JP6862845B2 - Method for measuring soluble interleukin-2 receptor and reagent for measurement - Google Patents
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Description
本発明は、可溶性インターロイキン−２受容体の測定方法、及び、可溶性インターロイキン−２受容体の測定用試薬に関する。 The present invention relates to a method for measuring a soluble interleukin-2 receptor and a reagent for measuring a soluble interleukin-2 receptor.
インターロイキン−２受容体（以下、ＩＬ−２Ｒと記す）はα鎖、β鎖、γ鎖から構成されているが、α鎖の一部が細胞上から遊離した可溶性インターロイキン−２受容体（以下、ｓＩＬ−２Ｒと記す）が血中に存在することが知られている（特許文献１、非特許文献１参照）。ｓＩＬ−２Ｒは活性化Ｔ細胞、Ｂ細胞によって産生されるために、生体の免疫防御機構の活性化、Ｔ細胞系及びＢ細胞系などの活性化に伴い血中のｓＩＬ−２Ｒが上昇することが報告されている。血清中のｓＩＬ−２Ｒ濃度は、慢性関節リウマチ、全身性エリテマトーデス（ＳＬＥ）などの自己免疫疾患や、ウイルス性肝炎、後天性免疫不全症候群（ＡＩＤＳ）などのウイルス感染症の患者で高値を示し、体内の活性化リンパ球量の指標の１つとなることが報告されている（非特許文献２参照）。また腫瘍細胞がｓＩＬ−２Ｒを産生し、成人Ｔ細胞白血病（ＡＴＬ）や非ホジキンリンパ腫の進行と血清中のｓＩＬ−２Ｒ濃度の変動が良く相関することが知られている（非特許文献３、非特許文献４参照）。このようにｓＩＬ−２Ｒに関して、様々な免疫系の疾患や病態との関連が報告されており、なかでも造血疾患の有望な血液中のマーカーと認識されている。血清中のｓＩＬ−２Ｒ濃度は成人Ｔ細胞白血病においては病態モニタリングの指標など、非ホジキンリンパ腫においては治療効果の判定、寛解後のモニタリング、再発の早期発見の指標などとして臨床的に有効活用されている。 The interleukin-2 receptor (hereinafter referred to as IL-2R) is composed of α chain, β chain, and γ chain, and a soluble interleukin-2 receptor (part of the α chain released from the cell) (hereinafter referred to as IL-2R). It is known that sIL-2R (hereinafter referred to as sIL-2R) is present in blood (see Patent Document 1 and Non-Patent Document 1). Since sIL-2R is produced by activated T cells and B cells, the sIL-2R in the blood increases with the activation of the immune defense mechanism of the living body and the activation of the T cell line and the B cell line. Has been reported. Serum sIL-2R levels were high in patients with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE) and viral infections such as viral hepatitis and acquired immunodeficiency syndrome (AIDS). It has been reported to be one of the indicators of the amount of activated lymphocytes in the body (see Non-Patent Document 2). It is also known that tumor cells produce sIL-2R, and that the progression of adult T-cell leukemia (ATL) and non-Hodgkin's lymphoma is well correlated with fluctuations in serum sIL-2R concentration (Non-Patent Document 3, Non-Patent Document 3, See Non-Patent Document 4). As described above, sIL-2R has been reported to be associated with various diseases and pathological conditions of the immune system, and is recognized as a promising marker in blood for hematopoietic diseases. The serum sIL-2R concentration is clinically effectively used as an index for pathological monitoring in adult T-cell leukemia, as an index for determining the therapeutic effect in non-Hodgkin's lymphoma, monitoring after remission, and as an index for early detection of recurrence. There is.
血清や血漿等の試料中のｓＩＬ−２Ｒの測定方法としては、ｓＩＬ−２Ｒに結合する抗体を用いる免疫測定法によるｓＩＬ−２Ｒの測定方法が知られており、ｓＩＬ−２Ｒに結合する抗体を担持する不溶性担体として、プレートを用いる方法（特許文献２）や磁性粒子を用いる方法（特許文献３）等が知られている。これらの方法においては、ｓＩＬ−２Ｒに結合する抗体とｓＩＬ−２Ｒとを抗原抗体反応させ免疫複合体を形成させたのち、免疫複合体中の標識の量を測定することにより、血清又は血漿等の試料中のｓＩＬ−２Ｒ濃度を測定することを原理とする標識法が用いられている。しかしながら、これらの標識法は、一般的に、免疫複合体中の標識の量を測定する前に、血清又は血漿等の試料中に含まれるｓＩＬ−２Ｒ以外の物質を取り除くために、洗浄液を用いて洗浄（Ｂ／Ｆ分離）を行う必要があり、測定に時間を要する。 As a method for measuring sIL-2R in a sample such as serum or plasma, a method for measuring sIL-2R by an immunoassay using an antibody that binds to sIL-2R is known, and an antibody that binds to sIL-2R is used. As the supported insoluble carrier, a method using a plate (Patent Document 2), a method using magnetic particles, and the like are known. In these methods, an antibody that binds to sIL-2R and sIL-2R are subjected to an antigen-antibody reaction to form an immune complex, and then the amount of the label in the immune complex is measured to obtain serum, plasma, or the like. A labeling method based on the principle of measuring the sIL-2R concentration in the sample is used. However, these labeling methods generally use a washing solution to remove substances other than sIL-2R contained in a sample such as serum or plasma before measuring the amount of labeling in the immune complex. It is necessary to perform cleaning (B / F separation), and it takes time for measurement.
一方、標識を使用しない免疫測定法の１つにラテックス凝集比濁法がある（特許文献４、５）。ラテックス凝集比濁法は、測定対象成分に結合する抗体または抗原を結合したラテックス粒子を用いて、検体中の測定対象成分と抗原抗体反応を起こさせ、ラテックス粒子の凝集による濁度変化を吸光度として測定する方法であり、Ｂ／Ｆ分離が不要な方法であるため、生化学検査用の汎用自動分析機での測定にも適用されている。しかしながら、ラテックス凝集比濁法は測定感度が悪いという問題があり、高感度が必要とされる測定対象成分の測定には適用が難しい。 On the other hand, one of the immunoassay methods that does not use a label is the latex agglutination turbidimetry method (Patent Documents 4 and 5). In the latex agglutination turbidimetry method, an antibody that binds to a component to be measured or a latex particle that binds an antigen is used to cause an antigen-antibody reaction with the component to be measured in a sample, and the change in turbidity due to aggregation of the latex particle is used as the absorbance. Since it is a method for measuring and does not require B / F separation, it is also applied to measurement with a general-purpose automatic analyzer for biochemical inspection. However, the latex agglutination turbidimetry method has a problem of poor measurement sensitivity, and is difficult to apply to the measurement of a component to be measured, which requires high sensitivity.
本発明の目的は、簡便で、かつ、高感度な試料中のｓＩＬ−２Ｒの測定方法及び測定用試薬を提供することにある。 An object of the present invention is to provide a simple and highly sensitive method for measuring sIL-2R in a sample and a reagent for measuring.
本発明者らは、上記課題を解決すべく鋭意検討した結果、試料を、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子、及び、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子と反応させる免疫凝集法により、簡便かつ高感度に、試料中のｓＩＬ−２Ｒを測定できる、という知見を見出して本発明を完成させた。すなわち、本発明は、以下の［１］〜［８］に関する。
［１］ 試料と、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子と、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子とを反応させ、抗原抗体反応によって生じた凝集を測定することを特徴とする、試料中のｓＩＬ−２Ｒの測定方法。
［２］ 第１抗体と不溶性担体粒子との結合、及び、第２抗体と不溶性担体粒子との結合が共に化学結合である、［１］に記載の方法。
［３］ 第1抗体及び第２抗体が共にモノクローナル抗体である、［１］又は［２］に記載の方法。
［４］ ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子、及び、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子が、共にラテックス粒子である、［１］〜［３］のいずれかに記載の方法。
As a result of diligent studies to solve the above problems, the present inventors have bound the sample to the first antibody that binds to sIL-2R and to the second antibody that binds to sIL-2R. Convenient by immunoaggregation method in which a non-insoluble carrier particle and an immunoaggregation method in which a second antibody that binds to sIL-2R is bound and the first antibody that binds to sIL-2R is not bound are reacted with the insoluble carrier particle. The present invention was completed by finding the finding that sIL-2R in a sample can be measured with high sensitivity. That is, the present invention relates to the following [1] to [8].
[1] The sample, the insoluble carrier particles to which the first antibody that binds to sIL-2R is bound, and the second antibody that binds to sIL-2R is not bound, and the second antibody that binds to sIL-2R. A sample in a sample, which comprises reacting with insoluble carrier particles to which an antibody is bound and to which a first antibody that binds to sIL-2R is not bound, and the aggregation generated by the antigen-antibody reaction is measured. Method for measuring sIL-2R.
[2] The method according to [1], wherein the binding between the first antibody and the insoluble carrier particles and the binding between the second antibody and the insoluble carrier particles are both chemical bonds.
[3] The method according to [1] or [2], wherein both the first antibody and the second antibody are monoclonal antibodies.
[4] The insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound, and the second antibody that binds to sIL-2R The method according to any one of [1] to [3], wherein the insoluble carrier particles that are bound and to which the first antibody that binds to sIL-2R is not bound are both latex particles.
［５］ ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子と、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子とを含むことを特徴とする、試料中のｓＩＬ−２Ｒの測定用試薬。
［６］ 第１抗体と不溶性担体粒子との結合、及び、第２抗体と不溶性担体粒子との結合が共に化学結合である、［５］に記載の試薬。
［７］ 第１抗体及び第２抗体が共にモノクローナル抗体である、［５］又は［６］に記載の試薬。
［８］ ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子、及び、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子が、共にラテックス粒子である、［５］〜［７］のいずれかに記載の試薬。
[5] The insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound are bound to the second antibody that binds to sIL-2R. A reagent for measuring sIL-2R in a sample, which comprises insoluble carrier particles to which the first antibody that binds to sIL-2R is not bound.
[6] The reagent according to [5], wherein the binding between the first antibody and the insoluble carrier particles and the binding between the second antibody and the insoluble carrier particles are both chemical bonds.
[7] The reagent according to [5] or [6], wherein both the first antibody and the second antibody are monoclonal antibodies.
[8] The insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound, and the second antibody that binds to sIL-2R The reagent according to any one of [5] to [7], wherein the insoluble carrier particles that are bound and to which the first antibody that binds to sIL-2R is not bound are both latex particles.
本発明により簡便で、かつ、高感度な試料中のｓＩＬ−２Ｒの測定方法及び測定用試薬が提供される。 The present invention provides a simple and highly sensitive method for measuring sIL-2R in a sample and a reagent for measuring.
（測定方法）
本発明の測定方法は、試料と、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子と、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子とを反応させ、抗原抗体反応によって生じた凝集を測定することを特徴とする、試料中のｓＩＬ−２Ｒの測定方法である。
(Measuring method)
In the measuring method of the present invention, the sample is bound to the insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that is bound to sIL-2R is not bound, and to sIL-2R. It is characterized by reacting with insoluble carrier particles to which the second antibody to be bound is bound and to which the first antibody to be bound to sIL-2R is not bound, and the aggregation generated by the antigen-antibody reaction is measured. , A method for measuring sIL-2R in a sample.
本発明のｓＩＬ−２Ｒの測定方法としては、例えば以下の工程を含有する方法等が挙げられる。
［１］試料と、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子とを反応させる工程；
［２］工程［１］の反応液に、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子を添加し、試料と、当該第１抗体、及び、当該第２抗体とを反応させ凝集を生成させる工程；
［３］工程［２］で生成した凝集を測定する工程;
［４］既知濃度のｓＩＬ−２Ｒを試料として用いて、上記［１］〜［３］を行うことにより、予め作成した、ｓＩＬ−２Ｒ濃度と凝集との関係を表す検量線と、工程［３］で測定された凝集の測定値とから、試料中のｓＩＬ−２Ｒ濃度を決定する工程。
Examples of the method for measuring sIL-2R of the present invention include a method including the following steps.
[1] A step of reacting a sample with insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and to which the second antibody that binds to sIL-2R is not bound;
[2] Insoluble carrier particles to which the second antibody binding to sIL-2R is bound and to which the first antibody binding to sIL-2R is not bound are added to the reaction solution of step [1]. A step of reacting a sample with the first antibody and the second antibody to generate agglutination;
[3] A step of measuring the agglomeration generated in the step [2];
[4] Using a known concentration of sIL-2R as a sample, by performing the above [1] to [3], a calibration curve prepared in advance showing the relationship between the sIL-2R concentration and aggregation and the step [3] ], The step of determining the sIL-2R concentration in the sample from the measured value of aggregation measured in.
工程［１］及び工程［２］は順次行っても、同時に行ってもよい。工程［１］及び工程［２］の反応、すなわち、抗原抗体反応は水性媒体中で行うことができる。水性媒体としては、本発明の測定方法を可能とする水性媒体であれば特に制限はなく、例えば後述の水性媒体等が挙げられる。水性媒体には、後述の金属イオン、糖類、防腐剤、蛋白質、界面活性剤、凝集促進剤等が含有されてもよい。また、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子は、抗原抗体反応の反応液中で生成されてもよく、この場合、一組の親和性物質の片方（Ａ）が結合した第１抗体と、一組の親和性物質のもう一方（ａ）が結合した不溶性担体粒子とを反応させることにより、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子を生成することができる。同様に、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子は、抗原抗体反応の反応液中で生成されてもよく、この場合、一組の親和性物質の片方（Ｂ）が結合した第２抗体と、一組の親和性物質のもう一方（ｂ）が結合した不溶性担体粒子とを反応させることにより、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子を生成することができる。ここで、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子と、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子とが同一の反応液中で生成する場合には、Ａ及びａからなる一組の親和性物質は、Ｂ及びｂからなる一組の親和性物質とは異なっていることが好ましい。一組の親和性物質としては、例えばアビジン類（アビジン、ストレプトアビジン、ニュートラアビジン等）とビオチンとの組み合わせ、抗体のFc領域と、Fc領域と結合する抗体との組み合わせ等が挙げられる。Ａ−ａの組み合わせとＢ−ｂの組み合わせとが異なる場合における、Ａ−ａの組み合わせ、及び、Ｂ−ｂの組み合わせとしては、例えば以下の組み合わせが挙げられる。
・ビオチン−アビジン類、又は、アビジン類−ビオチンの組み合わせ；第２抗体のFc領域と、当該Fc領域と結合する抗体との組み合わせ。
・第１抗体のFc領域と、当該Fc領域と結合する抗体との組み合わせ；ビオチン−アビジン類、又は、アビジン類−ビオチンの組み合わせ。
Steps [1] and [2] may be performed sequentially or simultaneously. The reaction of step [1] and step [2], that is, the antigen-antibody reaction can be carried out in an aqueous medium. The aqueous medium is not particularly limited as long as it is an aqueous medium that enables the measurement method of the present invention, and examples thereof include an aqueous medium described later. The aqueous medium may contain metal ions, sugars, preservatives, proteins, surfactants, aggregation promoters and the like, which will be described later. Further, even if the insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound are generated in the reaction solution of the antigen-antibody reaction. Often, in this case, sIL is caused by reacting the first antibody to which one of the set of affinity substances (A) is bound with the insoluble carrier particle to which the other (a) of the set of affinity substances is bound. It is possible to generate insoluble carrier particles in which the first antibody that binds to -2R is bound and the second antibody that binds to sIL-2R is not bound. Similarly, insoluble carrier particles to which the second antibody that binds to sIL-2R is bound and to which the first antibody that binds to sIL-2R is not bound are generated in the reaction solution of the antigen-antibody reaction. In this case, the second antibody to which one of the set of affinity substances (B) is bound is reacted with the insoluble carrier particle to which the other (b) of the set of affinity substances is bound. It is possible to generate insoluble carrier particles in which the second antibody that binds to sIL-2R is bound and the first antibody that binds to sIL-2R is not bound. Here, the insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound are bound to the second antibody that binds to sIL-2R. When the insoluble carrier particles to which the first antibody that binds to sIL-2R is not bound are produced in the same reaction solution, the set of affinity substances consisting of A and a is It is preferably different from the set of affinity substances consisting of B and b. Examples of the set of affinity substances include a combination of avidins (avidin, streptavidin, neutravidin, etc.) and biotin, a combination of an Fc region of an antibody and an antibody that binds to the Fc region, and the like. When the combination of Aa and the combination of Bb are different, the combination of Aa and the combination of Bb include, for example, the following combinations.
-Biotin-avidins or avidin-biotin combination; a combination of the Fc region of the second antibody and an antibody that binds to the Fc region.
-A combination of the Fc region of the first antibody and an antibody that binds to the Fc region; a combination of biotin-avidins or avidins-biotin.
工程［１］及び工程［２］における抗原抗体反応の反応温度は、本発明のｓＩＬ−２Ｒの測定を可能とする温度であれば特に制限はなく、通常、０〜５０℃であり、４〜４５℃が好ましく、２０〜４０℃が特に好ましい。工程［１］及び工程［２］における抗原抗体反応の反応時間は、本発明のｓＩＬ−２Ｒの測定を可能とする時間であれば特に制限はなく、通常、１分間〜1時間であり、２〜２０分間が好ましい。 The reaction temperature of the antigen-antibody reaction in the step [1] and the step [2] is not particularly limited as long as it is a temperature that enables the measurement of sIL-2R of the present invention, and is usually 0 to 50 ° C., 4 to 4. 45 ° C. is preferable, and 20 to 40 ° C. is particularly preferable. The reaction time of the antigen-antibody reaction in step [1] and step [2] is not particularly limited as long as it enables the measurement of sIL-2R of the present invention, and is usually 1 minute to 1 hour, and 2 -20 minutes is preferred.
工程［１］において、試料を予め後述の水性媒体で希釈して保持した後に抗原抗体反応に供することもできる。水性媒体には、後述の金属イオン、糖類、防腐剤、蛋白質、界面活性剤、凝集促進剤等が含有されてもよい。試料を予め水性媒体で希釈して保持する温度は、本発明のｓＩＬ−２Ｒの測定を可能とする温度であれば特に制限はなく、通常、０〜５０℃であり、４〜４５℃が好ましく、２０〜４０℃が特に好ましい。試料を予め水性媒体で希釈して保持する時間は、本発明のｓＩＬ−２Ｒの測定を可能とする時間であれば特に制限はなく、通常、１分間〜1時間であり、２〜２０分間が好ましい。 In the step [1], the sample can be subjected to an antigen-antibody reaction after being previously diluted and held in an aqueous medium described later. The aqueous medium may contain metal ions, sugars, preservatives, proteins, surfactants, aggregation promoters and the like, which will be described later. The temperature at which the sample is previously diluted with an aqueous medium and held is not particularly limited as long as it enables the measurement of sIL-2R of the present invention, and is usually 0 to 50 ° C, preferably 4 to 45 ° C. 20-40 ° C is particularly preferable. The time for pre-diluting and holding the sample in an aqueous medium is not particularly limited as long as it enables the measurement of sIL-2R of the present invention, and is usually 1 minute to 1 hour, and 2 to 20 minutes. preferable.
工程［３］において、工程［２］で生成した凝集を測定する方法としては、工程［２］で生成した凝集を測定し得る方法であれば特に制限はなく、例えば特定の波長での吸光度を測定する方法等が挙げられる。波長としては、工程［２］で生成した凝集を測定し得る波長であれば特に制限はなく、例えば５７０ｎｍ等が挙げられる。 In the step [3], the method for measuring the agglomeration generated in the step [2] is not particularly limited as long as it can measure the agglomeration generated in the step [2], and for example, the absorbance at a specific wavelength can be measured. Examples include a measuring method. The wavelength is not particularly limited as long as it can measure the aggregation generated in the step [2], and examples thereof include 570 nm.
工程［４］において、既知濃度のｓＩＬ−２Ｒを試料として用いて工程［１］〜［３］を行うことにより、予め作成した、ｓＩＬ−２Ｒ濃度と凝集との関係を表す検量線と、工程［３］で測定した凝集とから、試料中のｓＩＬ−２Ｒ濃度を決定することができる。 In step [4], a calibration curve showing the relationship between sIL-2R concentration and aggregation, which was prepared in advance by performing steps [1] to [3] using sIL-2R having a known concentration as a sample, and step The sIL-2R concentration in the sample can be determined from the aggregation measured in [3].
本発明における試料としては、本発明のｓＩＬ−２Ｒの測定を可能とする試料であれば特に制限はなく、例えば全血、血漿、血清、髄液、唾液、羊水、尿、汗、膵液等が挙げられるが、全血、血漿、血清等が好ましい。 The sample in the present invention is not particularly limited as long as it is a sample capable of measuring sIL-2R of the present invention, for example, whole blood, plasma, serum, cerebrospinal fluid, saliva, sheep water, urine, sweat, pancreatic fluid and the like. Although mentioned, whole blood, plasma, serum and the like are preferable.
ｓＩＬ−２Ｒに結合する抗体（第１抗体及び第２抗体）と不溶性担体粒子との間の結合としては、本発明のｓＩＬ−２Ｒの測定を可能とする結合であれば特に制限はなく、物理吸着、化学結合等が挙げられる。物理吸着としては、例えば静電的結合、水素結合、疎水結合等が挙げられる。化学結合としては、例えば共有結合、配位結合等が挙げられる。 The bond between the antibody (first antibody and second antibody) that binds to sIL-2R and the insoluble carrier particle is not particularly limited as long as it is a bond that enables measurement of sIL-2R of the present invention, and is physically limited. Examples include adsorption and chemical bonding. Examples of the physical adsorption include electrostatic bonds, hydrogen bonds, hydrophobic bonds and the like. Examples of the chemical bond include a covalent bond and a coordination bond.
ｓＩＬ−２Ｒに結合する抗体（第１抗体及び第２抗体）は、前述の物理吸着及び／又は化学結合を利用して、直接、不溶性担体粒子に固定化してもよいし、間接的に不溶性担体粒子に固定化してもよい。間接的な固定化方法としては、例えばビオチンとアビジン類（アビジン、ストレプトアビジン、ニュートラアビジン等）との特異的結合を介して、抗体を不溶性担体粒子に固定化する方法が挙げられる。あるいは、抗体は、リンカーを介した共有結合により不溶性担体粒子に固定化してもよい。 The antibody (first antibody and second antibody) that binds to sIL-2R may be directly immobilized on the insoluble carrier particles or indirectly by using the above-mentioned physical adsorption and / or chemical bond. It may be immobilized on particles. Examples of the indirect immobilization method include a method of immobilizing an antibody on insoluble carrier particles via a specific binding between biotin and avidins (avidin, streptavidin, neutravidin, etc.). Alternatively, the antibody may be immobilized on insoluble carrier particles by covalent bonding via a linker.
リンカーとしては、不溶性担体粒子表面の官能基と抗体の側鎖の官能基の両者を共有結合できる分子等が挙げられ、例えば、抗体が有する官能基と反応することができる第１の反応活性基と、不溶性担体粒子表面の官能基と反応することができる第２の反応活性基とを同時に持つ分子であって、第１の反応活性基と第２の反応活性基が異なる基である分子が好ましく用いられる。抗体の官能基および不溶性担体粒子がその表面に保持している官能基としては例えば、カルボキシ基、アミノ基、グリシジル基、スルフヒドリル基、水酸基、アミド基、イミノ基、Ｎ−ヒドロキシサクシニル基、マレイミド基等が挙げられる。リンカーにおける反応活性基としては、例えば、アリルアジド、カルボジイミド、ヒドラジド、アルデヒド、ヒドロキシメチルホスフィン、イミドエステル、イソシアネート、マレイミド、Ｎ−ヒドロキシスクシンイミド（ＮＨＳ）エステル、ペンタフルオロフェニル（ＰＦＰ）エステル、ソラレン、ピリジルジスルフィド、ビニルスルホン等の基が挙げられる。 Examples of the linker include molecules capable of covalently binding both the functional group on the surface of the insoluble carrier particle and the functional group on the side chain of the antibody. For example, a first reactive active group capable of reacting with the functional group of the antibody. And a molecule having a second reactive active group capable of reacting with a functional group on the surface of the insoluble carrier particles at the same time, and the first reactive group and the second reactive active group are different groups. It is preferably used. Examples of the functional group retained on the surface of the functional group of the antibody and the insoluble carrier particles include a carboxy group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, an N-hydroxysuccinyl group and a maleimide group. And so on. Examples of the reactive group in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, solarene, and pyridyl disulfide. , Vinyl sulfone and the like.
本発明における水性媒体としては、本発明のｓＩＬ−２Ｒの測定を可能とする水性媒体であれば特に制限はなく、例えば脱イオン水、蒸留水、緩衝液等が挙げられ、緩衝液が好ましい。緩衝液の調製に使用される緩衝剤としては、緩衝能を有するものならば特に制限されないが、ｐＨ１〜１１の例えば乳酸緩衝剤、クエン酸緩衝剤、酢酸緩衝剤、コハク酸緩衝剤、フタル酸緩衝剤、リン酸緩衝剤、トリエタノールアミン緩衝剤、ジエタノールアミン緩衝剤、リジン緩衝剤、バルビツール緩衝剤、イミダゾール緩衝剤、リンゴ酸緩衝剤、シュウ酸緩衝剤、グリシン緩衝剤、ホウ酸緩衝剤、炭酸緩衝剤、グリシン緩衝剤、グッド緩衝剤等が挙げられる。 The aqueous medium in the present invention is not particularly limited as long as it is an aqueous medium capable of measuring sIL-2R of the present invention, and examples thereof include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable. The buffer used for preparing the buffer is not particularly limited as long as it has a buffering ability, but for example, a lactic acid buffer, a citrate buffer, an acetate buffer, a succinic acid buffer, and a phthalic acid having a pH of 1 to 11 are not particularly limited. Buffers, phosphate buffers, triethanolamine buffers, diethanolamine buffers, lysine buffers, barbitur buffers, imidazole buffers, malic acid buffers, oxalate buffers, glycine buffers, borate buffers, Examples thereof include a carbon dioxide buffer, a glycine buffer, and a good buffer.
グッド緩衝剤としては、例えば２−モルホリノエタンスルホン酸（ＭＥＳ）緩衝剤、ビス（２−ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Ｂｉｓ−Ｔｒｉｓ）緩衝剤、トリス（ヒドロキシメチル）アミノメタン（Ｔｒｉｓ）緩衝剤、Ｎ−（２−アセトアミド）イミノ二酢酸（ＡＤＡ）緩衝剤、ピペラジン−Ｎ，Ｎ’−ビス（２−エタンスルホン酸）（ＰＩＰＥＳ）緩衝剤、２−［Ｎ−（２−アセトアミド）アミノ］エタンスルホン酸（ＡＣＥＳ）緩衝剤、３−モルホリノ−２−ヒドロキシプロパンスルホン酸（ＭＯＰＳＯ）緩衝剤、２−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］エタンスルホン酸（ＢＥＳ）緩衝剤、３−モルホリノプロパンスルホン酸（ＭＯＰＳ）緩衝剤、２−｛Ｎ−［トリス（ヒドロキシメチル）メチル］アミノ｝エタンスルホン酸（ＴＥＳ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−スルホエチル）ピペラジン（ＨＥＰＥＳ）緩衝剤、３−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］−２−ヒドロキシプロパンスルホン酸（ＤＩＰＳＯ）緩衝剤、２−ヒドロキシ−３−｛［Ｎ−トリス（ヒドロキシメチル）メチル］アミノ｝プロパンスルホン酸（ＴＡＰＳＯ）緩衝剤、ピペラジン−Ｎ，Ｎ’−ビス（２−ヒドロキシプロパン−３−スルホン酸）（ＰＯＰＳＯ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−ヒドロキシ−３−スルホプロピル）ピペラジン（ＨＥＰＰＳＯ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（３−スルホプロピル）ピペラジン（ＥＰＰＳ）緩衝剤、［Ｎ−トリス（ヒドロキシメチル）メチルグリシン］（Ｔｒｉｃｉｎｅ）緩衝剤、［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）グリシン］（Ｂｉｃｉｎｅ）緩衝剤、３−［Ｎ−トリス（ヒドロキシメチル）メチル］アミノプロパンスルホン酸（ＴＡＰＳ）緩衝剤、２−（Ｎ−シクロヘキシルアミノ）エタンスルホン酸（ＣＨＥＳ）緩衝剤、３−（Ｎ−シクロヘキシルアミノ）−２−ヒドロキシプロパンスルホン酸（ＣＡＰＳＯ）緩衝剤、３−（Ｎ−シクロヘキシルアミノ）プロパンスルホン酸（ＣＡＰＳ）緩衝剤等が挙げられる。 Good buffers include, for example, 2-morpholinoetan sulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, tris (hydroxymethyl) aminomethane (Tris). Buffer, N- (2-acetamide) iminodiacetic acid (ADA) buffer, piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES) buffer, 2- [N- (2-acetamide) Amino] ethanesulfonic acid (ACES) buffer, 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) buffer, 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) buffer Agent, 3-morpholinopropanesulfonic acid (MOPS) buffer, 2- {N- [tris (hydroxymethyl) methyl] amino} ethanesulfonic acid (TES) buffer, N- (2-hydroxyethyl) -N'- (2-sulfoethyl) piperazin (HEPES) buffer, 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N -Tris (Hydroxymethyl) Methyl] Amino} Propane Sulfonic Acid (TAPSO) Buffer, Piperazin-N, N'-Bis (2-Hydroxy Propane-3-Sulfonic Acid) (POPSO) Buffer, N- (2-Hydroxy) Ethyl) -N'-(2-hydroxy-3-sulfopropyl) piperazin (HEPPSO) buffer, N- (2-hydroxyethyl) -N'-(3-sulfopropyl) piperazin (EPPS) buffer, [N -Tris (hydroxymethyl) methylglycine] (Tricine) buffer, [N, N-bis (2-hydroxyethyl) glycine] (Bicine) buffer, 3- [N-Tris (hydroxymethyl) methyl] aminopropanesulfon Acid (TAPS) buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-N-) Cyclohexylamino) propanesulfonic acid (CAPS) buffers and the like can be mentioned.
本発明における金属イオンとしては、例えばマグネシウムイオン、マンガンイオン、亜鉛イオン等が挙げられる。本発明における糖類としては、例えばマンニトール、ソルビトール等が挙げられる。本発明における防腐剤としては、例えばアジ化ナトリウム、抗生物質（ストレプトマイシン、ペニシリン、ゲンタマイシン等）、バイオエース、プロクリン300、プロキセル（Proxel）ＧＸＬ等が挙げられる。本発明における蛋白質としては、例えばウシ血清アルブミン（ＢＳＡ）、ウシ胎児血清（ＦＢＳ）、カゼイン、ブロックエース（ＤＳファーマバイオメディカル社製）等が挙げられる。本発明における界面活性剤としては、例えば非イオン性界面活性剤、カチオン性界面活性剤、アニオン性界面活性剤、両性界面活性剤等が挙げられる。 Examples of the metal ion in the present invention include magnesium ion, manganese ion, zinc ion and the like. Examples of the saccharide in the present invention include mannitol, sorbitol and the like. Examples of the preservative in the present invention include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), bioace, Proclin 300, Proxel GXL, and the like. Examples of the protein in the present invention include bovine serum albumin (BSA), fetal bovine serum (FBS), casein, block ace (manufactured by DS Pharma Biomedical) and the like. Examples of the surfactant in the present invention include nonionic surfactants, cationic surfactants, anionic surfactants, amphoteric surfactants and the like.
本発明における凝集促進剤としては、ポリエチレングリコール、ヒドロキシエチルセルロース、デキストラン、Lipidure類（Lipidure BL-206等；日油社製）、Ficoll類（Ficoll 400等；ＧＥヘルスケア社製）等が挙げられる。 Examples of the aggregation accelerator in the present invention include polyethylene glycol, hydroxyethyl cellulose, dextran, Lipidures (Lipidure BL-206 and the like; manufactured by NOF CORPORATION), Ficolls (Ficoll 400 and the like; manufactured by GE Healthcare) and the like.
本発明における不溶性担体粒子としては、本発明のｓＩＬ−２Ｒの測定を可能とする不溶性担体粒子であれば特に制限はなく、例えば、ラテックス粒子、磁性粒子等が挙げられ、ラテックス粒子が好ましい。ラテックス粒子の素材としては、例えばポリスチレン、ポリ塩化ビニル、ポリプロピレン等が挙げられる。 The insoluble carrier particles in the present invention are not particularly limited as long as they are insoluble carrier particles capable of measuring sIL-2R of the present invention, and examples thereof include latex particles and magnetic particles, and latex particles are preferable. Examples of the material of the latex particles include polystyrene, polyvinyl chloride, polypropylene and the like.
本発明における反応溶液中の不溶性担体粒子の形状は、本発明のｓＩＬ−２Ｒの測定を可能とする形状であれば特に制限はなく、例えば球状、円柱状等が挙げられ、球状が好ましい。球状の粒子の粒径としては、通常、平均粒径３０〜８００ｎｍであり、平均粒径１００〜５００ｎｍが好ましく、平均粒径１５０〜４５０ｎｍがより好ましい。本発明における反応溶液中の不溶性担体粒子の濃度は、本発明のｓＩＬ−２Ｒの測定を可能とする濃度であれば特に制限はなく、通常、０．０００１〜１０重量％であり、０．０００５〜５重量％が好ましく、０．００１〜０．１重量％がより好ましい。 The shape of the insoluble carrier particles in the reaction solution in the present invention is not particularly limited as long as it is a shape that enables the measurement of sIL-2R of the present invention, and examples thereof include a spherical shape and a columnar shape, and a spherical shape is preferable. As the particle size of the spherical particles, the average particle size is usually 30 to 800 nm, the average particle size is preferably 100 to 500 nm, and the average particle size is more preferably 150 to 450 nm. The concentration of the insoluble carrier particles in the reaction solution in the present invention is not particularly limited as long as it is a concentration capable of measuring sIL-2R of the present invention, and is usually 0.0001 to 10% by weight, 0.0005. It is preferably ~ 5% by weight, more preferably 0.001 to 0.1% by weight.
ｓＩＬ−２Ｒに結合する第１抗体を結合させる不溶性担体粒子と、ｓＩＬ−２Ｒに結合する第２抗体を結合させる不溶性担体粒子とは同じであっても異なっていてもよいが、同じである方が好ましい。 The insoluble carrier particles that bind the first antibody that binds to sIL-2R and the insoluble carrier particles that bind the second antibody that binds to sIL-2R may be the same or different, but they are the same. Is preferable.
本発明におけるｓＩＬ−２Ｒに結合する抗体（第１抗体及び第２抗体）は、ｓＩＬ−２Ｒに結合し、本発明のｓＩＬ−２Ｒの測定を可能とする抗体であれば特に制限はなく、ポリクローナル抗体、モノクローナル抗体のいずれも使用できるが、モノクローナル抗体が好ましい。また、本発明においては、全長の抗体のみならず、抗体フラグメントを用いることもできる。抗体フラグメントとしては、例えば、抗体をパパイン処理により得られるＦａｂ、ペプシン処理により得られるＦ（ａｂ’）２、ペプシン処理−還元処理により得られるＦａｂ’等のＦｃ部分を除去した抗体フラグメント等が挙げられる。不溶性担体粒子として、アビジン、ニュートラアビジン又はストレプトアビジンがその表面に固定化された不溶性担体粒子を用いる場合には、第１抗体にビオチンが結合したビオチン結合第１抗体及び第２抗体にビオチンが結合したビオチン結合第２抗体を用いることができる。 The antibody (first antibody and second antibody) that binds to sIL-2R in the present invention is not particularly limited as long as it is an antibody that binds to sIL-2R and enables measurement of sIL-2R of the present invention. Both an antibody and a monoclonal antibody can be used, but a monoclonal antibody is preferable. Further, in the present invention, not only a full-length antibody but also an antibody fragment can be used. Examples of the antibody fragment include an antibody fragment obtained by removing Fc portions such as Fab obtained by papain treatment of an antibody, F (ab') 2 obtained by pepsin treatment, and Fab' obtained by pepsin treatment-reduction treatment. Be done. When insoluble carrier particles in which avidin, neutravidin or streptavidin are immobilized on the surface of the insoluble carrier particles are used, biotin is bound to the first antibody and biotin is bound to the first antibody and the second antibody. The biotin-bound second antibody can be used.
本発明において使用される、ｓＩＬ−２Ｒに結合する第１抗体が認識するｓＩＬ−２Ｒの部位と、ｓＩＬ−２Ｒに結合する第２抗体が認識するｓＩＬ−２Ｒの部位とは同じであっても、異なっていてもよいが、異なっていることが好ましい。 Even if the site of sIL-2R recognized by the first antibody that binds to sIL-2R and the site of sIL-2R recognized by the second antibody that binds to sIL-2R used in the present invention are the same. , May be different, but preferably different.
本発明において使用される、ｓＩＬ−２Ｒに結合する抗体は、ｓＩＬ−２Ｒそのもの、又は、ｓＩＬ−２Ｒ中のエピトープに相当するペプチドを抗原として用いる通常の抗体の製造方法により製造することができるが、市販品としても入手可能である。ｓＩＬ−２Ｒに結合する抗体の市販品としては、例えばモノクローナル抗体AM92.3（ピアース社製）、モノクローナル抗体7G7/B6（ピアース社製）等が挙げられる。 The antibody that binds to sIL-2R used in the present invention can be produced by the usual method for producing an antibody using sIL-2R itself or a peptide corresponding to an epitope in sIL-2R as an antigen. , Also available as a commercial product. Examples of commercially available antibodies that bind to sIL-2R include monoclonal antibodies AM92.3 (manufactured by Pierce) and monoclonal antibodies 7G7 / B6 (manufactured by Pierce).
（測定用試薬）
本発明の試料中のｓＩＬ−２Ｒ測定用試薬は、本発明のｓＩＬ−２Ｒの測定方法に使用される試薬であり、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子、及び、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子を含有する。
(Reagent for measurement)
The reagent for measuring sIL-2R in the sample of the present invention is a reagent used in the method for measuring sIL-2R of the present invention, to which the first antibody that binds to sIL-2R is bound, and sIL-. Insoluble carrier particles to which the second antibody binding to 2R is not bound, and insoluble to which the second antibody binding to sIL-2R is bound and the first antibody binding to sIL-2R is not bound. Contains carrier particles.
本発明の測定用試薬は凍結乾燥状態でも液状でもよい。凍結乾燥状態の測定用試薬を用いる場合には、予め水性媒体で溶解した後、測定に供する。水性媒体としては、例えば前述の水性媒体等が挙げられる。水性媒体には、前述の金属イオン、糖類、防腐剤、蛋白質、界面活性剤、凝集促進剤等が含有されてもよい。液状の測定用試薬には、水性媒体が含まれている。水性媒体としては、例えば前述の水性媒体等が挙げられる。水性媒体には、前述の金属イオン、糖類、防腐剤、蛋白質、界面活性剤、凝集促進剤等が含有されてもよい。 The measurement reagent of the present invention may be in a freeze-dried state or in a liquid state. When a reagent for measurement in a freeze-dried state is used, it is first dissolved in an aqueous medium and then subjected to measurement. Examples of the aqueous medium include the above-mentioned aqueous medium and the like. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins, surfactants, aggregation promoters and the like. The liquid measurement reagent contains an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium and the like. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins, surfactants, aggregation promoters and the like.
本発明の測定用試薬における、不溶性担体粒子、ｓＩＬ−２Ｒに結合する抗体（第１抗体、第２抗体）としては、前述の不溶性担体粒子、ｓＩＬ−２Ｒに結合する抗体（第１抗体、第２抗体）がそれぞれ挙げられる。本発明の測定用試薬における不溶性担体粒子の含量は、水性媒体で溶解された時の濃度または、水性媒体中の濃度が、通常０．０００１〜１０重量％となる含量であり、０．０００５〜５重量％となる含量が好ましく、０．００１〜０．１重量％となる含量がより好ましい。 Examples of the antibody (first antibody, second antibody) that binds to the insoluble carrier particles and sIL-2R in the measurement reagent of the present invention include the above-mentioned insoluble carrier particles and an antibody that binds to sIL-2R (first antibody, first antibody). 2 antibodies), respectively. The content of the insoluble carrier particles in the measurement reagent of the present invention is such that the concentration when dissolved in an aqueous medium or the concentration in the aqueous medium is usually 0.0001 to 10% by weight, 0.0005 to 50%. The content of 5% by weight is preferable, and the content of 0.001 to 0.1% by weight is more preferable.
本発明の測定用試薬において、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子は、抗原抗体反応液中で生成されてもよい。この場合、本発明の試薬には、ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子の代わりに、一組の親和性物質の片方（Ａ）が結合した第１抗体と、一組の親和性物質のもう一方（ａ）が結合した不溶性担体粒子を含む。同様に、本発明の測定用試薬において、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子は、抗原抗体反応液中で生成されてもよい。この場合、本発明の試薬には、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子の代わりに、一組の親和性物質の片方（Ｂ）が結合した第２抗体と、一組の親和性物質のもう一方（ｂ）が結合した不溶性担体粒子を含む。ここで、（Ａ）が結合した第１抗体と（ａ）が結合した不溶性担体粒子とから、第１抗体が結合した不溶性担体粒子を生成させる反応と、（Ｂ）が結合した第２抗体と（ｂ）が結合した不溶性担体粒子とから、第２抗体が結合した不溶性担体粒子を生成させる反応とが、同一の反応液中で行われる試薬の場合、Ａ及びａからなる一組の親和性物質は、Ｂ及びｂからなる一組の親和性物質とは異なっていることが好ましい。一組の親和性物質としては、例えば前述の親和性物質等が挙げられ、Ａ−ａの組み合わせとＢ−ｂの組み合わせとが異なる場合における、Ａ−ａの組み合わせ、及び、Ｂ−ｂの組み合わせとしては、例えば前述の組み合わせ等が挙げられる。 In the measurement reagent of the present invention, the insoluble carrier particles to which the first antibody binding to sIL-2R is bound and the second antibody binding to sIL-2R is not bound are present in the antigen-antibody reaction solution. It may be generated. In this case, the reagent of the present invention is replaced with a set of insoluble carrier particles in which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound. It contains a first antibody to which one of the affinity substances (A) of the above is bound and an insoluble carrier particle to which the other (a) of the set of affinity substances is bound. Similarly, in the measurement reagent of the present invention, the insoluble carrier particles to which the second antibody that binds to sIL-2R is bound and the first antibody that binds to sIL-2R is not bound are subjected to an antigen-antibody reaction. It may be produced in liquid. In this case, the reagent of the present invention is replaced with a set of insoluble carrier particles in which the second antibody that binds to sIL-2R is bound and the first antibody that binds to sIL-2R is not bound. It contains a second antibody to which one of the affinity substances (B) of the above is bound and an insoluble carrier particle to which the other (b) of the set of affinity substances is bound. Here, the reaction of producing the insoluble carrier particles to which the first antibody is bound from the first antibody to which (A) is bound and the insoluble carrier particles to which (a) is bound, and the second antibody to which (B) is bound. When the reaction for producing the insoluble carrier particles to which the second antibody is bound from the insoluble carrier particles to which (b) is bound is a reagent performed in the same reaction solution, a set of affinities consisting of A and a. The substance is preferably different from the set of affinity substances consisting of B and b. Examples of the set of affinity substances include the above-mentioned affinity substances, and when the combination of Aa and the combination of Bb are different, the combination of Aa and the combination of Bb. For example, the above-mentioned combination and the like can be mentioned.
本発明の測定用試薬において、試料を前処理するための試料前処理試薬が含まれていてもよい。前処理としては、例えば希釈、可溶化、変性等が挙げられる。試料前処理試薬としては、例えば前述の水性媒体等が挙げられる。水性媒体には、前述の金属イオン、糖類、防腐剤、蛋白質、界面活性剤、凝集促進剤等が含有されてもよい。試料前処理試薬は液状でも凍結乾燥状態でもよい。凍結乾燥状態の試料前処理試薬を用いる場合には、予め水性媒体で溶解した後、試料の前処理に用いることができる。水性媒体としては、例えば前述の水性媒体等が挙げられる。水性媒体には、前述の金属イオン、糖類、防腐剤、蛋白質、界面活性剤、凝集促進剤等が含有されてもよい。 The measurement reagent of the present invention may include a sample pretreatment reagent for pretreating a sample. Examples of the pretreatment include dilution, solubilization, denaturation and the like. Examples of the sample pretreatment reagent include the above-mentioned aqueous medium and the like. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins, surfactants, aggregation promoters and the like. The sample pretreatment reagent may be in a liquid state or in a lyophilized state. When a freeze-dried sample pretreatment reagent is used, it can be used for sample pretreatment after being previously dissolved in an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium and the like. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins, surfactants, aggregation promoters and the like.
本発明の測定用試薬は、保存、運搬、流通等の観点からキットの形態と取ることもできる。キットの形態としては、ｓＩＬ−２Ｒの測定を可能とする形態であれば特に制限はなく、例えば２試薬系のキット、３試薬系のキット等が挙げられる。ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子として、一組の親和性物質の片方（Ａ）が結合した第１抗体と、一組の親和性物質のもう一方（ａ）が結合した不溶性担体粒子を用いる場合には、一組の親和性物質の片方（Ａ）が結合した第１抗体と、もう一方の（ａ）が結合した不溶性担体粒子とは、同一の試薬に含まれても、別々の試薬に含まれてもよいが、別々の試薬に含まれることが好ましい。また、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子として、一組の親和性物質の片方（Ｂ）が結合した第２抗体と、一組の親和性物質のもう一方（ｂ）が結合した不溶性担体粒子を用いる場合には、一組の親和性物質の片方（Ｂ）が結合した第１抗体と、もう一方の（ｂ）が結合した不溶性担体粒子とは、同一の試薬に含まれても、別々の試薬に含まれてもよいが、別々の試薬に含まれることが好ましい。 The measurement reagent of the present invention can also be taken in the form of a kit from the viewpoint of storage, transportation, distribution and the like. The form of the kit is not particularly limited as long as it enables the measurement of sIL-2R, and examples thereof include a two-reagent kit and a three-reagent kit. One of the set of affinity substances (A) was bound as insoluble carrier particles to which the first antibody binding to sIL-2R was bound and the second antibody binding to sIL-2R was not bound. When an insoluble carrier particle in which the first antibody and the other (a) of the set of affinity substances are bound is used, the first antibody to which one of the set of affinity substances (A) is bound and the other The insoluble carrier particles to which (a) is bound may be contained in the same reagent or in different reagents, but are preferably contained in different reagents. Further, as insoluble carrier particles to which the second antibody that binds to sIL-2R is bound and the first antibody that binds to sIL-2R is not bound, one of a set of affinity substances (B) is used. When using the bound second antibody and the insoluble carrier particles to which the other (b) of the set of affinity substances is bound, the first antibody to which one of the set of affinity substances (B) is bound is used. The insoluble carrier particles to which the other (b) is bound may be contained in the same reagent or in different reagents, but are preferably contained in different reagents.
本発明の測定用キットの具体的態様を以下に例示する。
・測定用キット１
ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子と、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子とを含む第１試薬；及び、
試料前処理試薬を含む第２試薬
を含む、試料中のｓＩＬ−２Ｒ測定用キット。
・測定用キット２
ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子を含む第１試薬；及び、
ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子を含む第２試薬
を含む、試料中のｓＩＬ−２Ｒ測定用キット。
・測定用キット３
ｓＩＬ−２Ｒに結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していない不溶性担体粒子を含む第１試薬；
ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していない不溶性担体粒子を含む第２試薬；及び、
試料前処理試薬を含む第３試薬
を含む、試料中のｓＩＬ−２Ｒ測定用キット。
Specific embodiments of the measurement kit of the present invention are illustrated below.
・ Measurement kit 1
The insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound are bound to the second antibody that binds to sIL-2R. And a first reagent containing insoluble carrier particles to which the first antibody that binds to sIL-2R is not bound;
A kit for measuring sIL-2R in a sample, which comprises a second reagent containing a sample pretreatment reagent.
・ Measurement kit 2
A first reagent containing insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound;
For measuring sIL-2R in a sample, which comprises a second reagent containing insoluble carrier particles to which a second antibody that binds to sIL-2R is bound and to which a first antibody that binds to sIL-2R is not bound. kit.
・ Measurement kit 3
A first reagent containing insoluble carrier particles to which the first antibody that binds to sIL-2R is bound and the second antibody that binds to sIL-2R is not bound;
A second reagent containing insoluble carrier particles to which the second antibody that binds to sIL-2R is bound and to which the first antibody that binds to sIL-2R is not bound;
A kit for measuring sIL-2R in a sample, which comprises a third reagent containing a sample pretreatment reagent.
以下、実施例により本発明を実施例、比較例、参考例により、更に詳細に説明するが、これらは本発明の範囲を何ら制限するものではない。尚、以下の実施例、比較例、参考例においては、次のメーカーの試薬類を使用した。 Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples, and Reference Examples, but these do not limit the scope of the present invention at all. In the following examples, comparative examples, and reference examples, reagents of the following manufacturers were used.
カルボキシル基修飾ポリスチレンラテックス（粒径３３０ｎｍ、藤倉化成社製）、抗ｓＩＬ−２Ｒモノクローナル抗体AM92.3（ピアース社製）、抗ｓＩＬ−２Ｒモノクローナル抗体7G7/B6（ピアース社製）、Sulfo-NHS（サーモフィッシャーサイエンティフィック社製）、1-[3-（ジメチルアミノ）プロピル]-3-エチルカルボジイミド（ＷＳＣ）（和光純薬工業社製）、リン酸水素二ナトリウム・12水和物（関東化学社製）、リン酸二水素ナトリウム・2水和物（関東化学社製）、ＨＥＰＥＳ （同仁化学研究所社製）、塩化ナトリウム（和光純薬工業社製）、ＢＳＡ（Ｂｏｖｏｇｅｎ社製）、ツイーン２０（非イオン性界面活性剤；シグマ−アルドリッチ社製）、ヒドロキシエチルセルロース（同仁化学研究所社製）。 Carboxylic acid modified polystyrene latex (particle size 330 nm, manufactured by Fujikura Kasei Co., Ltd.), anti-sIL-2R monoclonal antibody AM92.3 (manufactured by Pierce), anti-sIL-2R monoclonal antibody 7G7 / B6 (manufactured by Pierce), Sulfo-NHS ( Thermo Fisher Scientific Co., Ltd.), 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (WSC) (Wako Pure Chemical Industries, Ltd.), disodium hydrogen phosphate, dodecahydrate (Kanto Chemical Co., Inc.) (Manufactured by), Sodium dihydrogen phosphate dihydrate (manufactured by Kanto Chemical Co., Ltd.), HEPES (manufactured by Dojin Chemical Research Institute), Sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.), BSA (manufactured by Bovogen), Tween 20 (Non-ionic surfactant; manufactured by Sigma-Aldrich), hydroxyethyl cellulose (manufactured by Dojin Chemical Research Institute).
以下の組成のｓＩＬ−２Ｒ測定用試薬を調製した。
〔第１試薬〕
リン酸緩衝液（ｐＨ８．０） ０．１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
塩化ナトリウム ０．１５ ｍｏｌ／Ｌ
ヒドロキシエチルセルロース ０．１５％
〔第２試薬〕
ＨＥＰＥＳ（ｐＨ７．７） ０．０１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
第１抗体結合ラテックス粒子１（参考例１で製造） ０．０１重量％
第２抗体結合ラテックス粒子１（参考例２で製造） ０．０１重量％
A reagent for measuring sIL-2R having the following composition was prepared.
[First reagent]
Phosphate buffer (pH 8.0) 0.1 mol / L
BSA 0.1%
Sodium chloride 0.15 mol / L
Hydroxyethyl cellulose 0.15%
[Second reagent]
HEPES (pH 7.7) 0.01 mol / L
BSA 0.1%
First antibody-bound latex particle 1 (manufactured in Reference Example 1) 0.01% by weight
Second antibody-bound latex particle 1 (manufactured in Reference Example 2) 0.01% by weight
［比較例１］
実施例１おける第１抗体結合ラテックス粒子１及び第２抗体結合ラテックス粒子１に代えて、同一のラテックス粒子上に第１抗体及び第２抗体が結合した、第１抗体及び第２抗体結合ラテックス粒子１を含有する以下の組成のｓＩＬ−２Ｒ測定用試薬を調製した。
〔第１試薬〕
リン酸緩衝液（ｐＨ８．０） ０．１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
塩化ナトリウム ０．１５ ｍｏｌ／Ｌ
ヒドロキシエチルセルロース ０．１５％
〔第２試薬〕
ＨＥＰＥＳ（ｐＨ７．７） ０．０１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
第１抗体及び第２抗体結合ラテックス粒子１（参考例３で製造） ０．０２重量％
[Comparative Example 1]
First antibody and second antibody-bound latex particles in which the first antibody and the second antibody are bound on the same latex particles instead of the first antibody-bound latex particles 1 and the second antibody-bound latex particles 1 in Example 1. A reagent for measuring sIL-2R having the following composition containing 1 was prepared.
[First reagent]
Phosphate buffer (pH 8.0) 0.1 mol / L
BSA 0.1%
Sodium chloride 0.15 mol / L
Hydroxyethyl cellulose 0.15%
[Second reagent]
HEPES (pH 7.7) 0.01 mol / L
BSA 0.1%
First antibody and second antibody-bound latex particles 1 (manufactured in Reference Example 3) 0.02% by weight
第１抗体結合ラテックス粒子１及び第２抗体結合ラテックス粒子１を含有する実施例１のｓＩＬ−２Ｒ測定用試薬と、第１抗体及び第２抗体結合ラテックス粒子１を含有する比較例１のｓＩＬ−２Ｒ測定用試薬を用いて、ｓＩＬ−２Ｒの測定感度を比較した。
（１）ｓＩＬ−２Ｒ溶液の調製
抗原用希釈液（４％ＢＳＡ、０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）を用いて３０００００Ｕ／ｍＬ ｓＩＬ−２Ｒ溶液を希釈し、１０００Ｕ／ｍＬのｓＩＬ−２Ｒ溶液を調製した。
（２）ｓＩＬ−２Ｒの測定
分光光度計Ｕ−３０００（日立ハイテクノロジーズ社製）を使用し、反応温度３７℃にて、前記ｓＩＬ−２Ｒ溶液５μＬと第１試薬１５０μＬとをセルに加えて均一攪拌した後、５分間保持した。その後、３７℃で保持しながら、第２試薬５０μＬを当該反応液に加えて均一攪拌した後、３０秒経過後の反応液の５７０ｎｍでの吸光度と３００秒経過後の反応液の５７０ｎｍでの吸光度との差（ΔＡｂｓ）を測定した。ΔＡｂｓが高い程、高感度で測定できることを意味する。実施例１の試薬又は比較例１の試薬を用いて測定した結果を表１に示す。表１から、本発明の実施例１のｓＩＬ−２Ｒ測定用試薬は、比較例１のｓＩＬ−２Ｒ測定用試薬と比較して、試料中のｓＩＬ−２Ｒを高感度で測定できることがわかる。
The reagent for measuring sIL-2R of Example 1 containing the first antibody-bound latex particle 1 and the second antibody-bound latex particle 1, and the sIL- of Comparative Example 1 containing the first antibody and the second antibody-bound latex particle 1. The measurement sensitivities of sIL-2R were compared using 2R measurement reagents.
(1) Preparation of sIL-2R solution 300,000 U / mL sIL- using a diluent for antigen (4% BSA, 0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4). The 2R solution was diluted to prepare a 1000 U / mL sIL-2R solution.
(2) Measurement of sIL-2R Using a spectrophotometer U-3000 (manufactured by Hitachi High-Technologies Corporation), 5 μL of the sIL-2R solution and 150 μL of the first reagent are added uniformly to the cell at a reaction temperature of 37 ° C. After stirring, it was held for 5 minutes. Then, while maintaining at 37 ° C., 50 μL of the second reagent was added to the reaction solution and stirred uniformly, and then the absorbance of the reaction solution after 30 seconds at 570 nm and the absorbance of the reaction solution after 300 seconds at 570 nm. The difference (ΔAbs) from the above was measured. The higher ΔAbs, the higher the sensitivity of measurement. Table 1 shows the results of measurement using the reagent of Example 1 or the reagent of Comparative Example 1. From Table 1, it can be seen that the reagent for measuring sIL-2R of Example 1 of the present invention can measure sIL-2R in a sample with higher sensitivity than the reagent for measuring sIL-2R of Comparative Example 1.
以下の組成のｓＩＬ−２Ｒ測定用試薬を調製した。
〔第１試薬〕
リン酸緩衝液（ｐＨ８．０） ０．１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
塩化ナトリウム ０．６ ｍｏｌ／Ｌ
ヒドロキシエチルセルロース ０．２２５％
〔第２試薬〕
ＨＥＰＥＳ（ｐＨ７．７） ０．０１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
第１抗体結合ラテックス粒子１（参考例１で製造） ０．０１重量％
第２抗体結合ラテックス粒子１（参考例２で製造） ０．０１重量％
A reagent for measuring sIL-2R having the following composition was prepared.
[First reagent]
Phosphate buffer (pH 8.0) 0.1 mol / L
BSA 0.1%
Sodium chloride 0.6 mol / L
Hydroxyethyl cellulose 0.225%
[Second reagent]
HEPES (pH 7.7) 0.01 mol / L
BSA 0.1%
First antibody-bound latex particle 1 (manufactured in Reference Example 1) 0.01% by weight
Second antibody-bound latex particle 1 (manufactured in Reference Example 2) 0.01% by weight
不溶性担体としてプレートを用いる、体外診断用医薬品であるｓＩＬ−２Ｒ測定用試薬「セルフリーＮ ＩＬ−２Ｒ」（協和メデックス社製）及び実施例３のｓＩＬ−２Ｒ測定用試薬を用いて、それぞれの試薬を用いた場合のｓＩＬ−２Ｒ濃度を比較した。
（１）測定試料
ｓＩＬ−２Ｒを含有する３種の血清（血清１、血清２、血清３：アリエス社より購入）、セルフリーＮ ＩＬ−２Ｒ付属の較正試薬レベルＩ、ＩＩを測定試料とした。
Using a plate as an insoluble carrier, the sIL-2R measurement reagent "Cellfree N IL-2R" (manufactured by Kyowa Medex Co., Ltd.), which is an in vitro diagnostic drug, and the sIL-2R measurement reagent of Example 3, respectively, were used. The sIL-2R concentrations when the reagents were used were compared.
(1) Measurement sample Three types of serum containing sIL-2R (serum 1, serum 2, serum 3: purchased from Aries) and calibration reagents levels I and II attached to self-free N IL-2R were used as measurement samples. ..
（２）セルフリーＮ ＩＬ−２Ｒを用いた試料中ｓＩＬ−２Ｒ濃度の測定
セルフリーＮ ＩＬ−２Ｒに付属の添付文書に記載の方法に従い、（１）の試料中のｓＩＬ−２Ｒ濃度を測定した。その結果を表２に示す。
(2) Measurement of sIL-2R concentration in sample using cell-free N IL-2R Measure sIL-2R concentration in sample according to the method described in the package insert attached to cell-free N IL-2R. did. The results are shown in Table 2.
（３）実施例３の試薬を用いた試料中ｓＩＬ−２Ｒ濃度の測定
（３）−Ａ 検量線の作成
WO2005/121795に記載された方法によって製造したｓＩＬ−２Ｒを、抗原用希釈液（４％ＢＳＡ、０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）により、ｓＩＬ−２Ｒ濃度が１８６ Ｕ／ｍＬ、５９０ Ｕ／ｍＬ、１７６３ Ｕ／ｍＬ、５３０７ Ｕ／ｍＬ、１０５６４ Ｕ／ｍＬになるように希釈したｓＩＬ−２Ｒ溶液、及び抗原用希釈液（ｓＩＬ−２Ｒ濃度 ０ Ｕ／ｍＬ）を標準試料とした。
(3) Measurement of sIL-2R concentration in sample using the reagent of Example 3 (3) -A Preparation of calibration curve
The sIL-2R produced by the method described in WO2005 / 121795 was prepared with a diluent for antigen (4% BSA, 0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4). , The sIL-2R solution diluted so that the sIL-2R concentration is 186 U / mL, 590 U / mL, 1763 U / mL, 5307 U / mL, 10564 U / mL, and the diluent for antigen (sIL-2R). Concentration 0 U / mL) was used as the standard sample.
日立７１７０型自動分析装置（日立ハイテクノロジーズ社製）を使用し、反応温度３７℃にて、前記標準試料５μＬと第１試薬１５０μＬとをセルに加えて均一攪拌した後、５分間保持した。その後、３７℃で保持しながら、第２試薬５０μＬを当該反応液に加えて均一攪拌した後、３６秒経過後の反応液の５７０ｎｍでの吸光度と３２４秒経過後の反応液の５７０ｎｍでの吸光度との差（ΔＡｂｓ）を測定した。各濃度のｓＩＬ−２Ｒ標準試料を測定した際のΔＡｂｓを縦軸、ｓＩＬ−２Ｒ標準試料の濃度を横軸にプロットし、ｓＩＬ−２Ｒ濃度とΔＡｂｓとの関係を示す検量線を作成した。 Using a Hitachi 7170 type automatic analyzer (manufactured by Hitachi High-Technologies Corporation), 5 μL of the standard sample and 150 μL of the first reagent were added to the cell at a reaction temperature of 37 ° C., and the mixture was uniformly stirred and then held for 5 minutes. Then, while maintaining at 37 ° C., 50 μL of the second reagent was added to the reaction solution and stirred uniformly, and then the absorbance of the reaction solution after 36 seconds at 570 nm and the absorbance of the reaction solution after 324 seconds at 570 nm. The difference (ΔAbs) from the above was measured. The ΔAbs when the sIL-2R standard sample of each concentration was measured was plotted on the vertical axis, and the concentration of the sIL-2R standard sample was plotted on the horizontal axis, and a calibration curve showing the relationship between the sIL-2R concentration and ΔAbs was prepared.
（３）−Ｂ （１）の試料の測定
（１）の試料について、実施例３のｓＩＬ−２Ｒ測定用試薬を用いて、以下の方法で、各試料のΔＡｂｓを測定した。
日立７１７０型自動分析装置を使用し、反応温度３７℃にて、（１）の試料５μＬと第１試薬１５０μＬとをセルに加えて均一攪拌した後、５分間保持した。その後、３７℃で保持しながら、第２試薬５０μＬを当該反応液に加えて均一攪拌した後、３６秒経過後の反応液の５７０ｎｍでの吸光度と３２４秒経過後の反応液の５７０ｎｍでの吸光度との差（ΔＡｂｓ）を測定した。
(3) -B Measurement of sample of (1) With respect to the sample of (1), ΔAbs of each sample was measured by the following method using the reagent for measuring sIL-2R of Example 3.
Using a Hitachi 7170 type automatic analyzer, 5 μL of the sample (1) and 150 μL of the first reagent were added to the cell at a reaction temperature of 37 ° C., and the mixture was uniformly stirred and then held for 5 minutes. Then, while maintaining at 37 ° C., 50 μL of the second reagent was added to the reaction solution and stirred uniformly, and then the absorbance of the reaction solution after 36 seconds at 570 nm and the absorbance of the reaction solution after 324 seconds at 570 nm. The difference (ΔAbs) from the above was measured.
（３）−Ｃ 試料中のｓＩＬ−２Ｒ濃度の算出
（３）−Ａで作成した検量線を用いて、（３）−Ｂにより得られた各試料のΔＡｂｓから、各試料中のｓＩＬ−２Ｒ濃度を算出した。その結果を表２に示す。
(3) Calculation of sIL-2R concentration in -C sample Using the calibration curve prepared in (3) -A, from ΔAbs of each sample obtained in (3) -B, sIL-2R in each sample The concentration was calculated. The results are shown in Table 2.
表２に示すように、実施例３の本発明のｓＩＬ−２Ｒ測定用試薬を用いて測定した場合のｓＩＬ−２Ｒ濃度は、セルフリーＮ ＩＬ−２Ｒを用いて測定した場合のｓＩＬ−２Ｒ濃度とほぼ一致しており、実施例３のｓＩＬ−２Ｒ測定用試薬を用いて、ｓＩＬ−２Ｒ濃度を正確に測定できることがわかる。 As shown in Table 2, the sIL-2R concentration when measured using the reagent for measuring sIL-2R of the present invention of Example 3 is the sIL-2R concentration when measured using self-free N IL-2R. It can be seen that the sIL-2R concentration can be accurately measured by using the reagent for measuring sIL-2R of Example 3.
以下の組成のｓＩＬ−２Ｒ測定用試薬を調製した。
〔第１試薬〕
リン酸緩衝液（ｐＨ８．０） ０．１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
塩化ナトリウム ０．３ ｍｏｌ／Ｌ
ヒドロキシエチルセルロース ０．０７５％
〔第２試薬〕
ＨＥＰＥＳ（ｐＨ７．７） ０．０１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
第１抗体結合ラテックス粒子２（参考例４で製造） ０．０１重量％
第２抗体結合ラテックス粒子２（参考例５で製造） ０．０１重量％
A reagent for measuring sIL-2R having the following composition was prepared.
[First reagent]
Phosphate buffer (pH 8.0) 0.1 mol / L
BSA 0.1%
Sodium chloride 0.3 mol / L
Hydroxyethyl cellulose 0.075%
[Second reagent]
HEPES (pH 7.7) 0.01 mol / L
BSA 0.1%
First antibody-bound latex particles 2 (manufactured in Reference Example 4) 0.01% by weight
Second antibody-bound latex particles 2 (manufactured in Reference Example 5) 0.01% by weight
本発明の、抗体とラテックス粒子とが物理吸着した抗体結合ラテックス粒子を含む、実施例５のｓＩＬ−２Ｒ測定用試薬を用いて、ｓＩＬ−２Ｒを測定した。
（１）ｓＩＬ−２Ｒ溶液の調製
抗原用希釈液（４％ＢＳＡ、０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）を用いて、１００００Ｕ／ｍＬのｓＩＬ−２Ｒ溶液を調製した。
（２）ｓＩＬ−２Ｒの測定
分光光度計Ｕ−３０００を使用し、波長５７０ｎｍおよび反応温度３７℃にて、前記ｓＩＬ−２Ｒ溶液５μＬと第１試薬１５０μＬとをセルに加えて均一攪拌した後、５分間保持した。その後、３７℃で保持しながら、第２試薬５０μＬを当該反応液に加えて均一攪拌した後、３０秒経過後の反応液の５７０ｎｍでの吸光度と３００秒経過後の反応液の５７０ｎｍでの吸光度との差（ΔＡｂｓ）を測定した。その結果を表３に示す。
The sIL-2R was measured using the reagent for measuring sIL-2R of Example 5, which contains the antibody-bound latex particles in which the antibody and the latex particles were physically adsorbed.
(1) Preparation of sIL-2R solution Using a diluent for antigen (4% BSA, 0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4), 10000 U / mL. A sIL-2R solution was prepared.
(2) Measurement of sIL-2R Using a spectrophotometer U-3000, 5 μL of the sIL-2R solution and 150 μL of the first reagent were added to the cell at a wavelength of 570 nm and a reaction temperature of 37 ° C., and then uniformly stirred. It was held for 5 minutes. Then, while maintaining at 37 ° C., 50 μL of the second reagent was added to the reaction solution and stirred uniformly, and then the absorbance of the reaction solution after 30 seconds at 570 nm and the absorbance of the reaction solution after 300 seconds at 570 nm. The difference (ΔAbs) from the above was measured. The results are shown in Table 3.
以下の組成のｓＩＬ−２Ｒ測定用試薬を調製した。
〔第１試薬〕
リン酸緩衝液（ｐＨ８．０） ０．１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
塩化ナトリウム ０．３ ｍｏｌ／Ｌ
ヒドロキシエチルセルロース ０．２％
〔第２試薬〕
ＨＥＰＥＳ（ｐＨ７．７） ０．０１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
第１抗体結合ラテックス粒子１（参考例１で製造） ０．０１重量％
第２抗体結合ラテックス粒子１（参考例２で製造） ０．０１重量％
A reagent for measuring sIL-2R having the following composition was prepared.
[First reagent]
Phosphate buffer (pH 8.0) 0.1 mol / L
BSA 0.1%
Sodium chloride 0.3 mol / L
Hydroxyethyl cellulose 0.2%
[Second reagent]
HEPES (pH 7.7) 0.01 mol / L
BSA 0.1%
First antibody-bound latex particle 1 (manufactured in Reference Example 1) 0.01% by weight
Second antibody-bound latex particle 1 (manufactured in Reference Example 2) 0.01% by weight
実施例７における第１抗体結合ラテックス粒子１及び第２抗体結合ラテックス粒子１に代えて、第１抗体結合ラテックス粒子２及び第２抗体結合ラテックス粒子２を含有する以下の組成のｓＩＬ−２Ｒ測定用試薬を調製した。
〔第１試薬〕
リン酸緩衝剤（ｐＨ８．０） ０．１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
塩化ナトリウム ０．３ ｍｏｌ／Ｌ
ヒドロキシエチルセルロース ０．２％
〔第２試薬〕
ＨＥＰＥＳ緩衝剤（ｐＨ７．７） ０．０１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
第１抗体結合ラテックス粒子２（参考例４で製造） ０．０１重量％
第２抗体結合ラテックス粒子２（参考例５で製造） ０．０１重量％
For sIL-2R measurement having the following composition containing the first antibody-bound latex particles 2 and the second antibody-bound latex particles 2 instead of the first antibody-bound latex particles 1 and the second antibody-bound latex particles 1 in Example 7. Reagents were prepared.
[First reagent]
Phosphate buffer (pH 8.0) 0.1 mol / L
BSA 0.1%
Sodium chloride 0.3 mol / L
Hydroxyethyl cellulose 0.2%
[Second reagent]
HEPES buffer (pH 7.7) 0.01 mol / L
BSA 0.1%
First antibody-bound latex particles 2 (manufactured in Reference Example 4) 0.01% by weight
Second antibody-bound latex particles 2 (manufactured in Reference Example 5) 0.01% by weight
［比較例２］
実施例７おける第１抗体結合ラテックス粒子１及び第２抗体結合ラテックス粒子１に代えて、同一のラテックス粒子上に第１抗体及び第２抗体が結合した、第１抗体及び第２抗体結合ラテックス粒子２を含有する以下の組成のｓＩＬ−２Ｒ測定用試薬を調製した。
〔第１試薬〕
リン酸緩衝剤（ｐＨ８．０） ０．１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
塩化ナトリウム ０．３ ｍｏｌ／Ｌ
ヒドロキシエチルセルロース ０．２％
〔第２試薬〕
ＨＥＰＥＳ緩衝剤（ｐＨ７．７） ０．０１ ｍｏｌ／Ｌ
ＢＳＡ ０．１％
第１抗体及び第２抗体結合ラテックス粒子２（参考例６で製造） ０．０２重量％
[Comparative Example 2]
First antibody and second antibody-bound latex particles in which the first antibody and the second antibody are bound on the same latex particles instead of the first antibody-bound latex particles 1 and the second antibody-bound latex particles 1 in Example 7. A reagent for measuring sIL-2R having the following composition containing 2 was prepared.
[First reagent]
Phosphate buffer (pH 8.0) 0.1 mol / L
BSA 0.1%
Sodium chloride 0.3 mol / L
Hydroxyethyl cellulose 0.2%
[Second reagent]
HEPES buffer (pH 7.7) 0.01 mol / L
BSA 0.1%
First antibody and second antibody-bound latex particles 2 (manufactured in Reference Example 6) 0.02% by weight
第１抗体結合ラテックス粒子１及び第２抗体結合ラテックス粒子１を含有する実施例７のｓＩＬ−２Ｒ測定用試薬と、第１抗体結合ラテックス粒子２及び第２抗体結合ラテックス粒子２を含有する実施例８のｓＩＬ−２Ｒ測定用試薬と、第１抗体及び第２抗体結合ラテックス粒子２を含有する比較例２のｓＩＬ−２Ｒ測定用試薬を用いて、ｓＩＬ−２Ｒの測定感度を比較した。 Example 7 containing the reagent for measuring sIL-2R of Example 7 containing the first antibody-bound latex particle 1 and the second antibody-binding latex particle 1, and the example containing the first antibody-binding latex particle 2 and the second antibody-binding latex particle 2. The measurement sensitivity of sIL-2R was compared using the reagent for measuring sIL-2R of No. 8 and the reagent for measuring sIL-2R of Comparative Example 2 containing the first antibody and the second antibody-bound latex particle 2.
（１）ｓＩＬ−２Ｒ溶液の調製
実施例２の抗原用希釈液を用いて３０００００Ｕ／ｍＬ ｓＩＬ−２Ｒ溶液を希釈し、１０００００Ｕ／ｍＬのｓＩＬ−２Ｒ溶液を調製した。
(1) Preparation of sIL-2R solution The sIL-2R solution was diluted with the diluent for antigen of Example 2 at 300,000 U / mL to prepare a 100,000 U / mL sIL-2R solution.
（２）ｓＩＬ−２Ｒの測定
分光光度計Ｕ−３０００（日立ハイテクノロジーズ社製）を使用し、波長５７０ｎｍおよび反応温度３７℃にて、前記ｓＩＬ−２Ｒ溶液５μＬと第１試薬１５０μＬとをセルに加えて均一攪拌した後、５分間保持した。その後、３７℃で保持しながら、第２試薬５０μＬを当該反応液に加えて均一攪拌した後、３０秒経過後の反応液の５７０ｎｍでの吸光度と３００秒経過後の反応液の５７０ｎｍでの吸光度との差（ΔＡｂｓ）を測定した。ΔＡｂｓが高い程、高感度で測定できることを意味する。実施例７、実施例８又は比較例２の各ｓＩＬ−２Ｒ測定用試薬を用いて測定した結果を表４に示す。表４から、本発明の実施例７および実施例８のｓＩＬ−２Ｒ測定用試薬は、比較例２のｓＩＬ−２Ｒ測定用試薬と比較して、試料中のｓＩＬ−２Ｒを高感度で測定できることがわかる。さらに、ラテックス粒子と抗体との結合が物理吸着であるラテックス粒子を含む実施例８のｓＩＬ−２Ｒ測定用試薬を用いるよりも、ラテックス粒子と抗体との結合が化学結合であるラテックス粒子を含む実施例７のｓＩＬ−２Ｒ測定用試薬を用いる方が、より高感度に、試料中のｓＩＬ−２Ｒを測定できることがわかる。
(2) Measurement of sIL-2R Using a spectrophotometer U-3000 (manufactured by Hitachi High-Technologies Corporation), 5 μL of the sIL-2R solution and 150 μL of the first reagent were placed in a cell at a wavelength of 570 nm and a reaction temperature of 37 ° C. After uniform stirring, the mixture was held for 5 minutes. Then, while maintaining at 37 ° C., 50 μL of the second reagent was added to the reaction solution and stirred uniformly, and then the absorbance of the reaction solution after 30 seconds at 570 nm and the absorbance of the reaction solution after 300 seconds at 570 nm. The difference (ΔAbs) from the above was measured. The higher ΔAbs, the higher the sensitivity of measurement. Table 4 shows the results of measurement using each sIL-2R measurement reagent of Example 7, Example 8 or Comparative Example 2. From Table 4, the reagents for measuring sIL-2R of Examples 7 and 8 of the present invention can measure sIL-2R in a sample with higher sensitivity than the reagents for measuring sIL-2R of Comparative Example 2. I understand. Further, rather than using the reagent for measuring sIL-2R of Example 8 containing the latex particles in which the bond between the latex particles and the antibody is physically adsorbed, the practice containing the latex particles in which the bond between the latex particles and the antibody is a chemical bond. It can be seen that the sIL-2R in the sample can be measured with higher sensitivity by using the reagent for measuring sIL-2R of Example 7.
このように、１つのラテックス粒子上に２種類の抗体が結合するラテックス粒子を用いてｓＩＬ−２Ｒ測定する方法に比べて、１つのラテックス粒子上に１種類の抗体のみが結合するラテックス粒子を用いてｓＩＬ−２Ｒ測定する方法の方が、試料中のｓＩＬ−２Ｒを高感度で測定できることが判明した。また、１つのラテックス粒子上に１種類の抗体のみが結合するラテックス粒子を用いてｓＩＬ−２Ｒを測定する方法において、ラテックス粒子と抗体との結合が化学結合である場合、さらに、試料中のｓＩＬ−２Ｒを高感度で測定できることが判明した。 As described above, as compared with the method of measuring sIL-2R using latex particles in which two types of antibodies are bound on one latex particle, latex particles in which only one type of antibody is bound on one latex particle are used. It was found that the method of measuring sIL-2R can measure sIL-2R in a sample with higher sensitivity. Further, in the method of measuring sIL-2R using latex particles in which only one type of antibody is bound on one latex particle, when the bond between the latex particles and the antibody is a chemical bond, further, sIL in the sample. It was found that -2R can be measured with high sensitivity.
［参考例１］
（第１抗体結合ラテックス粒子１の調製）
０．５％（ｗ／ｖ）カルボキシル基修飾ポリスチレンラテックス（粒径３３０ｎｍ）懸濁液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１ｍＬに２ｍｇ／ｍＬ ＷＳＣ溶液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１５０ μＬと４ ｍｇ／ｍＬ Ｓｕｌｆｏ−ＮＨＳ溶液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１５０ μＬを順に加え、２５℃で１５分間攪拌してカルボキシル基を活性化させた。当該反応液に、１．９ ｍｇ／ｍＬ抗ｓＩＬ−２Ｒモノクローナル抗体AM92.3（第１抗体）溶液（０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）を１５８ μＬ添加し、２５℃で６０分間攪拌しながら反応させ、ラテックス粒子に抗体を化学結合させた。当該反応液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿にブロッキング液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた後、２５℃で６０分間攪拌して、ラテックス粒子表面をブロッキングした。当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿物に洗浄液（０．１％ＢＳＡ、０．１％ツイーン２０を含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた。この後、当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）し、上清を除去した。この洗浄操作をもう一回行った後、沈殿物に希釈液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁し、第１抗体結合ラテックス粒子１の溶液とした。
[Reference example 1]
(Preparation of First Antibody-Binding Latex Particle 1)
0.5% (w / v) carboxyl group modified polystyrene latex (particle size 330 nm) suspension (0.01 mol / L HEPES buffer, pH 7.7) 2 mg / mL WSC solution (0.01 mol / L HEPES) in 1 mL Buffer solution, pH 7.7) 150 μL and 4 mg / mL Sulfo-NHS solution (0.01 mol / L HEPES buffer solution, pH 7.7) 150 μL are added in order, and the mixture is stirred at 25 ° C. for 15 minutes to activate the carboxyl group. I made it. A 1.9 mg / mL anti-sIL-2R monoclonal antibody AM92.3 (first antibody) solution (0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4) was added to the reaction solution. ) Was added, and the reaction was carried out at 25 ° C. for 60 minutes with stirring to chemically bond the antibody to the latex particles. After centrifuging the reaction solution (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed, and a blocking solution (10 mmol / L HEPES buffer solution containing 0.1% BSA, pH 7.7) is added to the precipitate. After adding mL and suspending by ultrasonic treatment (50 W, 30 seconds), the surface of the latex particles was blocked by stirring at 25 ° C. for 60 minutes. After centrifuging the suspension (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed and a 10 mmol / L HEPES buffer containing a wash solution (0.1% BSA, 0.1% Tween 20) in the precipitate. 1 mL of the solution, pH 7.7) was added and suspended by sonication (50 W, 30 seconds). Then, the suspension was centrifuged (15000 rpm, 4 ° C., 20 minutes) to remove the supernatant. After performing this washing operation once more, 1 mL of a diluent (10 mmol / L HEPES buffer containing 0.1% BSA, pH 7.7) was added to the precipitate, and sonication (50 W, 50 W, It was suspended by (30 seconds) to prepare a solution of the first antibody-bound latex particles 1.
［参考例２］
（第２抗体結合ラテックス粒子１の調製）
０．５％（ｗ／ｖ）カルボキシル基修飾ポリスチレンラテックス（粒径３３０ｎｍ）懸濁液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１ｍＬに２ｍｇ／ｍＬ ＷＳＣ溶液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１５０ μＬと４ ｍｇ／ｍＬ Ｓｕｌｆｏ−ＮＨＳ溶液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１５０ μＬを順に加え、２５℃で１５分間攪拌してカルボキシル基を活性化させた。当該反応液に、１．９ ｍｇ／ｍＬ抗ｓＩＬ−２Ｒモノクローナル抗体7G7/B6（第２抗体）溶液（０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）を１５８ μＬ添加し、２５℃で６０分間攪拌しながら反応させ、ラテックス粒子に抗体を化学結合させた。当該反応液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿にブロッキング液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた後、２５℃で６０分間攪拌して、ラテックス粒子表面をブロッキングした。当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿物に洗浄液（０．１％ＢＳＡ、０．１％ツイーン２０を含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた。この後、当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）し、上清を除去した。この洗浄操作をもう一回行った後、沈殿物に希釈液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁し、第２抗体結合ラテックス粒子１の溶液とした。
[Reference example 2]
(Preparation of Second Antibody-Binding Latex Particle 1)
0.5% (w / v) carboxyl group modified polystyrene latex (particle size 330 nm) suspension (0.01 mol / L HEPES buffer, pH 7.7) 2 mg / mL WSC solution (0.01 mol / L HEPES) in 1 mL Buffer solution, pH 7.7) 150 μL and 4 mg / mL Sulfo-NHS solution (0.01 mol / L HEPES buffer solution, pH 7.7) 150 μL are added in order, and the mixture is stirred at 25 ° C. for 15 minutes to activate the carboxyl group. I made it. A 1.9 mg / mL anti-sIL-2R monoclonal antibody 7G7 / B6 (second antibody) solution (0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4) was added to the reaction solution. ) Was added, and the reaction was carried out at 25 ° C. for 60 minutes with stirring to chemically bond the antibody to the latex particles. After centrifuging the reaction solution (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed, and a blocking solution (10 mmol / L HEPES buffer solution containing 0.1% BSA, pH 7.7) is added to the precipitate. After adding mL and suspending by ultrasonic treatment (50 W, 30 seconds), the surface of the latex particles was blocked by stirring at 25 ° C. for 60 minutes. After centrifuging the suspension (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed and a 10 mmol / L HEPES buffer containing a wash solution (0.1% BSA, 0.1% Tween 20) in the precipitate. 1 mL of the solution, pH 7.7) was added and suspended by sonication (50 W, 30 seconds). Then, the suspension was centrifuged (15000 rpm, 4 ° C., 20 minutes) to remove the supernatant. After performing this washing operation once more, 1 mL of a diluent (10 mmol / L HEPES buffer containing 0.1% BSA, pH 7.7) was added to the precipitate, and sonication (50 W, 50 W, It was suspended by (30 seconds) to prepare a solution of the second antibody-bound latex particles 1.
［参考例３］
（第１抗体及び第２抗体結合ラテックス粒子１の調製）
０．５％（ｗ／ｖ）カルボキシル基修飾ポリスチレンラテックス（粒径３３０ｎｍ）懸濁液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１ｍＬに２ｍｇ／ｍＬ ＷＳＣ溶液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１５０ μＬと４ ｍｇ／ｍＬ Ｓｕｌｆｏ−ＮＨＳ溶液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１５０ μＬを順に加え、２５℃で１５分間攪拌してカルボキシル基を活性化させた。当該反応液に、１．９ ｍｇ／ｍＬ抗ｓＩＬ−２Ｒモノクローナル抗体AM92.3溶液 ７９ μＬ（０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）と、１．９ ｍｇ／ｍＬ抗ｓＩＬ−２Ｒモノクローナル抗体7G7/B6溶液 ７９ μＬ（０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）とを添加し、２５℃で６０分間攪拌しながら反応させ、ラテックス粒子に抗体を化学結合させた。当該反応液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿にブロッキング液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた後、２５℃で６０分間攪拌して、ラテックス粒子表面をブロッキングした。当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿物に洗浄液（０．１％ＢＳＡ、０．１％ツイーン２０を含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた。この後、当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）し、上清を除去した。この洗浄操作をもう一回行った後、沈殿物に希釈液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁し、第１抗体及び第２抗体結合ラテックス粒子１の溶液とした。
[Reference example 3]
(Preparation of 1st antibody and 2nd antibody-bound latex particles 1)
0.5% (w / v) carboxyl group modified polystyrene latex (particle size 330 nm) suspension (0.01 mol / L HEPES buffer, pH 7.7) 2 mg / mL WSC solution (0.01 mol / L HEPES) in 1 mL Buffer solution, pH 7.7) 150 μL and 4 mg / mL Sulfo-NHS solution (0.01 mol / L HEPES buffer solution, pH 7.7) 150 μL are added in order, and the mixture is stirred at 25 ° C. for 15 minutes to activate the carboxyl group. I made it. 1.9 mg / mL anti-sIL-2R monoclonal antibody AM92.3 solution 79 μL (0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4) was added to the reaction solution. 1.9 mg / mL anti-sIL-2R monoclonal antibody 7G7 / B6 solution 79 μL (0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4) was added, and the temperature was 25 ° C. The reaction was carried out with stirring for 60 minutes, and the antibody was chemically bound to the latex particles. After centrifuging the reaction solution (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed, and a blocking solution (10 mmol / L HEPES buffer solution containing 0.1% BSA, pH 7.7) is added to the precipitate. After adding mL and suspending by ultrasonic treatment (50 W, 30 seconds), the surface of the latex particles was blocked by stirring at 25 ° C. for 60 minutes. After centrifuging the suspension (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed and a 10 mmol / L HEPES buffer containing a wash solution (0.1% BSA, 0.1% Tween 20) in the precipitate. 1 mL of the solution, pH 7.7) was added and suspended by sonication (50 W, 30 seconds). Then, the suspension was centrifuged (15000 rpm, 4 ° C., 20 minutes) to remove the supernatant. After performing this washing operation once more, 1 mL of a diluent (10 mmol / L HEPES buffer containing 0.1% BSA, pH 7.7) was added to the precipitate, and ultrasonic treatment (50 W, 50 W, It was suspended for 30 seconds) to prepare a solution of the first antibody and the second antibody-bound latex particles 1.
［参考例４］
（第１抗体結合ラテックス粒子２の調製）
０．５％（ｗ／ｖ）カルボキシル基修飾ポリスチレンラテックス（粒径３３０ｎｍ）懸濁液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１ｍＬに、１．９ ｍｇ／ｍＬ抗ｓＩＬ−２Ｒモノクローナル抗体AM92.3（第１抗体）溶液（０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）を１５８ μＬ添加し、２５℃で６０分間攪拌しながら反応させ、ラテックス粒子に抗体を物理吸着させた。当該反応液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿にブロッキング液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた後、２５℃で６０分間攪拌して、ラテックス粒子表面をブロッキングした。当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿物に洗浄液（０．１％ＢＳＡ、０．１％ツイーン２０を含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた。この後、当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）し、上清を除去した。この洗浄操作をもう一回行った後、沈殿物に希釈液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁し、第１抗体結合ラテックス粒子２の溶液とした。
[Reference example 4]
(Preparation of First Antibody-Binding Latex Particle 2)
1.9 mg / mL anti-sIL-2R monoclonal in 1 mL of 0.5% (w / v) carboxyl group modified polystyrene latex (particle size 330 nm) suspension (0.01 mol / L HEPES buffer, pH 7.7) Add 158 μL of antibody AM92.3 (first antibody) solution (0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4) and react with stirring at 25 ° C. for 60 minutes. The antibody was physically adsorbed on the latex particles. After centrifuging the reaction solution (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed, and a blocking solution (10 mmol / L HEPES buffer solution containing 0.1% BSA, pH 7.7) is added to the precipitate. After adding mL and suspending by ultrasonic treatment (50 W, 30 seconds), the surface of the latex particles was blocked by stirring at 25 ° C. for 60 minutes. After centrifuging the suspension (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed and a 10 mmol / L HEPES buffer containing a wash solution (0.1% BSA, 0.1% Tween 20) in the precipitate. 1 mL of the solution, pH 7.7) was added and suspended by sonication (50 W, 30 seconds). Then, the suspension was centrifuged (15000 rpm, 4 ° C., 20 minutes) to remove the supernatant. After performing this washing operation once more, 1 mL of a diluent (10 mmol / L HEPES buffer containing 0.1% BSA, pH 7.7) was added to the precipitate, and sonication (50 W, 50 W, It was suspended by (30 seconds) to prepare a solution of the first antibody-bound latex particles 2.
［参考例５］
（第２抗体結合ラテックス粒子２の調製）
０．５％（ｗ／ｖ）カルボキシル基修飾ポリスチレンラテックス（粒径３３０ｎｍ）懸濁液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１ｍＬに、１．９ ｍｇ／ｍＬ抗ｓＩＬ−２Ｒモノクローナル抗体7G7/B6（第２抗体）溶液（０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）を１５８ μＬ添加し、２５℃で６０分間攪拌しながら反応させ、ラテックス粒子に抗体を物理吸着させた。当該反応液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿にブロッキング液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた後、２５℃で６０分間攪拌して、ラテックス粒子表面をブロッキングした。当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿物に洗浄液（０．１％ＢＳＡ、０．１％ツイーン２０を含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた。この後、当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）し、上清を除去した。この洗浄操作をもう一回行った後、沈殿物に希釈液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁し、第２抗体結合ラテックス粒子２の溶液とした。
[Reference example 5]
(Preparation of 2nd antibody-bound latex particles 2)
1.9 mg / mL anti-sIL-2R monoclonal in 1 mL of 0.5% (w / v) carboxyl group modified polystyrene latex (particle size 330 nm) suspension (0.01 mol / L HEPES buffer, pH 7.7) Add 158 μL of antibody 7G7 / B6 (second antibody) solution (0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4) and react with stirring at 25 ° C. for 60 minutes. The antibody was physically adsorbed on the latex particles. After centrifuging the reaction solution (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed, and a blocking solution (10 mmol / L HEPES buffer solution containing 0.1% BSA, pH 7.7) is added to the precipitate. After adding mL and suspending by ultrasonic treatment (50 W, 30 seconds), the surface of the latex particles was blocked by stirring at 25 ° C. for 60 minutes. After centrifuging the suspension (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed and a 10 mmol / L HEPES buffer containing a wash solution (0.1% BSA, 0.1% Tween 20) in the precipitate. 1 mL of the solution, pH 7.7) was added and suspended by sonication (50 W, 30 seconds). Then, the suspension was centrifuged (15000 rpm, 4 ° C., 20 minutes) to remove the supernatant. After performing this washing operation once more, 1 mL of a diluent (10 mmol / L HEPES buffer containing 0.1% BSA, pH 7.7) was added to the precipitate, and sonication (50 W, 50 W, It was suspended by (30 seconds) to prepare a solution of the second antibody-bound latex particles 2.
［参考例６］
（第１抗体及び第２抗体結合ラテックス粒子２の調製）
０．５％（ｗ／ｖ）カルボキシル基修飾ポリスチレンラテックス（粒径３３０ｎｍ）懸濁液（０．０１ｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）１ｍＬに、１．９ ｍｇ／ｍＬ抗ｓＩＬ−２Ｒモノクローナル抗体AM92.3（第１抗体）溶液（０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）と、１．９ ｍｇ／ｍＬ抗ｓＩＬ−２Ｒモノクローナル抗体7G7/B6溶液 ７９ μＬ（０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ ｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．４）７９ μＬとを添加し、２５℃で６０分間攪拌しながら反応させ、ラテックス粒子に抗体を物理吸着させた。当該反応液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿にブロッキング液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた後、２５℃で６０分間攪拌して、ラテックス粒子表面をブロッキングした。当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）した後に上清を除去し、沈殿物に洗浄液（０．１％ＢＳＡ、０．１％ツイーン２０を含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁させた。この後、当該懸濁液を遠心分離(１５０００ｒｐｍ、４℃、２０分間）し、上清を除去した。この洗浄操作をもう一回行った後、沈殿物に希釈液（０．１％ＢＳＡを含有する１０ ｍｍｏｌ／Ｌ ＨＥＰＥＳ緩衝液、ｐＨ７．７）を１ ｍＬ添加し、超音波処理（５０ Ｗ、３０秒間）によって懸濁し、第１抗体及び第２抗体結合ラテックス粒子２の溶液とした。
[Reference example 6]
(Preparation of 1st antibody and 2nd antibody-bound latex particles 2)
1.9 mg / mL anti-sIL-2R monoclonal in 1 mL of 0.5% (w / v) carboxyl group modified polystyrene latex (particle size 330 nm) suspension (0.01 mol / L HEPES buffer, pH 7.7) Antibody AM92.3 (first antibody) solution (0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4) and 1.9 mg / mL anti-sIL-2R monoclonal antibody 7G7 / B6 solution 79 μL (0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.4) 79 μL was added and reacted at 25 ° C. for 60 minutes with stirring to produce latex particles. The antibody was physically adsorbed on the surface. After centrifuging the reaction solution (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed, and a blocking solution (10 mmol / L HEPES buffer solution containing 0.1% BSA, pH 7.7) is added to the precipitate. After adding mL and suspending by ultrasonic treatment (50 W, 30 seconds), the surface of the latex particles was blocked by stirring at 25 ° C. for 60 minutes. After centrifuging the suspension (15000 rpm, 4 ° C., 20 minutes), the supernatant is removed and a 10 mmol / L HEPES buffer containing a wash solution (0.1% BSA, 0.1% Tween 20) in the precipitate. 1 mL of the solution, pH 7.7) was added and suspended by sonication (50 W, 30 seconds). Then, the suspension was centrifuged (15000 rpm, 4 ° C., 20 minutes) to remove the supernatant. After performing this washing operation once more, 1 mL of a diluent (10 mmol / L HEPES buffer containing 0.1% BSA, pH 7.7) was added to the precipitate, and ultrasonic treatment (50 W, 50 W, It was suspended for 30 seconds) to prepare a solution of the first antibody and the second antibody-bound latex particles 2.
本発明により、成人Ｔ細胞白血病や非ホジキンリンパ腫における病態モニタリング等に有効な、試料中のｓＩＬ−２Ｒの簡便で、かつ、高感度な測定方法、及び、測定用試薬が提供される。
INDUSTRIAL APPLICABILITY The present invention provides a simple and highly sensitive measuring method of sIL-2R in a sample, and a reagent for measuring, which are effective for pathological monitoring and the like in adult T-cell leukemia and non-Hodgkin's lymphoma.
Claims (6)
前記第１手順の混合液と、
可溶性インターロイキン−２受容体（以下、ｓＩＬ−２Ｒと記す）に結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していないラテックス粒子と、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していないラテックス粒子と、第２緩衝剤と、を含む第２試薬と、
を混合し、反応させる第２手順；ここで、前記第２緩衝剤は、２−モルホリノエタンスルホン酸（ＭＥＳ）緩衝剤、ビス（２−ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Ｂｉｓ−Ｔｒｉｓ）緩衝剤、トリス（ヒドロキシメチル）アミノメタン（Ｔｒｉｓ）緩衝剤、Ｎ−（２−アセトアミド）イミノ二酢酸（ＡＤＡ）緩衝剤、ピペラジン−Ｎ，Ｎ’−ビス（２−エタンスルホン酸）（ＰＩＰＥＳ）緩衝剤、２−［Ｎ−（２−アセトアミド）アミノ］エタンスルホン酸（ＡＣＥＳ）緩衝剤、３−モルホリノ−２−ヒドロキシプロパンスルホン酸（ＭＯＰＳＯ）緩衝剤、２−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］エタンスルホン酸（ＢＥＳ）緩衝剤、３−モルホリノプロパンスルホン酸（ＭＯＰＳ）緩衝剤、２−｛Ｎ−［トリス（ヒドロキシメチル）メチル］アミノ｝エタンスルホン酸（ＴＥＳ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−スルホエチル）ピペラジン（ＨＥＰＥＳ）緩衝剤、３−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］−２−ヒドロキシプロパンスルホン酸（ＤＩＰＳＯ）緩衝剤、２−ヒドロキシ−３−｛［Ｎ−トリス（ヒドロキシメチル）メチル］アミノ｝プロパンスルホン酸（ＴＡＰＳＯ）緩衝剤、ピペラジン−Ｎ，Ｎ’−ビス（２−ヒドロキシプロパン−３−スルホン酸）（ＰＯＰＳＯ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−ヒドロキシ−３−スルホプロピル）ピペラジン（ＨＥＰＰＳＯ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（３−スルホプロピル）ピペラジン（ＥＰＰＳ）緩衝剤、［Ｎ−トリス（ヒドロキシメチル）メチルグリシン］（Ｔｒｉｃｉｎｅ）緩衝剤、［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）グリシン］（Ｂｉｃｉｎｅ）緩衝剤、３−［Ｎ−トリス（ヒドロキシメチル）メチル］アミノプロパンスルホン酸（ＴＡＰＳ）緩衝剤、２−（Ｎ−シクロヘキシルアミノ）エタンスルホン酸（ＣＨＥＳ）緩衝剤、３−（Ｎ−シクロヘキシルアミノ）−２−ヒドロキシプロパンスルホン酸（ＣＡＰＳＯ）緩衝剤及び３−（Ｎ−シクロヘキシルアミノ）プロパンスルホン酸（ＣＡＰＳ）緩衝剤から選択されるいずれか一以上のグッド緩衝剤であり、
抗原抗体反応によって生じた凝集を測定する第３手順と、
を含むことを特徴とする、試料中のｓＩＬ−２Ｒの測定方法。 The first step of mixing the sample with the first reagent containing the first buffer; where the first buffer is a lactic acid buffer, a citrate buffer, an acetate buffer, a succinate buffer, a phthal. Acid buffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, imidazole buffer, malic acid buffer, oxalate buffer, glycine buffer, borate buffer , One or more selected from carbon dioxide buffer and glycine buffer, and
With the mixed solution of the first step
Latex particles to which the first antibody that binds to the soluble interleukin-2 receptor (hereinafter referred to as sIL-2R) is bound and the second antibody that binds to sIL-2R is not bound, and sIL- A second reagent containing latex particles to which a second antibody that binds to 2R is bound and a first antibody that binds to sIL-2R is not bound, a second buffer, and a second buffer.
Mixing the second procedure Ru reacted; wherein said second buffer is 2-morpholino ethanesulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris ) Buffer, Tris (hydroxymethyl) aminomethane (Tris) buffer, N- (2-acetamido) iminodiacetic acid (ADA) buffer, piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES) ) Buffer, 2- [N- (2-acetamide) amino] ethanesulfonic acid (ACES) buffer, 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) buffer, 2- [N, N-bis ( 2-Hydroxyethyl) amino] ethanesulfonic acid (BES) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 2-{N- [tris (hydroxymethyl) methyl] amino} ethanesulfonic acid (TES) buffer Agent, N- (2-hydroxyethyl) -N'-(2-sulfoethyl) piperazin (HEPES) buffer, 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid ( DIPSO) Buffer, 2-Hydroxy-3-{[N-Tris (Hydroxymethyl) Methyl] Amino} Propane Sulfonic Acid (TAPSO) Buffer, Piperazin-N, N'-Bis (2-Hydroxy Propane-3-sulfon) Acid) (POPSO) buffer, N- (2-hydroxyethyl) -N'-(2-hydroxy-3-sulfopropyl) piperazin (HEPPSO) buffer, N- (2-hydroxyethyl) -N'-( 3-sulfopropyl) piperazin (EPPS) buffer, [N-tris (hydroxymethyl) methylglycine] (Tricine) buffer, [N, N-bis (2-hydroxyethyl) glycine] (Bicine) buffer, 3 -[N-Tris (hydroxymethyl) methyl] aminopropanesulfonic acid (TAPS) buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxy One or more good buffers selected from propanesulfonic acid (CAPSO) buffers and 3- (N-cyclohexylamino) propanesulfonic acid (CAPS) buffers.
The third step of measuring the aggregation caused by the antigen-antibody reaction, and
A method for measuring sIL-2R in a sample, which comprises.
可溶性インターロイキン−２受容体（以下、ｓＩＬ−２Ｒと記す）に結合する第１抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第２抗体が結合していないラテックス粒子と、ｓＩＬ−２Ｒに結合する第２抗体が結合しており、かつ、ｓＩＬ−２Ｒに結合する第１抗体が結合していないラテックス粒子と、第２緩衝剤と、を含む第２試薬；ここで、前記第２緩衝剤は、２−モルホリノエタンスルホン酸（ＭＥＳ）緩衝剤、ビス（２−ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Ｂｉｓ−Ｔｒｉｓ）緩衝剤、トリス（ヒドロキシメチル）アミノメタン（Ｔｒｉｓ）緩衝剤、Ｎ−（２−アセトアミド）イミノ二酢酸（ＡＤＡ）緩衝剤、ピペラジン−Ｎ，Ｎ’−ビス（２−エタンスルホン酸）（ＰＩＰＥＳ）緩衝剤、２−［Ｎ−（２−アセトアミド）アミノ］エタンスルホン酸（ＡＣＥＳ）緩衝剤、３−モルホリノ−２−ヒドロキシプロパンスルホン酸（ＭＯＰＳＯ）緩衝剤、２−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］エタンスルホン酸（ＢＥＳ）緩衝剤、３−モルホリノプロパンスルホン酸（ＭＯＰＳ）緩衝剤、２−｛Ｎ−［トリス（ヒドロキシメチル）メチル］アミノ｝エタンスルホン酸（ＴＥＳ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−スルホエチル）ピペラジン（ＨＥＰＥＳ）緩衝剤、３−［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ］−２−ヒドロキシプロパンスルホン酸（ＤＩＰＳＯ）緩衝剤、２−ヒドロキシ−３−｛［Ｎ−トリス（ヒドロキシメチル）メチル］アミノ｝プロパンスルホン酸（ＴＡＰＳＯ）緩衝剤、ピペラジン−Ｎ，Ｎ’−ビス（２−ヒドロキシプロパン−３−スルホン酸）（ＰＯＰＳＯ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（２−ヒドロキシ−３−スルホプロピル）ピペラジン（ＨＥＰＰＳＯ）緩衝剤、Ｎ−（２−ヒドロキシエチル）−Ｎ’−（３−スルホプロピル）ピペラジン（ＥＰＰＳ）緩衝剤、［Ｎ−トリス（ヒドロキシメチル）メチルグリシン］（Ｔｒｉｃｉｎｅ）緩衝剤、［Ｎ，Ｎ−ビス（２−ヒドロキシエチル）グリシン］（Ｂｉｃｉｎｅ）緩衝剤、３−［Ｎ−トリス（ヒドロキシメチル）メチル］アミノプロパンスルホン酸（ＴＡＰＳ）緩衝剤、２−（Ｎ−シクロヘキシルアミノ）エタンスルホン酸（ＣＨＥＳ）緩衝剤、３−（Ｎ−シクロヘキシルアミノ）−２−ヒドロキシプロパンスルホン酸（ＣＡＰＳＯ）緩衝剤及び３−（Ｎ−シクロヘキシルアミノ）プロパンスルホン酸（ＣＡＰＳ）緩衝剤から選択されるいずれか一以上のグッド緩衝剤である、
を含むことを特徴とする、試料中のｓＩＬ−２Ｒの測定用試薬。 First reagent containing a first buffer; where the first buffer is a lactic acid buffer, a citrate buffer, an acetate buffer, a succinate buffer, a phthalate buffer, a phosphate buffer, triethanol. Selected from amine buffers, diethanolamine buffers, lysine buffers, barbitur buffers, imidazole buffers, malic acid buffers, oxalate buffers, glycine buffers, borate buffers, carbon dioxide buffers and glycine buffers. One or more of them,
Latex particles to which the first antibody that binds to the soluble interleukin-2 receptor (hereinafter referred to as sIL-2R) is bound and the second antibody that binds to sIL-2R is not bound, and sIL- A second reagent containing latex particles to which the second antibody that binds to 2R is bound and the first antibody that binds to sIL-2R is not bound, and a second buffer; where the first is said. The two buffers are 2-morpholinoetan sulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, and tris (hydroxymethyl) aminomethane (Tris) buffer. , N- (2-acetamide) iminodiacetic acid (ADA) buffer, piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES) buffer, 2- [N- (2-acetamide) amino] Ethan sulfonic acid (ACES) buffer, 3-morpholino-2-hydroxypropane sulfonic acid (MOPSO) buffer, 2- [N, N-bis (2-hydroxyethyl) amino] ethane sulfonic acid (BES) buffer, 3-Molholino Propane Sulfonic Acid (MOPS) Buffer, 2- {N- [Tris (Hydroxymethyl) Methyl] Amino} Ethan Sulfonic Acid (TES) Buffer, N- (2-Hydroxyethyl) -N'-(2) -Sulfoethyl) piperazine (HEPES) buffer, 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N-Tris (Hydroxymethyl) Methyl] Amino} Propane Sulfonic Acid (TAPSO) Buffer, Piperazin-N, N'-Bis (2-Hydroxy Propane-3-Sulfonic Acid) (POPSO) Buffer, N- (2-Hydroxyethyl) -N'-(2-Hydroxy-3-sulfopropyl) piperazin (HEPPSO) buffer, N- (2-hydroxyethyl) -N'-(3-sulfopropyl) piperazin (EPPS) buffer, [N-Tris (Hydroxymethyl) methylglycine] (Tricine) buffer, [N, N-bis (2-hydroxyethyl) glycine] (Bicine) buffer, 3- [N-tris (hydroxymethyl) methyl] aminopropanesulfonic acid ( TAPS) buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) ) Any one or more good buffers selected from buffers and 3- (N-cyclohexylamino) propanesulfonic acid (CAPS) buffers.
A reagent for measuring sIL-2R in a sample, which comprises.
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