JP2016186498A - Hapten standard solution, and hapten determination reagent containing the same - Google Patents
Hapten standard solution, and hapten determination reagent containing the same Download PDFInfo
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本発明は、試料中に含まれるハプテンを定量する際用いるハプテン標準液、および前記標準液を含むハプテン定量試薬に関する。 The present invention relates to a hapten standard solution used when quantifying a hapten contained in a sample, and a hapten quantitative reagent containing the standard solution.
抗原抗体反応を利用して特定のハプテンを測定する場合には、当該ハプテンを認識する抗体の特異性の高さが測定系の正確度を左右する。 When measuring a specific hapten using an antigen-antibody reaction, the high specificity of the antibody that recognizes the hapten affects the accuracy of the measurement system.
通常、ハプテンに対して特異性の高い抗体を得るには、前記ハプテンの類似構造物質と交差反応性を示さない抗体を選択する。例えば、ハプテンがステロイドホルモンの場合は、試料（例えば血液、血清などの体液）中に存在する、ステロール骨格を有した物質やステロール骨格に類似した構造を有した物質等との交差反応を調査の上、前記物質に対する交差反応が極力低い抗体を選択する。ところが、ステロイドホルモンの場合、試料中に存在する当該ステロイドホルモンの類似構造物質の種類が多く、単一の特異性を有したモノクローナル抗体の技術をもってしても、当該ステロイドホルモンのみを認識する抗体を得るのは困難である。そのため一般的には、前記類似構造物質に対する交差反応率が５％以下であれば、当該ステロイドホルモンに対する抗体として良好な特異性を有する、とされている。しかしながら、前記良好な特異性を有した抗体であっても、前記類似構造物質の中には、当該ステロイドホルモンの１０倍以上の濃度で試料中に存在する物質があり、その場合は、前記抗体が有するわずかな交差反応により、ステロイドホルモンの定量値に大きく影響する可能性があった。また実際に抗原抗体反応を利用して試料（例えば血液、血清などの体液）中のハプテンを測定する場合、試料毎に交差反応の影響が異なるという問題もあった。 Usually, in order to obtain an antibody having high specificity for a hapten, an antibody that does not show cross-reactivity with a similar structural substance of the hapten is selected. For example, when the hapten is a steroid hormone, the cross-reaction with a substance having a sterol skeleton or a substance having a structure similar to the sterol skeleton present in a sample (for example, a body fluid such as blood or serum) is investigated. In addition, an antibody having the lowest possible cross-reactivity with the substance is selected. However, in the case of steroid hormones, there are many types of similar structural substances of the steroid hormone present in the sample, and even with the technology of a monoclonal antibody having a single specificity, an antibody that recognizes only the steroid hormone can be detected. It is difficult to get. Therefore, generally, if the cross-reaction rate with respect to the said similar structural substance is 5% or less, it will be said that it has the favorable specificity as an antibody with respect to the said steroid hormone. However, even in the case of an antibody having a good specificity, among the similar structural substances, there is a substance present in a sample at a concentration 10 times or more that of the steroid hormone, in which case the antibody The slight cross-reactivity that has a significant impact on steroid hormone quantification. In addition, when the hapten in a sample (for example, body fluid such as blood or serum) is actually measured by using an antigen-antibody reaction, there is a problem that the influence of the cross reaction differs for each sample.
前述した問題を解決する方法として、測定対象物質であるハプテンと前記ハプテンと交差反応性を有する物質とを共存させた標準液を用いて、前記ハプテンを抗原抗体反応により測定する方法がある（特許文献１）。当該方法では、標準液を測定する際、測定対象物質（ハプテン）の濃度に応じた交差反応が生じるため、交差反応を加味した検量線を作成することができる。したがって、ハプテンを含む試料を測定した結果を、前記検量線にあてはめることで、試料中に含まれる交差反応性を示す物質（前記ハプテンの類似構造物質）による影響を抑制した形で定量値を得ることができる。しかしながら、ハプテンがステロイドホルモンの場合では、
（１）標準液に添加する前記ステロイドホルモンの類似構造物質が高額であり、
（２）類似構造物質の標準液への添加濃度がｐｇ／ｍＬオーダーからｎｇ／ｍＬオーダーと非常に低濃度であり、
（３）類似構造物質が内分泌かく乱物質（環境ホルモン）として作用する可能性があり、標準液の製造、使用または廃棄におけるリスクを否定できないため、
前記方法を容易に適用できなかった。
As a method for solving the above-mentioned problem, there is a method for measuring the hapten by an antigen-antibody reaction using a standard solution in which a hapten as a measurement target substance and a substance having cross-reactivity with the hapten are used (patent) Reference 1). In this method, when a standard solution is measured, a cross-reaction according to the concentration of the substance to be measured (hapten) occurs, so a calibration curve that takes into account the cross-reaction can be created. Therefore, by applying the measurement result of the sample containing hapten to the calibration curve, a quantitative value can be obtained in a form that suppresses the influence of the cross-reactive substance (similar structure substance of the hapten) contained in the sample. be able to. However, if the hapten is a steroid hormone,
(1) The steroid hormone-like structural substance added to the standard solution is expensive,
(2) The concentration of the similar structure substance added to the standard solution is very low from the order of pg / mL to the order of ng / mL,
(3) Since similar structural substances may act as endocrine disrupting substances (environmental hormones), the risk of manufacturing, using or discarding standard solutions cannot be denied.
The method could not be easily applied.
そこで本発明の目的は、試料中に含まれるハプテンを定量する際用いるハプテン標準液であって、ハプテン濃度が低濃度域であっても、前記ハプテンの類似構造物質による影響を受けることなく、ハプテン濃度を正確に定量可能な標準液を提供することにある。 Therefore, an object of the present invention is a hapten standard solution used for quantifying a hapten contained in a sample, and even if the hapten concentration is in a low concentration range, the hapten is not affected by the hapten-like structural substance. The object is to provide a standard solution capable of accurately quantifying the concentration.
本発明者らは、前記目的を達成すべく鋭意検討を行なった結果、糖質を共存させた標準液を用いて検量線を作成し、それを用いて試料中のハプテン濃度を定量すると、試料中に含まれる前記ハプテンの類似構造物質による影響が抑えられ、結果として試料中のハプテン濃度を正確に定量できることを見出し、本発明を完成した。 As a result of intensive studies to achieve the above-mentioned object, the present inventors have prepared a calibration curve using a standard solution in which a saccharide is allowed to coexist, and quantified the hapten concentration in the sample using the standard curve. The present inventors have found that the influence of the hapten contained in the hapten by the similar structural substance can be suppressed, and as a result, the hapten concentration in the sample can be accurately determined.
すなわち本発明の第一の態様は、ハプテンを認識する抗体と標識した前記ハプテンを含むハプテン測定試薬を用いて試料中に含まれるハプテンを定量する際用いる、検量線を作成するための、糖類を共存させたハプテン標準液であって、ハプテンがエストラジオールであり、糖類が４％（ｗ／ｖ）から６％（ｗ／ｖ）のスクロースであり、ハプテンを含有しない前記標準液である。 That is, in the first aspect of the present invention, a saccharide for preparing a calibration curve for use in quantifying a hapten contained in a sample using a hapten-measuring reagent containing an hapten-recognizing antibody and the labeled hapten is used. A hapten standard solution coexisting, wherein the hapten is estradiol, the saccharide is 4% (w / v) to 6% (w / v) sucrose, and does not contain a hapten.
さらに本発明の第二の態様は、ハプテンを認識する抗体と標識した前記ハプテンを含むハプテン測定試薬と、糖類を共存させたハプテン標準液とを含む、ハプテン定量試薬であって、ハプテンがエストラジオールであり、糖類が４％（ｗ／ｖ）から６％（ｗ／ｖ）のスクロースであり、ハプテンを含有しない前記標準液である、前記定量試薬である。 Furthermore, the second aspect of the present invention is a hapten determination reagent comprising a hapten-measuring reagent containing an antibody recognizing a hapten and the labeled hapten, and a hapten standard solution in which a saccharide coexists, wherein the hapten is estradiol. Yes, the quantification reagent, wherein the saccharide is 4% (w / v) to 6% (w / v) sucrose and is a standard solution containing no hapten.
さらに本発明の第三の態様は、ハプテンを認識する抗体と標識した前記ハプテンを含むハプテン測定試薬を用いて、糖類を共存させたハプテン標準液を測定し、
前記標準液中のハプテン濃度に対する測定値から検量線を作成し、
前記ハプテン測定試薬を用いて試料を測定して得られた測定値から、前記検量線を用いて試料中の前記ハプテン量を定量する、
ハプテンの定量方法であって、
ハプテンがエストラジオールであり、糖類が４％（ｗ／ｖ）から６％（ｗ／ｖ）のスクロースであり、ハプテンを含有しない前記標準液を用いる、前記定量方法である。
Furthermore, the third aspect of the present invention is to measure a hapten standard solution in which saccharides coexist, using a hapten-measuring reagent containing the hapten labeled with an antibody that recognizes a hapten,
Create a calibration curve from the measured value for the hapten concentration in the standard solution,
From the measurement value obtained by measuring the sample using the hapten measurement reagent, the amount of the hapten in the sample is quantified using the calibration curve,
A method for quantifying a hapten,
In the quantification method, the hapten is estradiol, the saccharide is 4% (w / v) to 6% (w / v) sucrose, and the standard solution containing no hapten is used.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明におけるハプテンとは、通常競合法により測定する、低分子量の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（Ｔ３）、チロキシン（Ｔ４）、３，５−ジヨード−Ｌ−チロニン（Ｔ２）等の甲状腺ホルモンや、エストロン（Ｅ１）、エストラジオール（Ｅ２）、エストリオール（Ｅ３）、プロゲステロン（ｐｒｏｇｅｓｔｅｒｏｎｅ）、コルチゾール（ｃｏｒｔｉｓｏｌ）、テストステロン（ｔｅｓｔｏｓｔｅｒｏｎｅ）、デヒドロエピアンドロステロンサルフェート（ＤＨＥＡ−Ｓ）等のステロイドホルモンがあげられる。特にエストラジオール（Ｅ２）に代表されるステロイドホルモンが、本発明の試薬で測定するハプテンとして好ましい。 The hapten in the present invention is not particularly limited as long as it is a low molecular weight substance usually measured by a competition method. Examples thereof include triiodothyronine (T3), thyroxine (T4), 3,5-diiodo-L- Thyroid hormones such as thyronine (T2), estrone (E1), estradiol (E2), estriol (E3), progesterone (corgestol), testosterone, dehydroepiandrosterone sulfate (DHEA-S) ) And other steroid hormones. In particular, a steroid hormone represented by estradiol (E2) is preferable as a hapten to be measured with the reagent of the present invention.
本発明のハプテン標準液は、一定濃度のハプテンに糖質を共存させることを特徴としている。当該標準液は少なくとも使用時に液体状態であればよく、使用前は液体状態であってもよいし、凍結乾燥状態であってもよい。本発明のハプテン標準液に共存させる糖質は、水溶性であれば特に制限がなく、一例として、グルコース、フルクトース、ガラクトース等の単糖類、スクロース、ラクトース、マルトース、トレハロース等の二糖類、β−シクロデキストリン、メチル−β−シクロデキストリン等の多糖類、マンニトール、キシリトール、イノシトール、シクリトール等の糖アルコールがあげられる。なおハプテン測定に影響する、前記ハプテンと交差反応性を示す物質（前記ハプテンの類似構造物質）の種類およびその濃度は、測定対象試料によって異なる。そのため、本発明のハプテン標準液に共存させる糖質の種類およびその濃度は、測定対象試料に応じて適宜最適なものを選択するとよい。 The hapten standard solution of the present invention is characterized in that a saccharide is allowed to coexist in a hapten having a constant concentration. The standard solution may be in a liquid state at least during use, and may be in a liquid state before use or may be in a lyophilized state. The carbohydrate to be coexisted in the hapten standard solution of the present invention is not particularly limited as long as it is water-soluble, and examples include monosaccharides such as glucose, fructose, galactose, disaccharides such as sucrose, lactose, maltose, trehalose, β- Examples thereof include polysaccharides such as cyclodextrin and methyl-β-cyclodextrin, and sugar alcohols such as mannitol, xylitol, inositol and cyclitol. Note that the type and concentration of a substance that has a cross-reactivity with the hapten (a similar structural substance of the hapten) that affects hapten measurement differs depending on the sample to be measured. For this reason, the type and concentration of carbohydrates to coexist in the hapten standard solution of the present invention may be appropriately selected according to the sample to be measured.
本発明のハプテン標準液に共存させる糖類をスクリーニング（選択）する方法の一例を以下に示す。なお本発明のハプテン標準液に共存させる糖類の濃度をスクリーニングする際も、以下に示す方法と同様な方法で行なうことができる。
（１）各種濃度のハプテンに候補となる糖質を共存させた、ハプテン標準液を作製する。
（２）前記ハプテンを認識する抗体と標識した前記ハプテンとを含むハプテン測定試薬に、（１）で作製した標準液を添加し、競合法による測定を行なう。
（３）（２）の測定で得られた測定値（例えば、蛍光強度や発光強度）から、（１）で作製した標準液中のハプテン濃度に対する検量線をそれぞれ作成する。
（４）複数の測定対象試料（例えば血液、血清などの体液）について、（２）と同様の測定を行ない、（３）で作成した各検量線に基づき、試料中のハプテン濃度（定量値）をそれぞれ算出する。
（５）（４）で測定した試料と同じ試料を交差反応の影響がない測定方法（例えば、クロマトグラフ質量分析法）で測定して得られたハプテン濃度（定量値）と、（４）で算出した定量値とを比較し、両者の値がもっとも一致した糖質を、本発明のハプテン標準液に含ませる糖質として採用する。
An example of a method for screening (selecting) saccharides to coexist in the hapten standard solution of the present invention is shown below. In addition, when screening the concentration of the saccharide coexisting in the hapten standard solution of the present invention, the same method as shown below can be used.
(1) Prepare a hapten standard solution in which saccharides as candidates coexist with haptens at various concentrations.
(2) The standard solution prepared in (1) is added to a hapten measurement reagent containing an antibody that recognizes the hapten and the labeled hapten, and measurement is performed by a competition method.
(3) A calibration curve for the hapten concentration in the standard solution prepared in (1) is prepared from the measured values (for example, fluorescence intensity and luminescence intensity) obtained by the measurement in (2).
(4) For a plurality of samples to be measured (for example, body fluids such as blood and serum), the same measurement as in (2) is performed, and the hapten concentration (quantitative value) in the sample based on each calibration curve created in (3) Are calculated respectively.
(5) The hapten concentration (quantitative value) obtained by measuring the same sample as that measured in (4) with a measurement method (for example, chromatographic mass spectrometry) without the influence of cross-reaction, and (4) The calculated quantitative value is compared, and the saccharide having the best match between the two values is adopted as the saccharide to be included in the hapten standard solution of the present invention.
なお、共存させる糖質の影響が強いと、ハプテン標準液を測定した時の応答が試料を測定した時の応答より低くなり、検量線から算出した、試料中のハプテン濃度（定量値）がマイナス値となるおそれがある。そのため糖質を本発明のハプテン標準液に共存させる際、前記定量値がマイナス値とならない糖類およびその濃度を検討する必要がある。具体的には、ハプテンがステロイドホルモンのひとつであるエストラジオール（Ｅ２）であり、測定対象試料が血清である場合の、本発明のハプテン標準液に共存させる糖質の好ましい例として、４％（ｗ／ｖ）から６％（ｗ／ｖ）のスクロースがあげられる。 In addition, if the influence of coexisting carbohydrates is strong, the response when measuring the hapten standard solution is lower than the response when measuring the sample, and the hapten concentration (quantitative value) in the sample calculated from the calibration curve is negative. May be a value. Therefore, when a saccharide is allowed to coexist in the hapten standard solution of the present invention, it is necessary to examine a saccharide whose quantitative value is not a negative value and its concentration. Specifically, when the hapten is estradiol (E2), which is one of steroid hormones, and the sample to be measured is serum, 4% (w / V) to 6% (w / v) sucrose.
本発明のハプテン標準液は、前記ハプテンを認識する抗体と標識した前記ハプテンとを含むハプテン測定試薬と組み合わせることで、試料中に含まれるハプテン濃度を、前記ハプテンと交差反応性を示す物質の影響を受けることなく正確に定量することができる。測定対象試料に特に限定はなく、一例として、血清、血漿、全血等の血液検体、尿、リンパ液、羊水、唾液、乳汁といった体液があげられる。前記ハプテン測定試薬に含まれる、ハプテンに標識させる物質は、当該物質の存否を検出器により検出可能な物質であればよく、一例として、酵素、放射性同位元素、蛍光物質、発光物質があげられる。前記物質は前記ハプテンに直接標識してもよく、前記ハプテンと特異的に結合する物質（例えば抗ハプテン抗体）を利用して間接的に標識してもよい。 The hapten standard solution of the present invention is a combination of a hapten measurement reagent containing an antibody that recognizes the hapten and a labeled hapten, whereby the hapten concentration contained in the sample is influenced by the substance that is cross-reactive with the hapten. Can be accurately quantified without exposure. The sample to be measured is not particularly limited, and examples include blood samples such as serum, plasma, and whole blood, and body fluids such as urine, lymph, amniotic fluid, saliva, and milk. The substance to be labeled on the hapten contained in the hapten measurement reagent may be any substance that can detect the presence or absence of the substance with a detector. Examples thereof include enzymes, radioisotopes, fluorescent substances, and luminescent substances. The substance may be directly labeled on the hapten, or may be indirectly labeled using a substance that specifically binds to the hapten (for example, an anti-hapten antibody).
尚、本発明のハプテン標準液は、前記ハプテンを認識する抗体と標識した前記ハプテンとを含むハプテン測定試薬を用いた測定におけるコントロールとしても用いることができる。 The hapten standard solution of the present invention can also be used as a control in measurement using a hapten measuring reagent containing an antibody that recognizes the hapten and the labeled hapten.
本発明は試料中に含まれるハプテンを定量する際用いるハプテン標準液であって、糖類を共存させることを特徴としている。本発明のハプテン標準液を、ハプテンを認識する抗体と標識した前記ハプテンを含むハプテン測定試薬と組み合わせることで、前記ハプテンに対する交差反応性を示す物質（前記ハプテンの類似構造物質）を多く含む試料に対しても交差反応の影響を抑制することができ、結果として前記試料中の前記ハプテン濃度を正確に定量することができる。 The present invention is a hapten standard solution used for quantifying a hapten contained in a sample, and is characterized in that a saccharide is allowed to coexist. By combining the hapten standard solution of the present invention with a hapten-measuring reagent containing a labeled hapten and an antibody that recognizes the hapten, a sample containing a large amount of a substance that exhibits cross-reactivity to the hapten (a substance having a similar structure to the hapten). In contrast, the influence of the cross reaction can be suppressed, and as a result, the hapten concentration in the sample can be accurately quantified.
以下、血清試料中に含まれるエストラジオール（Ｅ２）量の測定を例として、本発明をさらに詳細に説明するが、本発明は本実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail by taking the measurement of the amount of estradiol (E2) contained in a serum sample as an example, but the present invention is not limited to this example.
実施例１ 標準液に共存させる糖類濃度の検討
Ｅ２標準液に共存させる最適な糖類濃度を検討した。
（１）ステロイドホルモンを除去した血清（ＴＲＩＮＡ ＢＩＯＲＥＡＣＴＩＶＥ ＡＧ社製）に対して、スクロースを０％（ｗ／ｖ）、１％（ｗ／ｖ）、２％（ｗ／ｖ）、３％（ｗ／ｖ）、４％（ｗ／ｖ）、５％（ｗ／ｖ）、６％（ｗ／ｖ）および７％（ｗ／ｖ）共存させたベース血清（Ｅ２未添加のＥ２標準液）をそれぞれ作製した。
（２）Ｅ２を認識する抗体を磁性ビーズに結合して得られた固相抗体に、Ｅ２誘導体にウシ小腸由来アルカリホスファターゼ（ＡＬＰ）（オリエンタル酵母社製）を化学結合により標識して得られた酵素標識抗原と牛皮由来ペプタイド（ニッピ社製）とを含む５０ｍＭトリス緩衝液（ｐＨ７．５）を添加後、凍結乾燥することで、Ｅ２測定試薬を作製した。
（３）（２）で作製したＥ２測定試薬および市販の全自動エンザイムイムノアッセイ装置（ＡＩＡ−２０００、東ソー社製）を用いて、下記に示す方法で試料を測定した。測定試料としては、（１）で作製したベース血清または同位体希釈ガスクロマトグラフ質量分析法（ＩＤ−ＧＣ／ＭＳ法）によりＥ２濃度がゼロと値付けされたヒト血清試料（リファレンス検体）を用いた。
（３−１）（２）で作製したＥ２測定試薬に前記測定試料を添加し、３７℃で１０分間反応させた。
（３−２）Ｂ／Ｆ分離操作後、ＡＬＰの基質である４−メチルウンベリフェリルリン酸（４ＭＵＰ）を添加した。
（３−３）ＡＬＰによって４ＭＵＰが分解されて生成する４−メチルウンベリフェロン（４ＭＵ）の蛍光強度から４ＭＵの増加速度（ｎｍｏｌ／秒）（ＦＵ）を算出した。
Example 1 Examination of saccharide concentration coexisting in standard solution The optimum saccharide concentration coexisting in the E2 standard solution was examined.
(1) For serum from which steroid hormones have been removed (manufactured by TRINA BIOREACTIVE AG), sucrose is 0% (w / v), 1% (w / v), 2% (w / v), 3% (w / V) 4% (w / v), 5% (w / v), 6% (w / v) and 7% (w / v) coexisting base serum (E2 non-added E2 standard solution) Each was produced.
(2) Obtained by labeling a solid-phase antibody obtained by binding an antibody recognizing E2 to magnetic beads with E2 derivative by bovine small intestine-derived alkaline phosphatase (ALP) (manufactured by Oriental Yeast Co., Ltd.) by chemical bonding. After adding 50 mM Tris buffer (pH 7.5) containing enzyme-labeled antigen and cowhide-derived peptide (Nippi), the reagent for E2 measurement was prepared by lyophilization.
(3) Using the E2 measurement reagent prepared in (2) and a commercially available fully automatic enzyme immunoassay device (AIA-2000, manufactured by Tosoh Corporation), a sample was measured by the method described below. As the measurement sample, the base serum prepared in (1) or the human serum sample (reference sample) whose E2 concentration was marked as zero by the isotope dilution gas chromatograph mass spectrometry (ID-GC / MS method) was used. .
(3-1) The measurement sample was added to the E2 measurement reagent prepared in (2) and reacted at 37 ° C. for 10 minutes.
(3-2) After the B / F separation operation, 4-methylumbelliferyl phosphate (4MUP), which is an ALP substrate, was added.
(3-3) The rate of increase in 4MU (nmol / sec) (FU) was calculated from the fluorescence intensity of 4-methylumbelliferone (4MU) produced by the decomposition of 4MUP by ALP.
結果を表１および図１に示す。スクロースの共存量が増えるに従い、ＦＵ値は減少し、スクロースを４％（ｗ／ｖ）から６％（ｗ／ｖ）共存させた時にリファレンス検体のＦＵ値とほぼ同等（１００±２％以内）の値を示した。すなわち、Ｅ２標準液にスクロースを４％（ｗ／ｖ）から６％（ｗ／ｖ）共存させることで、血清試料中に含まれる、Ｅ２と交差反応性を示す物質（Ｅ２類似構造物質）の影響を反映させることができる。 The results are shown in Table 1 and FIG. As the coexistence amount of sucrose increases, the FU value decreases. When sucrose coexists with 4% (w / v) to 6% (w / v), it is almost the same as the FU value of the reference sample (within 100 ± 2%). The value of was shown. That is, by allowing sucrose to coexist in the E2 standard solution at 4% (w / v) to 6% (w / v), a substance (E2 similar structure substance) that is cross-reactive with E2 contained in the serum sample is contained. The impact can be reflected.
スクロースが共存しないＥ２標準液、またはスクロースを共存させたＥ２標準液で検量線を作成し、当該作成した検量線を基に、ヒト血清試料を測定した。
（１）（１）で作製した各ベース血清のうち、スクロースが共存しないベース血清（０％（ｗ／ｖ））、またはスクロースを５％（ｗ／ｖ）共存させたベース血清に対し、エタノールに溶解したＥ２をそれぞれ添加し、６点濃度のＥ２標準液をそれぞれ作製した。
（２）実施例１（３）に記載の方法により、Ｅ２標準液を測定し、ＦＵ値を算出した。ＦＵ値は試料中のＥ２濃度に比例するため、標準液中のＥ２濃度に対するＦＵ値をプロットすることで検量線を作成した。
（３）実施例１（３）に記載の方法により、ヒト血清試料３８試料（あらかじめ同位体希釈ガスクロマトグラフ質量分析計法（ＩＤ−ＧＣ／ＭＳ法）でＥ２濃度の値付けを行なった試料）を測定し、（２）で作成した検量線を用いて、各試料中に含まれるＥ２濃度を定量した。
(1) Of the base sera prepared in (1), ethanol is used for the base serum in which sucrose does not coexist (0% (w / v)) or in the presence of 5% (w / v) sucrose. E2 dissolved in each was added to prepare a 6-point E2 standard solution.
(2) The E2 standard solution was measured by the method described in Example 1 (3), and the FU value was calculated. Since the FU value is proportional to the E2 concentration in the sample, a calibration curve was created by plotting the FU value against the E2 concentration in the standard solution.
(3) According to the method described in Example 1 (3), 38 human serum samples (samples for which E2 concentration was priced in advance by an isotope dilution gas chromatograph mass spectrometer method (ID-GC / MS method)) Was measured, and the E2 concentration contained in each sample was quantified using the calibration curve prepared in (2).
結果を表２および図２に示す。糖類であるスクロースが共存しないＥ２標準液で検量線を作成した場合は、特にＥ２濃度の低いヒト血清試料において、ＩＤ−ＧＣ／ＭＳ法で値付けされたＥ２濃度との乖離が大きく、Ｅ２濃度が５０ｐｇ／ｍＬ以下のヒト血清検体では２倍以上高値となっていた。すなわち、スクロースが共存しないＥ２標準液で検量線を作成した場合は、特に低濃度域において、ヒト血清試料中に含まれる、Ｅ２と交差反応性を示す物質の影響を受けていることがわかる。一方、スクロースを５％（ｗ／ｖ）共存させたＥ２標準液で検量線を作成した場合は、Ｅ２濃度が５０ｐｇ／ｍＬ以下のヒト血清検体であっても、ＩＤ−ＧＣ／ＭＳ法で値付けされたＥ２濃度との乖離は最大でも３０％程度に抑えることができた。つまり、検量線を作成する際に使用するＥ２標準液に糖類であるスクロースを一定量共存させることで、試料（検体）中に含まれるＥ２と交差反応性を示す物質の影響を低減させることができ、結果として、前記物質の影響を受けやすい、低濃度のＥ２を含む試料であってもＥ２を正確に定量できることがわかる。 The results are shown in Table 2 and FIG. When a calibration curve is prepared with an E2 standard solution in which sucrose, which is a saccharide, does not coexist, especially in a human serum sample with a low E2 concentration, the difference from the E2 concentration determined by the ID-GC / MS method is large, and the E2 concentration However, in human serum samples of 50 pg / mL or less, the value was more than twice as high. That is, it can be seen that when a calibration curve is prepared with an E2 standard solution in which sucrose does not coexist, it is influenced by a substance that cross-reacts with E2 contained in a human serum sample, particularly in a low concentration range. On the other hand, when a calibration curve is prepared with an E2 standard solution in which 5% (w / v) sucrose coexists, even if it is a human serum sample with an E2 concentration of 50 pg / mL or less, the value is obtained by the ID-GC / MS method. The deviation from the attached E2 concentration could be suppressed to about 30% at the maximum. In other words, by making a certain amount of sucrose, a saccharide, coexist in the E2 standard solution used when preparing a calibration curve, it is possible to reduce the influence of substances that cross-react with E2 contained in the sample (specimen). As a result, it can be seen that E2 can be accurately quantified even in a sample containing a low concentration of E2, which is easily affected by the substance.
Claims (3)
前記標準液中のハプテン濃度に対する測定値から検量線を作成し、
前記ハプテン測定試薬を用いて試料を測定して得られた測定値から、前記検量線を用いて試料中の前記ハプテン量を定量する、
ハプテンの定量方法であって、
ハプテンがエストラジオールであり、糖類が４％（ｗ／ｖ）から６％（ｗ／ｖ）のスクロースであり、ハプテンを含有しない前記標準液を用いる、前記定量方法。 Using a hapten measurement reagent containing the labeled hapten and an antibody that recognizes a hapten, a hapten standard solution in which a saccharide is present is measured,
Create a calibration curve from the measured value for the hapten concentration in the standard solution,
From the measurement value obtained by measuring the sample using the hapten measurement reagent, the amount of the hapten in the sample is quantified using the calibration curve,
A method for quantifying a hapten,
The quantification method, wherein the hapten is estradiol, the saccharide is 4% (w / v) to 6% (w / v) sucrose, and the standard solution containing no hapten is used.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07507871A (en) * | 1992-06-10 | 1995-08-31 | アボツト・ラボラトリーズ | Determination of estradiol by competitive immunoassay |
| US6103536A (en) * | 1997-05-02 | 2000-08-15 | Silver Lake Research Corporation | Internally referenced competitive assays |
| JP2001264328A (en) * | 2000-03-14 | 2001-09-26 | Tosoh Corp | Enzyme immunoassay for ethynylestradiol |
| JP2003344411A (en) * | 2002-05-22 | 2003-12-03 | Toyobo Co Ltd | Immobilization method of receptor, and receptor composition manufactured by the method |
| JP2004065107A (en) * | 2002-08-06 | 2004-03-04 | Toyobo Co Ltd | Method for assaying drug-metabolizing enzyme activity, method for evaluating activity inhibition of drug-metabolizing enzyme, and composition for the methods |
| JP2011117779A (en) * | 2009-12-01 | 2011-06-16 | Tosoh Corp | Method for measuring steroid hormones or vitamins in blood |
-
2016
- 2016-06-23 JP JP2016124428A patent/JP6222295B2/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07507871A (en) * | 1992-06-10 | 1995-08-31 | アボツト・ラボラトリーズ | Determination of estradiol by competitive immunoassay |
| US6103536A (en) * | 1997-05-02 | 2000-08-15 | Silver Lake Research Corporation | Internally referenced competitive assays |
| JP2001264328A (en) * | 2000-03-14 | 2001-09-26 | Tosoh Corp | Enzyme immunoassay for ethynylestradiol |
| JP2003344411A (en) * | 2002-05-22 | 2003-12-03 | Toyobo Co Ltd | Immobilization method of receptor, and receptor composition manufactured by the method |
| JP2004065107A (en) * | 2002-08-06 | 2004-03-04 | Toyobo Co Ltd | Method for assaying drug-metabolizing enzyme activity, method for evaluating activity inhibition of drug-metabolizing enzyme, and composition for the methods |
| JP2011117779A (en) * | 2009-12-01 | 2011-06-16 | Tosoh Corp | Method for measuring steroid hormones or vitamins in blood |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022116782A1 (en) * | 2020-12-04 | 2022-06-09 | 江南大学 | Ethyl maltol monoclonal antibody hybridoma cell line and application thereof |
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