアにより決定し、得られた結合曲線から、ｙ＝５０になるときのｘの値（＝Ｐ１）をＩＣ５０とする。また、抗体の特異性はカドミウムや水銀に対するＩＣ５０と他の金属に対するＩＣ５０との比を交差反応性として求める。
<Evaluation of monoclonal antibodies>
The affinity and specificity of the obtained monoclonal antibody to the antigen are evaluated by comparing the affinity of the cadmium complex or mercury complex with the complex of another metal. Affinity is assessed by the antigen concentration (IC 50 ) that inhibits the binding of the antibody to the antigen by 50%. The specificity is evaluated by comparing the IC 50 for cadmium and mercury with the IC 50 for other metals. The IC 50 may be calculated by any method, or may be obtained by analyzing the measurement result using software (for example, Origin Version 6.0). For example, a measurement value obtained by a fluorescence sensor method or the like is converted into a relative value with a measurement value when no antigen is added as 100%, and then an approximate expression of a binding curve between an antibody and an antigen is obtained as follows:
y = 99 / (1+ (x / P1) P2 ) +0.5
Wherein x is the amount of the antibody, y is the amount (%) of the binding between the antibody and the antigen, and P1 and P2 are approximate parameters. Also, P1 and P2 are determined by the soft Fair, from the resulting binding curves and IC 50 values (= P1) of x that causes the y = 50. The specificity of the antibody is determined by determining the ratio of the IC 50 for cadmium or mercury to the IC 50 for another metal as cross-reactivity.
＜免疫クロマトグラフィー＞
抗体を用いた免疫学的測定法の一例として免疫クロマトグラフィーを挙げる。この方法は、試料を試験紙上に滴下するだけで目的物質の有無を数分から数十分の間に判定できるため簡便性に優れ、かつ特別な機械装置を必要としないため非常に安価である。本発明において、免疫クロマトグラフィーは、キレート剤−タンパク質複合体を利用することにより所望の金属イオンを効果的に検出するものである。図７にその実施形態の一例を示す。プラスチックバッキングシート１の上に、メンブレン２と吸収パッド３を一部で重なるように配置し、メンブレン２の先端の試料滴下位置５に試料を滴下すると試料が吸収パッド３に向かってメンブレン２上を流動し、標識された抗金属ＥＤＴＡモノクロナール抗体あるいは標識されたＥＤＴＡ−タンパク質複合体が固定化された領域４を通過する際に、金属ＥＤＴＡ錯体が抗体に捕捉されあるいは抗金属ＥＤＴＡモノクロナール抗体がＥＤＴＡ−タンパク質複合体のＥＤＴＡと錯体を形成してその錯体に抗金属ＥＤＴＡが捕捉されて、固定化領域４が目視可能となることで検出対象物の有無を簡易に検出可能としている。なお、ＥＤＴＡなどのキレート剤を直接標識することは困難であるため、キレート剤にタンパク質を付加する前あるいは後に、タンパク質に色素粒子を付加することで間接的にキレート剤を標識するのが好ましい。あるいは、モノクローナル抗体自体を標識することもできる。これらタンパク質の標識は通常行われている手法によって行うことができる。
<Immunochromatography>
An example of an immunoassay using an antibody is immunochromatography. This method is excellent in simplicity because the presence or absence of the target substance can be determined in a few minutes to tens of minutes by simply dropping a sample on a test paper, and is very inexpensive because no special mechanical device is required. In the present invention, immunochromatography is to effectively detect a desired metal ion by utilizing a chelating agent-protein complex. FIG. 7 shows an example of the embodiment. When the membrane 2 and the absorption pad 3 are partially overlapped on the plastic backing sheet 1 and a sample is dropped on the sample dropping position 5 at the tip of the membrane 2, the sample flows on the membrane 2 toward the absorption pad 3. When flowing through the region 4 where the labeled anti-metal EDTA monoclonal antibody or the labeled EDTA-protein complex is immobilized, the metal EDTA complex is captured by the antibody or the anti-metal EDTA monoclonal antibody is A complex is formed with EDTA of the EDTA-protein complex, and the antimetal EDTA is captured by the complex, and the immobilization region 4 is made visible so that the presence or absence of the detection target can be easily detected. Since it is difficult to directly label a chelating agent such as EDTA, it is preferable to label the chelating agent indirectly by adding dye particles to the protein before or after adding the protein to the chelating agent. Alternatively, the monoclonal antibody itself can be labeled. Labeling of these proteins can be performed by a commonly used technique.
（１）試験法１
本発明のモノクローナル抗体を試験紙の一部分に試料の流れを横切るように帯状に固定化する。次いで、試験試料中にキレート剤−タンパク質−色素粒子（キレート剤−標識タンパク質）複合体を添加して、カドミウムイオンまたは水銀イオンと結合させたのち試験紙に滴下させる（図９（Ａ），（Ｂ）参照）。目的の金属イオンが存在する場合には、金属−キレート剤錯体が形成され、金属錯体と標識タンパク質複合体が試験紙上に帯状に固定化したモノクローナル抗体によって補足され、その結果として色素粒子が帯状に密集して試料中の金属イオンが可視化する（図９（Ｃ），（Ｄ）参照）。試料中に金属イオンが存在しない場合は、キレート剤−タンパク質−色素粒子複合体は試験紙上に固定化されたモノクローナル抗体に補足されないため、色素粒子により可視化されない。
(1) Test method 1
The monoclonal antibody of the present invention is immobilized on a part of the test paper in a band shape so as to cross the flow of the sample. Next, a chelating agent-protein-dye particle (chelating agent-labeled protein) complex is added to the test sample and allowed to bind to cadmium ions or mercury ions and then dropped on a test paper (FIGS. 9A and 9B). B)). When the target metal ion is present, a metal-chelator complex is formed, and the metal complex and the labeled protein complex are captured by the monoclonal antibody immobilized in a band on the test paper, and as a result, the dye particles are banded. The metal ions in the sample are densely visualized (see FIGS. 9C and 9D). If no metal ions are present in the sample, the chelator-protein-dye particle complex will not be captured by the monoclonal antibody immobilized on the test paper and will not be visualized by the dye particles.
（２）試験法２
キレート剤−タンパク質複合体を利用して金属イオンを検出する免疫クロマトグラフィーとして次の方法がある。まず、試験法１における抗体の代わりにキレート剤−タンパク質を試験紙上に帯状に固定化する（図１０（Ｂ）参照）。色素粒子はモノクローナル抗体に付加する。この標識抗体と試験試料を混合してからともに試験紙に滴下させると（図１０（Ａ），（Ｂ）参照）、金属イオンが試験紙上のキレート剤−タンパク質複合体に捕捉され金属−キレート剤錯体を形成し、結果として標識されたモノクロナール抗体が金属−キレート剤錯体を介して試験紙上に補足され、帯状に密集した色素粒子により試料中の金属イオンが可視化される（図１０（Ｃ），（Ｄ）参照）。
(2) Test method 2
The following method is used as immunochromatography for detecting metal ions using a chelating agent-protein complex. First, a chelating agent-protein is immobilized on a test paper in a band shape instead of the antibody in the test method 1 (see FIG. 10B). Dye particles are added to the monoclonal antibody. When the labeled antibody and the test sample are mixed and then dropped together on a test paper (see FIGS. 10A and 10B), metal ions are captured by the chelating agent-protein complex on the test paper and the metal-chelating agent A complex is formed, and as a result, the labeled monoclonal antibody is captured on the test paper via the metal-chelating agent complex, and the metal ions in the sample are visualized by the dye particles densely packed in a band shape (FIG. 10 (C)). , (D)).
これら２つの試験法の利点は、タンパク質を介することでキレート剤を帯状に試験紙に固定することが容易になること、およびタンパク質１分子当たりに複数のキレート剤を付加するすることができ、単純に試験紙上にキレート剤を固定した場合に比べて表面積を大きく取ることが可能になり、結果として検出感度を上げることができることである。 The advantages of these two test methods are that it is easy to immobilize the chelating agent on the test paper by the intermediation of the protein, and that a plurality of chelating agents can be added per molecule of the protein. In addition, it is possible to increase the surface area as compared with the case where the chelating agent is fixed on the test paper, and as a result, the detection sensitivity can be increased.
本発明を下記の実施例によって具体的に説明するが、本発明はこれらに限定されるものではない。なお、本実施例では抗原としてカドミウムとＥＤＴＡの錯体（Ｃｄ−ＥＤＴＡ）、キャリアタンパク質としてヘモシアニンを用いて、カドミウムとしてＣｄ−ＥＤＴＡを特異的に認識する抗体を作製した。 The present invention will be specifically described by the following examples, but the present invention is not limited to these examples. In this example, an antibody that specifically recognizes Cd-EDTA as cadmium was prepared using cadmium and EDTA complex (Cd-EDTA) as an antigen and hemocyanin as a carrier protein.
実施例１ 抗カドミウムモノクローナル抗体
（１）抗原の作製
１ｍｇのイソチオシアノベンジル−ＥＤＴＡ（同仁化学社製）を２００μｌの５０ｍＭ ＭＥＳ緩衝液（ｐＨ５）に溶解した後、２００μｌの１０ｍＭ カドミウム溶液を添加した。３０分間静置して錯体を形成させ、２ｍｇ／ｍｌのスカシガイ由来のへモシアニン（以下ＫＬＨと略す）を１ｍｌ添加した。次いで１０Ｎ〜１Ｎ 水酸化ナトリウム溶液によりｐＨを９.５に調整した。これを一晩緩やかに撹拌し、ゲル濾過カラムにより溶液を２５ｍＭ ＨＥＰＥＳ緩衝液（ｐＨ７.０）に置換し、抗原を精製した。得られたＣｄ−ＥＤＴＡとヘモシアニンの複合体を免疫用の抗原とした。また、ヘモシアニンの代わりにオバルブミンを用いて同様な操作を行ってＣｄ−ＥＤＴＡとオバルブミンの複合体を作製し、これをスクリーニング用抗原とした。
Example 1 Preparation of Anti-Cadmium Monoclonal Antibody (1) Antigen After 1 mg of isothiocyanobenzyl-EDTA (manufactured by Dojindo) was dissolved in 200 μl of 50 mM MES buffer (pH 5), 200 μl of 10 mM cadmium solution was added. . The mixture was allowed to stand for 30 minutes to form a complex, and 1 ml of 2 mg / ml limpet-derived hemocyanin (hereinafter abbreviated as KLH) was added. Then the pH was adjusted to 9.5 with a 10N to 1N sodium hydroxide solution. This was gently stirred overnight, and the solution was replaced with a 25 mM HEPES buffer (pH 7.0) using a gel filtration column to purify the antigen. The obtained complex of Cd-EDTA and hemocyanin was used as an antigen for immunization. A similar operation was performed using ovalbumin instead of hemocyanin to produce a complex of Cd-EDTA and ovalbumin, which was used as a screening antigen.
（２）抗カドミウム抗体の作製
ａ）マウスの免疫
６匹のＢＡＬＢ／Ｃ近交系のマウス（雌、５週齢、日本クレア）を１週間程度飼育環境に慣らしたのち、１回目の免疫を行った。免疫は、実施例１の免疫用抗原と等量のアジュバントを十分に混合してエマルジョンとした。１回の免疫につき、タンパク質量にして約０.３ｍｇの抗原を用いて１回につき２カ所、１００μｌずつ皮下注射した。２回目以降の免疫は２週間程度おきに３回または４回行った。３回目の免疫後、４〜７目の間にマウスの尾部より数滴の血液を採取し、スクリーニング用抗原を用いて抗体価を測定し、抗体の産生を確認した。
(2) Preparation of anti-cadmium antibody a) Immunization of mice Six BALB / C inbred mice (female, 5 weeks old, CLEA Japan) were acclimated to the breeding environment for about 1 week, and the first immunization was performed. went. For immunization, an emulsion was prepared by sufficiently mixing the antigen for immunization of Example 1 and an equal amount of adjuvant. For one immunization, 100 μl of the antigen was injected subcutaneously in two places at a time using about 0.3 mg of antigen as a protein amount. The second and subsequent immunizations were performed three or four times about every two weeks. After the third immunization, several drops of blood were collected from the tail of the mouse between the fourth and seventh eyes, and the antibody titer was measured using a screening antigen to confirm the production of the antibody.
ｂ）ハイブリドーマの作製
最後の免疫から４日〜６日経過したマウスから、脾臓を摘出し、脾臓細胞とミエローマ細胞（ＮＳＯ株、理化学研究所）と融合させた。細胞を融合する際は、重合度１５００のポリエチレングリコールで脾臓細胞とミエローマを処理した。細胞融合に関する一連の操作は３７℃にて行った。得られたハイブリドーマは９６穴プレートに分注し、ＨＡＴ培地中で３７℃，５％ＣＯ２の条件下で培養した。ハイブリドーマは、融合後約２週間培養し、その間は３〜４日に１回培地を交換した。なお、１０日目前後まではＨＡＴ培地を用い、その後はＨＴ培地を用いた。
b) Preparation of hybridoma A spleen was excised from a mouse 4-6 days after the last immunization, and spleen cells were fused with myeloma cells (NSO strain, RIKEN). When cells were fused, spleen cells and myeloma were treated with polyethylene glycol having a degree of polymerization of 1500. A series of operations regarding cell fusion were performed at 37 ° C. The obtained hybridoma was dispensed into a 96-well plate and cultured in a HAT medium under the conditions of 37 ° C. and 5% CO 2 . The hybridomas were cultured for about 2 weeks after fusion, during which time the medium was changed once every 3 to 4 days. The HAT medium was used until about day 10, and the HT medium was used thereafter.
ｃ）スクリーニング
スクリーニングは、フロー式蛍光センサー（キネクサ３０００）を用いて蛍光センサー法にて行った。また、抗原を固定化する担体としてアガロースビーズ（ＮＨＳ修飾済み，ファルマシア）を用いた。２ｍｌのアガロースビーズ懸濁液に対して、上記（１）の作製方法で得たスクリーニング用抗原１００μｇを添加して抗原をビーズに固定化した。
９６穴プレートの培養液の上清を約１０μｌずつ取り、１本の試験管にまとめた（約１ｍｌ）。一次スクリーニングでは、この試験管に固定化抗原を添加し、固定化抗原に結合した抗体は蛍光物質Ｃｙ５にて標識された二次抗体（抗マウスＩｇＧヤギ抗体）を用いて間接的に蛍光標識し、上清中の抗体と固定化抗原との結合を蛍光センサーにより検出した。二次スクリーニングでは、培養上清にＣｄ−ＥＤＴＡを１μＭとなるように加え、平衡化したのち、錯体と結合しなかった抗体のビーズへの結合を同様に測定した。二次スクリーニングによって、Ｃｄ−ＥＤＴＡと特異的に結合する抗体を含むと判定されたプレートについては，１本の試験管にまとめる培養上清を１２穴分〜１穴分と段階的に絞り込みながら、一次スクリーニング同様、上清中の抗体のビーズへの結合を測定し、目的の抗体を産生するハイブリドーマとして、Ｓｏ２２Ｂ１１、Ｓｏ２１Ｄ５、Ｓｏ２５Ａ１、Ｓｏ２６Ｇ８を特定した。スクリーニングによって陽性と判断された抗体生産細胞は、メチルセルロース培地を用いて単一の細胞に由来するコロニーを形成させた後、コロニーを液体培地に移し培養を続けた。
c) Screening Screening was performed by a fluorescence sensor method using a flow-type fluorescence sensor (Kynexa 3000). Agarose beads (NHS-modified, Pharmacia) were used as a carrier for immobilizing the antigen. To 2 ml of the agarose bead suspension, 100 μg of the screening antigen obtained by the preparation method (1) was added to immobilize the antigen on the beads.
Approximately 10 μl of the supernatant of the culture solution in the 96-well plate was taken and combined into one test tube (about 1 ml). In the primary screening, an immobilized antigen was added to this test tube, and the antibody bound to the immobilized antigen was indirectly fluorescently labeled using a secondary antibody (anti-mouse IgG goat antibody) labeled with a fluorescent substance Cy5. The binding between the antibody in the supernatant and the immobilized antigen was detected by a fluorescent sensor. In the secondary screening, Cd-EDTA was added to the culture supernatant to a concentration of 1 μM, and after equilibration, the binding of the antibody not bound to the complex to the beads was measured in the same manner. For the plate determined to contain an antibody that specifically binds to Cd-EDTA by the secondary screening, the culture supernatant to be collected in one test tube is gradually narrowed down to 12 to 1 wells. As in the primary screening, the binding of the antibody in the supernatant to the beads was measured, and So22B11, So21D5, So25A1, and So26G8 were identified as hybridomas producing the desired antibody. The antibody-producing cells determined to be positive by the screening were allowed to form a colony derived from a single cell using a methylcellulose medium, and then transferred to a liquid medium to continue the culture.
ｄ）モノクローナル抗体の生産
単離されたコロニーを９６穴プレート、２４穴プレート、２５ｃｍ２フラスコの順に培地量を増やしながら継代した。継代の後３〜５日の間に培養上清中の抗体量を蛍光センサーにて測定し、抗体量の多いものを次の培地に移した。なお、９６穴プレートの培地量は２００μｌ、２４穴プレートの培地量は１０００μｌ、２５ｃｍ２フラスコの培地量は１０ｍｌである。また、培養後の培地は脱塩カラムを用いて培地に含まれる若干の重金属を除去し、２５ｍＭ ＨＥＰＥＳ緩衝液（ｐＨ７.０）に置換した。さらに、プロテインＡアフィニティーカラムに供して、得られたモノクローナル抗体を精製した。
d) Production of monoclonal antibody The isolated colonies were subcultured in the order of a 96-well plate, a 24-well plate, and a 25 cm 2 flask while increasing the amount of medium. Between 3 and 5 days after the passage, the amount of antibody in the culture supernatant was measured with a fluorescent sensor, and those with a large amount of antibody were transferred to the next medium. The medium volume of the 96-well plate is 200 μl, the medium volume of the 24-well plate is 1000 μl, and the medium volume of the 25 cm 2 flask is 10 ml. Further, the medium after the cultivation was replaced with a 25 mM HEPES buffer (pH 7.0) by removing some heavy metals contained in the medium using a desalting column. Further, the obtained monoclonal antibody was applied to a protein A affinity column to purify the obtained monoclonal antibody.
（３）抗カドミウム抗体の評価
Ｃｄ−ＥＤＴＡ及び他の金属とＥＤＴＡの錯体に対する親和性を測定・比較することで得られた抗カドミウム抗体Ｓｏ２２Ｂ１１、Ｓｏ２１Ｄ５、Ｓｏ２５Ａ１、Ｓｏ２６Ｇ８を評価した。なお、カドミウム以外の金属として、カルシウム（Ｃａ−ＥＤＴＡ）、銅（Ｃｕ−ＥＤＴＡ）、鉄（Ｆｅ−ＥＤＴＡ）、水銀（Ｈｇ−ＥＤＴＡ）、マグネシウム（Ｍｇ−ＥＤＴＡ）、ニッケル（Ｎｉ−ＥＤＴＡ）、鉛（Ｐｂ−ＥＤＴＡ）および亜鉛（Ｚｎ−ＥＤＴＡ）を用い、フリーのＥＤＴＡをコントロールとした。
(3) Evaluation of anti-cadmium antibody The anti-cadmium antibodies So22B11, So21D5, So25A1, and So26G8 obtained by measuring and comparing the affinity of Cd-EDTA and the complex of other metals with EDTA were evaluated. In addition, as metals other than cadmium, calcium (Ca-EDTA), copper (Cu-EDTA), iron (Fe-EDTA), mercury (Hg-EDTA), magnesium (Mg-EDTA), nickel (Ni-EDTA), Lead (Pb-EDTA) and zinc (Zn-EDTA) were used, and free EDTA was used as a control.
まず、蛍光センサーに導入して２Ｖ前後の蛍光強度を得られる濃度の抗体溶液（約１ｎＭ）を作製した。この抗体溶液に、金属ＥＤＴＡ錯体溶液を１０μＭになるように加え、抗体と十分平衡化させた。平衡化した溶液を蛍光センサーに導入して固定化抗原に結合する抗体の量を測定した。この結果、抗体Ｓｏ２２Ｂ１１が最も強い親和性を示した錯体は、Ｃａ−ＥＤＴＡでありＣｄ−ＥＤＴＡ、Ｍｇ−ＥＤＴＡ、Ｈｇ−ＥＤＴＡおよびコントロールのＥＤＴＡにも同じような親和性を示しており、モノクローナル抗体Ｓｏ２２Ｂ１１は特定の金属の定量には不向きな抗体であると評価される。これに対して、Ｓｏ２１Ｄ５、Ｓｏ２５Ａ１、Ｓｏ２６Ｇ８はいずれも、Ｃｄ−ＥＤＴＡに対して最も強い親和性を示し、２番目に強い親和性を示すＣｕ−ＥＤＴＡとは頭著な差が見られた。これらの結果を図１に示す。なお、図１において、Freeは金属を配位していないＥＤＴＡ、他は表示金属とＥＤＴＡとの錯体を示す。蛍光強度は抗原を加えなかったときの蛍光強度の値を１００％として相対値を示している。 First, an antibody solution (about 1 nM) having a concentration capable of obtaining a fluorescence intensity of about 2 V when introduced into a fluorescent sensor was prepared. To this antibody solution, a metal EDTA complex solution was added so as to have a concentration of 10 μM, and sufficiently equilibrated with the antibody. The equilibrated solution was introduced into a fluorescent sensor, and the amount of antibody bound to the immobilized antigen was measured. As a result, the complex in which the antibody So22B11 showed the strongest affinity was Ca-EDTA, and showed similar affinity to Cd-EDTA, Mg-EDTA, Hg-EDTA, and control EDTA. So22B11 is evaluated as an antibody unsuitable for quantification of a specific metal. In contrast, So21D5, So25A1, and So26G8 all exhibited the strongest affinity for Cd-EDTA, and a marked difference was found from Cu-EDTA, which exhibited the second strongest affinity. These results are shown in FIG. In FIG. 1, Free indicates EDTA to which no metal is coordinated, and the other indicates a complex of the indicated metal and EDTA. The fluorescence intensity is shown as a relative value with the value of the fluorescence intensity when no antigen was added as 100%.
（４）抗カドミウム抗体の詳細な評価
上記（３）にてカドミウムに対して特異性が高いと評価されたモノクローナル抗体Ｓｏ２１Ｄ５、Ｓｏ２５Ａ１、Ｓｏ２６Ｇ８についてはさらに詳細な分析した。なお、本実施例では、各種全属ＥＤＴＡ錯体に対するＩＣ５０を求め、抗体の錯体ヘの親和性を厳密に評価した。
各金属錯体の濃度（０.１ｎＭ〜１００ｍＭ）を１０倍ずつ変化させて、金属錯体濃度を変化させたときの各モノクローナル抗体と金属錯体との結合量を蛍光センサーで測定した。その結果を結合曲線として図２に示す。また、得られた測定結果をソフトフェア（Origin Version 6.0）により解析して、各金属錯体に対するＩＣ５０を求め、抗体の交差反応性をカドミウムに対するＩＣ５０と他の金属に対するＩＣ５０との比として求めた。モノクローナル抗体Ｓｏ２６Ｇ８の各金属錯体とのＩＣ５０および交差反応性を下記の表に示す。尚、図２において、Freeは金属を配位していないＥＤＴＡ、他は表示金属とＥＤＴＡとの錯体を示す。蛍光強度は抗原を加えなかったときの蛍光強度の値を１００％として相対値を示している。
(4) Detailed evaluation of anti-cadmium antibody The monoclonal antibodies So21D5, So25A1, and So26G8 evaluated to have high specificity for cadmium in (3) above were further analyzed in detail. In this embodiment, it obtains the IC 50 for various total genus EDTA complex was rigorously evaluate the affinity of the antibody complexes f.
The concentration (0.1 nM to 100 mM) of each metal complex was changed by 10 times, and the amount of binding between each monoclonal antibody and the metal complex when the metal complex concentration was changed was measured with a fluorescent sensor. The result is shown in FIG. 2 as a binding curve. The obtained measurement results were analyzed by software (Origin Version 6.0) to determine the IC 50 for each metal complex, and the antibody cross-reactivity was determined as the ratio of the IC 50 for cadmium to the IC 50 for other metals. I asked. The IC 50 and cross-reactivity with each metal complex of monoclonal antibodies So26G8 shown in the table below. In FIG. 2, Free represents EDTA to which no metal is coordinated, and the other represents a complex of the indicated metal and EDTA. The fluorescence intensity is shown as a relative value with the value of the fluorescence intensity when no antigen was added as 100%.
モノクローナル体Ｓｏ２６Ｇ８はＣｄ−ＥＤＴＡに対して最も強い親和性を示し、他の金属錯体と１％以上の反応交差性を示さなかったことから、カドミウムに対して非常に特異性が高いことが明らかとなった。また、モノクローナル抗体Ｓｏ２１Ｄ５、Ｓｏ２５Ａ１についても同様に評価したが、結果はＳｏ２６Ｇ８と一致していた。このため、モノクローナル抗体Ｓｏ２１Ｄ５、Ｓｏ２５Ａ１およびＳｏ２６Ｇ８を産生するハイブリドーマは同一の親細胞に由来すると考えられる。 Monoclonal So26G8 exhibited the strongest affinity for Cd-EDTA, and did not show a cross-reactivity of 1% or more with other metal complexes, indicating that it is very specific for cadmium. became. The monoclonal antibodies So21D5 and So25A1 were evaluated in the same manner, and the results were consistent with those of So26G8. Therefore, hybridomas producing monoclonal antibodies So21D5, So25A1 and So26G8 are considered to be derived from the same parent cell.
実施例２ 抗水銀モノクローナル抗体
（１）抗原の作製
１ｍｇのイソチオシアノベンジル−ＥＤＴＡ（同仁化学社製）を２００μｌの５０ｍＭ ＭＥＳ緩衝液（ｐＨ５）に溶解した後、最終濃度が５ｍＭになるように塩化水銀溶液を添加した。次いで、実施例１−（１）と同様にして水銀−ＥＤＴＡ錯体（Ｈｇ−ＥＤＴＡ）−ヘモシアニン複合体を得て、これを免疫用の抗原とした。また、ヘモシアニンの代わりにオバルブミンを用いて同様な操作を行ってＨｇ−ＥＤＴＡとオバルブミンの複合体を作製し、これをスクリーニング用抗原とした。
Example 2 Preparation of Anti-Mercury Monoclonal Antibody (1) Antigen 1 mg of isothiocyanobenzyl-EDTA (manufactured by Dojindo) was dissolved in 200 μl of 50 mM MES buffer (pH 5), and the final concentration was adjusted to 5 mM. A mercury chloride solution was added. Next, a mercury-EDTA complex (Hg-EDTA) -hemocyanin complex was obtained in the same manner as in Example 1- (1), and this was used as an antigen for immunization. A similar operation was performed using ovalbumin instead of hemocyanin to produce a complex of Hg-EDTA and ovalbumin, which was used as a screening antigen.
（１）抗水銀抗体の作成
前記（１）で調製した抗原を用いたこと以外は実施例１−（２）と同様にして、１０００株以上のハイブリドーマを作製し、これらのハイブリドーマの中から、Ｈｇ−ＥＤＴＡとの結合強度を指標にしてスクリーニングし、Ｈｇ−ＥＤＴＡに強い結合性を有する株としてＮｘ２２Ｃ３株を得た。Ｎｘ２２Ｃ３株はメチルセルロース培地を用いてシングルコロニーとした後、再度液体培地に戻した。次いで、単離されたコロニーを９６穴プレート、２４穴プレート、２５ｃｍ２フラスコの順に培地量を増やしながら継代し、培養後の培地からモノクローナル抗体Ｎｘ２２Ｃ３を精製した。
(1) Preparation of anti-mercury antibody In the same manner as in Example 1- (2) except that the antigen prepared in the above (1) was used, 1,000 or more hybridomas were prepared, and from among these hybridomas, Screening was performed using the binding strength to Hg-EDTA as an index to obtain an Nx22C3 strain as a strain having strong binding to Hg-EDTA. The Nx22C3 strain was converted into a single colony using a methylcellulose medium, and then returned to a liquid medium again. Next, the isolated colonies were subcultured while increasing the medium volume in the order of a 96-well plate, a 24-well plate, and a 25 cm 2 flask, and the monoclonal antibody Nx22C3 was purified from the cultured medium.
（３）抗水銀抗体の評価
抗カドミウム抗体と同様にして、得られたモノクローナル抗体とＨｇ−ＥＤＴＡおよび他の金属とＥＤＴＡの錯体に対する親和性を測定・比較することで得られた抗水銀抗体Ｎｘ２２Ｃ３株を評価した。なお、水銀以外の金属として、Ｃｄ−ＥＤＴＡ、Ｃｕ−ＥＤＴＡ、Ｚｎ−ＥＤＴＡ、Ｍｇ−ＥＤＴＡ、Ｆｅ−ＥＤＴＡ、Ｃａ−ＥＤＴＡおよびＰｂ−ＥＤＴＡを用い、フリーのＥＤＴＡをコントロールとした。なお、ＩＣ５０値に対して抗体濃度が十分に小さい（１０分の１以下の）場合にＩＣ５０値と結合解離定数がほぼ一致することが知られているため、本実施例では評価として結合解離定数を用いた。
その結果、モノクローナル抗体Ｎｘ２２Ｃ３は、Ｃｄ−ＥＤＴＡとは１９.２％の交差反応性を示したが、これはＨｇ−ＥＤＴＡとＣｄ−ＥＤＴＡの立体構造がよく似ているためであると考えられる。Ｃｄ−ＥＤＴＡ以外の錯体に対する交差反応性は０.２６％以下であり、Ｎｘ２２Ｃ３抗体がＨｇ−ＥＤＴＡとＣｄ−ＥＤＴＡに対して強い特異性を有することが確認された。これらの結果を下記の表２に示す。
(3) Evaluation of anti-mercury antibody The anti-mercury antibody Nx22C3 obtained by measuring and comparing the affinity of the obtained monoclonal antibody with the complex of Hg-EDTA and other metals and EDTA in the same manner as the anti-cadmium antibody. The strain was evaluated. In addition, Cd-EDTA, Cu-EDTA, Zn-EDTA, Mg-EDTA, Fe-EDTA, Ca-EDTA, and Pb-EDTA were used as metals other than mercury, and free EDTA was used as a control. Since the antibody concentration is known to bond dissociation constant as an IC 50 value coincides substantially if sufficiently small (less than one tenth) relative to an IC 50 value, binding as evaluated in this example The dissociation constant was used.
As a result, the monoclonal antibody Nx22C3 showed a cross-reactivity of 19.2% with Cd-EDTA, which is considered to be because the three-dimensional structures of Hg-EDTA and Cd-EDTA are very similar. The cross-reactivity with complexes other than Cd-EDTA was 0.26% or less, confirming that the Nx22C3 antibody has strong specificity for Hg-EDTA and Cd-EDTA. The results are shown in Table 2 below.
試験例１ 抗カドミウム抗体を用いた測定試験
カドミウムに対する法規制には、環境基準、水質基準と排水基準があり、規制値はそれぞれ１０ｐｐｂと１００ｐｐｂである。このため、本発明のモノクローナル抗体Ｓｏ２６Ｇ８がこれらの規制値の濃度のカドミウムを検出しうるかを実施例１−（４）と同様に評価した。なお、本試験例では法規制値の標記単位に合わせて濃度をｐｐｂにて表す。
その結果、抗体のＣｄ−ＥＤＴＡに対する結合曲線は１０ｐｐｂよりも２桁ほど低い濃度から曲線が下り始めており、この抗体Ｓｏ２６Ｇ８がカドミウムの環境基準と排出基準の判定に利用できることが示された（図３）。また、Ｃｕ−ＥＤＴＡとＺｎ−ＥＤＴＡに関しては，結合曲線が１００ｐｐｂ付近から下り始めている（図３）。この結果は測定試料中に銅または亜鉛が１００ｐｐｂ以上存在している場合、銅または亜鉛によってカドミウムの擬陽性となる可能性を示す。しかし、この抗体で陰性であった場合はカドミウムも銅も亜鉛も規制値以上は存在しないことが示される。また、抗体で陽性の場合には、カドミウム、銅および亜鉛の濃度を従来の方法で厳密に測定すればよいため、本発明のモノクローナル抗体は調査現場での簡易測定に非常に有用であることが示された。
Test Example 1 Measurement Test Using Anti-Cadmium Antibody Laws and regulations on cadmium include an environmental standard, a water quality standard and a drainage standard, and the regulated values are 10 ppb and 100 ppb, respectively. Therefore, it was evaluated in the same manner as in Example 1- (4) whether the monoclonal antibody So26G8 of the present invention could detect cadmium at these regulated concentrations. In this test example, the concentration is represented by ppb in accordance with the unit of the regulation value.
As a result, the binding curve of the antibody to Cd-EDTA began to fall from a concentration about two orders of magnitude lower than 10 ppb, indicating that this antibody So26G8 can be used to determine the environmental standards and elimination standards for cadmium (FIG. 3). ). For Cu-EDTA and Zn-EDTA, the binding curve starts to fall from around 100 ppb (FIG. 3). This result indicates that when copper or zinc is present in the measurement sample in an amount of 100 ppb or more, there is a possibility that copper or zinc causes false positive of cadmium. However, a negative result with this antibody indicates that neither cadmium nor copper or zinc is present above regulatory levels. In addition, when the antibody is positive, since the concentration of cadmium, copper and zinc can be strictly measured by a conventional method, the monoclonal antibody of the present invention may be very useful for simple measurement at a survey site. Indicated.
試験例２ 抗水銀抗体を用いた測定試験
水銀に対する環境基準は０.５ｐｐｂである。このため、本発明のモノクローナル抗体Ｎｘ２２Ｃ３がこの規制値の濃度の水銀を検出し得るかを試験例１に従って評価した。また、モノクローナル抗体Ｎｘ２２Ｃ３はカドミウムとも反応するため、カドミウムについても同様に評価した。
Ｎｘ２２Ｃ３抗体の結合分析のグラフについて、蛍光強度がおよそ８０％の時の錯体濃度が検出下限とし、Ｎｘ２２Ｃ３抗体のＨｇ−ＥＤＴＡとＣｄ−ＥＤＴＡに対する検出限界を決定した（図４を参照）。その結果、Ｎｘ２２Ｃ３抗体のＨｇ−ＥＤＴＡに対する検出限界は０.３ｐｐｂ（１.５ｎＭ）であり、Ｃｄ−ＥＤＴＡ錯体に対する検出限界は０.３ｐｐｂ（２.７ｎＭ）であった。よってＮｘ２２Ｃ３抗体は水銀の環境基準である０.５ｐｐｂおよびカドミウムに対する環境基準１０ｐｐｂの測定が可能であることが確認された。尚、図４において横軸はＮｘ２２Ｃ３抗体に加えたＣｄ−ＥＤＴＡまたはＨｇ−ＥＤＴＡの濃度を示しており、縦軸は蛍光強度の相対値を示している。蛍光強度は、ＥＤＴＡ錯体の濃度が０の時（ＥＤＴＡ錯体を抗体に加えなかった時）の蛍光強度を１００とした。曲線は各データの近似曲線を示す。
Test Example 2 Measurement test using anti-mercury antibody The environmental standard for mercury is 0.5 ppb. Therefore, it was evaluated according to Test Example 1 whether the monoclonal antibody Nx22C3 of the present invention could detect mercury at the concentration of this regulation value. Since the monoclonal antibody Nx22C3 also reacts with cadmium, cadmium was similarly evaluated.
Regarding the graph of the binding analysis of the Nx22C3 antibody, the detection concentration was determined based on the complex concentration when the fluorescence intensity was about 80%, and the detection limit of the Nx22C3 antibody for Hg-EDTA and Cd-EDTA was determined (see Fig. 4). As a result, the detection limit of the Nx22C3 antibody for Hg-EDTA was 0.3 ppb (1.5 nM), and the detection limit for the Cd-EDTA complex was 0.3 ppb (2.7 nM). Therefore, it was confirmed that the Nx22C3 antibody can measure the environmental standard of mercury of 0.5 ppb and the environmental standard of cadmium of 10 ppb. In FIG. 4, the horizontal axis represents the concentration of Cd-EDTA or Hg-EDTA added to the Nx22C3 antibody, and the vertical axis represents the relative value of the fluorescence intensity. The fluorescence intensity was 100 when the concentration of the EDTA complex was 0 (when the EDTA complex was not added to the antibody). The curve shows an approximate curve of each data.
しかし、Ｎｘ２２Ｃ３抗体は水銀とカドミウムに両方と結合するため、これらの金属を両方含む試料については正確な濃度を測定できない可能性がある。そこで、水銀とカドミウムの化学的な性質の差を利用して一方の金属がＥＤＴＡと錯体を形成することを阻害することにより、水銀とカドミウムを区別して測定することを試みた。具体的には、水銀イオンの水溶液にグルタチオンやメルカプトエタノール、あるいは牛血清アルブミンなどシステイン残基を含むタンパク質を添加すると、これらに含まれるＳＨ基と水銀が反応し、ＳＨ基の硫黄と水銀が共有結合することが知られているため、試料にＳＨ基含有物質例えばグルタチオンを添加する場合と添加しない場合で測定する。カドミウムイオンが添加したグルタチオンと反応せず、かつグルタチオンがＮｘ２２Ｃ３抗体とＣｄ−ＥＤＴＡと結合を阻害しなければ、Ｎｘ２２Ｃ３抗体によって水銀とカドミウムを区別して測定することが可能となる。
そこで、試料にグルタチオンを添加した場合と添加しない場合でカドミウムと水銀について結合分析を行った結果、カドミウムは低濃度でもグルタチオンの影響をほとんど受けることなくＮｘ２２Ｃ３抗体により測定できることが確認された（図５を参照）。この抗体のカドミウムに対する感度は、Ｓｏ２６Ｇ８抗体を５０倍ほど上回るものであった。一方、水銀はグルタチオンによってＥＤＴＡとの錯体形成が阻害される。その結果、Ｎｘ２２Ｃ３抗体とＨｇ−ＥＤＴＡとの結合は検出されなかった（図６を参照）。これらの結果から、グルタチオンで前処理した場合と前処理しない場合について測定することにより、試料中に水銀およびカドミウムが存在するか否かを区別して判定しうることのみならず、水銀とカドミウムの各々について同時に濃度を測定しうることが示された。尚、図５及び図６において、各試料のＥＤＴＡの濃度は１００μＭである。グルタチオンを加えた試料のグルタチオン濃度は１０μＭであり、ＥＤＴＡを加える前に金属と反応させた。蛍光強度は金属の濃度が０の試料（ＥＤＴＡ濃度は１００μＭ）の蛍光強度を１００％として相対値で示した。
However, since the Nx22C3 antibody binds to both mercury and cadmium, accurate concentrations may not be measured for samples containing both of these metals. Thus, an attempt was made to separately measure mercury and cadmium by inhibiting the formation of a complex between EDTA and one metal by utilizing the difference in the chemical properties of mercury and cadmium. Specifically, when a protein containing a cysteine residue such as glutathione, mercaptoethanol, or bovine serum albumin is added to an aqueous solution of mercury ions, the SH groups contained therein react with mercury, and the sulfur and SH of the SH group are shared. Since binding is known, the measurement is performed with and without the addition of an SH group-containing substance such as glutathione to the sample. If the cadmium ion does not react with the added glutathione and glutathione does not inhibit the binding between the Nx22C3 antibody and Cd-EDTA, mercury and cadmium can be measured separately by the Nx22C3 antibody.
Then, a binding analysis was performed on cadmium and mercury in the case where glutathione was added to the sample and in the case where glutathione was not added. As a result, it was confirmed that cadmium can be measured by the Nx22C3 antibody even at a low concentration with little influence of glutathione (FIG. 5). See). The sensitivity of this antibody to cadmium was about 50 times higher than that of the So26G8 antibody. On the other hand, mercury inhibits complex formation with EDTA by glutathione. As a result, no binding between the Nx22C3 antibody and Hg-EDTA was detected (see FIG. 6). From these results, by measuring the case of pretreatment with glutathione and the case without pretreatment, it is possible to determine whether or not mercury and cadmium are present in the sample, as well as to determine whether mercury and cadmium are present. It was shown that the concentration could be measured simultaneously for. In FIGS. 5 and 6, the EDTA concentration of each sample is 100 μM. The glutathione concentration of the sample to which glutathione was added was 10 μM, and the sample was reacted with the metal before adding EDTA. The fluorescence intensity was shown as a relative value with the fluorescence intensity of a sample having a metal concentration of 0 (EDTA concentration of 100 μM) as 100%.
実施例３ 免疫クロマトグラフィー
免疫クロマトグラフィー装置を次のように構成した。プラスチックバッキングシート
（商品コード：３１４−００４.０２０，
ニップンテクノクラスタ）上にメンブレン（５ｍｍ×５０ｍｍ）
（ＦＦ６０，ニップンテクノクラスタ
）を配置させ、さらにメンブレンの一端と重複するように吸収パッド
（吸収紙３００，ニップンテクノクラスタ
）を配置させる（図７を参照）。次いで、メンブレン（試験紙）の一部分に１ｍｇ／ｍｌの抗カドミウム抗体Ｓｏ２６Ｇ８ 約２μｌを塗布し、３７℃で１時間乾燥して固定化し、試験紙を１００ｍｇ／ｍｌのＢＳＡ溶液に浸し、３７℃で１時間乾燥してブロッキングした。
１０ｐｐｂ〜１ｐｐｍの濃度の塩化カドミウム溶液１００μｌを１０μｌのＥＤＴＡ−ＯＶＡ−青色ラテックスと試験管中で混合し、この混合液に試験紙の先端を浸した。試料とＥＤＴＡ−ＯＶＡ−青色ラテックスの混合液は試験紙に吸い取られ、３０分後に抗カドミウムＥＤＴＡ抗体を固定した位置に青色のバンドが検出された（図８）。この結果から本実施例の免疫クロマトグラフィーでは、１００ｐｐｂのカドミウムを再現性よく検出しうることが確認された。因みに、カドミウムに対する感度は、Ｓｏ２６Ｇ８よりもＮｘ２２Ｃ３抗体の方が５０倍程上回るため、Ｎｘ２２Ｃ３抗体を用いる免疫クロマトグラフィーの場合の方がより再現性よく低濃度まで検出できるものと推定できる。
Example 3 Immunochromatography An immunochromatography apparatus was configured as follows. Plastic backing sheet (product code: 314-004.020,
Membrane (5mm x 50mm) on Nipple Techno Cluster
(FF60, nipple techno cluster) are arranged, and furthermore, an absorbent pad (absorbent paper 300, nipple techno cluster) is arranged so as to overlap with one end of the membrane (see FIG. 7). Then, about 2 μl of 1 mg / ml anti-cadmium antibody So26G8 was applied to a part of the membrane (test paper), dried at 37 ° C. for 1 hour to immobilize, and the test paper was immersed in a 100 mg / ml BSA solution at 37 ° C. Dry for 1 hour and block.
100 μl of a cadmium chloride solution having a concentration of 10 ppb to 1 ppm was mixed with 10 μl of EDTA-OVA-blue latex in a test tube, and the tip of a test paper was dipped in this mixture. The mixture of the sample and EDTA-OVA-blue latex was blotted on a test paper, and after 30 minutes, a blue band was detected at the position where the anti-cadmium EDTA antibody was immobilized (FIG. 8). From these results, it was confirmed that 100 ppb of cadmium could be detected with good reproducibility in the immunochromatography of this example. Incidentally, since the sensitivity to cadmium is about 50 times higher with the Nx22C3 antibody than with So26G8, it can be estimated that immunochromatography using the Nx22C3 antibody can detect reproducibly down to low concentrations.
Claims (7)
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