JP5963984B2 - Sample pretreatment method for thyroid stimulating antibody measurement - Google Patents
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Description
本発明は、甲状腺刺激抗体測定において、サンプルをあらかじめ活性炭処理することにより、甲状腺刺激抗体を精度良く測定するための方法、又はそのための材料に関するものである。 The present invention relates to a method for measuring a thyroid stimulating antibody with high accuracy by pre-treating a sample with activated carbon in thyroid stimulating antibody measurement, or a material therefor.
甲状腺刺激抗体（thyroid stimulation antibody：TSAb）は、バセドウ病をはじめとする甲状腺疾患の患者血清中に存在することが知られ、主にバセドウ病の診断、治療経過観察用マーカーとして有用であることが報告されている。従来、患者血中のTSAbを測定する方法としてはいくつかの方法が知られている。例えば、患者血清をブタ甲状腺細胞と反応させ、血清中のTSAbと、ブタ甲状腺細胞膜上に存在する甲状腺刺激ホルモンレセプターの反応によって産生されるサイクリックAMP（cAMP）の量をラジオイムノアッセイ（RIA）等で測定することにより、血清検体中のTSAb量を測定する方法などである（例えば、非特許文献１）。このとき、患者血中にはもともと内因性のcAMPが含まれていることから、正確な定量を行うために、患者血清から得た抗体画分をポリエチレングリコール（PEG）により精製し、内因性のcAMPを除去してから測定を行っていた。 Thyroid stimulation antibody (TSAb) is known to be present in the serum of patients with thyroid diseases such as Graves' disease, and may be useful mainly as a marker for diagnosing Grace's disease and monitoring the course of treatment. It has been reported. Conventionally, several methods are known as methods for measuring TSAb in patient blood. For example, reacting patient serum with porcine thyroid cells, and measuring the amount of cyclic AMP (cAMP) produced by the reaction of TSAb in the serum with the thyroid stimulating hormone receptor present on the porcine thyroid cell membrane, radioimmunoassay (RIA), etc. And measuring the amount of TSAb in a serum sample by measuring (see Non-Patent Document 1, for example). At this time, since endogenous cAMP was originally contained in the patient's blood, the antibody fraction obtained from the patient's serum was purified with polyethylene glycol (PEG) to perform accurate quantification. Measurements were taken after cAMP was removed.
しかしながら、非特許文献１の方法において、患者血清をPEG処理して沈澱させ、遠心操作により上清を除き、再度抗体画分を溶解する操作は、測定対象の検体数が増えるほど測定者の負担も大きく、結果の判定までに時間を要する原因となっていた。また本願発明者らは、この方法を行ってみたところ、PEG処理によって沈澱した抗体画分が溶解しにくく、操作が煩雑であり、さらには測定値にばらつきが生じていることに気がついた（後述の実験データ参照）。 However, in the method of Non-Patent Document 1, the patient serum is precipitated by PEG treatment, the supernatant is removed by centrifugation, and the antibody fraction is dissolved again. The burden on the measurer increases as the number of samples to be measured increases. It was also a cause that took time to judge the result. Further, the inventors of the present application conducted this method, and found that the antibody fraction precipitated by PEG treatment was difficult to dissolve, the operation was complicated, and further, the measurement values varied (described later). See experimental data).
即ち、従来のTSAbの測定法においてはPEG処理などの前処理によって内因性のcAMPを除去することにより、TSAbによるレセプター刺激活性を検出することが可能であったが、PEG処理の操作性が悪いだけでなく、沈澱したIgG画分は溶解しにくく、測定精度に改善の余地を有していた。 That is, in the conventional TSAb measurement method, it was possible to detect receptor stimulating activity by TSAb by removing endogenous cAMP by pretreatment such as PEG treatment, but the operability of PEG treatment is poor. Not only the precipitated IgG fraction was difficult to dissolve, but there was room for improvement in measurement accuracy.
本発明は上記事情に鑑みてなされたものであり、TSAbの測定が簡便に、又は短時間に、精度良く行うことができる方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method capable of accurately measuring TSAb simply or in a short time.
本発明者らは、上述の問題点を解決するために鋭意研究を重ねた結果、後述する実施例に記載のように、TSAb測定の際に測定対象検体を活性炭処理することにより内因性cAMPの影響を除くことで、操作方法が簡便化するだけでなく、従来法に比べて精度の高いTSAbの定量が可能となることを発見した。この方法を用いると、後述する実施例では、従来のPEG処理法を行った場合に比べてアッセイ間の測定値のばらつきが抑制された。さらに、カットオフ値を下げることができ、PEG処理法では検出できない弱陽性の症例を検出することが可能であった。またさらに、活性炭をゼラチンやアガロースなどのゲル状物質によりチューブやマイクロプレートなどの底部に固化させ、そこにサンプルを入れて撹拌又は静置するという簡便な操作により、内因性cAMPの除去を十分に行うことが出来ることを発見した。 As a result of intensive studies in order to solve the above-mentioned problems, the present inventors, as described in the Examples described later, by treating the analyte to be measured with activated carbon during TSAb measurement, the endogenous cAMP It was discovered that removing the effect not only simplified the operation method, but also made it possible to quantify TSAb with higher accuracy than the conventional method. When this method was used, in the examples described later, variations in measured values between assays were suppressed as compared to the case where the conventional PEG treatment method was performed. Furthermore, the cut-off value could be lowered, and weak positive cases that could not be detected by the PEG treatment method could be detected. Furthermore, endogenous cAMP can be sufficiently removed by a simple operation of solidifying the activated carbon on the bottom of a tube or microplate with gelatinous material such as gelatin or agarose, and then stirring and leaving the sample in it. I found something I could do.
即ち本発明によれば、測定対象検体中の甲状腺刺激抗体の測定方法において、（ａ）測定対象検体と、多孔性物質とを接触させる工程、（ｂ）上記工程（ａ）を経て得られる、多孔性物質で処理済の測定対象検体と、甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞と、を接触させる工程、及び（ｃ）上記工程（ｂ）によって生じた甲状腺刺激抗体刺激反応を検出する工程、を含む、方法が提供される。この方法を用いれば、TSAb測定の前処理工程において、影響物質の除去を多孔性物質による吸着操作により行うことによって、TSAb測定の操作性及び測定精度を向上させることができる。なお、好ましい実施形態は後述する。 That is, according to the present invention, in the method for measuring a thyroid stimulating antibody in a sample to be measured, (a) a step of contacting the sample to be measured with a porous substance, (b) obtained through the step (a), A step of contacting an analyte to be measured that has been treated with a porous substance and a thyroid cell or a thyroid stimulating hormone receptor-expressing cell; and (c) a step of detecting a thyroid stimulating antibody stimulation reaction generated by the step (b), A method is provided comprising: If this method is used, the operability and measurement accuracy of TSAb measurement can be improved by removing the influence substance by adsorption operation with a porous substance in the pretreatment step of TSAb measurement. A preferred embodiment will be described later.
また本発明によれば、多孔性物質を含む、TSAbの測定用キットが提供される。このキットを用いれば、簡便に、精度よく測定対象検体中のTSAb濃度を測定できる。 Moreover, according to the present invention, a kit for measuring TSAb containing a porous substance is provided. By using this kit, the TSAb concentration in the sample to be measured can be measured easily and accurately.
また本発明によれば、多孔性物質を含む、TSAb測定の前処理用試薬が提供される。この試薬を用いれば、簡便に、精度よく測定対象検体中のTSAb濃度を測定できる。 In addition, according to the present invention, a pretreatment reagent for TSAb measurement containing a porous substance is provided. By using this reagent, the TSAb concentration in the sample to be measured can be measured easily and accurately.
また本発明によれば、多孔性物質を含む、甲状腺疾患の診断用キットが提供される。このキットを用いれば、簡便に、精度よく甲状腺疾患の診断ができる。 Moreover, according to this invention, the kit for a diagnosis of a thyroid disease containing a porous substance is provided. If this kit is used, thyroid disease can be diagnosed easily and accurately.
また本発明によれば、多孔性物質を含有するゲル状固形物質を、容器の内側に固定化している、TSAb測定の前処理用容器が提供される。この容器を用いれば、簡便に、精度よく測定対象検体中のTSAb濃度を測定できる。 In addition, according to the present invention, there is provided a pretreatment container for TSAb measurement, in which a gel-like solid substance containing a porous substance is immobilized inside the container. If this container is used, the TSAb concentration in the sample to be measured can be measured easily and accurately.
以下、本発明の実施の形態について詳細に説明する。なお、同様な内容については繰り返しの煩雑を避けるために、適宜説明を省略する。 Hereinafter, embodiments of the present invention will be described in detail. In addition, in order to avoid the repetition complexity about the same content, description is abbreviate | omitted suitably.
本発明の一実施形態は、TSAbの測定方法において、測定対象検体を多孔性物質で処理する工程を含む、方法である。この測定方法は、例えば、（ａ）測定対象検体と、多孔性物質とを接触させる工程、（ｂ）上記工程（ａ）を経て得られる、多孔性物質で処理済の測定対象検体と、甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞と、を接触させる工程、及び（ｃ）上記工程（ｂ）によって生じた甲状腺刺激抗体刺激反応を検出する工程、を経て行ってもよい。またこの測定方法は、例えば、以下の工程を経て行ってもよい。
（i）多孔性物質処理工程：測定対象検体と、多孔性物質を含む溶液とを混合、撹拌する。これにより、多孔性物質に測定対象検体中の内因性cAMPが吸着する。その後、遠心分離し、上清を取り出す。
（ii）cAMP産生工程：上記の上清と、甲状腺細胞を含む溶液とを混合、静置する。これにより、上清中のTSAbと甲状腺細胞とが反応し、cAMPが産生する。静置後、上清を取り出す。
（iii）cAMP RIA濃度測定工程：上記の上清と、標識cAMP液と、cAMP抗体液とを混合、撹拌、静置する。さらに、cAMP第二抗体を加えた後、Ｂ／Ｆ分離を行い、標識cAMP由来の放射線をカウントする。これにより、上記（ii）で産生したcAMP濃度がわかる。そして、測定対象検体とコントロール検体のcAMP生産量の比率から、測定対象検体中のTSAb活性、及びTSAb濃度を算出することができる。
One embodiment of the present invention is a method including a step of treating a specimen to be measured with a porous substance in a TSAb measurement method. This measurement method includes, for example, (a) a step of bringing a sample to be measured into contact with a porous substance, (b) a sample to be measured that has been treated with a porous substance, obtained through the above step (a), and a thyroid gland. You may perform through the process of making a cell or a thyroid stimulating hormone receptor expression cell contact, and the process of detecting the thyroid stimulating antibody stimulation reaction produced by the said process (b). Moreover, you may perform this measuring method through the following processes, for example.
(I) Porous substance treatment step: A sample to be measured and a solution containing a porous substance are mixed and stirred. Thereby, endogenous cAMP in the analyte to be measured is adsorbed to the porous substance. Thereafter, it is centrifuged and the supernatant is removed.
(Ii) cAMP production step: The above supernatant and a solution containing thyroid cells are mixed and allowed to stand. As a result, TSAb in the supernatant reacts with thyroid cells to produce cAMP. After standing, remove the supernatant.
(Iii) cAMP RIA concentration measurement step: The above supernatant, labeled cAMP solution, and cAMP antibody solution are mixed, stirred, and allowed to stand. Further, after adding the cAMP second antibody, B / F separation is performed, and the radiation derived from the labeled cAMP is counted. Thereby, the cAMP concentration produced in the above (ii) is known. Then, the TSAb activity and the TSAb concentration in the measurement target sample can be calculated from the ratio of the cAMP production amount between the measurement target sample and the control sample.
またこの測定方法は、例えば、（ｄ）測定対象検体と、多孔性物質とを接触させ、上記測定対象検体と上記多孔性物質とを含む溶液を得る工程、（ｅ）上記工程（ｄ）で得られた溶液を、沈殿と上清に分離する工程、（ｆ）上記工程（ｅ）で得られた上清と、甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞とを接触させ、上記上清と上記甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞とを含む溶液を得る工程、（ｇ）上記工程（ｆ）で得られた溶液を、沈殿と上清に分離する工程、及び（ｈ）上記工程（ｇ）で得られた上清中のcAMP濃度を測定する工程、を経て行ってもよい。ここで、上記工程（ｈ）のcAMP濃度は、測定対象検体中のTSAb濃度の指標とすることができる。また例えば、複数の検体のTSAb%値（（測定対象血清検体のcAMP濃度）／（健常者血清検体のcAMP濃度）×100）を測定後、その測定結果に基づきカットオフ値を設定してもよい。そして、特定の測定対象検体におけるTSAb%値が、上記カットオフ値以上の場合、甲状腺疾患陽性と診断することができる。 In addition, this measurement method includes, for example, (d) a step of bringing a sample to be measured into contact with a porous substance to obtain a solution containing the sample to be measured and the porous substance, and (e) the step (d). A step of separating the resulting solution into a precipitate and a supernatant; (f) contacting the supernatant obtained in step (e) with a thyroid cell or a thyroid stimulating hormone receptor-expressing cell; A step of obtaining a solution containing thyroid cells or thyroid-stimulating hormone receptor-expressing cells, (g) a step of separating the solution obtained in the step (f) into a precipitate and a supernatant, and (h) the step (g). The step of measuring the cAMP concentration in the supernatant obtained in step 1 may be performed. Here, the cAMP concentration in the step (h) can be used as an index of the TSAb concentration in the measurement target sample. Alternatively, for example, after measuring the TSAb% value ((cAMP concentration of a serum sample to be measured) / (cAMP concentration of a healthy subject serum sample) × 100) of a plurality of samples, a cut-off value may be set based on the measurement results. Good. Then, when the TSAb% value in a specific measurement target sample is equal to or higher than the cut-off value, it can be diagnosed that the thyroid disease is positive.
そして、TSAbの測定方法において、測定対象検体を多孔性物質で処理する工程を含む方法によれば、後述する実施例で実証されているように、測定対象検体中のTSAb濃度を簡便に、且つ高精度で測定することができる。そのため、この方法を用いれば、簡便に、且つ高精度に甲状腺疾患を診断することができる。 And, in the method for measuring TSAb, according to the method including the step of treating the sample to be measured with a porous substance, the TSAb concentration in the sample to be measured can be simply and It can be measured with high accuracy. Therefore, if this method is used, a thyroid disease can be diagnosed simply and with high accuracy.
測定対象検体中の内因性cAMP、又はcAMP産生を刺激する因子を除去する多孔性物質としては、活性炭を使用できるが、多孔質で低分子物質を吸着する能力を有するものであれば、活性炭以外の物質を用いても良い。例えば、多孔質樹脂、又は多孔質シリカ化合物等であってもよい。活性炭は、粉末状のものでも顆粒状のものでもよい。活性炭は、内因性cAMP、又はcAMP産生を刺激する因子の除去効率向上の観点からは、デキストラン被膜処理されたもの（デキストラン被膜処理活性炭、Dextran Coated Charcoal、以下「DCC」）を用いることがより好ましい。また、処理後に容易に分離が可能なように、一定の粒子径のものを使用して、自然に下方に沈澱させて上清を用いてもよい。さらに、より分離を容易にするため、多孔性物質による処理を、ゲル状物質（ゲル状固形物質、又は単にゲルと称することもある）で担体に固定化した多孔性物質を用いて行ってもよい。即ち、多孔性物質をゼラチンやアガロースなどのゲル状物質に溶解した後、低温で固化させたものを用いてもよいし、さらにチューブやマイクロプレートなどの担体の底部で固化させて固定化し、遠心操作を省略できるようにしても良い。 Activated carbon can be used as a porous substance that removes endogenous cAMP or a factor that stimulates cAMP production in the sample to be measured, but other than activated carbon as long as it is porous and has the ability to adsorb low-molecular substances. These materials may be used. For example, a porous resin or a porous silica compound may be used. Activated carbon may be powdery or granular. From the viewpoint of improving the efficiency of removing endogenous cAMP or a factor that stimulates cAMP production, it is more preferable to use activated carbon that has been treated with dextran coating (dextran coated activated carbon, hereinafter referred to as “DCC”). . In addition, a supernatant having a certain particle size may be used and the supernatant may be naturally precipitated so that separation can be easily performed after the treatment. Furthermore, in order to facilitate separation, the porous material may be treated with a porous material immobilized on a carrier with a gel material (also referred to as a gel solid material or simply a gel). Good. That is, a porous substance dissolved in a gelatinous substance such as gelatin or agarose and then solidified at a low temperature may be used, or it may be solidified at the bottom of a carrier such as a tube or a microplate and fixed, and then centrifuged. The operation may be omitted.
多孔性物質で測定対象検体を処理するとき、多孔性物質及び測定対象検体を含む混合液中における多孔性物質の終濃度は０．２％（ｗ／ｗ）以上が好ましく、１％（ｗ／ｗ）以上がより好ましい。この場合、測定対象検体中のTSAb濃度を特に高精度で測定することができる。またこの濃度は、２０％（ｗ/ｗ）以下が好ましい。この場合、溶液の粘度が適度に滑らかであり、分注操作がし易くなる。この場合、懸濁のし易さや、ピペット等での吸引操作の滑らかさなどの操作面が特に良好である。またこの濃度は特に限定されないが、例えば、0.05、0.1、0.2、0.5、1、2、4、5、6、8、10、15、18、20、又は30％(w/w)であってもよく、それらいずれかの値以上、又は範囲内であってもよい。 When the sample to be measured is treated with the porous substance, the final concentration of the porous substance in the mixed solution containing the porous substance and the sample to be measured is preferably 0.2% (w / w) or more, preferably 1% (w / w w) or more is more preferable. In this case, the TSAb concentration in the sample to be measured can be measured with particularly high accuracy. The concentration is preferably 20% (w / w) or less. In this case, the viscosity of the solution is moderately smooth, and the dispensing operation is easy. In this case, operation surfaces such as ease of suspension and smoothness of suction operation with a pipette or the like are particularly favorable. Further, this concentration is not particularly limited, but for example, 0.05, 0.1, 0.2, 0.5, 1, 2, 4, 5, 6, 8, 10, 15, 18, 20, or 30% (w / w) It may also be greater than or within any of these values.
活性炭は、細孔を有する多孔質の炭素質物質を含む。この活性炭の平均粒子径は、10〜25μmが特に好ましい。この場合、適度に分散し、遠心すると沈澱しやすく、使用し易い。また、測定対象検体中のTSAb濃度を特に高精度に測定することができる。この平均粒子径は特に限定されないが、例えば、0.5、1、3、5、8、10、15、20、25、30、40、60、100、300、又は500μmであってもよく、それらいずれかの値以下、又は範囲内であってもよい。本明細書において「平均粒子径」は、体積平均粒子径を意味する。体積平均粒子径は、レーザー回折・散乱法によって求めた粒度分布における積算値５０％での粒径として求めることができる。 Activated carbon includes a porous carbonaceous material having pores. The average particle diameter of the activated carbon is particularly preferably 10 to 25 μm. In this case, it disperses moderately, and when centrifuged, it tends to precipitate and is easy to use. In addition, the TSAb concentration in the sample to be measured can be measured with particularly high accuracy. The average particle diameter is not particularly limited, and may be, for example, 0.5, 1, 3, 5, 8, 10, 15, 20, 25, 30, 40, 60, 100, 300, or 500 μm. It may be less than or within that value. In the present specification, the “average particle size” means a volume average particle size. The volume average particle diameter can be determined as a particle diameter at an integrated value of 50% in the particle size distribution determined by the laser diffraction / scattering method.
ゲル状固形物質に含まれる多孔性物質の濃度は、１％（ｗ／ｗ）以上が好ましく、１０％（ｗ／ｗ）以上がより好ましい。この場合、測定対象検体中のTSAb濃度を特に高精度に測定することができる。この濃度は特に限定されないが、例えば、0.1、0.5、1、2、5、8、10、15、20、30、又は50％(w/w)であってもよく、それらいずれかの値以上、又は範囲内であってもよい。 The concentration of the porous substance contained in the gel-like solid substance is preferably 1% (w / w) or more, and more preferably 10% (w / w) or more. In this case, the TSAb concentration in the sample to be measured can be measured with particularly high accuracy. This concentration is not particularly limited, but may be, for example, 0.1, 0.5, 1, 2, 5, 8, 10, 15, 20, 30, or 50% (w / w), and any one or more of these values Or within range.
ゲル状固形物質に含まれる多孔性物質で測定対象検体を処理するとき、ゲル状固形物質と測定対象検体の容量比（ゲル状固形物質／測定対象検体）は、０．０５以上が好ましく、０．４以上がより好ましい。この場合、測定対象検体中のTSAb濃度を特に高精度に測定することができる。この容量比は特に限定されないが、例えば、0.01、0.05、0.1、0.2、0.5、0.8、1、2、5、8、又は10であってもよく、それらいずれかの値以上、又は範囲内であってもよい。 When the sample to be measured is treated with a porous material contained in the gel solid material, the volume ratio of the gel solid material to the sample to be measured (gel solid material / sample to be measured) is preferably 0.05 or more, 0 .4 or more is more preferable. In this case, the TSAb concentration in the sample to be measured can be measured with particularly high accuracy. The capacity ratio is not particularly limited, and may be, for example, 0.01, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, or 10, and any one of those values or within the range. There may be.
ゲル状固形物質に含まれる多孔性物質以外の成分は特に限定されないが、例えば、多糖、蛋白質、又はそれらいずれかを主成分とする組成物を含んでいてもよい。多糖は、例えばアガロース、又はデキストランを含む。蛋白質は、例えばコラーゲン、ケラチンを含む。上記組成物は、例えばゼラチンを含む。なおゲル状固形物質は、外力を加えられない状態で所定の形状を維持でき、外力に応じて任意の形状に変形できる物質である。またゲル状固形物質は、より低粘度で流動性のゾルと区別できる。 Ingredients other than the porous substance contained in the gel-like solid substance are not particularly limited. For example, a polysaccharide, a protein, or a composition containing either of them as a main component may be included. Polysaccharides include, for example, agarose or dextran. The protein includes, for example, collagen and keratin. The composition includes, for example, gelatin. The gel-like solid substance is a substance that can maintain a predetermined shape in a state where an external force is not applied and can be deformed into an arbitrary shape according to the external force. Gel-like solid substances can also be distinguished from lower viscosity and flowable sols.
ゲル状固形物質に含まれるアガロースの濃度は、１％（ｗ/ｗ）以上が好ましい。この場合、攪拌等によってゲルが破壊され難くなる。また、このアガロース濃度は、１０％（ｗ/ｗ）以下が好ましい。この場合、溶液の粘度が適度に滑らかであり、分注操作がし易くなる。このアガロース濃度は特に限定されないが、例えば、0.5、0.8、1、2、4、6、8、10、又は15％(w/w)であってもよく、それらいずれかの値の範囲内であってもよい。 The concentration of agarose contained in the gel-like solid substance is preferably 1% (w / w) or more. In this case, the gel is hardly broken by stirring or the like. The agarose concentration is preferably 10% (w / w) or less. In this case, the viscosity of the solution is moderately smooth, and the dispensing operation is easy. The agarose concentration is not particularly limited, and may be, for example, 0.5, 0.8, 1, 2, 4, 6, 8, 10, or 15% (w / w). There may be.
ゲル状固形物質に含まれるゼラチンの濃度は、３％（ｗ/ｗ）以上が好ましい。この場合、攪拌等によってゲルが破壊され難くなる。また、このゼラチン濃度は、２０％（ｗ/ｗ）以下が好ましい。この場合、溶液の粘度が適度に滑らかであり、分注操作がし易くなる。このゼラチン濃度は特に限定されないが、例えば、0.5、1、2、3、4、6、8、10、15、20、又は25％(w/w)であってもよく、それらいずれかの値の範囲内であってもよい。なお、ゲル状固形物質に含まれる多糖、蛋白質、又はそれらいずれかを主成分とする組成物の濃度は、ここで列挙したゼラチン濃度と同程度であってもよい。 The concentration of gelatin contained in the gel-like solid substance is preferably 3% (w / w) or more. In this case, the gel is hardly broken by stirring or the like. The gelatin concentration is preferably 20% (w / w) or less. In this case, the viscosity of the solution is moderately smooth, and the dispensing operation is easy. The gelatin concentration is not particularly limited, and may be, for example, 0.5, 1, 2, 3, 4, 6, 8, 10, 15, 20, or 25% (w / w). It may be within the range. The concentration of the polysaccharide, protein contained in the gel-like solid substance, or a composition containing either of them as a main component may be the same as the gelatin concentration listed here.
多孔性物質で測定対象検体を処理する時間は特に限定されないが、例えば、0.5、1、2、3、4、5、6、10、20、30、又は60分であってもよく、それらいずれかの値以上、又は範囲内であってもよい。 The time for processing the analyte to be measured with the porous material is not particularly limited, and may be, for example, 0.5, 1, 2, 3, 4, 5, 6, 10, 20, 30, or 60 minutes. These values may be greater than or within the range.
本実施形態に係るTSAbの測定方法は、多孔性物質で処理した後の測定対象検体を、ＰＥＧ共存下において甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞と反応させる工程、およびこの反応によって生じた甲状腺刺激抗体刺激反応を検出する工程、を含む。共存させるＰＥＧの分子量は特に限定されないが、例えば、重量平均分子量で1000、5000、10000、15000、20000、25000、30000、35000、40000、45000、50000、55000、60000、65000、又は70000であってもよく、それらいずれかの値の範囲内であってもよい。また、ＰＥＧを共存させた反応液中のＰＥＧ濃度は4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0、13.0、14.0、又は15.0重量％であってもよく、それらいずれかの値以上、又は範囲内であってもよい。 The method for measuring TSAb according to this embodiment comprises a step of reacting a specimen to be measured after treatment with a porous substance with thyroid cells or thyroid stimulating hormone receptor-expressing cells in the presence of PEG, and thyroid stimulation caused by this reaction. Detecting an antibody stimulation response. The molecular weight of the coexisting PEG is not particularly limited.For example, the weight average molecular weight is 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, or 70000. It may be within the range of any one of these values. The PEG concentration in the reaction solution in which PEG is present may be 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, or 15.0% by weight, either It may be greater than or within the range.
TSAbの検出には、ブタ甲状腺細胞の他、ヒト、ブタなどの甲状腺刺激ホルモン（ＴＳＨ）受容体を動物由来の細胞に形質転換して発現させた培養細胞を用いることができる。活性炭等により前処理した検体と、甲状腺細胞あるいはＴＳＨ受容体発現細胞とを反応させ、一定時間経過後に、産生されたｃＡＭＰの量をラジオイムノアッセイ（RIA）や酵素免疫測定法（EIA）、蛍光免疫測定法などの方法によって測定できる。 For detection of TSAb, cultured cells obtained by transforming and expressing animal-derived thyroid stimulating hormone (TSH) receptors such as humans and pigs in addition to porcine thyroid cells can be used. A specimen pretreated with activated carbon or the like is reacted with thyroid cells or TSH receptor-expressing cells, and after a certain period of time, the amount of cAMP produced is determined by radioimmunoassay (RIA), enzyme immunoassay (EIA), or fluorescence immunization. It can be measured by a method such as a measurement method.
甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞は、メッシュで処理してもよい。このメッシュの開口径は、30〜500μｍが好ましく、100〜250μｍがより好ましい。この場合、測定対象検体中のTSAb濃度を特に高精度に測定することができる。この開口径は特に限定されないが、例えば、5、10、20、30、40、50、80、100、130、150、180、200、230、250、300、400、500、600、又は800μｍであってもよく、それらいずれかの値の範囲内であってもよい。メッシュ処理することにより、実施例に示すようにcAMP測定値のばらつきを小さくする効果が得られる。 Thyroid cells or thyroid stimulating hormone receptor-expressing cells may be treated with a mesh. The opening diameter of this mesh is preferably 30 to 500 μm, and more preferably 100 to 250 μm. In this case, the TSAb concentration in the sample to be measured can be measured with particularly high accuracy. The opening diameter is not particularly limited. For example, it is 5, 10, 20, 30, 40, 50, 80, 100, 130, 150, 180, 200, 230, 250, 300, 400, 500, 600, or 800 μm. It may be within the range of any of these values. By performing mesh processing, an effect of reducing variation in cAMP measurement values can be obtained as shown in the embodiment.
甲状腺刺激抗体刺激反応は、TSAbと、甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞との間で生じる反応を含む。この反応は、例えば、TSAbと、甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞とが反応することによって産生するcAMPを検出することによって、測定してもよい。 The thyroid stimulating antibody stimulation response includes a reaction that occurs between TSAb and thyroid cells or thyroid stimulating hormone receptor-expressing cells. This reaction may be measured, for example, by detecting cAMP produced by the reaction of TSAb with thyroid cells or thyroid stimulating hormone receptor-expressing cells.
また、TSAbの測定法として、ＴＳＨ受容体とTSAbの反応によって生じたｃＡＭＰによってルシフェラーゼが発現されるよう遺伝子操作により形質転換された細胞を用い、発光を検出することによって刺激活性を測定する方法（特表２００４−５００５８０、特表２０１０−５３９９７５）や、産生されたｃＡＭＰによってカルシウムチャネルが活性化し、これによって生じるカルシウムイオンの細胞内流入を検出することでｃＡＭＰの産生量を測定する方法（ＷＯ２０１１／００１８８５）などが報告されているが、本発明の実施形態に係る前処理法はこれらの測定系にも応用が可能である。 In addition, as a method for measuring TSAb, a method for measuring stimulating activity by detecting luminescence using cells transformed by genetic manipulation so that luciferase is expressed by cAMP generated by the reaction of TSH receptor and TSAb ( JP-T-2004-500580, JP-T 2010-539975), and a method of measuring the amount of cAMP produced by detecting the intracellular inflow of calcium ions caused by activation of calcium channels by the cAMP produced (WO2011 / However, the pretreatment method according to the embodiment of the present invention can be applied to these measurement systems.
また本発明の一実施形態は、多孔性物質を含む、TSAbの測定用キットである。このキットは、本実施形態の活性炭を含む試薬、あるいはチューブやマイクロプレートなどをキットの構成試薬（アッセイ緩衝液、前処理用チューブ又はプレート等）として含有していてもよい。また、キットの他の構成試薬としては、採用した測定法により必要な試薬を適宜添付すればよい。 One embodiment of the present invention is a TSAb measurement kit containing a porous material. This kit may contain a reagent containing activated carbon according to the present embodiment, a tube, a microplate, or the like as a constituent reagent of the kit (such as an assay buffer, a pretreatment tube or a plate). Moreover, what is necessary is just to attach a reagent required as another component reagent of a kit suitably by the employ | adopted measuring method.
例えば、以下のような構成試薬を含むキットを例示することができる。
前処理用ＤＣＣ溶液（多孔性物質を含む）又は前処理用プレート（多孔性物質をアガロースにより固定化したプレート）
ブタ甲状腺細胞液
細胞洗浄液
ｃＡＭＰ標準液
ｃＡＭＰ抗体液
標識ｃＡＭＰ液
また、キットにはアッセイ緩衝液、反応停止液、メッシュなどを含んでいてもよい。さらにｃＡＭＰの測定をＲＩＡによって行う場合には第二抗体液など、ＥＩＡによって行う場合には固相化プレート、発色液、発色停止液、洗浄液などを含んでいてもよい。
For example, a kit containing the following constituent reagents can be exemplified.
DCC solution for pretreatment (including porous material) or pretreatment plate (plate with porous material immobilized with agarose)
Porcine thyroid cell fluid cell washing solution cAMP standard solution cAMP antibody solution labeled cAMP solution The kit may contain an assay buffer solution, a reaction stop solution, a mesh and the like. Further, when cAMP is measured by RIA, a second antibody solution or the like may be included, and when cAMP is measured by EIA, a solid phase plate, a color developing solution, a color developing stop solution, a washing solution or the like may be included.
上記キットにおいて、前処理用プレート（又はＤＣＣ溶液）は非特異的なｃＡＭＰ産生能を有する物質を吸着することができる活性炭などの多孔質物質を含む。測定対象の測定対象検体としては、例えば、甲状腺疾患患者血清、バセドウ病患者血清、又は血漿サンプルの他、TSAb（又はＴＳＨ、あるいはＴＳＨと類似の物質）を含むサンプルで、不特定多数の低分子物質等の不純物を含むものであってもよい。 In the kit, the pretreatment plate (or DCC solution) contains a porous substance such as activated carbon that can adsorb a substance having nonspecific cAMP production ability. Samples to be measured include, for example, samples containing thyroid disease patient serum, Graves' disease patient serum, or plasma samples, as well as TSAb (or TSH, or a substance similar to TSH), and an unspecified number of small molecules. It may contain impurities such as substances.
また本発明の一実施形態は、多孔性物質を含む、TSAb測定の前処理用試薬である。この試薬で前処理を行えば、測定対象検体中のTSAb濃度を簡便に、且つ高精度で測定することができる。 One embodiment of the present invention is a reagent for pretreatment of TSAb measurement, which includes a porous substance. If pretreatment is performed with this reagent, the TSAb concentration in the sample to be measured can be measured easily and with high accuracy.
また本発明の一実施形態は、多孔性物質を含む、甲状腺疾患の診断用キットである。このキットを利用すれば、簡便に、且つ高精度に甲状腺疾患を診断することができる。 One embodiment of the present invention is a kit for diagnosing thyroid disease comprising a porous substance. By using this kit, thyroid diseases can be diagnosed easily and with high accuracy.
また本発明の一実施形態は、多孔性物質を含有するゲル状固形物質を、容器の内側に固定化している、TSAb測定の前処理用容器である。TSAbの測定の前処理工程において、この容器に血清検体を入れて撹拌又は静置するという非常に簡便な操作により、cAMPを十分に除去できる。またこの容器を用いることによって、遠心分離やフィルター濾過などによる活性炭除去操作を経ずに、非常に簡便な操作により、甲状腺疾患を診断することができる。なお、上記内側は、容器内部の底面、又は側面であってもよい。本明細書において「容器」の形態は特に限定されないが、例えばチューブ、プレート等であってもよい。また本明細書において「容器」に収容可能な容量は特に限定されないが、例えば、0.01、0.1、0.5、1.0、1.5、3、5、10、又は30mlであってもよく、それらいずれかの値以上、又は範囲内であってもよい。また、この容器を含む、TSAbの測定用キットも本発明の一実施形態に含まれる。 One embodiment of the present invention is a pretreatment container for TSAb measurement, in which a gel-like solid substance containing a porous substance is immobilized on the inside of the container. In the pretreatment step of TSAb measurement, cAMP can be sufficiently removed by a very simple operation of putting a serum specimen in this container and stirring or standing. Further, by using this container, thyroid disease can be diagnosed by a very simple operation without performing an operation for removing activated carbon by centrifugation or filter filtration. The inner side may be a bottom surface or a side surface inside the container. In the present specification, the form of the “container” is not particularly limited, but may be, for example, a tube, a plate, or the like. In addition, the capacity that can be accommodated in the “container” in the present specification is not particularly limited, and may be, for example, 0.01, 0.1, 0.5, 1.0, 1.5, 3, 5, 10, or 30 ml. It may be within the above or range. Further, a TSAb measurement kit including this container is also included in one embodiment of the present invention.
また本発明の一実施形態は、多孔性物質を含有するゲル状固形物質を内側に固定化している容器に、測定対象検体を投入する工程を含む、TSAbの測定方法である。この方法によれば、TSAbの測定の前処理工程において、非常に簡便な操作により、cAMPを十分に除去できる。またこの方法を用いることによって、遠心分離やフィルター濾過などによる活性炭除去操作を経ずに、非常に簡便な操作により、甲状腺疾患を診断することができる。 In addition, an embodiment of the present invention is a method for measuring TSAb, which includes a step of putting a specimen to be measured into a container in which a gel-like solid substance containing a porous substance is immobilized. According to this method, cAMP can be sufficiently removed by a very simple operation in the TSAb measurement pretreatment step. Further, by using this method, thyroid disease can be diagnosed by a very simple operation without performing an operation for removing activated carbon by centrifugation, filter filtration, or the like.
また本発明の一実施形態は、多孔性物質を含有するゲル状固形物質を含む、TSAb測定の前処理用基剤、又はcAMP補足用基剤である。TSAb測定において、この基剤で測定対象検体に前処理を行えば、測定対象検体中のTSAb濃度を簡便に、且つ高精度で測定することができる。この基剤を用いたTSAb濃度の測定方法も、本発明の一実施形態に含まれる。本明細書において、「基剤」又は「担体」の形態は特に限定されないが、例えばチューブ、プレート、ビーズ、カラム等であってもよい。 Moreover, one embodiment of the present invention is a base for pretreatment for TSAb measurement or a base for supplementing cAMP, which includes a gel-like solid substance containing a porous substance. In the TSAb measurement, if the sample to be measured is pretreated with this base, the TSAb concentration in the sample to be measured can be measured easily and with high accuracy. A method for measuring the TSAb concentration using this base is also included in one embodiment of the present invention. In the present specification, the form of “base” or “carrier” is not particularly limited, and may be, for example, a tube, a plate, a bead, a column, or the like.
また本発明の一実施形態は、多孔性物質を含有するゲル状固形物質を、容器の内側に固定化している、cAMP補足用容器である。TSAb測定において、この容器で測定対象検体に前処理を行えば、測定対象検体中のTSAb濃度を簡便に、且つ高精度で測定することができる。 Moreover, one embodiment of the present invention is a cAMP supplement container in which a gel-like solid substance containing a porous substance is immobilized inside the container. In TSAb measurement, if the sample to be measured is pretreated in this container, the TSAb concentration in the sample to be measured can be measured easily and with high accuracy.
また本発明の一実施形態は、上記の実施形態に係るTSAbの測定方法を利用して、甲状腺疾患を診断する方法である。また本発明の一実施形態は、上記の実施形態に係るTSAbの測定方法によって、測定対象検体中のTSAb濃度を検出することで、測定対象検体における甲状腺疾患の指標を検出する方法である。 One embodiment of the present invention is a method of diagnosing thyroid disease using the method for measuring TSAb according to the above-described embodiment. One embodiment of the present invention is a method for detecting an index of thyroid disease in a measurement target sample by detecting a TSAb concentration in the measurement target sample by the TSAb measurement method according to the above embodiment.
以上、本発明の実施形態について述べたが、これらは本発明の例示であり、上記以外の様々な構成を採用することもできる。また、上記実施形態に記載の構成を組み合わせて採用することもできる。 As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above are also employable. Moreover, it is also possible to adopt a combination of the configurations described in the above embodiments.
以下、本発明を実施例をあげて具体的に説明するが、本発明はこれらによって何ら限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
＜実施例１：ＴＳＡｂ測定系の確立＞
１）ＤＣＣ処理時間の検討
まず、測定系におけるＤＣＣ処理時間の適切な範囲を決定すべく、標準溶液中におけるDCCへのcAMPの吸着を測定することにより、液相DCC処理時間によるcAMP除去への影響を検討した。なお以下、各試薬等の濃度に使用する％は、特に明記しない限り％（ｗ／ｗ）を意味する。
<Example 1: Establishment of TSAb measurement system>
1) Examination of DCC treatment time First, in order to determine the appropriate range of DCC treatment time in the measurement system, by measuring the adsorption of cAMP to DCC in the standard solution, it is possible to eliminate cAMP by the liquid phase DCC treatment time. The impact was examined. Hereinafter,% used for the concentration of each reagent, etc. means% (w / w) unless otherwise specified.
１．５ｍｌチューブにcAMP標準液と、5%Norit SX (活性炭、Norit製造、和光純薬工業販売)、0.3%Dextran40、及び2%BSAとを４：１で加え、０、１、５、１０、又は３０分間撹拌した。5000rpm、５分間遠心分離し、上清をサンプルとして使用し、RIAによりcAMP濃度を測定した。ＲＩＡでは、まず試験管にDCC処理サンプル２５μｌを分注し、さらに１２５Ｉ−標識cAMP液１００μｌと、cAMP抗体液１００μｌとを加えて良く撹拌した。次に４℃、２４時間静置した。cAMP第二抗体液５００μｌを各試験管に加え、良く撹拌し、４℃、３０分間静置した。さらに3000rpm、４℃、２０分間遠心分離した。上清を吸引除去した後、沈殿由来のγ線をガンマカウンター（アロカ社製）を用いてカウントした。その結果、処理時間５分間で、35pmol/ml のcAMPのほぼ全量を十分に吸収できることが分かった（図１）。 Add cAMP standard solution, 5% Norit SX (activated carbon, manufactured by Norit, Wako Pure Chemical Industries), 0.3% Dextran40, and 2% BSA to a 1.5 ml tube at a ratio of 0, 1, 5, 10, Or stirred for 30 minutes. The mixture was centrifuged at 5000 rpm for 5 minutes, the supernatant was used as a sample, and the cAMP concentration was measured by RIA. In RIA, first, 25 μl of a DCC-treated sample was dispensed into a test tube, and further 100 μl of 125 I-labeled cAMP solution and 100 μl of cAMP antibody solution were added and stirred well. Next, it left still at 4 degreeC for 24 hours. 500 μl of cAMP second antibody solution was added to each test tube, stirred well, and allowed to stand at 4 ° C. for 30 minutes. Furthermore, it centrifuged at 3000 rpm and 4 degreeC for 20 minutes. After removing the supernatant by aspiration, γ-rays derived from the precipitate were counted using a gamma counter (Aloka). As a result, it was found that almost all of 35 pmol / ml of cAMP could be sufficiently absorbed in the treatment time of 5 minutes (FIG. 1).
２）ＤＣＣ活性炭濃度の検討
続いて、測定系におけるＤＣＣ活性炭濃度の適切な範囲を決定すべく、処理時間を５分間に設定したときの、液相DCCの活性炭濃度によるcAMP除去への影響について検討した（図２）。その結果、DCC濃度が５％で35pmol/mlのcAMPのほぼ全量を吸収した。DCC濃度が１％では７０％、DCC濃度が０．２％では３０％のcAMPを吸収した。なお、さらにDCC濃度を変えて行ってみたところ、DCC濃度が２０％以下のときに、懸濁のし易さや、ピペット等での吸引操作の滑らかさなどの操作面が特に良好であることが分かった。
以上の結果をもとに、DCC処理工程を含むＴＳＡｂの測定系が確立された。
2) Examination of DCC activated carbon concentration Next, to determine the appropriate range of DCC activated carbon concentration in the measurement system, we examined the effect of activated carbon concentration of liquid phase DCC on cAMP removal when the processing time was set to 5 minutes. (FIG. 2). As a result, almost all cAMP of 35 pmol / ml was absorbed at a DCC concentration of 5%. When the DCC concentration was 1%, 70% was absorbed, and when the DCC concentration was 0.2%, 30% of cAMP was absorbed. Furthermore, when the DCC concentration was changed, when the DCC concentration was 20% or less, the operation surface such as the ease of suspension and the smoothness of the suction operation with a pipette or the like was particularly good. I understood.
Based on the above results, a TSAb measurement system including a DCC treatment process was established.
［方法］
３−１）DCC処理
１．５ｍｌチューブにヒト血清検体と、5%Norit SX-Plus、0.3%Dextran40、及び2%BSAからなる溶液とを容量比４：１で加え、５分間撹拌した。5000rpm、５分間遠心分離し、上清をDCC処理液として使用した。
[Method]
3-1) DCC treatment A human serum specimen and a solution consisting of 5% Norit SX-Plus, 0.3% Dextran40, and 2% BSA were added to a 1.5 ml tube at a volume ratio of 4: 1 and stirred for 5 minutes. Centrifugation was performed at 5000 rpm for 5 minutes, and the supernatant was used as a DCC treatment solution.
３−２）細胞調製液
冷凍状態のブタ甲状腺細胞液（J Clin Endocrinol Metab 1982;54:108参照）１ｍＬを、２５℃水浴中で２.５分間解凍した。解凍後の細胞液を１０ｍｌのHAM培地に加え、混和して懸濁した。１０００ｒｐｍ、２５℃、５分間遠心分離した後、上清を吸引除去した。沈殿した細胞にHAM培地を５ｍＬ加え、混和して均一に懸濁した。さらに、懸濁後の溶液を径100μｍのメッシュに通し、細胞調整液とした。
3-2) Cell Preparation Solution 1 mL of frozen porcine thyroid cell fluid (see J Clin Endocrinol Metab 1982; 54: 108) was thawed in a 25 ° C. water bath for 2.5 minutes. The cell solution after thawing was added to 10 ml of HAM medium, mixed and suspended. After centrifugation at 1000 rpm and 25 ° C. for 5 minutes, the supernatant was removed by suction. To the precipitated cells, 5 mL of HAM medium was added, mixed and suspended uniformly. Further, the suspended solution was passed through a mesh having a diameter of 100 μm to obtain a cell preparation solution.
３−３）PEG処理
１．５ｍｌチューブに血清検体と30%PEGとを容量比１：３で加えてよく撹拌し、４℃、５分間静置した。４℃、3000rpm、２０分間遠心分離し、上清を除去した。Hanks'バッファーにより沈澱を溶解し、PEG処理液とした。
3-3) PEG treatment A serum sample and 30% PEG were added to a 1.5 ml tube at a volume ratio of 1: 3, and the mixture was well stirred and left at 4 ° C. for 5 minutes. Centrifugation was performed at 4 ° C. and 3000 rpm for 20 minutes, and the supernatant was removed. The precipitate was dissolved with Hanks' buffer to prepare a PEG treatment solution.
３−４）DCC処理液、又はPEG処理液と細胞調製液の反応（cAMP産生反応）
未処理の９６穴マイクロプレートにサンプル希釈液（1mM IBMX、10%PEG含有Hanks' buffer）を50μｌ分注した。さらに、DCC処理液を25μｌずつ加え、プレートシェイカーで１分間撹拌した。さらに、上記３−２）で調製した細胞調整液を50μｌずつ加えた。プレートを軽くたたき、細胞を均一に懸濁した後、３７℃、４時間静置し、0.5%Triton X-100を100μｌずつ加え、プレートミキサーで５分間、混合した。その後、上清をとり、DCC処理サンプルとした。また、DCC処理液をPEG処理液に代えて同様の手順を行い、PEG処理サンプルを調整した。
3-4) Reaction of DCC treatment solution or PEG treatment solution and cell preparation solution (cAMP production reaction)
50 μl of a sample diluent (1 mM IBMX, Hanks' buffer containing 10% PEG) was dispensed into an untreated 96-well microplate. Furthermore, 25 μl of DCC treatment solution was added and stirred for 1 minute with a plate shaker. Further, 50 μl of the cell preparation solution prepared in 3-2) above was added. After tapping the plate and suspending the cells uniformly, the plate was allowed to stand at 37 ° C. for 4 hours, 100 μl of 0.5% Triton X-100 was added and mixed for 5 minutes with a plate mixer. Thereafter, the supernatant was taken and used as a DCC-treated sample. In addition, a PEG-treated sample was prepared by replacing the DCC-treated solution with a PEG-treated solution and performing the same procedure.
３−５）cAMP濃度測定（RIA）
試験管にDCC処理サンプル２５μｌを分注し、さらに１２５Ｉ−標識cAMP液１００μｌと、cAMP抗体液１００μｌとを加えて良く撹拌した。次に４℃、２４時間静置した。cAMP第二抗体液５００μｌを各試験管に加え、良く撹拌し、４℃、３０分間静置した。さらに3000rpm、４℃、２０分間遠心分離した。上清を吸引除去した後、沈殿由来のγ線をガンマカウンター（アロカ社製）を用いてカウントした。また、DCC処理サンプルを、PEG処理サンプル又はcAMP標準液に代えて同様の手順を行い、沈殿をカウントした。なお本明細書では、DCC処理工程を経てTSAb濃度を測定する方法を、改良法と称することもある。
3-5) cAMP concentration measurement (RIA)
25 μl of a DCC-treated sample was dispensed into a test tube, and further 100 μl of 125 I-labeled cAMP solution and 100 μl of cAMP antibody solution were added and stirred well. Next, it left still at 4 degreeC for 24 hours. 500 μl of cAMP second antibody solution was added to each test tube, stirred well, and allowed to stand at 4 ° C. for 30 minutes. Furthermore, it centrifuged at 3000 rpm and 4 degreeC for 20 minutes. After removing the supernatant by aspiration, γ-rays derived from the precipitate were counted using a gamma counter (Aloka). Moreover, the DCC process sample was replaced with the PEG process sample or the cAMP standard solution, the same procedure was performed, and precipitation was counted. In the present specification, the method of measuring the TSAb concentration through the DCC treatment step may be referred to as an improved method.
４）メッシュ使用による効果の検討
細胞をメッシュ処理する工程の効果を調べるため、上記（３−２）の細胞調整液の作製工程において、径100μｍのメッシュを通した場合と通さなかった場合での、cAMPの濃度測定の結果を比較した。結果、下記表１のようになった。なお、F検定による分散分析により、Ｐ値＜０．０５であった。したがって、メッシュを使用した場合では、使用しなかった場合と比較して、測定値のばらつきを有意に抑制できることが明らかになった。
4) Examination of effect by using mesh In order to investigate the effect of the process of meshing cells, in the preparation process of the cell adjustment solution of (3-2) above, the case of passing through the mesh with a diameter of 100 μm The results of cAMP concentration measurement were compared. As a result, it became as shown in Table 1 below. In addition, P value <0.05 by analysis of variance by F test. Therefore, it has been clarified that the variation in measured values can be significantly suppressed when the mesh is used, compared to the case where the mesh is not used.
＜実施例２：従来法との比較＞
１）サンプル前処理法の違いによるアッセイ間差の測定値のばらつきの比較
甲状腺疾患の患者血清検体（測定対象血清検体）を用いて、PEG処理サンプル、あるいはDCC処理サンプルを５回に分けて調製した。また、健常者血清検体を用いて、同様にサンプルを調整した。それらのサンプルについて同時にTSAb%値（（測定対象血清検体のcAMP濃度）／（健常者血清検体のcAMP濃度）×100）を測定し、サンプル調製によるばらつきへの影響を検討した。その結果、図３に示す通り、測定値はDCC処理サンプルの方が高くなったが、ばらつきはPEG処理の方が大きくなった。DCC処理サンプルによるCV（coefficient of variation）値は7.8%であったのに対し、PEG処理サンプルによるCV値は20.5%となり、PEG処理と比較してDCC処理はばらつきが小さく、精度の高い測定が可能となることを示している。
<Example 2: Comparison with conventional method>
1) Comparison of variation in measured values of inter-assay differences due to differences in sample pretreatment methods Prepared by dividing PEG-treated samples or DCC-treated samples into 5 samples using thyroid patient serum samples (measuring serum samples) did. In addition, samples were prepared in the same manner using serum samples from healthy subjects. The TSAb% value ((cAMP concentration of serum sample to be measured) / (cAMP concentration of healthy subject serum sample) × 100) was simultaneously measured for these samples, and the influence on variation due to sample preparation was examined. As a result, as shown in FIG. 3, the measured value was higher in the DCC-treated sample, but the variation was larger in the PEG-treated sample. The CV (coefficient of variation) value for the DCC-treated sample was 7.8%, while the CV value for the PEG-treated sample was 20.5%. It shows that it becomes possible.
２）PEG処理による現行キット法（従来法）とDCC処理による改良法の測定値の比較
TSAbキット「ヤマサ」（ヤマサ醤油株式会社製、80063）を用いた操作法（以下、「従来法」と称する）と、改良法（上記実施例１参照）との２種類の方法により、健常者血清、及びTRAb陽性あるいは弱陽性患者血清のTSAb濃度を測定し、測定値及び陽性率を比較した。なお、従来法は、前処理をPEGを添加することで行い、ブタ甲状腺細胞によるcAMP産生反応時はPEGを添加せずに行った。
2) Comparison of measured values between the current kit method using PEG processing (conventional method) and the improved method using DCC processing
By using two methods, an operation method (hereinafter referred to as “conventional method”) using a TSAb kit “Yamasa” (manufactured by Yamasa Shoyu Co., Ltd., 80063) and an improved method (see Example 1 above) TSAb concentrations of serum and TRAb positive or weakly positive patient serum were measured, and measured values and positive rates were compared. In the conventional method, pretreatment was performed by adding PEG, and the cAMP production reaction by porcine thyroid cells was performed without adding PEG.
改良法にて健常者110例のTSAb濃度を測定した。その結果、健常者コントロール血清のTSAb濃度を100%とした場合の、上記健常者110例のTSAb%値は平均107.0%、標準偏差（SD）8.5となり、平均＋2SDから計算した仮のカットオフ値は124.0％となった。一方で、従来法で同様に健常者110例を測定した結果、TSAb%値は平均153.4%、標準偏差（SD）34.2となり、平均＋2SDから計算したカットオフ値は221.7％となった。 The TSAb concentration in 110 healthy subjects was measured by the improved method. As a result, when the TSAb concentration of the healthy subject control serum is 100%, the TSAb% value of the above 110 healthy subjects is 107.0% on average, and the standard deviation (SD) is 8.5, which is a provisional cutoff value calculated from the average + 2SD Became 124.0%. On the other hand, as a result of measuring 110 healthy subjects in the same manner by the conventional method, the average TSAb% value was 153.4% and the standard deviation (SD) was 34.2, and the cut-off value calculated from the average + 2SD was 221.7%.
血清中のTSHレセプター抗体（TRAb）＝0.5IU/L以上の６例の患者血清のTSAb濃度を、従来法および改良法で測定したところ、従来法では１例のみが陽性となったが、改良法では５例が陽性となった（表２）。このように、従来法では検出できない弱陽性の症例を、改良法では検出できるようになっており、DCC処理を用いた方法がPEG処理を用いる方法より優れていることが示された。 The serum TSH receptor antibody (TRAb) = 0.5 IU / L or more of 6 patients' serum TSAb concentrations measured by the conventional method and the improved method. Five cases were positive by the method (Table 2). Thus, weak positive cases that cannot be detected by the conventional method can be detected by the improved method, and it was shown that the method using DCC processing is superior to the method using PEG processing.
３）未治療バセドウ病患者及び再発バセドウ病患者における陽性率の評価
血清中のTSHレセプター抗体（TRAb）＝0.5IU/L以上の未治療バセドウ病患者110例、及び再発バセドウ病患者19例の血清のTSAb濃度を、従来法及び改良法で測定した。なおこのとき、cAMP濃度の測定はEIAにより行った。その後、上記２）で算出したカットオフ値を用いて、陽性、又は陰性に分類した。
3) Evaluation of positive rate in patients with untreated Graves 'disease and patients with recurrent Graves' disease Serum of 110 patients with untreated Graves 'disease with TSH receptor antibody (TRAb) = 0.5 IU / L and 19 patients with recurrent Graves' disease The TSAb concentration was measured by conventional and improved methods. At this time, the cAMP concentration was measured by EIA. Then, it classified into positive or negative using the cut-off value calculated by said 2).
表３及び４は、未治療バセドウ病患者の分類結果である。表３は分類された患者数を、表４は陽性率を示している。表５及び６は、再発バセドウ病患者の分類結果である。表５は分類された患者数を、表６は陽性率を示している。その結果、未治療バセドウ病患者及び再発バセドウ病患者のいずれにおいても、改良法の陽性率は、従来法の陽性率よりも高い値となっていた。この結果は、改良法は従来法よりも高精度に、未治療バセドウ病及び再発バセドウ病を検出できることを意味している。また、特に再発バセドウ病を検査する場合に、改良法と従来法との差が大きくなっていた。 Tables 3 and 4 are the classification results of untreated Graves' disease patients. Table 3 shows the number of patients classified, and Table 4 shows the positive rate. Tables 5 and 6 show the classification results of patients with recurrent Graves' disease. Table 5 shows the number of patients classified, and Table 6 shows the positive rate. As a result, the positive rate of the improved method was higher than the positive rate of the conventional method in both untreated Graves 'disease patients and recurrent Graves' disease patients. This result means that the improved method can detect untreated Graves 'disease and recurrent Graves' disease with higher accuracy than the conventional method. Also, especially when examining recurrent Graves' disease, the difference between the improved method and the conventional method was large.
＜実施例３：ゼラチン・アガロース固化ＤＣＣチューブの検討＞
活性炭固相化チューブを調整し、活性炭固相化チューブを用いたときの検体処理時間を検討した（図４）。まず、2.5%アガロースを70℃に加温溶解した。次に、12mmチューブに70℃に加温溶解した2.5%アガロースと、5%ゼラチン、10%Charcoal、及び0.3%Dextranを200μL分注し、冷蔵してゲル化した。さらに、cAMP溶液100、200、又は500μLを加え、一定時間撹拌し、上清をサンプルとして使用し、RIA（上記実施例１（３−５）参照）によりcAMP濃度を測定した。
<Example 3: Examination of gelatin-agarose solidified DCC tube>
The activated carbon solid-phased tube was prepared, and the specimen processing time when the activated carbon solid-phased tube was used was examined (FIG. 4). First, 2.5% agarose was dissolved by heating at 70 ° C. Next, 200 μL of 2.5% agarose, 5% gelatin, 10% Charcoal, and 0.3% Dextran dissolved in a 12 mm tube by heating at 70 ° C. was dispensed and gelled by refrigeration. Furthermore, cAMP solution 100, 200, or 500 μL was added, stirred for a certain period of time, the supernatant was used as a sample, and the cAMP concentration was measured by RIA (see Example 1 (3-5) above).
サンプル量100、200μLでは、５分間の処理で50pmol/ml のcAMPを完全に除去することができた。サンプル量500μLでも約１時間で、50pmol/ml のcAMPのほぼ全量を吸収した。この結果から、活性炭固相化チューブを用いれば、活性炭固相化チューブに血清検体を入れて撹拌又は静置するという非常に簡便な操作により、cAMPを十分に除去できることがわかった。この方法は、遠心分離やフィルター濾過などによる活性炭除去操作を必要とせず、非常に簡便な操作により、甲状腺疾患を診断することを可能にする優れた方法である。 With sample amounts of 100 and 200 μL, 50 pmol / ml of cAMP could be completely removed by treatment for 5 minutes. Even in a sample volume of 500 μL, almost all of cAMP at 50 pmol / ml was absorbed in about 1 hour. From this result, it was found that if an activated carbon solid-phased tube was used, cAMP could be sufficiently removed by a very simple operation of putting a serum sample in the activated carbon solid-phased tube and stirring or standing. This method is an excellent method that makes it possible to diagnose a thyroid disease by a very simple operation without requiring an operation for removing activated carbon by centrifugation or filter filtration.
なお、上記ゲル化を行う際の各種条件を、いくつか変えて行った結果を以下に示す。アガロース濃度が1％以上の条件下では、ゲルが特に適度な強度、固さを有しており、攪拌時にゲルが破壊されにくくなっていた。アガロース濃度が2％超の条件下では、ゲルがチューブやプレートの担体に対して、特に適度に吸着し、上清への活性炭の混入も特に抑えられた。アガロースのみを用いるのではなく、ゼラチンを混合することによって、ゲルがチューブやプレートの担体に対して、特に適度に吸着し、上清への活性炭の混入も特に抑えられた。 In addition, the result of having changed several conditions at the time of performing the said gelatinization is shown below. Under conditions where the agarose concentration was 1% or more, the gel had particularly appropriate strength and hardness, and the gel was less likely to be destroyed during stirring. Under conditions where the agarose concentration exceeded 2%, the gel adsorbed moderately to the carrier of the tube or plate, and mixing of activated carbon into the supernatant was particularly suppressed. When gelatin was mixed instead of using only agarose, the gel was adsorbed particularly moderately to the carrier of the tube or plate, and mixing of activated carbon into the supernatant was particularly suppressed.
ゼラチン濃度が3％以上の条件下では、ゲルが特に適度な強度、固さを有しており、攪拌時にゲルが破壊されにくくなっていた。また、処理中の温度が25℃未満の条件下では、ゲルが膨張し難く、ゲルが水分を吸収して液量が変動することが抑えられ、さらに、攪拌時にゲルが破壊されにくくなっていた。 Under conditions where the gelatin concentration was 3% or more, the gel had particularly appropriate strength and hardness, and the gel was less likely to be destroyed during stirring. In addition, under conditions where the temperature during the treatment was less than 25 ° C., the gel did not easily expand, the gel absorbed moisture, and the liquid volume was prevented from fluctuating, and the gel was not easily destroyed during stirring. .
また、ゼラチンとアガロースを混合して使用することにより、温度の影響を受けにくくなり、撹拌時の強度が保たれるようになった。濃度は、例えば5%ゼラチンと2.5%アガロースを混合して使用した場合、良好な結果が得られた。 In addition, the mixture of gelatin and agarose makes it less susceptible to the influence of temperature, and the strength during stirring can be maintained. For example, when 5% gelatin and 2.5% agarose were mixed and used, good results were obtained.
以上のとおり、血清検体を活性炭で前処理することによって、血清検体中のTSAb濃度を、高精度に測定できた。この改良法は、従来のＰＥＧ法に比べて操作が簡便であり、作業時間、工程の削減が可能である。また、実施例からも明らかなように、改良法では、従来のＰＥＧ処理法を行った場合に比べてアッセイ間の測定値のばらつきが抑制される。さらに、改良法を用いた場合ではカットオフ値を下げることができるため、従来法では検出できない弱陽性の症例をも検出することが可能である。
即ち、改良法を用いることにより、患者サンプル中のTSAbをより正確に、かつより簡便に測定することが可能となる。従って、改良法は、バセドウ病などの甲状腺疾患の診断あるいは経過観察などに有用である。
As described above, the TSAb concentration in the serum sample could be measured with high accuracy by pretreating the serum sample with activated carbon. This improved method is simpler in operation than the conventional PEG method, and can reduce working time and processes. Further, as is clear from the examples, the improved method suppresses the variation in measured values between assays as compared to the conventional PEG treatment method. Furthermore, since the cut-off value can be lowered when the improved method is used, it is possible to detect weakly positive cases that cannot be detected by the conventional method.
That is, by using the improved method, TSAb in a patient sample can be measured more accurately and more easily. Therefore, the improved method is useful for diagnosis or follow-up of thyroid diseases such as Graves' disease.
以上、本発明を実施例に基づいて説明した。この実施例はあくまで例示であり、種々の変形例が可能なこと、またそうした変形例も本発明の範囲にあることは当業者に理解されるところである。 In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.
Claims (2)
（ａ）測定対象検体と、活性炭とを接触させる工程、
（ｂ）前記工程（ａ）を経て得られる、活性炭で処理済の測定対象検体と、メッシュで処理された、甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞と、をＰＥＧ共存下において接触させる工程、及び
（ｃ）前記工程（ｂ）によって生じた甲状腺刺激抗体刺激反応を検出する工程、
を含み、
前記メッシュで処理は、甲状腺細胞又は甲状腺刺激ホルモンレセプター発現細胞を含む溶液をメッシュに通す工程を経ることを含む、
方法。 In the method for measuring a thyroid stimulating antibody in a sample to be measured,
(A) a step of bringing a specimen to be measured into contact with activated carbon;
(B) a step of contacting the measurement target specimen treated with activated carbon obtained through the step (a) with a thyroid cell or a thyroid stimulating hormone receptor-expressing cell treated with a mesh in the presence of PEG; and (C) detecting the thyroid stimulating antibody stimulation reaction generated by the step (b),
Only including,
The treatment with the mesh includes a step of passing a solution containing thyroid cells or thyroid stimulating hormone receptor-expressing cells through the mesh.
Method.
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