JP2021081317A - Measuring method using anti-immunocomplex antibody - Google Patents
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Abstract
Description
本発明は、抗イムノコンプレックス抗体を用いた測定方法に関するものである。 The present invention relates to a measurement method using an anti-immunocomplex antibody.
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、放射性同位元素や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 usually measured by an immunoassay method called a competitive method. The competitive method is a method in which a hapten contained in a sample and a labeled hapten labeled with a radioisotope or an enzyme are competitively reacted with a certain amount of anti-hapten antibody. As the amount of hapten contained in the sample increases, the amount of labeled hapten binding to the anti-hapten antibody decreases. From this, the amount of hapten contained in the sample can be estimated based on the ratio of the labeled hapten binding to the anti-hapten antibody. The measurement sensitivity in the competitive method depends on the affinity constant of the anti-hapten antibody used. However, it is difficult to obtain an anti-hapten antibody having a high affinity constant, and therefore it is extremely difficult to measure a very small amount 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。抗イムノコンプレックス抗体を用いたハプテン測定系は、例えば以下に示す方法で行なわれ、試料中に含まれるハプテンを競合法によらず測定することができる。
（１）プレート等に抗イムノコンプレックス抗体を固定化する。
（２）標的ハプテンを含む試料と、酵素で標識した抗ハプテンン抗体を混合した後、（１）に添加する。
（３）過剰量の標識抗ハプテン抗体を洗浄し分離する。
（４）標識酵素に対する基質を添加し、酵素と基質の反応に由来するシグナルを検出する。
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。
A non-competitive immunoassay method using an anti-immunocomplex antibody has been proposed as a measurement method that can solve the problem of immunoassay by such a competitive method. The hapten measurement system using the anti-immunocomplex antibody is carried out by, for example, the method shown below, and the hapten contained in the sample can be measured without competing methods.
(1) Immobilize the anti-immunocomplex antibody on a plate or the like.
(2) The sample containing the target hapten and the enzyme-labeled anti-hapten antibody are mixed and then added to (1).
(3) Excessive amount of labeled anti-hapten antibody is washed and separated.
(4) A substrate for the labeling enzyme is added, and a signal derived from the reaction between the enzyme and the substrate is detected.
Since the anti-immunocomplex antibody selectively binds to an immune complex consisting of a hapten and an anti-hapten antibody, an increase in signal is observed as the amount of hapten contained in the sample increases.
抗イムノコンプレックス抗体を用いた測定系の具体例として特許文献１に示すようなエストラジオール（Ｅ２）測定系が上げられる。特許文献１に開示された技術では、用手法により酵素標識抗Ｅ２抗体とＥ２を含む測定対象液を事前に混合した後、抗イムノコンプレックス抗体が固定化された担体と反応させる方法であった。特許文献１に記載の方法では、類似物質に対する反応性（交叉反応性）が低いことが好ましい。 As a specific example of the measurement system using the anti-immunocomplex antibody, an estradiol (E2) measurement system as shown in Patent Document 1 can be mentioned. The technique disclosed in Patent Document 1 is a method in which an enzyme-labeled anti-E2 antibody and a solution to be measured containing E2 are mixed in advance by a conventional method, and then the anti-immunocomplex antibody is reacted with an immobilized carrier. In the method described in Patent Document 1, it is preferable that the reactivity (cross-reactivity) with a similar substance is low.
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系において、交叉反応性を抑えた測定方法を提供することである。 An object of the present invention is to provide a measurement method in which cross-reactivity is suppressed in a measurement system using an anti-immunocomplex antibody.
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）・抗ハプテ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、
（但し、一方は水不溶担体に固定化されており、他方は標識されている）
を用いた免疫測定方法であって、以下の（ｉ）〜（ｉｉｉ）の工程を含む方法：
（ｉ）抗ハプテンウサギモノクローナル抗体と測定対象溶液中のハプテンとを、吸収抗体の共存下、反応させる工程、
（ｉｉ）抗イムノコンプレックス抗体を、ハプテン−抗ハプテ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。
（２）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）に記載の方法。
（３）標識がアルカリホスファターゼである、（１）又は（２）に記載の方法。
（４）ハプテンがエストラジオールである、（１）〜（３）のいずれかに記載の方法。
The present invention made in view of the above problems includes the following aspects.
(1) ・ Anti-hapten rabbit monoclonal antibody, and ・ Anti-immunocomplex antibody,
(However, one is immobilized on a water-insoluble carrier and the other is labeled)
A method for measuring immunoassay using the above, which comprises the following steps (i) to (iii):
(I) A step of reacting an anti-hapten rabbit monoclonal antibody with a hapten in a solution to be measured in the coexistence of an absorbing antibody.
(Ii) A step of reacting an anti-immunocomplex antibody with an immune complex of a hapten-anti-hapten rabbit monoclonal antibody,
(Iii) A step of detecting a signal derived from a label.
(2) The method according to (1), wherein the anti-immunocomplex antibody is a mouse monoclonal antibody.
(3) The method according to (1) or (2), wherein the label is alkaline phosphatase.
(4) The method according to any one of (1) to (3), wherein the hapten is estradiol.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
（１）ハプテン
本発明におけるハプテンは、通常は競合法により測定されるような分子量の小さい物質であれば特に限定はなく、一例として、トリヨードサイロニン、チロキシン、３，５−ジヨード−Ｌ−チロニン等の甲状腺ホルモンや、エストロン、エストラジオール、エストリオール、プロゲステロン、コルチゾール等のステロイドホルモンがあげられる。特に本発明ではステロイドホルモンが好ましく、その中でもＥ２が更に好ましい。また甲状腺ホルモンの中ではチロキシンが好ましい。
(1) Hapten The hapten in the present invention is not particularly limited as long as it is a substance having a small molecular weight as usually measured by a competitive method. As an example, triiodothyronine, thyroxine, 3,5-diiodo-L- Examples include thyroid hormones such as thyronine and steroid hormones such as estrone, estradiol, estriol, progesterone and cortisol. In particular, steroid hormones are preferable in the present invention, and E2 is more preferable among them. Among the thyroid hormones, thyroxine is preferable.
（２)測定対象溶液
測定対象溶液としては、測定対象の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）、尿などの体液が好ましい。
(2) Solution to be measured The solution to be measured is not particularly limited as long as it contains a hapten to be measured. For example, human blood (whole blood, plasma, serum, etc.), body fluids such as urine are preferable.
（３)抗ハプテンウサギモノクローナル抗体
本発明では、抗ハプテン抗体としてウサギモノクローナル抗体を用いる。その理由は、ウサギモノクローナル抗体は、マウスモノクローナル抗体と比較して、ハプテンに対して高い親和性を有するものが得られるからである。
(3) Anti-Hapten Rabbit Monoclonal Antibody In the present invention, a rabbit monoclonal antibody is used as the anti-hapten antibody. The reason is that a rabbit monoclonal antibody can be obtained having a high affinity for a hapten as compared with a mouse monoclonal antibody.
（４）抗イムノコンプレックス抗体
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はウサギモノクローナル抗体とハプテンの複合体に対する特異的な抗体を取得する観点から、ウサギ以外の動物種から得ることが好ましい。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から抗イムノコンプレックスモノクローナル抗体を取得することが好ましい。
(4) Anti-immunocomplex antibody An anti-immunocomplex antibody is neither an antibody against a hapten nor an antibody against an antibody against a hapten, but an antibody against a complex of a hapten and an antibody against it. In the present invention, the anti-immunocomplex antibody is preferably obtained from an animal species other than rabbit from the viewpoint of obtaining a specific antibody against a complex of a rabbit monoclonal antibody and a hapten. Examples of animal species include mice, rats, goats, and sheep. In the present invention, it is particularly preferable to obtain an anti-immunocomplex monoclonal antibody from a mouse.
（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。水不溶性担体は、抗ハプテ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のいずれか一方が固定化される。固定化の方法には特に限定はなく、また直接固定化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）固定化してもよい。
(5) Water-insoluble carrier In the present invention, the water-insoluble carrier is not particularly limited, and for example, fine particles such as resin, glass, and polystyrene, particles, beads, and plates are used. As the water-insoluble carrier, either an anti-hapten rabbit monoclonal antibody or an anti-immunocomplex antibody is immobilized. The method of immobilization is not particularly limited, and may be immobilized directly or indirectly (for example, via an avidin-biotin bond or the like).
（６）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、抗ハプテ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の他方（水不溶性担体に固定化されない方）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。
(6) Labeling In the present invention, labeling is not particularly limited, but for example, enzymes, radioisotopes, dyes and the like are used. As the enzyme, alkaline phosphatase or the like can be preferably used. The label is attached to the other of the anti-hapten rabbit monoclonal antibody and the anti-immunocomplex antibody (the one that is not immobilized on the water-insoluble carrier). The labeling method is not particularly limited, and may be labeled directly or indirectly (for example, via an avidin-biotin bond).
（７）交叉反応
交叉反応は、測定対象物質の類似物質（交叉反応性物質）が測定対象物質に対する抗体と反応することである。免疫測定試薬において交叉反応性の大きな測定系を使用した場合、交叉反応により測定結果が偽高値となる。通常競合法を用いて測定するハプテンを、抗イムノコンプレックス抗体を用いてサンドイッチ型のアッセイを行った場合、サンドイッチ型アッセイの方が、交叉反応性が大きくなる場合があることを、本発明者らは見出した。そしてこの点について本発明者らが検討した結果、サンドイッチ型アッセイを行う場合、競合法に比べて多くの抗ハプテン抗体を用いることが一因と考えられた。また、抗ハプテン抗体とハプテンとの免疫複合体、抗ハプテン抗体と交叉反応性物質との複合体の表面構造が同一であり、抗イムノコンプレックス抗体がどちらも認識してしまうことも原因として考えられた。
(7) Cross-reaction A cross-reaction is a reaction in which a substance similar to a substance to be measured (cross-reactive substance) reacts with an antibody against the substance to be measured. When a measurement system having high cross-reactivity is used in the immunoassay reagent, the measurement result becomes a false high value due to the cross-reactivity. The present inventors have stated that when a hapten usually measured by a competitive method is subjected to a sandwich-type assay using an anti-immunocomplex antibody, the sandwich-type assay may have higher cross-reactivity. Found. As a result of the investigation by the present inventors on this point, it was considered that one of the reasons is that more anti-hapten antibodies are used when performing the sandwich type assay as compared with the competitive method. Another possible cause is that the surface structure of the immune complex between the anti-hapten antibody and the hapten and the complex between the anti-hapten antibody and the cross-reactive substance are the same, and the anti-immunocomplex antibody recognizes both of them. It was.
（８）吸収抗体
吸収抗体は、測定対象のハプテンにはほとんど反応せず、交叉反応性物質に特異的に反応する抗体である。測定対象ハプテンへの反応性は全くないことが好ましいが、測定感度に影響のでない範囲の弱い反応性を有していても問題ない。具体的には、吸収抗体の測定対象ハプテンへの反応性は、交叉反応性物質への反応性の約１/１００以下であることが好ましい。
(8) Absorbing antibody An absorbing antibody is an antibody that hardly reacts with the hapten to be measured and specifically reacts with a cross-reactive substance. It is preferable that there is no reactivity with the hapten to be measured, but there is no problem even if it has a weak reactivity within a range that does not affect the measurement sensitivity. Specifically, the reactivity of the absorbed antibody to the hapten to be measured is preferably about 1/100 or less of the reactivity to the cross-reactive substance.
（９）工程（ｉ）〜（ｉｉｉ）
上述の工程（ｉ）〜（ｉｉｉ）は、この順に行われることが好ましいが、工程（ｉ）（ｉｉ）を同時に行ってもよい。また工程（ｉ）と（ｉｉ）の間や、工程（ｉｉ）と（ｉｉｉ）の間に他の工程が存在してもよい。例えば間に分離／洗浄工程を有することにより、測定感度を高めることができる。
(9) Steps (i) to (iii)
The above steps (i) to (iii) are preferably performed in this order, but steps (i) and (ii) may be performed at the same time. Further, another step may exist between the steps (i) and (ii) or between the steps (ii) and (iii). For example, by having a separation / cleaning step in between, the measurement sensitivity can be increased.
（１０）自動分析系
本発明の方法では、自動分析系、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分析を行うことが好ましい。
(10) Automatic analysis system In the method of the present invention, an automatic analysis system, for example, biological sample analysis Vol. It is preferable to use a fully automatic immunoassay device as shown in 39, No. 4 (2016). For example, it is preferable to perform the following automatic analysis using a reagent cup having two cells as shown in FIG.
（１０−１）２セルカップの一方のセルに抗ハプテン抗体を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。
（１０−２）試薬カップ、測定対象液を自動免疫測定装置にセットする。
（１０−３）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に分注し、反応させる。この際、吸収抗体を共存させる。
（１０−４）反応終了後、抗ハプテン抗体を固定化した水不溶性担体に未吸着の成分を洗い流す。
（１０−５）試薬カップの酵素標識抗イムノコンプレックス抗体を、水不溶性担体側へ移す。
（１０−６）反応終了後、抗ハプテン抗体を固定化した水不溶性担体に未吸着の成分を洗い流す。
（１０−７）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。
(10-1) A water-insoluble carrier on which an anti-hapten antibody is immobilized is dispensed into one cell of a 2-cell cup, and an enzyme-labeled anti-immunocomplex antibody is dispensed into the other cell to prepare a reagent cup.
(10-2) Set the reagent cup and the liquid to be measured in the automatic immunoassay device.
(10-3) In the automatic immunoassay device, the solution to be measured containing hapten is dispensed to the water-insoluble carrier side of the reagent cup and reacted. At this time, the absorbing antibody is allowed to coexist.
(10-4) After completion of the reaction, the unadsorbed components are washed away on a water-insoluble carrier on which an anti-hapten antibody is immobilized.
(10-5) Transfer the enzyme-labeled anti-immunocomplex antibody of the reagent cup to the water-insoluble carrier side.
(10-6) After completion of the reaction, the unadsorbed components are washed away on a water-insoluble carrier on which an anti-hapten antibody is immobilized.
(10-7) The substrate of the enzyme is added, and the luminescence intensity of the product derived from the enzyme reaction is measured.
抗イムノコンプレックス抗体を用いて対象ハプテンを測定する際に、交叉反応性の低い方法が望まれる。本発明の測定方法では、抗ハプテン抗体と測定対象ハプテンとの反応の際に、吸収抗体を共存させることで、交叉反応を抑えた測定系が提供可能である。 When measuring a target hapten using an anti-immunocomplex antibody, a method with low cross-reactivity is desired. In the measuring method of the present invention, it is possible to provide a measuring system in which the crossover reaction is suppressed by coexisting the absorbing antibody when the anti-hapten antibody and the hapten to be measured react.
［実施例１］
以下、本発明の実施例をエストラジオール（Ｅ２）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に関して詳細に説明するが、本発明はこれらに限定されるものではない。
[Example 1]
Hereinafter, examples of the present invention will be described in detail with respect to rabbit monoclonal antibodies against estradiol (E2) and anti-immunoplex mouse monoclonal antibodies against their immune complexes, but the present invention is not limited thereto.
（１）抗Ｅ２ウサギモノクローナル抗体
抗Ｅ２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-E2 Rabbit Monoclonal Antibody The anti-E2 rabbit monoclonal antibody was obtained by the method described in JP-A-2009-240300.
（２）抗イムノコンプレックスマウスモノクローナル抗体
Ｅ２と抗Ｅ２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-Immuno Complex Mouse Monoclonal Antibody An antibody against the immune complex of E2 and an anti-E2 rabbit monoclonal antibody (anti-immunocomplex mouse monoclonal antibody) was obtained by the method described in Japanese Patent No. 6031944.
（３）吸収抗体（エチニルエストラジオール（ＥＥ２）に対する抗体）
吸収抗体、即ちＥ２の類似物質であるＥＥ２に対する抗体は以下に示す方法で取得した。
(3) Absorption antibody (antibody against ethinyl estradiol (EE2))
Absorbent antibody, that is, antibody against EE2, which is a similar substance to E2, was obtained by the method shown below.
（３−１）動物への免疫
免疫動物としては、マウス５週齢メス６匹を使用した。抗原は１，３，５（１０）−ＥＳＴＲＡＴＲＩＥＮ−１７α−ＥＴＨＹＮＹＬ−３，１７−ｂｅｔａ−ＤＩＯＬ−６−ＯＮＥ−６−ＣＡＲＢＯＸＹＭＥＴＨＹＬＯＸＩＭＥ：ＢＳＡ（ＳＴＥＲＡＬＯＩＤＳ社製）を用い、抗原溶液とアジュバンドとを等量混合したエマルジョンを作製し、それをマウスに対して１週間間隔で４回免疫した。マウス１匹あたりの免疫量は抗原量として５０μｇを免疫した。またアジュバントは、初回の免疫ではフロイント完全アジュバントを、二回目以降の免疫ではフロイント不完全アジュバントをそれぞれ用いた。
(3-1) Immunity to animals As immunized animals, 6 5-week-old female mice were used. As the antigen, 1,3,5 (10) -ESTRATRIEN-17α-ETHYNYL-3,17-beta-DIOL-6-ONE-6-CARBOXYMETHYLOXIME: BSA (manufactured by STARALOIDS) was used, and the antigen solution and the adjuvant were equalized. Emulsions mixed in volume were made and immunized with mice four times at weekly intervals. The amount of immunity per mouse was 50 μg as the amount of antigen. As the adjuvant, Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second and subsequent immunizations.
（３−２）抗体価の確認
以下に示すＥＬＩＳＡで抗体価の上昇を確認した。
（３−２−１）
ＫＬＨに化学的に結合したＥＥ２（ＫＬＨ−ＥＥ２）を１μｇ／ｍＬでＥＬＩＳＡプレートに固定化後、１％のスキムミルク溶液でブロッキングした。
（３−２−２）
取得したマウスの抗血清を１０００倍希釈の状態から希釈系列（２倍希釈）を作製し、ＥＬＩＳＡプレートに固相化したＫＬＨ−ＥＥ２と反応させた。
（３−２−３）
Ｂ／Ｆ（Ｂｏｕｎｄ／Ｆｒｅｅ）分離後、アルカリホスファターゼ（以下、ＡＬＰとする）標識抗体であるαＭｏｕｓｅＩｇＧ−ＡＬＰ（Ｍｅｒｃｋ社製）をプレートに添加して、プレート上のマウス抗体と反応させた。
（３−２−４）
未反応のＡＬＰ標識抗体をＢ／Ｆ分離後、ＡＬＰの基質である４−メチルウンベリフェリルリン酸（４−ＭＵＰ）をプレートに分注し、蛍光強度を測定することで検出し、抗体価の上昇したマウスを選別した。
(3-2) Confirmation of antibody titer An increase in antibody titer was confirmed by ELISA shown below.
(3-2-1)
EE2 (KLH-EE2) chemically bound to KLH was immobilized on an ELISA plate at 1 μg / mL and then blocked with a 1% skim milk solution.
(3-2-2)
A dilution series (2-fold dilution) was prepared from the obtained mouse antiserum diluted 1000-fold, and reacted with KLH-EE2 immobilized on an ELISA plate.
(3-2-3)
After B / F (Bound / Free) separation, αMouseIgG-ALP (manufactured by Merck), which is an alkaline phosphatase (hereinafter referred to as ALP) -labeled antibody, was added to the plate and reacted with the mouse antibody on the plate.
(3-2-4)
After B / F separation of unreacted ALP-labeled antibody, 4-methylumbelliferyl phosphate (4-MUP), which is a substrate of ALP, was dispensed into a plate and detected by measuring the fluorescence intensity, and the antibody titer was detected. Elevated mice were screened.
（３−３）吸収抗体生産ハイブリドーマの作製
（３−２）で選択したマウスから、以下に示す方法で抗体産生細胞を作製した。
（３−３−１）抗体価の上昇したマウスの脾臓を摘出し、定法に従い脾臓細胞を調製した。調製した脾臓細胞を電気融合法によりマウスミエローマ細胞（ＳＰ２／０）と融合させ、ハイブリドーマを作製した。
（３−３−２）融合後のハイブリドーマ浮遊液を１０％ＦＣＳ（Ｆｅｔａｌ ｃａｌｆ ｓｅｒｕｍ）と１×ＨＡＴ（ｓｉｇｕｍａ製）を含むＥ−ＲＤＦ培地（極東製薬製）に懸濁後、マイクロタイタープレートにまいて８日間培養し、培養上清を取得した。
(3-3) Preparation of Absorbing Antibody Producing Hybridoma Antibody-producing cells were prepared from the mice selected in (3-2) by the method shown below.
(3-3-1) The spleen of a mouse having an increased antibody titer was excised, and spleen cells were prepared according to a conventional method. The prepared spleen cells were fused with mouse myeloma cells (SP2 / 0) by an electric fusion method to prepare a hybridoma.
(3-3-2) The hybridoma suspension after fusion is suspended in an E-RDF medium (manufactured by Far East Pharmaceutical Co., Ltd.) containing 10% FCS (fetal calf serum) and 1 × HAT (manufactured by siguma), and then placed on a microtiter plate. The cells were cultivated for 8 days to obtain a culture supernatant.
（３−４）吸収抗体生産ハイブリドーマのスクリーニング
吸収抗体はＥＥ２と強く反応し、Ｅ２との反応性は低いことが好ましい。そこで本発明における吸収抗体のスクリーニングでは、Ｅ２の存在下でもＥＥ２と選択的に反応する抗体を選別すればよい。具体的には、以下に示すＥＬＩＳＡでスクリーニングを行った。
（３−４−１）
ＫＬＨ−ＥＥ２を１μｇ／ｍＬでＥＬＩＳＡプレートに固定化後、１％のスキムミルク溶液でブロッキングした。
（３−４−２）
培養上清をＥ２の存在下、又は非存在下でそれぞれＥＬＩＳＡプレートに固相化したＫＬＨ−ＥＥ２と反応させた。
（３−４−３）
Ｂ／Ｆ分離後、ＡＬＰ標識抗体であるαＭｏｕｓｅＩｇＧ−ＡＬＰ（Ｍｅｒｃｋ社製）をプレートに添加して、プレート上のマウス抗体と反応させた。
（３−４−４）
未反応のＡＬＰ標識抗体をＢ／Ｆ分離後、ＡＬＰの基質である４−メチルウンベリフェリルリン酸（４−ＭＵＰ）をプレートに分注し、蛍光強度を測定することで検出し、Ｅ２の存在下でＥＥ２と反応する抗体を生産するハイブリドーマを選別した。
(3-4) Screening for Absorbent Antibody Production Hybridoma Absorbent antibody preferably reacts strongly with EE2 and has low reactivity with E2. Therefore, in the screening of the absorbed antibody in the present invention, an antibody that selectively reacts with EE2 even in the presence of E2 may be selected. Specifically, screening was performed by the ELISA shown below.
(3-4-1)
KLH-EE2 was immobilized on an ELISA plate at 1 μg / mL and then blocked with 1% skim milk solution.
(3-4-2)
The culture supernatant was reacted with KLH-EE2 immobilized on an ELISA plate in the presence or absence of E2, respectively.
(3-4-3)
After B / F separation, αMouseIgG-ALP (manufactured by Merck), which is an ALP-labeled antibody, was added to the plate and reacted with the mouse antibody on the plate.
(3-4-4)
After B / F separation of the unreacted ALP-labeled antibody, 4-methylumbelliferyl phosphate (4-MUP), which is a substrate of ALP, was dispensed into a plate and detected by measuring the fluorescence intensity of E2. Hybridomas that produce antibodies that react with EE2 in the presence were selected.
（３−５）吸収抗体の生産及び精製
（３−４）で選別した吸収抗体生産ハイブリドーマを、１０％ＦＣＳを含むＥ−ＲＤＦ培地（極東製薬製）で培養し、培養上清を得た。培養上清に存在する抗体を硫安沈殿により濃縮し、Ｔｓｋｇｅｌ Ｅｔｈｅｒ−５ｐｗカラムを用いて精製し、吸収抗体を得た。
(3-5) Production and purification of absorbed antibody The absorbed antibody-producing hybridoma selected in (3-4) was cultured in an E-RDF medium (manufactured by Far East Pharmaceutical Co., Ltd.) containing 10% FCS to obtain a culture supernatant. The antibody present in the culture supernatant was concentrated by ammonium sulfate precipitation and purified using a Tskgel Ether-5pw column to obtain an absorbed antibody.
（４）自動分析
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) Automatic analysis The automatic analysis was performed by the following method using a fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation).
（４−１）試薬カップの作製
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Ｅ２ウサギモノクローナル抗体を固定化した微粒子を含む溶液に、吸収抗体を１０μｇ／ｍＬになるよう添加し、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抗体と、特許文献１を参考に、β−エストラジオール−６−オン６−（Ｏ−カルボキシメチルオキシム）を含む溶液を分注した。溶液を凍結乾燥し、アルミシールをし、Ｅ２測定用試薬カップとした。
(4-1) Preparation of Reagent Cup A reagent cup used for automatic analysis was prepared using a cup having two cells. (Hereinafter, according to the contents, one cell of the cup is referred to as a cell on the fine particle side, and the other cell is referred to as a cell on the conjugate side.)
The absorbed antibody was added to a solution containing the fine particles on which the anti-E2 rabbit monoclonal antibody was immobilized at 10 μg / mL, and the antibody was dispensed into the cells on the fine particle side. A solution containing an alkaline phosphatase-labeled anti-immunocomplex antibody and β-estradiol-6-one-6- (O-carboxymethyloxime) was dispensed into the conjugate-side cell with reference to Patent Document 1. The solution was lyophilized, sealed with aluminum, and used as a reagent cup for E2 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Ｅ２測定用試薬カップをセットし、Ｅ２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。測定結果を図２に示す。Ｅ２の濃度に応じて発光強度が大きくなることが確認され、吸収抗体存在条件下で、抗イムノコンプレックス抗体を用いたＥ２の自動分析系を構築できたことを確認した。
(4-2) Measurement Set the reagent cup for E2 measurement prepared in (4-1) in a fully automatic chemiluminescent enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation), and 6 types of samples with known E2 concentration. Measurements (cal1 to cal6) were performed, and a calibration curve was prepared. The measurement results are shown in FIG. It was confirmed that the luminescence intensity increased according to the concentration of E2, and it was confirmed that an automatic analysis system for E2 using an anti-immunocomplex antibody could be constructed under the condition of the presence of an absorbing antibody.
（４−３）交叉反応性の評価
ＥＥ２を１０ｎｇ／ｍＬに調整した溶液を用いて、（４−１）で作製したＥ２測定用試薬カップ及び、ＡＩＡ−ＣＬ２４００を用いて発光強度を測定した。また、（４−２）で作成した検量線から交叉反応率を計算した。結果を表１に示す。
(4-3) Evaluation of cross-reactivity The emission intensity was measured using the reagent cup for measuring E2 prepared in (4-1) and AIA-CL2400 using a solution prepared by adjusting EE2 to 10 ng / mL. In addition, the cross-reactivity rate was calculated from the calibration curve prepared in (4-2). The results are shown in Table 1.
［比較例１］
以下、実施例１で作製したＥ２測定用試薬カップ中に吸収抗体を含まない場合を比較例として説明する。
[Comparative Example 1]
Hereinafter, the case where the absorbing antibody is not contained in the reagent cup for E2 measurement prepared in Example 1 will be described as a comparative example.
（１）吸収抗体を含まないＥ２用測定カップ（比較用カップ）
実施例１（４−１）と同様にして、但し吸収抗体を添加せずに、比較用カップを製造した。
(1) Measurement cup for E2 (comparison cup) that does not contain absorbed antibody
A comparative cup was produced in the same manner as in Example 1 (4-1), except that no absorbing antibody was added.
（２）測定
ＡＩＡ−ＣＬ２４００に（１）で作製した比較用カップをセットし、Ｅ２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。測定結果を図３に示す。図２（実施例１）と比較して、吸収抗体の有無で検量線の形に大きな変化はないことが確認された。
(2) Measurement The comparison cup prepared in (1) was set in AIA-CL2400, and 6 types of samples (cal1 to cal6) having known E2 concentrations were measured to prepare a calibration curve. The measurement results are shown in FIG. Compared with FIG. 2 (Example 1), it was confirmed that there was no significant change in the shape of the calibration curve depending on the presence or absence of the absorbed antibody.
（３）交叉反応性の評価
（１）で作製した比較用カップとＥＥ２を１０ｎｇ／ｍＬに調整した溶液を用いて、ＡＩＡ−ＣＬ２４００で測定した。（２）で作成した検量線から交叉反応率を計算した。
実施例１及び比較例１で測定した交叉反応率を表１に示す。
(3) Evaluation of cross-reactivity The comparison cup prepared in (1) and the solution prepared by adjusting EE2 to 10 ng / mL were used for measurement with AIA-CL2400. The cross-reactivity rate was calculated from the calibration curve prepared in (2).
Table 1 shows the cross-reactivity rates measured in Example 1 and Comparative Example 1.
交叉反応率の結果から、吸収抗体の存在により、交叉反応を大幅に改善可能であることが示された。以上の結果から、本発明の方法により抗イムノコンプレックス抗体を用いた測定系において、交叉反応の抑制が可能であることが確認された。 The results of the cross-reactivity rate showed that the presence of the absorbed antibody could significantly improve the cross-reactivity. From the above results, it was confirmed that the cross-reactivity can be suppressed in the measurement system using the anti-immunocomplex antibody by the method of the present invention.
Claims (4)
・抗イムノコンプレックス抗体、
（但し、一方は水不溶担体に固定化されており、他方は標識されている）
を用いた免疫測定方法であって、以下の（ｉ）〜（ｉｉｉ）の工程を含む方法：
（ｉ）抗ハプテンウサギモノクローナル抗体と測定対象溶液中のハプテンとを、吸収抗体の共存下、反応させる工程、
（ｉｉ）抗イムノコンプレックス抗体を、ハプテン−抗ハプテ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。 -Anti-hapten rabbit monoclonal antibody, and-Anti-immunocomplex antibody,
(However, one is immobilized on a water-insoluble carrier and the other is labeled)
A method for measuring immunoassay using the above, which comprises the following steps (i) to (iii):
(I) A step of reacting an anti-hapten rabbit monoclonal antibody with a hapten in a solution to be measured in the coexistence of an absorbing antibody.
(Ii) A step of reacting an anti-immunocomplex antibody with an immune complex of a hapten-anti-hapten rabbit monoclonal antibody,
(Iii) A step of detecting a signal derived from a label.
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