JP7803086B2 - Immunoassay method with improved cross-reactivity - Google Patents
Immunoassay method with improved cross-reactivityInfo
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Description
本発明は、抗イムノコンプレックス抗体を用いる測定法の交叉反応の改善方法に関するものである。 The present invention relates to a method for improving cross-reactivity in assays using anti-immunocomplex antibodies.
低分子化合物であるハプテンは、通常、競合法とよばれる免疫測定方法によって測定される。競合法は、試料中に含まれるハプテンと、酵素などで標識した標識ハプテンとを、一定量の抗ハプテン抗体に対して競合的に反応させる方法である。試料中に含まれるハプテン量が多くなると、標識ハプテンが抗ハプテン抗体へ結合する量が低下する。このことから、標識ハプテンが抗ハプテン抗体へ結合する割合を基にして、試料中に含まれるハプテン量を推定することができる。競合法における測定感度は、用いられる抗ハプテン抗体の親和定数に依存する。しかし親和定数の高い抗ハプテン抗体を得ることは困難であり、そのため、ごく微量のハプテンを測定することは極めて困難であった（非特許文献１）。 Haptens, which are low-molecular-weight compounds, are typically measured using an immunoassay known as a competitive method. In a competitive method, a hapten contained in a sample is reacted competitively with a fixed amount of anti-hapten antibody, which is labeled with an enzyme or other reagent. As the amount of hapten contained in a sample increases, the amount of labeled hapten that binds to the anti-hapten antibody decreases. Therefore, the amount of hapten contained in a sample can be estimated based on the rate at which labeled hapten binds to the anti-hapten antibody. The measurement sensitivity of a competitive method depends on the affinity constant of the anti-hapten antibody used. However, it is difficult to obtain an anti-hapten antibody with a high affinity constant, making it extremely difficult to measure even minute amounts of hapten (Non-Patent Document 1).
このような競合法による免疫測定の課題を解決し得る測定方法として、抗イムノコンプレックス抗体を用いた非競合型の免疫測定法が提案されている（特許文献１）。抗イムノコンプレックス抗体は、ハプテンと抗ハプテン抗体からなる免疫複合体に選択的に結合するため、試料中に含まれるハプテン量の増加に伴って、シグナルの増加が観測される。抗イムノコンプレックス抗体を用いたサンドイッチ型のアッセイは、競合法に比べ大量の抗ハプテン抗体を測定に用いることが可能であり、これにより測定の高感度化が可能であった。しかし、大量の抗ハプテン抗体を利用することで交叉反応性が悪化する場合があった。 A non-competitive immunoassay using anti-immunocomplex antibodies has been proposed as a measurement method that can solve the problems associated with competitive immunoassays (Patent Document 1). Anti-immunocomplex antibodies selectively bind to immune complexes consisting of haptens and anti-hapten antibodies, so an increase in signal is observed as the amount of hapten contained in the sample increases. Sandwich assays using anti-immunocomplex antibodies allow for the use of larger amounts of anti-hapten antibodies in measurements compared to competitive methods, thereby enabling higher measurement sensitivity. However, using large amounts of anti-hapten antibodies can sometimes result in worsening cross-reactivity.
本発明の目的は、抗イムノコンプレックス抗体を用いる測定方法において、交叉反応性を改善する方法を提供することである。 The object of the present invention is to provide a method for improving cross-reactivity in assays using anti-immunocomplex antibodies.
上記課題に鑑みてなされた本発明は、以下の態様を包含する。
（１）以下の（ｉ）～（ｉｉｉ）の工程を含む免疫測定方法：
（ｉ）水不溶性担体に間接的に固定化された抗ハプテン抗体と、測定対象溶液中のハプテンとを、反応させる工程、
（ｉｉ）標識化された抗イムノコンプレックス抗体を、ハプテン－抗ハプテン抗体の免疫複合体と反応させる工程、
（ｉｉｉ）標識に由来するシグナルを検出する工程。
（２）抗ハプテン抗体がウサギモノクローナル抗体である、（１）に記載の方法。
（３）抗イムノコンプレックス抗体がマウスモノクローナル抗体である、（１）または（２）に記載の方法。
（４）ハプテンがジゴキシンであり、抗ハプテン抗体が抗ジゴキシンウサギモノクローナル抗体である、（１）～（３）いずれか１項に記載の方法。
（５）抗ハプテン抗体がフルオレッセインで修飾され、抗フルオレッセイン抗体により間接的に水不溶担体に固定化されている、（１）～（４）いずれか１項に記載の方法。
The present invention, which has been made in view of the above problems, includes the following aspects.
(1) An immunoassay method comprising the following steps (i) to (iii):
(i) reacting an anti-hapten antibody indirectly immobilized on a water-insoluble carrier with a hapten in a solution to be measured;
(ii) reacting a labeled anti-immunocomplex antibody with the hapten-antihapten antibody immune complex;
(iii) detecting a signal derived from the label.
(2) The method according to (1), wherein the anti-hapten antibody is a rabbit monoclonal antibody.
(3) The method according to (1) or (2), wherein the anti-immunocomplex antibody is a mouse monoclonal antibody.
(4) The method according to any one of (1) to (3), wherein the hapten is digoxin and the anti-hapten antibody is an anti-digoxin rabbit monoclonal antibody.
(5) The method according to any one of (1) to (4), wherein the anti-hapten antibody is modified with fluorescein and indirectly immobilized on the water-insoluble carrier via the anti-fluorescein antibody.
以下、本発明を詳細に説明する。 The present invention is described in detail below.
（１）ハプテン
本発明におけるハプテンは、通常競合法で測定される低分子の物質を指す。ハプテンとしては、甲状腺ホルモン（チロキシン、サイロキシン）、女性ホルモン（エストラジオール、エストリオール）や薬剤（ジゴキシン、ジギトキシン）などを例示可能である。本発明では特にジゴキシンを測定対象ハプテンとして用いることが好ましい。
(1) Hapten In the present invention, a hapten refers to a low-molecular-weight substance that is usually measured by a competitive method. Examples of haptens include thyroid hormones (thyroxine, thyroxine), female hormones (estradiol, estriol), and drugs (digoxin, digitoxin). In the present invention, it is particularly preferable to use digoxin as the hapten to be measured.
（２）測定対象溶液
測定対象溶液としては、ハプテンを含有するものであれば特に限定されるものではない。例えば人の血液（全血、血漿、血清等）、尿などの体液などが例示可能である。特に本発明においては血清等の血液が好ましい。
(2) Measurement Solution The measurement solution is not particularly limited as long as it contains a hapten. Examples include human blood (whole blood, plasma, serum, etc.) and body fluids such as urine. In particular, blood such as serum is preferred in the present invention.
（３）抗イムノコンプレックス抗体
本発明における抗イムノコンプレックス抗体は、ハプテンに対する抗体ではなく、ハプテンに対する抗体への抗体でもなく、ハプテンとそれに対する抗体との複合体に対する抗体である。本発明においては、抗イムノコンプレックス抗体の動物種は特に限定されない。動物種としてはマウス、ラット、ヤギ、ヒツジなどを例示できる。本発明においては特に、マウス由来の抗イムノコンプレックスモノクローナル抗体を用いることが好ましい。
(3) Anti-immunocomplex antibody The anti-immunocomplex antibody of the present invention is not an antibody against a hapten, nor an antibody to an antibody against an antibody against a hapten, but an antibody against a complex of a hapten and its antibody. In the present invention, the animal species of the anti-immunocomplex antibody is not particularly limited. Examples of animal species include mouse, rat, goat, and sheep. In the present invention, it is particularly preferable to use an anti-immunocomplex monoclonal antibody derived from a mouse.
（４）抗ハプテン抗体
本発明で、抗ハプテン抗体は高い親和性を有することが好ましい。高い親和性を有する抗体としてモノクローナル抗体が好ましく、それを取得可能な動物種として、ヤギ、ヒツジ、ウサギなどを例示可能である。親和性としては解離定数（ＫＤ）が１０－１０（Ｍ）以下であることが好ましく、更には１０－１１（Ｍ）以下であることが好ましい。特に本発明においてはウサギ由来のモノクローナル抗体を用いることが好ましい。
(4) Anti-hapten antibody In the present invention, it is preferable that the anti-hapten antibody has high affinity. Monoclonal antibodies are preferred as antibodies with high affinity, and examples of animal species from which they can be obtained include goats, sheep, and rabbits. The affinity is preferably such that the dissociation constant (KD) is 10 −10 (M) or less, and more preferably 10 −11 (M) or less. In particular, it is preferable to use a rabbit-derived monoclonal antibody in the present invention.
（５）水不溶性担体
本発明において水不溶性担体は特に限定されるものではないが、例えば、樹脂、ガラス、ポリスチレンなどの微粒子、粒子、ビーズ、プレート等が用いられる。水不溶性担体には、抗ハプテン抗体が間接的に固定化される。
(5) Water-insoluble carrier In the present invention, the water-insoluble carrier is not particularly limited, and examples thereof include fine particles, particles, beads, plates, etc. made of resin, glass, polystyrene, etc. An anti-hapten antibody is indirectly immobilized on the water-insoluble carrier.
（６）間接的な固定化法
間接的な固定化法は例えば、アビジン－ビオチン結合等を介して固定化してもよいし、水不溶担体に固相化した抗フルオレッセイン抗体を介してフルオレッセイン修飾した抗ハプテン抗体を固定化してもよい。また、抗ハプテン抗体の動物種に対する抗体（例えば、抗マウス抗体、抗ラット抗体、抗ウサギ抗体など）を固定化して、その抗体を介して抗ハプテン抗体を固定化してもよい。本発明においては、特に抗フルオレッセイン抗体を水不溶性担体に固定化し、それを介してフルオレッセイン修飾した抗ハプテン抗体を固定化することが好ましい。
(6) Indirect Immobilization Methods Indirect immobilization methods may involve immobilization via an avidin-biotin bond or the like, or immobilization of a fluorescein-modified anti-hapten antibody via an anti-fluorescein antibody solid-phased on a water-insoluble carrier. Alternatively, an antibody against the animal species of the anti-hapten antibody (e.g., an anti-mouse antibody, an anti-rat antibody, an anti-rabbit antibody, etc.) may be immobilized, and the anti-hapten antibody may be immobilized via that antibody. In the present invention, it is particularly preferred to immobilize an anti-fluorescein antibody on a water-insoluble carrier and then immobilize the fluorescein-modified anti-hapten antibody via the antibody.
（７）標識
本発明において標識は特に限定されるものではないが、例えば酵素、放射性同位元素、色素等が用いられる。酵素としては、好ましくはアルカリホスファターゼ等が使用できる。標識は、抗イムノコンプレックス抗体に結合される。標識の方法には特に限定はなく、また直接標識してもよく又は間接的に（例えばアビジン－ビオチン結合等を介して）標識してもよい。
(7) Labeling In the present invention, the label is not particularly limited, but examples thereof include enzymes, radioisotopes, dyes, etc. As the enzyme, alkaline phosphatase or the like can be preferably used. The label is bound to an anti-immunocomplex antibody. There is no particular limitation on the labeling method, and labeling may be performed directly or indirectly (for example, via an avidin-biotin bond, etc.).
（８）測定方法
本発明の方法では、例えば生物試料分析 Ｖｏｌ．３９，Ｎｏ４（２０１６）に示されるような、全自動免疫測定装置を用いることが好ましい。例えば図１に示すような２つのセルを有する試薬カップを用いて以下のような自動測定を行うことが好ましい。
（８－１）２セルカップの一方のセルに抗ハプテン抗体を間接的に固定化した水不溶性担体を分注し、他方のセルに酵素標識抗イムノコンプレックス抗体を分注し、試薬カップとする。
（８－２）試薬カップ、測定対象溶液を自動免疫測定装置にセットする。
（８－３）自動免疫測定装置において、ハプテンを含む測定対象溶液を試薬カップの水不溶性担体側のセルに分注し、反応させる。
（８－４）反応終了後、抗ハプテン抗体を間接的に固定化した水不溶性担体に未吸着の成分を洗浄する。
（８－５）試薬カップの酵素標識抗イムノコンプレックス抗体を、水不溶性担体側のセルへ移す。
（８－６）反応終了後、抗ハプテン抗体を間接的に固定化した水不溶性担体に未吸着の成分を洗浄する。
（８－７）酵素の基質を添加して、酵素反応に由来する生成物の発光強度などを測定する。
(8) Measurement Method In the method of the present invention, it is preferable to use a fully automated immunoassay device such as that described in Biological Sample Analysis, Vol. 39, No. 4 (2016). For example, it is preferable to perform the following automated measurement using a reagent cup having two cells as shown in FIG.
(8-1) A water-insoluble carrier on which an anti-hapten antibody is indirectly immobilized is dispensed into one cell of a two-cell cup, and an enzyme-labeled anti-immunocomplex antibody is dispensed into the other cell to form a reagent cup.
(8-2) Place the reagent cup and the solution to be measured into the automatic immunoassay device.
(8-3) In an automatic immunoassay device, a solution to be measured containing a hapten is dispensed into a cell on the water-insoluble carrier side of a reagent cup and allowed to react.
(8-4) After the reaction is completed, the components not adsorbed onto the water-insoluble carrier on which the anti-hapten antibody is indirectly immobilized are washed away.
(8-5) The enzyme-labeled anti-immunocomplex antibody in the reagent cup is transferred to the cell on the water-insoluble carrier side.
(8-6) After the reaction is completed, the components not adsorbed onto the water-insoluble carrier on which the anti-hapten antibody is indirectly immobilized are washed away.
(8-7) The substrate of the enzyme is added, and the luminescence intensity of the product resulting from the enzyme reaction is measured.
（９）交叉反応性
本発明において特異性が高い（交叉反応物質をほとんど認識しない）とは、交叉反応性が５％以下、さらに好ましくは１％以下であることが好ましい。
(9) Cross-reactivity In the present invention, high specificity (hardly recognizing cross-reactive substances) means that the cross-reactivity is preferably 5% or less, more preferably 1% or less.
ハプテン濃度を測定する際、特異性の高い測定系が望まれる。本発明の測定法では、抗イムノコンプレックス抗体を用いた測定系において、抗ハプテン抗体を水不溶担体に間接的に固定化することで、特異性を向上することが可能である。 When measuring hapten concentrations, a highly specific measurement system is desired. In the measurement method of the present invention, specificity can be improved in a measurement system using an anti-immunocomplex antibody by indirectly immobilizing the anti-hapten antibody on a water-insoluble carrier.
［実施例１］抗ハプテン抗体を間接的に固定化した微粒子による測定
以下、本発明の実施例をジゴキシンに対するウサギモノクローナル抗体と、それらの免疫複合体に対する抗イムノコンプレックスマウスモノクローナル抗体を用いて詳細に説明するが、本発明はこれに限定されるものではない。
Example 1 Measurement using microparticles on which anti-hapten antibodies are indirectly immobilized Below, examples of the present invention will be described in detail using a rabbit monoclonal antibody against digoxin and an anti-immunocomplex mouse monoclonal antibody against the immune complex thereof, but the present invention is not limited to this.
（１）抗ジゴキシンウサギモノクローナル抗体
抗ジゴキシンウサギモノクローナル抗体は特開２００９－２４０３００号公報に記載の方法で取得した。
(1) Anti-digoxin rabbit monoclonal antibody Anti-digoxin rabbit monoclonal antibody was obtained by the method described in JP 2009-240300 A.
（２）抗イムノコンプレックスマウスモノクローナル抗体
ジゴキシンと抗ジゴキシンウサギモノクローナル抗体の免疫複合体に対する抗体（抗イムノコンプレックスマウスモノクローナル抗体）は特許第６０３１９４４号公報に記載の方法で取得した。
(2) Anti-immunocomplex mouse monoclonal antibody An antibody against the immune complex of digoxin and anti-digoxin rabbit monoclonal antibody (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent No. 6,031,944.
（３）フルオレッセイン修飾抗ジゴキシンウサギモノクローナル抗体
フルオレッセインで修飾した抗ジゴキシンウサギモノクローナル抗体は、Ｆｌｕｏｒｅｓｃｅｉｎ Ｌａｂｅｌｉｎｇ Ｋｉｔ－ＮＨ２（株式会社 同仁化学研究所製）と、（１）で取得した抗ジゴキシンウサギモノクローナル抗体とを用いて作製した。
(3) Fluorescein-modified anti-digoxin rabbit monoclonal antibody Fluorescein-modified anti-digoxin rabbit monoclonal antibody was prepared using Fluorescein Labeling Kit-NH2 (manufactured by Dojindo Laboratories, Inc.) and the anti-digoxin rabbit monoclonal antibody obtained in (1).
（４）測定
自動分析は、全自動化学発光酵素免疫測定装置（ＡＩＡ－ＣＬ２４００、東ソー（株）製）を用いて、以下の方法で行った。
(4) Measurement Automated analysis was carried out using a fully automated chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) according to the following method.
（４－１）試薬カップの作製
図１に示す２つのセルを有するカップを用いて、自動分析に用いる試薬カップを作製した。（以下、中身に即して、カップの一方のセルを微粒子側のセル、他方のセルをコンジュゲート側のセルと呼ぶ。）
抗フルオレッセイン抗体を固定化した微粒子に、フルオレッセイン修飾抗ジゴキシンウサギモノクローナル抗体を混合し、抗フルオレッセイン抗体及びフルオレッセインを介して間接的に抗ジゴキシン抗体が固定化された微粒子を作製した。この間接固定化微粒子を含む溶液を、微粒子側のセルに分注した。コンジュゲート側のセルには、アルカリホスファターゼ標識した抗イムノコンプレックスマウスモノクローナル抗体を分注した。アルミシールをし、ジゴキシン測定用試薬カップとした。
(4-1) Preparation of Reagent Cup A reagent cup for automatic analysis was prepared using a cup having two cells as shown in Figure 1. (Hereinafter, one cell of the cup will be referred to as the particle side cell and the other cell as the conjugate side cell, depending on the contents.)
Microparticles immobilized with anti-fluorescein antibody were mixed with fluorescein-modified anti-digoxin rabbit monoclonal antibody to produce microparticles with anti-digoxin antibody immobilized indirectly via the anti-fluorescein antibody and fluorescein. A solution containing these indirectly immobilized microparticles was dispensed into the cell containing the microparticles. An alkaline phosphatase-labeled anti-immunocomplex mouse monoclonal antibody was dispensed into the cell containing the conjugate. An aluminum seal was placed over the cup to prepare a reagent cup for digoxin measurement.
（４－２）測定
全自動化学発光酵素免疫測定装置（ＡＩＡ－ＣＬ２４００、東ソー（株）製）に（４－１）で作製したジゴキシン測定用試薬カップをセットし、ジゴキシン濃度既知のサンプル６種（ｃａｌ１～ｃａｌ６）の測定を行い、検量線を作成した。測定は１０μＬの濃度既知サンプル（血清ベース）をジゴキシン測定用試薬カップの微粒子側のセルに分注し、５分間反応（一次反応）させた後、洗浄液による洗浄（Ｂ／Ｆ分離）を行い、次にコンジュゲート側のセルの試薬（５０μＬ）を微粒子側のセルに移し反応させた（二次反応、３分間）。二次反応の終了後、二回目の洗浄液による洗浄を行った。その後、酵素の基質（３（－５－ｔｅｒｔ－ブチル－４，４－ジメチル－２，６，７－トリオキサビシクロ［３，２，０］ヘプト－1-イル）フェニルリン酸エステルジナトリウム塩）を添加し、発光強度を測定した。測定結果を表１及び図２に示す。ジゴキシンの濃度に応じて発光強度が大きくなることが確認され、抗イムノコンプレックス抗体を用いたジゴキシンの測定系を構築できたことを確認した。
(4-2) Measurement: The digoxin assay reagent cup prepared in (4-1) was placed in a fully automated chemiluminescent enzyme immunoassay system (AIA-CL2400, Tosoh Corporation), and six samples with known digoxin concentrations (cal 1 to cal 6) were measured to create a calibration curve. For the measurement, 10 μL of a known-concentration sample (serum-based) was dispensed into the cell on the microparticle side of the digoxin assay reagent cup and allowed to react for 5 minutes (primary reaction), followed by washing with a cleaning solution (B/F separation). Next, the reagent (50 μL) from the conjugate cell was transferred to the microparticle cell and allowed to react (secondary reaction, 3 minutes). After completion of the secondary reaction, a second wash with a cleaning solution was performed. The enzyme substrate (3(-5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3,2,0]hept-1-yl)phenyl phosphate ester disodium salt) was then added, and the luminescence intensity was measured. The measurement results are shown in Table 1 and Figure 2. It was confirmed that the luminescence intensity increased depending on the concentration of digoxin, confirming that a digoxin measurement system using an anti-immunocomplex antibody had been established.
（５）交叉反応性評価
交叉反応性の評価は、ジゴキシンのゼロ濃度サンプルに表３に記載した交叉反応物質をそれぞれ添加し、測定することで行った。交叉反応率は次式に示す計算式で計算した。
交叉反応率（％）＝［（交叉反応性物質添加時の検量線からの算出濃度（ｎｇ／ｍＬ））／（交叉反応物質の分子量）］／［（同量のジゴキシン添加時の検量線からの算出濃度（ｎｇ／ｍＬ））／（ジゴキシンの分子量）］×１００
［比較例１］抗ハプテン抗体を直接固定化した微粒子による測定
（１－１）試薬カップの作製
図１に示す２つのセルを有するカップを用いて、自動分析に用いる試薬カップを作製した。抗ジゴキシンウサギモノクローナル抗体及び、抗イムノコンプレックス抗体は実施例１で示したものと同じものを使用した。
(5) Evaluation of cross-reactivity Cross-reactivity was evaluated by adding each of the cross-reacting substances listed in Table 3 to a zero concentration sample of digoxin and measuring the results. The cross-reactivity rate was calculated using the following formula:
Cross-reactivity rate (%) = [(calculated concentration (ng/mL) from the calibration curve when the cross-reacting substance was added) / (molecular weight of the cross-reacting substance)] / [(calculated concentration (ng/mL) from the calibration curve when the same amount of digoxin was added) / (molecular weight of digoxin)] × 100
Comparative Example 1 Measurement using microparticles directly immobilized with anti-hapten antibodies (1-1) Preparation of reagent cup A reagent cup for use in automatic analysis was prepared using a cup having two cells as shown in Figure 1. The anti-digoxin rabbit monoclonal antibody and anti-immunocomplex antibody used were the same as those used in Example 1.
抗ジゴキシンウサギモノクローナル抗体を微粒子に直接固定化した。この直接固定化微粒子を含む溶液を、微粒子側のセルに分注した。コンジュゲート側のセルには、アルカリホスファターゼ標識した抗イムノコンプレックスマウスモノクローナル抗体を分注した。アルミシールをし、ジゴキシン測定用試薬カップとした。 Anti-digoxin rabbit monoclonal antibody was directly immobilized onto microparticles. A solution containing these directly immobilized microparticles was dispensed into the cell on the microparticle side. Alkaline phosphatase-labeled anti-immunocomplex mouse monoclonal antibody was dispensed into the cell on the conjugate side. An aluminum seal was placed over the cup, creating a reagent cup for digoxin measurement.
（１－２）測定
全自動化学発光酵素免疫測定装置（ＡＩＡ－ＣＬ２４００、東ソー（株）製）に（１－１）で作製したジゴキシン測定用試薬カップをセットし、ジゴキシン濃度既知のサンプル６種（ｃａｌ１～ｃａｌ６）の測定を行い、検量線を作成した。測定は１０μＬの濃度既知サンプル（血清ベース）をジゴキシン測定用試薬カップの微粒子側のセルに分注し、５分間反応（一次反応）させた後、洗浄液による洗浄（Ｂ／Ｆ分離）を行い、次にコンジュゲート側のセルの試薬（５０μＬ）を微粒子側のセルに移し反応させた（二次反応、３分間）。二次反応の終了後、二回目の洗浄液による洗浄を行った。その後、酵素の基質を添加し、発光強度を測定した。測定結果を表２及び図３に示す。ジゴキシンの濃度に応じて発光強度が大きくなることが確認され、抗イムノコンプレックス抗体を用いたジゴキシンの測定系を構築できたことを確認した。
(1-2) Measurement: The digoxin assay reagent cup prepared in (1-1) was placed in a fully automated chemiluminescent enzyme immunoassay system (AIA-CL2400, Tosoh Corporation), and six samples with known digoxin concentrations (cal 1 to cal 6) were measured to create a calibration curve. For the measurement, 10 μL of a known-concentration sample (serum-based) was dispensed into the cell on the microparticle side of the digoxin assay reagent cup and allowed to react for 5 minutes (primary reaction), followed by washing with a cleaning solution (B/F separation). Next, the reagent (50 μL) from the conjugate cell was transferred to the microparticle cell and allowed to react (secondary reaction, 3 minutes). After the secondary reaction, a second wash with a cleaning solution was performed. The enzyme substrate was then added, and the luminescence intensity was measured. The measurement results are shown in Table 2 and Figure 3. It was confirmed that the luminescence intensity increased with digoxin concentration, confirming that a digoxin assay system using an anti-immunocomplex antibody had been successfully constructed.
（２）交叉反応性評価
交叉反応性の評価は、ジゴキシンのゼロ濃度サンプルに表３に記載した交叉反応物質をそれぞれ添加し、測定することで行った。交叉反応率は実施例１と同様に計算した。
(2) Evaluation of Cross-Reactivity The evaluation of cross-reactivity was carried out by adding each of the cross-reacting substances listed in Table 3 to a zero concentration sample of digoxin and measuring the results. The cross-reactivity rate was calculated in the same manner as in Example 1.
表３に実施例１及び比較例１で測定した交叉反応性を示す。実施例１のように、微粒子に間接的に抗体を固定化した方が高い特異性であることを確認した。 Table 3 shows the cross-reactivity measured in Example 1 and Comparative Example 1. It was confirmed that indirect immobilization of antibodies to microparticles, as in Example 1, resulted in higher specificity.
Claims (1)
マウスモノクローナル抗体である抗イムノコンプレックス抗体とを用いる以下の（ｉ）～（ｉｉｉ）の工程を含むジゴキシンの免疫測定方法であって、
（ｉ）水不溶性担体に間接的に固定化された抗ジゴキシンウサギモノクローナル抗体と、測定対象溶液中のジゴキシンとを、反応させる工程、
（ｉｉ）標識化された抗イムノコンプレックス抗体を、ジゴキシン－抗ジゴキシンウサギモノクローナル抗体の免疫複合体と反応させる工程、
（ｉｉｉ）標識に由来するシグナルを検出する工程、
前記抗ジゴキシンウサギモノクローナル抗体を水不溶性担体に間接的に固定化する方法が、フルオレッセインで修飾された抗ジゴキシンウサギモノクローナル抗体を、水不溶担体に固定化された抗フルオレッセイン抗体により間接的に水不溶担体に固定化する方法であることを特徴とする、
免疫測定方法。 anti-digoxin rabbit monoclonal antibody, which is a rabbit monoclonal antibody;
A method for immunoassay of digoxin using an anti-immunocomplex antibody which is a mouse monoclonal antibody, the method comprising the following steps (i) to (iii) :
(i) reacting an anti-digoxin rabbit monoclonal antibody indirectly immobilized on a water-insoluble carrier with digoxin in a solution to be measured;
(ii) reacting a labeled anti-immunocomplex antibody with the digoxin-anti-digoxin rabbit monoclonal antibody immune complex;
(iii) detecting a signal derived from the label;
The method for indirectly immobilizing an anti-digoxin rabbit monoclonal antibody on a water-insoluble carrier is characterized in that the anti-digoxin rabbit monoclonal antibody modified with fluorescein is indirectly immobilized on a water-insoluble carrier by an anti-fluorescein antibody immobilized on a water-insoluble carrier.
Immunoassay method.
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| JP2001174460A (en) | 1999-12-17 | 2001-06-29 | Tosoh Corp | Immunoassay method and immunoassay reagent |
| JP2002277464A (en) | 2000-08-21 | 2002-09-25 | F Hoffmann La Roche Ag | Improved binding assay |
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| JP2021081317A (en) | 2019-11-20 | 2021-05-27 | 東ソー株式会社 | Measuring method using anti-immunocomplex antibody |
| WO2021100459A1 (en) | 2019-11-20 | 2021-05-27 | 東ソー株式会社 | Measurement method using anti-immunocomplex antibody |
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| JP2001174460A (en) | 1999-12-17 | 2001-06-29 | Tosoh Corp | Immunoassay method and immunoassay reagent |
| JP2002277464A (en) | 2000-08-21 | 2002-09-25 | F Hoffmann La Roche Ag | Improved binding assay |
| US20130231462A1 (en) | 2012-02-27 | 2013-09-05 | Bio-Rad Laboratories, Inc. | Anti-immune complex antibodies |
| JP2021081317A (en) | 2019-11-20 | 2021-05-27 | 東ソー株式会社 | Measuring method using anti-immunocomplex antibody |
| WO2021100459A1 (en) | 2019-11-20 | 2021-05-27 | 東ソー株式会社 | Measurement method using anti-immunocomplex antibody |
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