JP6221466B2 - Hapten measurement method - Google Patents
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本発明はハプテンの測定法に関する。 The present invention relates to a method for measuring a hapten.
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。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）。 A hapten, which is a low molecular weight compound, is 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 reacted competitively with a certain amount of anti-hapten antibody. As the amount of hapten contained in the sample increases, the amount of labeled hapten bound 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 competition method depends on the affinity constant of the anti-hapten antibody used. It is difficult to obtain an anti-hapten antibody with a high affinity constant, and therefore it was 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、酵素などで標識した抗ハプテン抗体を添加する。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。そのため、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. A hapten measurement system using an anti-immunocomplex antibody is performed, for example, by the method shown below, and can measure a hapten contained in a sample without using a competition method.
(1) Immobilize an anti-immunocomplex antibody on a plate or the like.
(2) A sample containing the target hapten and an anti-hapten antibody labeled with an enzyme or the like are added. The anti-immunocomplex antibody selectively binds to an immune complex composed of a hapten and an anti-hapten antibody. Therefore, 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非競合型の免疫測定法の具体例としては、テロラヒドロカンアビノールの測定系（特許文献１）や、プロゲステロンの測定系（特許文献２）があげられる。 Specific examples of the non-competitive immunoassay method using an anti-immunocomplex antibody include a terahydrocanbininol measurement system (Patent Document 1) and a progesterone measurement system (Patent Document 2).
特許文献３に記載のように、抗イムノコンプレックス抗体は遊離の（免疫複合体を形成していない）抗ハプテン抗体との反応性もわずかながら有している。そのため、標識抗イムノコンプレックス抗体が、ハプテンと未反応の抗ハプテン抗体へ結合し、測定のノイズ、すなわちハプテンの存在に依存しないシグナルの原因となることがあった。 As described in Patent Document 3, the anti-immunocomplex antibody has a slight reactivity with a free (non-immunocomplex) anti-hapten antibody. Therefore, the labeled anti-immunocomplex antibody may bind to the hapten and an unreacted anti-hapten antibody, and may cause measurement noise, that is, a signal independent of the presence of the hapten.
本発明の目的は、抗ハプテン抗体、抗イムノコンプレックス抗体を含む、ハプテン測定系において、測定ノイズを低減することができる測定法を提供することにある。 An object of the present invention is to provide a measurement method capable of reducing measurement noise in a hapten measurement system including an anti-hapten antibody and an anti-immunocomplex antibody.
前記課題を鑑みてなされた本発明は、以下の態様を包含する。
（１）ハプテンと抗ハプテン抗体を反応させ、生成したハプテン−抗ハプテン抗体複合体と抗イムノコンプレックス抗体を反応させる際に、ハプテン類似物質を共存させることを特徴とする、ハプテンの測定方法。
（２）ハプテン類似物質を、抗イムノコンプレックス抗体より先に反応系中に共存させる、上述（１）に記載の方法。
（３）ハプテン類似物質を、抗イムノコンプレックス抗体と同時に反応系中に共存させる、上述（１）に記載の方法。
（４）ハプテンがステロイドホルモンである、上述（１）〜（３）のいずれかに記載の方法。
（５）抗ハプテン抗体がウサギモノクローナル抗体である、上述（１）〜（４）のいずれかに記載の方法。
（６）ハプテンがエストラジオールである、上述（１）〜（５）のいずれかに記載方法。
以下、本発明を詳細に説明する。
This invention made | formed in view of the said subject includes the following aspects.
(1) A method for measuring a hapten, comprising reacting a hapten with an anti-hapten antibody and reacting the generated hapten-anti-hapten antibody complex with an anti-immunocomplex antibody, wherein a hapten-like substance coexists.
(2) The method according to (1) above, wherein the hapten-like substance is allowed to coexist in the reaction system prior to the anti-immunocomplex antibody.
(3) The method according to (1) above, wherein the hapten-like substance is allowed to coexist in the reaction system simultaneously with the anti-immunocomplex antibody.
(4) The method according to any one of (1) to (3) above, wherein the hapten is a steroid hormone.
(5) The method according to any one of (1) to (4) above, wherein the anti-hapten antibody is a rabbit monoclonal antibody.
(6) The method according to any one of (1) to (5) above, wherein the hapten is estradiol.
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（以下、Ｅ２とする）、エストリオール、プロゲステロン（ｐｒｏｇｅｓｔｅｒｏｎｅ）、コルチゾール（ｃｏｒｔｉｓｏｌ）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。
(1) Hapten The hapten in the present invention is not particularly limited as long as it is a substance having a low molecular weight as measured by a competitive method. For example, triiodothyronine, thyroxine, 3,5-diiodo-L- Thyroid hormones such as thyronine and steroid hormones such as estrone, estradiol (hereinafter referred to as E2), estriol, progesterone, and cortisol. In the present invention, steroid hormones are particularly preferable, and E2 is more preferable among them.
（２）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。
(2) Anti-immunocomplex antibody An anti-immunocomplex antibody is not an antibody against a hapten but an antibody against an antibody against a hapten, and is an antibody against a complex of a hapten and an antibody against it.
（３）ハプテン類似物質
ハプテン類似物質は抗ハプテン抗体のエピトープの構造を含む分子であり、抗ハプテン抗体とは反応するが、その複合体、即ち、ハプテン類似物質と抗ハプテン抗体との複合体が抗イムノコンプレックス抗体と反応しない物質であればよい。例えば、ハプテン類似物質は、抗ハプテン抗体が認識するエピトープに影響を与えない位置に、測定対象のハプテンとは異なる構造を持つ物質であり、それによってハプテン類似物質と抗ハプテン抗体との複合体は、その表面にハプテン類似物質に由来するハプテンとは異なる構造が存在し、それが立体障害を引き起こして、抗イムノコンプレックス抗体と反応しないというものであってもよい。またハプテン類似物質は、抗イムノコンプレックス抗体と反発しあう電荷を帯びていて、その反発力によって、ハプテン類似物質と抗ハプテン抗体との複合体が抗イムノコンプレックス抗体と反応しないというものであってもよい。Ｅ２類似物質としては、例えばβ−エストラジオール−６−オン、６α−ヒドロキシエストラジオール、フルベストラント、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）（以下、６−ｃｍｏとする）、Ｅ２とＢＳＡとの結合物であるβ−エストラジオール６−（Ｏ−カルボキシメチル）オキシム：ＢＳＡ（以下、Ｅ２−ＢＳＡとする）などがあげられる。
(3) Hapten-like substance A hapten-like substance is a molecule containing the epitope structure of an anti-hapten antibody and reacts with an anti-hapten antibody, but its complex, that is, a complex of a hapten-like substance and an anti-hapten antibody Any substance that does not react with the anti-immunocomplex antibody may be used. For example, a hapten-like substance is a substance having a structure different from that of the hapten to be measured at a position that does not affect the epitope recognized by the anti-hapten antibody, so that the complex of the hapten-like substance and the anti-hapten antibody is The surface may have a structure different from a hapten derived from a hapten-like substance, which causes steric hindrance and does not react with an anti-immunocomplex antibody. A hapten-like substance has a charge repelling with an anti-immunocomplex antibody, and due to the repulsive force, a complex of the hapten-like substance and the anti-hapten antibody does not react with the anti-immunocomplex antibody. Good. Examples of E2 analogs include β-estradiol-6-one, 6α-hydroxyestradiol, fulvestrant, β-estradiol-6-one 6- (O-carboxymethyloxime) (hereinafter referred to as 6-cmo), Β-estradiol 6- (O-carboxymethyl) oxime that is a conjugate of E2 and BSA: BSA (hereinafter referred to as E2-BSA) and the like.
（４）ハプテン類似物質の測定系への共存
抗イムノコンプレックス抗体は遊離の（免疫複合体を形成していない）抗ハプテン抗体との反応性もわずかながら有しているため、この弱い反応性が測定時のノイズ、すなわちハプテンの存在に依存しないシグナルの原因となることがある。この測定ノイズは以下に示すような反応系で、ハプテン類似物質を共存させることで抑えることが可能となる。
（４−１）抗ハプテン抗体とハプテンを含む試料とを接触させ、当該ハプテンを抗ハプテン抗体に補足させる。
（４−２）（４−１）で得られた抗体−ハプテン複合体を、抗イムノコンプレックス抗体と反応させる。この時、反応液中にハプテン類似物質を抗ハプテン抗体に対して過剰量共存させておく。
(4) Coexistence of hapten-like substances in the measurement system The anti-immunocomplex antibody has a slight reactivity with a free (non-immunocomplex) anti-hapten antibody. It can cause noise during measurement, ie a signal that does not depend on the presence of a hapten. This measurement noise can be suppressed by the coexistence of a hapten-like substance in a reaction system as shown below.
(4-1) An anti-hapten antibody and a sample containing a hapten are brought into contact with each other, and the hapten is supplemented with the anti-hapten antibody.
(4-2) The antibody-hapten complex obtained in (4-1) is reacted with an anti-immunocomplex antibody. At this time, an excess amount of the hapten-like substance is allowed to coexist with the anti-hapten antibody in the reaction solution.
過剰量共存するハプテン類似物質は、測定対象のハプテンと反応しなかった抗ハプテン抗体と反応し、免疫複合体を形成する。前述のように、このハプテン類似物質と抗ハプテン抗体との免疫複合体は抗イムノコンプレックス抗体と反応しない。このように過剰量のハプテン類似物質の共存により、遊離の抗ハプテン抗体は反応系中にはほとんど存在しなくなるため、遊離の抗ハプテン抗体と抗イムノコンプレックス抗体との反応はほとんど起こらず、その結果、それに由来する測定ノイズを低減することができる。 The hapten-like substance coexisting in an excess amount reacts with an anti-hapten antibody that has not reacted with the hapten to be measured, and forms an immune complex. As described above, the immune complex of this hapten analog and anti-hapten antibody does not react with the anti-immunocomplex antibody. As a result of the coexistence of an excessive amount of the hapten-like substance, almost no free anti-hapten antibody is present in the reaction system, so that the reaction between the free anti-hapten antibody and the anti-immunocomplex antibody hardly occurs. Measurement noise derived therefrom can be reduced.
ハプテン類似物質を反応系中に共存させる量は２ｎｇ／ｍＬ以上２０μｇ／ｍＬ以下が好ましい。 The amount of the hapten-like substance to coexist in the reaction system is preferably 2 ng / mL or more and 20 μg / mL or less.
（５）ハプテン類似物質の共存の時期
ハプテン類似物質を共存させる時期は、測定対象のハプテンと抗ハプテン抗体が反応した後であればよく、特に限定されない。具体的には、ハプテン類似物質は、ハプテン−抗ハプテン抗体免疫複合体と抗イムノコンプレックス抗体とを反応させる際に、抗イムノコンプレックス抗体と同時に共存させてもよいし、抗イムノコンプレックス抗体より先に共存させてもよい。
(5) Timing of coexistence of hapten-like substances The time of coexistence of hapten-like substances is not particularly limited as long as the hapten to be measured reacts with the anti-hapten antibody. Specifically, the hapten-like substance may coexist with the anti-immunocomplex antibody when reacting the hapten-anti-hapten antibody immune complex and the anti-immunocomplex antibody, or before the anti-immunocomplex antibody. You may coexist.
（６）検出方法
本発明の測定方法は、ＥＬＩＳＡ法、ＲＩＡ法、蛍光偏向法等の検出方法を適宜採用することができ、必要に応じて抗体を固相化又は標識化して用いればよい。例えば、抗ハプテン抗体または抗イムノコンプレックス抗体のどちらか一方を固相に固定化して、他方を酵素等で標識することが好ましい。どちらの抗体を標識するかは特に限定されない。具体的には、抗イムノコンプレックス抗体を不溶性のビーズに固定化して、抗ハプテン抗体をアルカリホスファターゼ（ＡＬＰ）などの酵素で標識してもよい。また実施例で使用したＡＩＡ−６００ＩＩ（東ソー社製）などのエンザイムイムノアッセイ装置を用いて行うこともできる。
(6) Detection method As the measurement method of the present invention, a detection method such as an ELISA method, an RIA method, or a fluorescence deflection method can be appropriately employed, and the antibody may be used after being immobilized or labeled as necessary. For example, it is preferable that either one of the anti-hapten antibody or the anti-immunocomplex antibody is immobilized on a solid phase and the other is labeled with an enzyme or the like. Which antibody is labeled is not particularly limited. Specifically, the anti-immunocomplex antibody may be immobilized on insoluble beads, and the anti-hapten antibody may be labeled with an enzyme such as alkaline phosphatase (ALP). Moreover, it can also carry out using enzyme immunoassay apparatuses, such as AIA-600II (made by Tosoh Corporation) used in the Example.
（７）ノイズ低減の評価方法
ハプテン類似物質の共存によるノイズ低減は、標的ハプテンを含まない溶液の測定シグナルが、ハプテン類似物質の共存により減少することで確認される。
(7) Evaluation Method for Noise Reduction Noise reduction due to the coexistence of a hapten-like substance is confirmed by a decrease in the measurement signal of a solution not containing the target hapten due to the coexistence of the hapten-like substance.
本発明はハプテン測定時に反応液中にハプテン類似物質を共存させることを特徴としている。従来の抗イムノコンプレックス抗体を用いたハプテン測定系では、抗イムノコンプレックス抗体が遊離の抗ハプテン抗体との反応性もわずかながら有しているため、測定時のノイズの要因となっていた。本発明では、ハプテン類似物質を反応系中に共存させることで、測定ノイズを低減することができる。 The present invention is characterized in that a hapten-like substance is allowed to coexist in a reaction solution at the time of hapten measurement. In a conventional hapten measurement system using an anti-immunocomplex antibody, the anti-immunocomplex antibody has a slight reactivity with a free anti-hapten antibody, which causes noise during measurement. In the present invention, measurement noise can be reduced by allowing a hapten-like substance to coexist in the reaction system.
以下、実施例により、本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。なお、ハプテンとしてはエストラジオール（Ｅ２）を用い、抗ハプテン抗体、抗イムノコンプレックス抗体、ハプテン類似物質は以下のものを用いた。
（１）抗ハプテン抗体
抗ハプテン抗体は特開２００９−２４０３００号公報に記載の方法で得た、抗Ｅ２ウサギモノクローナル抗体を用いた。
（２）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、上記抗Ｅ２ウサギモノクローナル抗体とＥ２との複合体をマウスに免疫することで得た、マウス抗イムノコンプレックス抗体を用いた。
（３）ハプテン類似物質
ハプテン類似物質として６−ｃｍｏまたはＥ２−ＢＳＡを用いた。
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these. Estradiol (E2) was used as the hapten, and the following were used as the anti-hapten antibody, anti-immunocomplex antibody, and hapten-like substance.
(1) Anti-hapten antibody The anti-hapten antibody used was an anti-E2 rabbit monoclonal antibody obtained by the method described in JP-A-2009-240300.
(2) Anti-immunocomplex antibody As the anti-immunocomplex antibody, a mouse anti-immunocomplex antibody obtained by immunizing a mouse with the complex of the anti-E2 rabbit monoclonal antibody and E2 was used.
(3) Hapten-like substance 6-cmo or E2-BSA was used as a hapten-like substance.
［実施例１］ ハプテン類似物質濃度の検討（６−ｃｍｏ）
ハプテン類似物質の濃度の検討は、以下に示すＡＩＡ−６００ＩＩ（東ソー社製）を用いた測定系中に、ハプテン類似物質として６−ｃｍｏ（ｓｉｇｍａ社製）を共存させることで行った。
（１）水不溶性担体に抗イムノコンプレックス抗体を５００ｎｇ／担体となるよう物理的に吸着させ、ＢＳＡを用いてブロッキング処理を行った。
（２）６−ｃｍｏを共存させる濃度を変化させて、抗イムノコンプレックス抗体固定化担体と共に容器中に分注した。
（３）アルカリフォスファターゼ（ＡＬＰ）標識した抗Ｅ２ウサギモノクローナル抗体を上述の容器に加え反応させた。
（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ン酸（４−ＭＵＰ）を分注し、経時的に蛍光強度を測定することで４メチルウンベリフェロンの（４−ＭＵ）の生成速度を検出した。
[Example 1] Examination of hapten-like substance concentration (6-cmo)
The concentration of the hapten-like substance was examined by allowing 6-cmo (made by Sigma) as a hapten-like substance to coexist in a measurement system using AIA-600II (made by Tosoh Corporation) shown below.
(1) An anti-immunocomplex antibody was physically adsorbed on a water-insoluble carrier so as to be 500 ng / carrier, and a blocking treatment was performed using BSA.
(2) The concentration at which 6-cmo was allowed to coexist was changed and dispensed into a container together with the anti-immunocomplex antibody immobilized carrier.
(3) Alkaline phosphatase (ALP) -labeled anti-E2 rabbit monoclonal antibody was added to the container and allowed to react.
(4) After the B / F separation of the unreacted ALP-labeled antibody, 4-methylumbelliferic acid (4-MUP), which is a substrate of ALP, is dispensed, and the fluorescence intensity is measured over time to obtain 4 methyl. The production rate of umbelliferone (4-MU) was detected.
結果を図１に示す。横軸に６−ｃｍｏ濃度、縦軸にＡＩＡ−６００ＩＩでの検出値（ｒａｔｅ（ｎｍｏｌ／Ｌ・ｓ））をプロットし、ハプテン類似物質の共存によるノイズの低減を評価した。６−ｃｍｏ非共存下では、標的抗原であるＥ２が存在しないにも関わらず、３１．９ｎｍｏｌ／Ｌ・ｓと高いｒａｔｅを示す。一方で、６−ｃｍｏを２０ｎｇ／ｍＬ以上共存させるとｒａｔｅが１．６ｎｍｏｌ／Ｌ・ｓ程度に減少した。このことから、Ｅ２の類似物質である６−ｃｍｏを共存させることで、遊離の抗Ｅ２抗体と抗イムノコンプレックス抗体間の結合が阻害されることが示唆された。 The results are shown in FIG. The 6-cmo concentration was plotted on the horizontal axis and the detection value (rate (nmol / L · s)) on AIA-600II was plotted on the vertical axis, and noise reduction due to the coexistence of hapten-like substances was evaluated. In the absence of 6-cmo, a high rate of 31.9 nmol / L · s is exhibited despite the absence of E2 as the target antigen. On the other hand, when 6-cmo was allowed to coexist for 20 ng / mL or more, the rate was reduced to about 1.6 nmol / L · s. From this, it was suggested that the binding between free anti-E2 antibody and anti-immunocomplex antibody is inhibited by the coexistence of 6-cmo, which is a similar substance of E2.
［実施例２］ Ｅ２測定系の構築（６−ｃｍｏ共存）
２０ｎｇ／ｍＬの６−ｃｍｏ存在下で、Ｅ２濃度測定のための検量線作成を以下に示す方法で行った。
（１）水不溶性担体に抗イムノコンプレックス抗体を５００ｎｇ／担体となるよう物理的に吸着させ、ＢＳＡを用いてブロッキング処理を行った。
（２）２０ｎｇ／ｍＬの６−ｃｍｏを、抗イムノコンプレックス抗体固定化担体と共に容器中に分注した。
（３）ＡＬＰ標識した抗Ｅ２ウサギモノクローナル抗体と濃度既知のＥ２溶液を反応させた後、上述の容器に加え反応させた。
（４）未反応のＡＬＰ標識抗体をＢ／Ｆ分離後、４−ＭＵＰを分注し、経時的に蛍光強度を測定することで４−ＭＵの生成速度を検出した。
[Example 2] Construction of E2 measurement system (6-como coexistence)
In the presence of 20 ng / mL 6-cmo, a calibration curve for measuring E2 concentration was prepared by the method shown below.
(1) An anti-immunocomplex antibody was physically adsorbed on a water-insoluble carrier so as to be 500 ng / carrier, and a blocking treatment was performed using BSA.
(2) 20 ng / mL of 6-cmo was dispensed into a container together with the anti-immunocomplex antibody-immobilized carrier.
(3) After reacting an ALP-labeled anti-E2 rabbit monoclonal antibody with an E2 solution having a known concentration, it was added to the above-described container and allowed to react.
(4) After the B / F separation of the unreacted ALP-labeled antibody, 4-MUP was dispensed, and the fluorescence intensity was measured over time to detect the production rate of 4-MU.
結果を図２、３に示す。横軸にＥ２濃度、縦軸にＡＩＡ−６００ＩＩでの検出値（ｒａｔｅ（ｎｍｏｌ／Ｌ・ｓ））をプロットし、検量線を作成した。結果より、６−ｃｍｏの存在下においても、Ｅ２濃度に応じてｒａｔｅが上昇することが確認でき、特に低濃度域においても同様のことが確認できた。 The results are shown in FIGS. A calibration curve was created by plotting the E2 concentration on the horizontal axis and the detected value (rate (nmol / L · s)) on AIA-600II on the vertical axis. From the results, it was confirmed that the rate was increased according to the E2 concentration even in the presence of 6-cmo, and the same was confirmed particularly in the low concentration region.
［実施例３］ ハプテン類似物質濃度の検討（Ｅ２−ＢＳＡ）
ハプテン類似物質の濃度の検討は、以下に示すＡＩＡ−６００ＩＩ（東ソー社製）を用いた測定系中に、ハプテン類似物質としてＥ２−ＢＳＡ（ｓｉｇｍａ社製）を共存させることで行った。
（１）水不溶性担体に抗イムノコンプレックス抗体を５００ｎｇ／担体となるよう物理的に吸着させ、ＢＳＡを用いてブロッキング処理を行った。
（２）Ｅ２−ＢＳＡを共存させる濃度を変化させて、抗イムノコンプレックス抗体固定化担体と共に容器中に分注した。
（３）アルカリフォスファターゼ（ＡＬＰ）標識した抗Ｅ２ウサギモノクローナル抗体を上述の容器に加え反応させた。
（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ン酸（４−ＭＵＰ）を分注し、経時的に蛍光強度を測定することで４メチルウンベリフェロンの（４−ＭＵ）の生成速度を検出した。
[Example 3] Examination of hapten-like substance concentration (E2-BSA)
The concentration of the hapten-like substance was examined by allowing E2-BSA (made by Sigma) to coexist as a hapten-like substance in a measurement system using AIA-600II (made by Tosoh Corporation) shown below.
(1) An anti-immunocomplex antibody was physically adsorbed on a water-insoluble carrier so as to be 500 ng / carrier, and a blocking treatment was performed using BSA.
(2) The concentration at which E2-BSA was allowed to coexist was changed and dispensed into a container together with the anti-immunocomplex antibody immobilized carrier.
(3) Alkaline phosphatase (ALP) -labeled anti-E2 rabbit monoclonal antibody was added to the container and allowed to react.
(4) After the B / F separation of the unreacted ALP-labeled antibody, 4-methylumbelliferic acid (4-MUP), which is a substrate of ALP, is dispensed, and the fluorescence intensity is measured over time to obtain 4 methyl. The production rate of umbelliferone (4-MU) was detected.
結果を図４に示す。横軸にＥ２−ＢＳＡ濃度、縦軸にＡＩＡ−６００ＩＩでの検出値（ｒａｔｅ）をプロットし、ハプテン類似物質の共存によるノイズの低減を評価した。Ｅ２−ＢＳＡ非共存下では、標的抗原であるＥ２が存在しないにも関わらず、８．１ｎｍｏｌ／Ｌ・ｓと高いｒａｔｅを示す。一方で、Ｅ２−ＢＳＡを１００ｎｇ／ｍＬ以上共存させるとｒａｔｅが２．０ｎｍｏｌ／Ｌ・ｓ程度まで減少した。このことから、Ｅ２の類似物質であるＥ２−ＢＳＡを共存させることで、遊離の抗Ｅ２抗体と抗イムノコンプレックス抗体間の結合が阻害されることが示唆された。 The results are shown in FIG. The E2-BSA concentration was plotted on the horizontal axis, and the detection value (rate) of AIA-600II was plotted on the vertical axis, and the reduction of noise due to the coexistence of hapten-like substances was evaluated. In the absence of E2-BSA, a high rate of 8.1 nmol / L · s is exhibited despite the absence of E2 as the target antigen. On the other hand, when E2-BSA was allowed to coexist for 100 ng / mL or more, the rate decreased to about 2.0 nmol / L · s. This suggests that the binding between free anti-E2 antibody and anti-immunocomplex antibody is inhibited by the coexistence of E2-BSA, which is a similar substance of E2.
［実施例４］ Ｅ２測定系の構築（Ｅ２−ＢＳＡ共存）
１００ｎｇ／ｍＬのＥ２−ＢＳＡ存在下で、Ｅ２濃度測定のための検量線作成を以下に示す方法で行った。
（１）水不溶性担体に抗イムノコンプレックス抗体を５００ｎｇ／担体となるよう物理的に吸着させ、ＢＳＡを用いてブロッキング処理を行った。
（２）１００ｎｇ／ｍＬのＥ２−ＢＳＡを、抗イムノコンプレックス抗体固定化担体と共に容器中に分注した。
（３）ＡＬＰ標識した抗Ｅ２ウサギモノクローナル抗体と濃度既知のＥ２溶液を反応させた後、上述の容器に加え反応させた。
（４）未反応のＡＬＰ標識抗体をＢ／Ｆ分離後、４−ＭＵＰを分注し、経時的に蛍光強度を測定することで４−ＭＵの生成速度を検出した。
[Example 4] Construction of E2 measurement system (coexistence with E2-BSA)
In the presence of 100 ng / mL E2-BSA, a calibration curve for measuring E2 concentration was prepared by the method shown below.
(1) An anti-immunocomplex antibody was physically adsorbed on a water-insoluble carrier so as to be 500 ng / carrier, and a blocking treatment was performed using BSA.
(2) 100 ng / mL E2-BSA was dispensed into a container together with an anti-immunocomplex antibody-immobilized carrier.
(3) After reacting an ALP-labeled anti-E2 rabbit monoclonal antibody with an E2 solution having a known concentration, it was added to the above-described container and allowed to react.
(4) After the B / F separation of the unreacted ALP-labeled antibody, 4-MUP was dispensed, and the fluorescence intensity was measured over time to detect the production rate of 4-MU.
［比較例１］ Ｅ２−ＢＳＡなしでのＥ２測定系
実施例４と同様にして、但し、Ｅ２−ＢＳＡを共存させずに反応を行った。
結果を表１及び図５、６に示す。表１にはＥ２−ＢＳＡの有無による、各Ｅ２濃度でのｒａｔｅを示す。図５、６は横軸にＥ２濃度、縦軸にＡＩＡ−６００ＩＩでのｒａｔｅをプロットし作成した検量線を示す。
[Comparative Example 1] E2 measurement system without E2-BSA The reaction was performed in the same manner as in Example 4 except that E2-BSA was not present.
The results are shown in Table 1 and FIGS. Table 1 shows the rate at each E2 concentration with and without E2-BSA. 5 and 6 show calibration curves created by plotting E2 concentration on the horizontal axis and rate on AIA-600II on the vertical axis.
以上の結果から、抗イムノコンプレックス抗体を用いた競合法によらないＥ２測定系において、ハプテン類似物質を共存させることで、測定ノイズを低減できたことがわかる。 From the above results, it can be seen that measurement noise can be reduced by coexisting a hapten-like substance in an E2 measurement system that does not rely on a competition method using an anti-immunocomplex antibody.
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