JP6566697B2 - Novel calciprotein particle (CPP) measurement method using fluorescent probe - Google Patents
Novel calciprotein particle (CPP) measurement method using fluorescent probe Download PDFInfo
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Description
本発明は、例えば腎機能低下と共に血中に増加するカルシプロテイン粒子(calciprotein particle；CPP)の新規測定方法、並びに当該測定方法により測定することができる慢性腎臓病診断マーカーに関する。 The present invention relates to a novel method for measuring calciprotein particles (CPP) that increase in blood with a decrease in renal function, for example, and a diagnostic marker for chronic kidney disease that can be measured by the measurement method.
腎機能低下と共に、血中にリン酸カルシウム結晶と血清タンパク質フェチュインA(Fetuin-A)との複合体ナノ粒子であるカルシプロテイン粒子(以下、「CPP」と称する)が増えてくることが知られている。 It is known that calciprotein particles (hereinafter referred to as “CPP”), which are complex nanoparticles of calcium phosphate crystals and serum protein fetuin-A, increase in the blood with decreased renal function. .
血清中のCPPは、高速(16,000g)で長時間(2時間)の遠心により沈殿するため、従来においては、当該高速での長時間の遠心前後で血清フェチュインA濃度をELISAで測定し、その差をもって血清CPPを間接的に測定してきた(非特許文献１及び２)。しかしながら、この方法では、CPPが低濃度になる程、遠心前後の血清フェチュインA濃度の差が小さくなるために測定誤差が大きくなるという原理的な限界が問題となっていた。 CPP in serum is precipitated by centrifugation at a high speed (16,000g) for a long time (2 hours), so conventionally, serum fetuin A concentration was measured by ELISA before and after the long-time centrifugation at the high speed, Serum CPP has been indirectly measured with a difference (Non-patent Documents 1 and 2). However, in this method, the lower the CPP concentration, the smaller the difference between the serum fetuin A concentration before and after centrifugation, and thus the principle limitation that the measurement error increases becomes a problem.
上述のように、従来において、血中のCPP濃度を直接的に、且つ高感度で測定できる方法は知られていなかった。 As described above, conventionally, no method has been known that can measure the CPP concentration in blood directly and with high sensitivity.
そこで、本発明は、これらの実情に鑑み、血中のCPP濃度を直接的に測定できる新規な方法を提供することを目的とする。 In view of these circumstances, an object of the present invention is to provide a novel method capable of directly measuring the CPP concentration in blood.
上記課題を解決するため鋭意研究を行った結果、CPP中のリン酸カルシウム結晶に特異的に結合する蛍光プローブを使用することで、血中のCPP濃度を直接的に測定できることを見出した。また、見出した当該CPP測定方法によれば、血中には物性の異なる2種類のCPPが存在し、そのうちの高密度(大きくて重い)のCPPが慢性腎臓病患者において特異的に増加していることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, it was found that the concentration of CPP in blood can be directly measured by using a fluorescent probe that specifically binds to a calcium phosphate crystal in CPP. In addition, according to the CPP measurement method that has been found, there are two types of CPPs with different physical properties in blood, of which high density (large and heavy) CPPs are specifically increased in patients with chronic kidney disease. And the present invention has been completed.
すなわち、本発明は以下を包含する。
（１）血清とリン酸カルシウム結晶特異的結合性蛍光プローブとを接触させる工程を含み、該蛍光プローブの蛍光強度を指標としてCPP量を測定する、CPPの測定方法。
（２）リン酸カルシウム結晶特異的結合性蛍光プローブが、ビスホスホネートと蛍光色素とのコンジュゲートである、（１）記載の方法。
（３）血清を前記工程に供することで総CPP量を測定し、且つ血清を16,000〜24,000gで1.5〜2時間遠心分離に供することにより得られた上清を前記工程に供することで低密度CPP量を測定し、該総CPP量から該低密度CPP量を引いて高密度CPP量を算出する工程をさらに含む、（１）又は（２）記載の方法。
（４）リン酸カルシウム結晶特異的結合性蛍光プローブを含む、CPPの測定キット。
（５）リン酸カルシウム結晶特異的結合性蛍光プローブが、ビスホスホネートと蛍光色素とのコンジュゲートである、（４）記載のキット。
（６）血清とリン酸カルシウム結晶特異的結合性蛍光プローブとを接触させる工程を含み、該蛍光プローブの蛍光強度を指標としてCPP量を測定する、慢性腎臓病の検査方法。
（７）リン酸カルシウム結晶特異的結合性蛍光プローブが、ビスホスホネートと蛍光色素とのコンジュゲートである、（６）記載の方法。
（８）血清を前記工程に供することで総CPP量を測定し、且つ血清を16,000〜24,000gで1.5〜2時間遠心分離に供することにより得られた上清を前記工程に供することで低密度CPP量を測定し、該総CPP量から該低密度CPP量を引いて高密度CPP量を算出する工程をさらに含む、（６）又は（７）記載の方法。
（９）リン酸カルシウム結晶特異的結合性蛍光プローブを含む、慢性腎臓病診断キット。
（１０）リン酸カルシウム結晶特異的結合性蛍光プローブが、ビスホスホネートと蛍光色素とのコンジュゲートである、（９）記載のキット。
（１１）血清を16,000〜24,000gで1.5〜2時間遠心分離に供することで沈殿する高密度CPPから成る慢性腎臓病診断マーカー。
That is, the present invention includes the following.
(1) A method for measuring CPP, which comprises a step of contacting serum with a calcium phosphate crystal-specific binding fluorescent probe, and measuring the amount of CPP using the fluorescence intensity of the fluorescent probe as an index.
(2) The method according to (1), wherein the calcium phosphate crystal-specific binding fluorescent probe is a conjugate of a bisphosphonate and a fluorescent dye.
(3) The total amount of CPP is measured by subjecting the serum to the above step, and the supernatant obtained by subjecting the serum to centrifugation at 16,000 to 24,000 g for 1.5 to 2 hours is subjected to the above step to reduce the density. The method according to (1) or (2), further comprising a step of measuring a CPP amount and subtracting the low-density CPP amount from the total CPP amount to calculate a high-density CPP amount.
(4) A CPP measurement kit comprising a calcium phosphate crystal-specific binding fluorescent probe.
(5) The kit according to (4), wherein the calcium phosphate crystal-specific binding fluorescent probe is a conjugate of a bisphosphonate and a fluorescent dye.
(6) A method for examining chronic kidney disease, comprising a step of contacting serum with a calcium phosphate crystal-specific binding fluorescent probe, and measuring the amount of CPP using the fluorescence intensity of the fluorescent probe as an index.
(7) The method according to (6), wherein the calcium phosphate crystal-specific binding fluorescent probe is a conjugate of a bisphosphonate and a fluorescent dye.
(8) The total amount of CPP is measured by subjecting the serum to the above step, and the supernatant obtained by subjecting the serum to centrifugation at 16,000 to 24,000 g for 1.5 to 2 hours is subjected to the above step to reduce the density. The method according to (6) or (7), further comprising a step of measuring a CPP amount and subtracting the low-density CPP amount from the total CPP amount to calculate a high-density CPP amount.
(9) A chronic kidney disease diagnostic kit comprising a calcium phosphate crystal-specific binding fluorescent probe.
(10) The kit according to (9), wherein the calcium phosphate crystal-specific binding fluorescent probe is a conjugate of bisphosphonate and a fluorescent dye.
(11) A diagnostic marker for chronic kidney disease comprising high-density CPP that is precipitated by subjecting serum to centrifugation at 16,000-24,000 g for 1.5-2 hours.
本発明によれば、血中のCPP濃度を直接的に、且つ高感度で測定でき、また慢性腎臓病を正確に検査することができる。また、本発明によれば、慢性腎臓病の診断に有用なマーカーが提供される。 According to the present invention, CPP concentration in blood can be measured directly and with high sensitivity, and chronic kidney disease can be accurately examined. In addition, according to the present invention, a marker useful for diagnosis of chronic kidney disease is provided.
本発明に係るCPPの測定方法(以下、「本方法」と称する)は、血清とリン酸カルシウム結晶特異的結合性蛍光プローブとを接触させる工程を含むものである。本方法では、CPP中のリン酸カルシウム結晶に当該蛍光プローブが特異的に結合し、結合した当該蛍光プローブの蛍光強度を指標としてCPP量(濃度)を直接的に且つ高感度で測定することができる。また、CPPは慢性腎臓病患者の血中において増加することから、本方法は、慢性腎臓病の検査方法、慢性腎臓病の評価又は判定方法、慢性腎臓病を検出するためのin vitroにおけるデータ収集方法等ということができる。 The CPP measurement method according to the present invention (hereinafter referred to as “the present method”) includes a step of contacting serum with a calcium phosphate crystal-specific binding fluorescent probe. In this method, the fluorescent probe specifically binds to the calcium phosphate crystal in CPP, and the amount (concentration) of CPP can be measured directly and with high sensitivity using the fluorescence intensity of the bound fluorescent probe as an index. In addition, since CPP increases in the blood of patients with chronic kidney disease, this method is used to test for chronic kidney disease, to evaluate or determine chronic kidney disease, and to collect data in vitro to detect chronic kidney disease. It can be said that it is a method.
本方法で使用する血清が由来する被験体は、例えばヒトを含む霊長類、ウシ、ブタ、ウマ、ヤギ、ヒツジ等の家畜動物、イヌ、ネコ等のペット動物、マウス、モルモット、ウサギ等の実験動物、を含めた哺乳動物であって良く、好ましくはヒトである。また、血清は、当該被験体から得られた血液を例えば約3,000gで約10分間遠心分離に供することで、上清として得ることができる。 The subject from which the serum used in this method is derived is, for example, primates including humans, domestic animals such as cattle, pigs, horses, goats and sheep, pet animals such as dogs and cats, experiments such as mice, guinea pigs and rabbits. Mammals, including animals, may be used, and preferably humans. Serum can be obtained as a supernatant by subjecting blood obtained from the subject to centrifugation, for example, at about 3,000 g for about 10 minutes.
本方法で使用するリン酸カルシウム結晶特異的結合性蛍光プローブとしては、リン酸カルシウム結晶に特異的に結合し、且つ蛍光を発するプローブであれば特に限定されないが、例えば、リン酸カルシウム結晶に特異的に結合するビスホスホネートと蛍光色素(例えば、Rhodamin, FITC, IRDye, Alexa Fluoraなど)とのコンジュゲート等が挙げられる。さらに、当該コンジュゲートとしては、例えば下記の式：
本方法では、血清とリン酸カルシウム結晶特異的結合性蛍光プローブとを接触させるように混合し、血清中のCPPと当該蛍光プローブとを結合させる。例えば、5〜50μM(好ましくは50μM)の蛍光プローブ溶液10〜100μl(好ましくは45μl)に、血清0.5〜5μl(好ましくは5μl)を加えて、緩やかに振盪しながら室温(約24℃)で0.5〜2時間(好ましくは1時間)反応させる。 In this method, serum and calcium phosphate crystal-specific binding fluorescent probe are mixed in contact with each other, and CPP in serum and the fluorescent probe are bound to each other. For example, add 0.5 to 5 μl (preferably 5 μl) of serum to 10 to 100 μl (preferably 45 μl) of a fluorescent probe solution of 5 to 50 μM (preferably 50 μM) and add 0.5 to 0.5 μl at room temperature (about 24 ° C.) while gently shaking. The reaction is allowed for ~ 2 hours (preferably 1 hour).
反応後、例えば以下のいずれかの方法によりリン酸カルシウム結晶特異的結合性蛍光プローブに基づく蛍光強度を測定し、血中(血清中)のCPP量(濃度)を測定することができる。
(1) 反応後、遊離の蛍光プローブを除くように、反応物をゲル濾過スピンカラム(例えばMicro Bio-Spin P-30(Bio-rad社製))に供し、次いで800〜1,200g(好ましくは1,000g)で1.5〜3分間(好ましくは2分間)遠心分離に供し、溶出画分を得る。さらに、CPPを溶解するように、得られた溶出画分4〜40μl(好ましくは40μl)に250〜500mM(好ましくは500 mM) EDTAを1〜10μl(好ましくは10μl)、5〜10％(好ましくは10％)SDSを1〜10μl(好ましくは10μl)加えて、室温(約24℃)で5〜10分間(好ましくは5分間)放置する。放置後、使用する蛍光プローブの励起波長と蛍光波長(近赤外線蛍光色素の場合、励起波長： 668 nm、蛍光波長：687 nm)に準じて、蛍光プローブの蛍光強度を読み取る。読み取った蛍光強度から血中(血清中)CPP量(濃度)を定量する。
(2) 反応後、反応物を、抗フェチュインA抗体を用いるELISAに供した後、上記同様に蛍光プローブの蛍光強度を読み取る。
After the reaction, for example, the fluorescence intensity based on the calcium phosphate crystal-specific binding fluorescent probe can be measured by any of the following methods, and the amount (concentration) of CPP in blood (serum) can be measured.
(1) After the reaction, the reaction product is applied to a gel filtration spin column (for example, Micro Bio-Spin P-30 (manufactured by Bio-rad)) so as to remove the free fluorescent probe, and then 800 to 1,200 g (preferably 1,000 g) for 1.5 to 3 minutes (preferably 2 minutes) to obtain an elution fraction. Furthermore, in order to dissolve CPP, the obtained elution fraction 4 to 40 μl (preferably 40 μl) is added to 250 to 500 mM (preferably 500 mM) EDTA 1 to 10 μl (preferably 10 μl), 5 to 10% (preferably 10%) SDS is added in an amount of 1 to 10 μl (preferably 10 μl) and left at room temperature (about 24 ° C.) for 5 to 10 minutes (preferably 5 minutes). Then, the fluorescence intensity of the fluorescent probe is read in accordance with the excitation wavelength and fluorescence wavelength of the fluorescent probe to be used (in the case of a near-infrared fluorescent dye, excitation wavelength: 668 nm, fluorescence wavelength: 687 nm). The amount (concentration) of CPP in blood (serum) is quantified from the read fluorescence intensity.
(2) After the reaction, the reaction product is subjected to ELISA using an anti-fetuin A antibody, and the fluorescence intensity of the fluorescent probe is read in the same manner as described above.
また、リン酸カルシウム結晶であるヒドロキシアパタイトを使用して、本方法の工程同様にヒドロキシアパタイトに結合させたリン酸カルシウム結晶特異的結合性蛍光プローブに基づく蛍光強度を測定し、検量線を作成する。当該検量線から、ヒドロキシアパタイトに結合する蛍光プローブ量を算出し、血中(血清中)CPP濃度を、ヒドロキシアパタイト濃度として換算することもできる。 Further, using a hydroxyapatite that is a calcium phosphate crystal, the fluorescence intensity based on the calcium phosphate crystal-specific binding fluorescent probe bound to the hydroxyapatite is measured in the same manner as in the present method, and a calibration curve is created. From the calibration curve, the amount of fluorescent probe that binds to hydroxyapatite can be calculated, and the blood (serum) CPP concentration can be converted as the hydroxyapatite concentration.
血中には2種類のCPP(すなわち、小さくて軽い低密度CPPと大きくて重い高密度CPP)が存在し、そのうちの高密度のCPPが慢性腎臓病患者において特異的に増加する。すなわち、高密度CPPは、慢性腎臓病診断マーカーとして有用である。 There are two types of CPP in the blood (ie, small and light low density CPP and large and heavy high density CPP), of which high density CPP is specifically increased in patients with chronic kidney disease. That is, high density CPP is useful as a marker for diagnosing chronic kidney disease.
低密度CPPは、血清を例えば16,000〜24,000g(好ましくは16,000g)で1.5〜2時間(好ましくは2時間)遠心分離に供することで得られた上清に存在し、一方、高密度CPPは当該遠心分離により得られた沈殿物に存在する。 Low density CPP is present in the supernatant obtained by subjecting the serum to centrifugation, for example at 16,000-24,000 g (preferably 16,000 g) for 1.5-2 hours (preferably 2 hours), whereas high-density CPP is Present in the precipitate obtained by centrifugation.
そこで、本方法により血清から低密度CPPと高密度CPPとを含む総CPP量を測定し、且つ本方法により血清由来の上記上清から低密度CPP量を測定する。当該総CPP量から当該低密度CPP量を引くことで、高密度CPP量を算出することができる。 Therefore, the amount of total CPP including low density CPP and high density CPP is measured from serum by this method, and the amount of low density CPP is measured from the serum-derived supernatant by this method. By subtracting the low density CPP amount from the total CPP amount, the high density CPP amount can be calculated.
あるいは、本方法において、血清とリン酸カルシウム結晶特異的結合性蛍光プローブとを反応後、反応物を16,000〜24,000g(好ましくは16,000g)で1.5〜2時間(好ましくは2時間)遠心分離に供し、得られた沈殿物から蛍光プローブの蛍光強度を読み取ることで、高密度CPP量を直接測定することもできる。 Alternatively, in this method, after reacting serum with a calcium phosphate crystal-specific binding fluorescent probe, the reaction product is subjected to centrifugation at 16,000-24,000 g (preferably 16,000 g) for 1.5-2 hours (preferably 2 hours), The amount of high-density CPP can also be directly measured by reading the fluorescence intensity of the fluorescent probe from the obtained precipitate.
また、本方法において、血清とリン酸カルシウム結晶特異的結合性蛍光プローブとを反応後、反応物を、高密度CPPと低密度CPPとを分離できるポアサイズのフィルターに供し、高密度CPPを分離する。分離した高密度CPPから蛍光プローブの蛍光強度を読み取ることで、高密度CPP量を直接測定することができる。 In this method, after reacting serum with a calcium phosphate crystal-specific binding fluorescent probe, the reaction product is subjected to a pore size filter capable of separating high-density CPP and low-density CPP to separate high-density CPP. By reading the fluorescence intensity of the fluorescent probe from the separated high-density CPP, the amount of high-density CPP can be directly measured.
本方法において、被験体由来の血中(血清中)総CPP又は高密度CPP量(濃度)が、健常者の血中(血清中)総CPP又は高密度CPP量(濃度)と比較して有意に(例えば、1.1〜10倍、好ましくは1.5〜10倍)高い場合に、被験体は慢性腎臓病であると判断することができる。 In this method, the blood (serum) total CPP or high-density CPP level (concentration) from the subject is significantly higher than the blood (serum) total CPP or high-density CPP level (concentration) of healthy subjects. (Eg, 1.1 to 10 times, preferably 1.5 to 10 times), the subject can be determined to have chronic kidney disease.
また、本発明は、上述のリン酸カルシウム結晶特異的結合性蛍光プローブを含む、CPP測定又は慢性腎臓病診断キットに関する。当該キットは、当該蛍光プローブ以外に、さらにCPP測定又は慢性腎臓病診断のための取り扱い説明書、容器、試薬等を適宜含むことができる。 The present invention also relates to a CPP measurement or chronic kidney disease diagnosis kit comprising the above-mentioned calcium phosphate crystal-specific binding fluorescent probe. In addition to the fluorescent probe, the kit can further include an instruction manual, a container, a reagent, etc. for CPP measurement or chronic kidney disease diagnosis.
以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
〔実施例１〕血中CPP測定法
１．原理
血中CPP測定法の原理は以下の通りである：
(1) リン酸カルシウム結晶に特異的に結合する近赤外線蛍光プローブOsteosenseを血清に加えてCPPに結合させる；
(2) 次いで、ゲル濾過スピンカラムでFreeのOsteosenseを除く；
(3) CPPに結合したOsteosenseの蛍光を近赤外線スキャナーで定量する。
[Example 1] Blood CPP measurement method Principle The principle of blood CPP measurement is as follows:
(1) A near-infrared fluorescent probe Osteosense that specifically binds to calcium phosphate crystals is added to serum to bind to CPP;
(2) Next, remove the free Osteosense by gel filtration spin column;
(3) The fluorescence of Osteosense bound to CPP is quantified with a near infrared scanner.
２．試薬
血中CPP測定法に使用する試薬は以下の通りである：
(i) Osteosense 680EX(Perkin Elmer #NE10010EX)(Krebs-Ringer-Hepes Bufferで50 μMの濃度に溶解)；
(ii) 500 mM EDTA, pH 8.0；
(iii) 10％ SDS；
(iv) DMEM(Gibco 12100-046)(1 M HEPES(pH 7.4)を終濃度25 mMになるように加える)；
(v) Micro Bio-Spin P-30(Bio-rad 732-6226)；
(vi) 96 well optical bottom plate(Nunc 165305)；
(vii) Hydroxyapatite nanoparticles(Sigma-Aldrich 677418）。
2. Reagents Reagents used for blood CPP measurement are as follows:
(i) Osteosense 680EX (Perkin Elmer # NE10010EX) (dissolved in Krebs-Ringer-Hepes Buffer to a concentration of 50 μM);
(ii) 500 mM EDTA, pH 8.0;
(iii) 10% SDS;
(iv) DMEM (Gibco 12100-046) (add 1 M HEPES (pH 7.4) to a final concentration of 25 mM);
(v) Micro Bio-Spin P-30 (Bio-rad 732-6226);
(vi) 96 well optical bottom plate (Nunc 165305);
(vii) Hydroxyapatite nanoparticles (Sigma-Aldrich 677418).
３．方法
３−１．総CPPの測定
総CPPの測定を、以下の工程により行った：
(1) (i)Osteosense溶液を(iv)DMEMで10倍希釈した；
(2) 上記Osteosense希釈液45 μlに血清5 μlを加えた；
(3) 緩やかに振盪しながら室温(24℃)で1時間反応させた；
(4) この間に、(v)スピンカラムを(iv)DMEMで平衡化しておいた；
(5) 上記反応液30 μlと(iv)DMEM 30 μlを(v)スピンカラムにapplyした；
(6) 1,000 gで2分間遠心し、溶出画分を回収した；
(7) 溶出画分40 μlを(vi)96穴プレートに移し、(ii)500 mM EDTAを10 μl、(iv)DMEMを40 μl、(iii)10％ SDSを10 μl加えて室温で5分間放置し、CPPを溶かした；
(8) 近赤外線スキャナー(LI-COR Odyssey CLx)でOsteosenseの蛍光強度を定量し、血中CPP濃度とした。
3. Method 3-1. Measurement of total CPP Measurement of total CPP was performed by the following steps:
(1) (i) Osteosense solution was diluted (iv) 10 times with DMEM;
(2) 5 μl of serum was added to 45 μl of the above Osteosense diluent;
(3) The reaction was allowed to proceed at room temperature (24 ° C) for 1 hour with gentle shaking;
(4) During this time, (v) the spin column was (iv) equilibrated with DMEM;
(5) 30 μl of the above reaction solution and (iv) 30 μl of DMEM were applied to (v) a spin column;
(6) Centrifuge at 1,000 g for 2 minutes to collect the eluted fraction;
(7) Transfer 40 μl of the eluted fraction to (vi) 96-well plate, add (ii) 10 μl of 500 mM EDTA, (iv) 40 μl of DMEM, and (iii) 10 μl of 10% SDS at room temperature. Left for minutes to dissolve the CPP;
(8) The fluorescence intensity of Osteosense was quantified with a near-infrared scanner (LI-COR Odyssey CLx) to obtain the blood CPP concentration.
３−２．L-CPP(低密度CPP)の測定
L-CPPの測定の工程は、上記総CPPの測定における工程(2)で、血清5 μlの代わりに、血清を16,000 gで2時間遠心した上清5 μlを用いた以外は、当該総CPPの測定の工程と同じであった。
3-2. Measurement of L-CPP (low density CPP)
The L-CPP measurement step was the same as the total CPP measurement step (2) except that 5 μl of the supernatant obtained by centrifuging the serum at 16,000 g for 2 hours was used instead of 5 μl of the serum. It was the same as the measurement process.
３−３．H-CPP(高密度CPP)の測定
上記総CPPの測定による総CPP値から、上記L-CPPの測定によるL-CPP値を引いて算出した。
3-3. Measurement of H-CPP (High Density CPP) It was calculated by subtracting the L-CPP value measured by the L-CPP from the total CPP value measured by the total CPP.
３−４．血中CPP濃度の別の表示方法
血中CPP濃度のもう一つの表示方法として、上記総CPPの測定における工程(8)で得たOsteosenseの蛍光強度に等しい蛍光強度を発する市販のリン酸カルシウム結晶の標準品(vii)Hydroxyapatite(HA)の濃度として表示する方法がある。この場合、以下のように、HAを使った検量線を作成する：
(1) HAを(iv)DMEMで懸濁し、希釈系列(1000, 500, 150, 125, 62.5 μg/ml)を作成する；
(2) 上記総CPPの測定における工程(2)で、血清5 μlの代わりにHA懸濁液5 μlを加える；
(3) 緩やかに振盪しながら室温(24℃)で1時間反応させる；
(4) 16,000 gで2時間遠心する；
(5) 上清40 μlを(vi)96穴プレートに移し、(ii)500 mM EDTAを10 μl、(iv)DMEMを40 μl、(iii)10％ SDSを10 μl加えて室温で5分間放置する；
(6) 近赤外線スキャナー(LI-COR Odyssey CLx)でOsteosenseの蛍光強度を定量して検量線を作成し、HA 1 μg/mlに結合するOsteosense量を算出する；
(7) Osteosenseの蛍光強度で表した血中CPP濃度を、同量のOsteosenseが結合できるHA濃度(μg/ml)に換算する。
3-4. Another method of displaying blood CPP concentration As another method of displaying blood CPP concentration, a standard of commercially available calcium phosphate crystals that emit fluorescence intensity equal to the Osteosense fluorescence intensity obtained in step (8) in the above total CPP measurement. (Vii) There is a method of displaying as the concentration of Hydroxyapatite (HA). In this case, create a calibration curve using HA as follows:
(1) Suspend HA in (iv) DMEM to create a dilution series (1000, 500, 150, 125, 62.5 μg / ml);
(2) In step (2) in the above total CPP measurement, 5 μl of HA suspension is added instead of 5 μl of serum;
(3) React for 1 hour at room temperature (24 ° C) with gentle shaking;
(4) Centrifuge for 2 hours at 16,000 g;
(5) Transfer 40 μl of supernatant to (vi) 96-well plate, add (ii) 10 μl of 500 mM EDTA, (iv) 40 μl of DMEM, (iii) 10 μl of 10% SDS for 5 minutes at room temperature put;
(6) Quantify the fluorescence intensity of Osteosense with a near-infrared scanner (LI-COR Odyssey CLx) to create a calibration curve, and calculate the amount of Osteosense bound to HA 1 μg / ml;
(7) The blood CPP concentration represented by the fluorescence intensity of Osteosense is converted to the HA concentration (μg / ml) that can be bound by the same amount of Osteosense.
〔実施例２〕透析患者(慢性腎臓病患者)と健常者由来の血清におけるCPPの測定
透析患者5例と健常者4例の血清を用いて、実施例１に記載の方法で総CPP、L-CPP及びH-CPPを測定した。
[Example 2] Measurement of CPP in serum from dialysis patients (chronic kidney disease patients) and healthy subjects Using the sera of 5 dialysis patients and 4 healthy subjects, total CPP, L -CPP and H-CPP were measured.
結果を図１〜３に示す。
図１は、透析患者(実線)と健常者(破線)の血清における総CPPの測定結果を示すグラフである。
The results are shown in FIGS.
FIG. 1 is a graph showing the measurement results of total CPP in sera of dialysis patients (solid line) and healthy subjects (broken line).
図２は、透析患者(実線)と健常者(破線)の血清におけるL-CPPの測定結果を示すグラフである。 FIG. 2 is a graph showing the measurement results of L-CPP in the sera of dialysis patients (solid line) and healthy persons (broken line).
図３は、透析患者(黒塗り棒グラフ)と健常者(白抜き棒グラフ)の血清におけるH-CPPの測定結果を示すグラフである。 FIG. 3 is a graph showing the measurement results of H-CPP in the sera of dialysis patients (black bar graph) and healthy subjects (white bar graph).
本実施例では、上記総CPPの測定における工程(6)の後に300 μlの(iv)DMEMで7回分画し、CPPに結合しなかった過剰のOsteosenseが溶出するところまで確認した。最初の画分(図１及び２における矢印)がCPPに相当した。 In this example, after step (6) in the measurement of the total CPP, fractionation was performed 7 times with 300 μl of (iv) DMEM, and it was confirmed that excess Osteosense that did not bind to CPP was eluted. The first fraction (arrow in FIGS. 1 and 2) corresponded to CPP.
図１に示すように、透析患者の血中総CPP値は、健常者よりも高値であることが分かった。 As shown in FIG. 1, it was found that the blood total CPP value of the dialysis patient was higher than that of the healthy subject.
同じ症例でL-CPPを測定した。図２に示すように、L-CPP値は透析患者と健常者で差がなかった。 L-CPP was measured in the same case. As shown in FIG. 2, there was no difference in L-CPP values between dialysis patients and healthy subjects.
総CPP値からL-CPP値を引いてH-CPPを算出した。図３に示すように、透析患者では主にH-CPPが上昇していることが分かった。 H-CPP was calculated by subtracting L-CPP value from total CPP value. As shown in FIG. 3, it was found that H-CPP mainly increased in dialysis patients.
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