JPH10337198A - Assay of osteoclast-derived acid phosphatase - Google Patents
Assay of osteoclast-derived acid phosphataseInfo
- Publication number
- JPH10337198A JPH10337198A JP14961597A JP14961597A JPH10337198A JP H10337198 A JPH10337198 A JP H10337198A JP 14961597 A JP14961597 A JP 14961597A JP 14961597 A JP14961597 A JP 14961597A JP H10337198 A JPH10337198 A JP H10337198A
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- Japan
- Prior art keywords
- acid phosphatase
- derived
- osteoclast
- activity
- substrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
Abstract
Description
【０００１】[0001]
【発明が属する技術分野】本発明は、破骨細胞由来酸性
ホスファターゼ活性の測定方法およびそれに使用するキ
ットに関する。本発明によれば簡便で特異的に破骨細胞
由来酸性ホスファターゼ活性の測定が可能であり、臨床
検査測定用の方法として医学的治療や臨床検査の分野に
おいて極めて有効である。TECHNICAL FIELD [0001] The present invention relates to a method for measuring osteoclast-derived acid phosphatase activity and a kit used therefor. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to simply and specifically measure osteoclast-derived acid phosphatase activity, and it is extremely effective in the field of medical treatment and clinical tests as a method for measuring clinical tests.
【０００２】[0002]
【従来の技術】酸性ホスファターゼは、至適ｐＨが酸性
で有機モノリン酸エステルを加水分解する酵素であり、
前立腺由来酸性ホスファターゼ、破骨細胞由来酸性ホス
ファターゼ、赤血球由来酸性ホスファターゼおよび血小
板由来酸性ホスファターゼなどの種々の由来のものがあ
る。酒石酸の添加によってもその酵素活性が阻害されな
い血清中の酒石酸抵抗性酸性ホスファターゼは、その大
部分が破骨細胞由来酸性ホスファターゼとされ、その測
定は、破骨細胞の機能を評価する指標として有用とされ
ており、骨吸収マーカーとして興味が持たれている（骨
代謝マーカー，福永仁夫，中村利孝，松本俊夫編，メデ
ィカルレビュー社，１９９５）。2. Description of the Related Art Acid phosphatase is an enzyme having an acidic pH and hydrolyzing an organic monophosphate.
There are various sources such as prostate-derived acid phosphatase, osteoclast-derived acid phosphatase, erythrocyte-derived acid phosphatase, and platelet-derived acid phosphatase. Tartrate-resistant acid phosphatase in serum, whose enzyme activity is not inhibited even by the addition of tartaric acid, is predominantly osteoclast-derived acid phosphatase, and its measurement is useful as an index for evaluating the function of osteoclasts. It is of interest as a bone resorption marker (bone metabolism marker, edited by Yoshio Fukunaga, Toshitaka Nakamura, Toshio Matsumoto, Medical Review, 1995).
【０００３】一般的に酵素活性測定法による検体中の酒
石酸抵抗性酸性ホスファターゼの測定は、酒石酸の存在
下で合成基質としてリン酸エステルを用いて酵素反応に
より生ずる反応生成物（アルコールやフェノール類）を
比色定量することにより酵素活性を求めている。その
際、酒石酸は検体中に主として共存する前立腺由来酸性
ホスファターゼの酵素活性を阻害し、従って残存した酸
性ホスファターゼ活性を基質を用いて測定することによ
り、破骨細胞由来酸性ホスファターゼ活性の指標として
酒石酸抵抗性酸性ホスファターゼ活性を求めている。さ
らに、より特異的な破骨細胞由来酸性ホスファターゼ活
性測定法の改善法としては、血清を５倍に希釈した液を
３７℃で１時間インキュベートする前処理をした後、残
りの酒石酸抵抗性酸性ホスファターゼ活性を、酒石酸存
在下、基質としてｐ−ニトロフェニルリン酸を用いて測
定する方法が知られている（日大医誌，４９巻，９０４
−９１１頁（１９９０）；クリニカル・ケミストリー，
３３巻，４５８−４６２頁（１９８７））。一方、免疫
学的測定法による酒石酸抵抗性酸性ホスファターゼの測
定方法として、モノクローナル抗体を用いた酵素免疫測
定法（ＥＩＡ法）による測定方法が知られている（臨床
検査，３７巻，３７３−３７７頁（１９９３））。In general, the measurement of tartaric acid-resistant acid phosphatase in a sample by an enzyme activity measurement method is performed by using a phosphate ester as a synthetic substrate in the presence of tartaric acid to produce a reaction product (alcohol or phenol) produced by an enzymatic reaction. The enzyme activity is determined by colorimetric determination of At that time, tartaric acid inhibits the enzymatic activity of prostate-derived acid phosphatase, which mainly coexists in the sample, and therefore, by measuring the remaining acid phosphatase activity using a substrate, tartaric acid resistance is used as an indicator of osteoclast-derived acid phosphatase activity. Seeking acid phosphatase activity. Further, as a more specific method for improving the method for measuring osteoclast-derived acid phosphatase activity, a pretreatment of incubating a 5-fold dilution of serum at 37 ° C. for 1 hour, followed by the remaining tartrate-resistant acid phosphatase was performed. A method of measuring the activity using p-nitrophenyl phosphate as a substrate in the presence of tartaric acid is known (Nihon University Medical Journal, Vol. 49, 904).
-911 (1990); Clinical chemistry,
33, 458-462 (1987)). On the other hand, as a method for measuring tartrate-resistant acid phosphatase by an immunoassay, a measurement method by an enzyme immunoassay (EIA method) using a monoclonal antibody is known (Clinical Inspection, Vol. 37, pp. 373-377). (1993)).
【０００４】[0004]
【発明が解決しようとする課題】破骨細胞由来酸性ホス
ファターゼの指標として酒石酸抵抗性酸性ホスファター
ゼ活性を求める従来の活性測定法は、特異性、感度、測
定の煩雑さ及び測定時間の点で問題を有している。検体
中には、酒石酸抵抗性酸性ホスファターゼとして、破骨
細胞由来酸性ホスファターゼ以外に赤血球由来酸性ホス
ファターゼや血小板由来酸性ホスファターゼが存在す
る。すなわち、検体の採取により溶血が生じたとき、赤
血球由来酸性ホスファターゼは検体中に含まれてくる
し、検体として血清を用いる場合、血清製造の際の血液
凝固の過程で血小板が破壊されて血小板由来酸性ホスフ
ァターゼが検体中に含まれてくる。そのため、従来の酒
石酸抵抗性酸性ホスファターゼ活性測定法は、特異的に
破骨細胞由来酸性ホスファターゼ活性を測定していると
は言えない。The conventional method for measuring tartrate-resistant acid phosphatase activity as an index of osteoclast-derived acid phosphatase has problems in terms of specificity, sensitivity, complexity of measurement, and measurement time. Have. In the specimen, red blood cell-derived acid phosphatase and platelet-derived acid phosphatase other than osteoclast-derived acid phosphatase exist as tartrate-resistant acid phosphatase. That is, red blood cell-derived acid phosphatase is included in the sample when hemolysis occurs due to collection of the sample, and when serum is used as the sample, platelets are destroyed during the blood coagulation process during serum production and platelet-derived acid phosphatase is generated. Phosphatase is included in the sample. Therefore, the conventional method for measuring tartrate-resistant acid phosphatase activity cannot be said to specifically measure osteoclast-derived acid phosphatase activity.
【０００５】さらに、前記した日大医誌，４９巻，９０
４−９１１頁（１９９０）記載の改善法でも、血小板由
来酸性ホスファターゼによる影響を除くことはできな
い。また、この改善法では、基質としてｐ−ニトロフェ
ニルリン酸を用いているが、その反応生成物であるｐ−
ニトロフェノールは、酸性ホスファターゼの反応の至適
ｐＨである酸性域では発色しないため、アルカリを加え
反応を停止し発色させる必要がある。そのため、この改
善法では、レートアッセイが不可能なため、現代の臨床
検査の主流である自動分析装置での測定に適応できな
い。また、この改善法では、感度が十分でないため、酵
素反応の反応時間も３０分と長時間必要である。一方、
免疫学的測定法による酒石酸抵抗性酸性ホスファターゼ
の測定法は、モノクローナル抗体を使用するためさらに
特異性には優れている。しかしながら、操作が煩雑であ
り、抗原抗体反応は酵素反応に比べさらに長時間を必要
とするため、測定に時間を必要とする。測定するための
試薬も高価でありコスト面でも問題を有する。本発明
は、かかる問題に鑑み、簡便で特異的な破骨細胞由来酸
性ホスファターゼの測定方法及びそれに使用するキット
を提供することを目的とする。Further, the aforementioned Nihon University Medical Journal, Vol.
Even the improvement method described on page 4-911 (1990) cannot eliminate the effects of platelet-derived acid phosphatase. Further, in this improved method, p-nitrophenyl phosphate is used as a substrate, and the reaction product p-nitrophenyl phosphate is used.
Nitrophenol does not develop color in the acidic range, which is the optimum pH for the acid phosphatase reaction, so it is necessary to stop the reaction by adding an alkali to develop color. Therefore, this improved method cannot be applied to the measurement using an automatic analyzer, which is the mainstream of modern clinical tests, because a rate assay is impossible. Further, in this improved method, since the sensitivity is not sufficient, the reaction time of the enzymatic reaction requires a long time of 30 minutes. on the other hand,
The method for measuring tartrate-resistant acid phosphatase by an immunoassay is more excellent in specificity because a monoclonal antibody is used. However, the operation is complicated, and the antigen-antibody reaction requires a longer time than the enzyme reaction, so that the measurement requires time. Reagents for measurement are also expensive and have a problem in cost. In view of the above problems, an object of the present invention is to provide a simple and specific method for measuring osteoclast-derived acid phosphatase and a kit used for the method.
【０００６】[0006]
【課題を解決するための手段】本発明は、ｉ）検体中の
酸性ホスファターゼに、酒石酸の存在下で酸性ホスファ
ターゼ用基質を作用させて酸性ホスファターゼ活性を測
定し（以下、測定１と記載することもある）、それとは
別にii）検体中の酸性ホスファターゼに、赤血球および
血小板由来酸性ホスファターゼを阻害しない破骨細胞由
来酸性ホスファターゼ阻害剤と酒石酸との存在下で酸性
ホスファターゼ用基質を作用させて酸性ホスファターゼ
活性を測定し（以下、測定２と記載することもある）、
次いで、iii)上記ｉ）の活性から上記ii）の活性を差し
引くことにより測定することを特徴とする破骨細胞由来
酸性ホスファターゼ活性の測定方法である。更に本発明
は、ｉ）酒石酸を含む試薬、ii）赤血球および血小板由
来酸性ホスファターゼを阻害しない破骨細胞由来酸性ホ
スファターゼ阻害剤と酒石酸とを含む試薬、及びiii)酸
性ホスファターゼ用基質を含む試薬を必須構成試薬とす
る破骨細胞由来酸性ホスファターゼ活性測定用キットで
ある。According to the present invention, i) an acid phosphatase in a sample is allowed to act on a substrate for acid phosphatase in the presence of tartaric acid to measure acid phosphatase activity (hereinafter referred to as measurement 1). Ii) In addition, ii) acid phosphatase in a sample is allowed to act on an acid phosphatase substrate in the presence of an osteoclast-derived acid phosphatase inhibitor that does not inhibit erythrocyte and platelet-derived acid phosphatase and tartaric acid. Activity was measured (hereinafter, sometimes referred to as measurement 2),
Next, iii) a method for measuring osteoclast-derived acid phosphatase activity, wherein the activity is measured by subtracting the activity of ii) from the activity of i). Further, the present invention essentially requires i) a reagent containing tartaric acid, ii) a reagent containing an osteoclast-derived acid phosphatase inhibitor that does not inhibit erythrocyte and platelet-derived acid phosphatase, and tartaric acid, and iii) a reagent containing a substrate for acid phosphatase. This is a kit for measuring osteoclast-derived acid phosphatase activity as a constituent reagent.
【０００７】[0007]
【発明の実施の形態】本発明で対象とする検体は、ヒト
の血液、血清、血漿などが代表的なものである。本発明
の測定方法における測定１の酒石酸存在下では、検体中
の前立腺由来酸性ホスファターゼ活性は阻害されるが、
破骨細胞由来酸性ホスファターゼ、赤血球由来酸性ホス
ファターゼおよび血小板由来酸性ホスファターゼは阻害
されず、従ってその阻害されない３種類の酸性ホスファ
ターゼ活性の和が主な測定１での測定値となると考えら
れる。測定２では、酒石酸に加え、赤血球および血小板
由来酸性ホスファターゼを阻害しない破骨細胞由来酸性
ホスファターゼ阻害剤も存在するので、前立腺由来酸性
ホスファターゼに加え、破骨細胞由来酸性ホスファター
ゼも阻害されるが、赤血球由来酸性ホスファターゼおよ
び血小板由来酸性ホスファターゼは阻害されず、従って
その阻害されない２種類の酸性ホスファターゼ活性の和
が主な測定２での測定値となると考えられる。したがっ
て、測定１での測定値から測定２での測定値を差し引く
ことにより、破骨細胞由来酸性ホスファターゼ活性を正
確に求めることができると考えられる。BEST MODE FOR CARRYING OUT THE INVENTION The specimens targeted in the present invention are typically human blood, serum, plasma and the like. In the presence of tartaric acid in Measurement 1 in the measurement method of the present invention, the activity of prostate-derived acid phosphatase in the sample is inhibited,
The osteoclast-derived acid phosphatase, the erythrocyte-derived acid phosphatase and the platelet-derived acid phosphatase are not inhibited, and thus it is considered that the sum of the three non-inhibited acid phosphatase activities is the measurement value in the main measurement 1. In measurement 2, since there is also an osteoclast-derived acid phosphatase inhibitor that does not inhibit erythrocyte and platelet-derived acid phosphatase in addition to tartaric acid, in addition to prostate-derived acid phosphatase, osteoclast-derived acid phosphatase is also inhibited. It is considered that the acid phosphatase derived from platelets and the acid phosphatase derived from platelets are not inhibited, and thus the sum of the activities of the two non-inhibited acid phosphatases is the main measurement in measurement 2. Therefore, it is considered that the osteoclast-derived acid phosphatase activity can be accurately determined by subtracting the measurement value of measurement 2 from the measurement value of measurement 1.
【０００８】本発明で用いられる赤血球および血小板由
来酸性ホスファターゼを阻害しない破骨細胞由来酸性ホ
スファターゼ阻害剤（以下、単に阻害剤と記載すること
もある）としては、赤血球および血小板由来酸性ホスフ
ァターゼを阻害せずかつ破骨細胞由来酸性ホスファター
ゼを阻害するものであれば特に限定されない。そのよう
な阻害剤として、例えばフッ化ナトリウム、フッ化カリ
ウム等のフッ化塩を例示できる。本発明では、酸性ホス
ファターゼに合成基質を作用させるとき、その阻害剤の
濃度は、好ましくは５〜５００ｍＭ、さらに好ましくは
２０から２００ｍＭである。本発明で用いられる酒石酸
としては、Ｌ（＋）−酒石酸、またはそのナトリウム塩
またはカリウム塩等の塩が好ましい。本発明では、酒石
酸の存在下で酸性ホスファターゼに基質を作用させると
き、その酒石酸の濃度は、好ましくは５〜５００ｍＭ、
さらに好ましくは２０〜２００ｍＭである。[0008] The osteoclast-derived acid phosphatase inhibitor which does not inhibit erythrocyte- and platelet-derived acid phosphatase used in the present invention (hereinafter sometimes simply referred to as an inhibitor) includes those which inhibit erythrocyte- and platelet-derived acid phosphatase. It is not particularly limited as long as it inhibits osteoclast-derived acid phosphatase. Examples of such inhibitors include fluoride salts such as sodium fluoride and potassium fluoride. In the present invention, when a synthetic substrate is allowed to act on acid phosphatase, the concentration of the inhibitor is preferably 5 to 500 mM, more preferably 20 to 200 mM. As the tartaric acid used in the present invention, L (+)-tartaric acid or a salt thereof such as a sodium salt or a potassium salt is preferable. In the present invention, when the substrate is allowed to act on acid phosphatase in the presence of tartaric acid, the concentration of tartaric acid is preferably 5 to 500 mM,
More preferably, it is 20 to 200 mM.
【０００９】本発明に用いられる酸性ホスファターゼ用
基質は、酸性ホスファターゼ、特に、破骨細胞由来酸性
ホスファターゼと反応し、酸性ホスファターゼ活性を測
定できるものであればいずれでもかまわないが、レート
アッセイ可能な基質が好ましい。レートアッセイ可能な
基質として、α−ナフチルリン酸およびその塩、２，６
−ジクロロ−４−ニトロフェニルリン酸およびその塩、
２−クロロ−４−ニトロフェニルリン酸およびその塩、
２，６−ジクロロ−４−アセチルフェニルリン酸および
その塩を例示できる。そのなかでも、２，６−ジクロロ
−４−アセチルフェニルリン酸およびその塩が、感度が
高く測定波長３４０ｎｍで測定できる点から好ましい。
反応時の基質濃度は、各基質の酸性ホスファターゼに対
する親和性により異なるが、酸性ホスファターゼに対す
るＫｍの５〜１０倍が好ましい。本発明において、検体
中の酸性ホスファターゼに基質を作用させて酸性ホスフ
ァターゼ活性を測定するときは、生成するアルコールや
フェノールの生成量を分光光度計や自動分析装置を用
い、適当な波長の吸光度変化を測定することにより目的
の活性を測定できる。この場合、レートアッセイで酸性
ホスファターゼ活性を測定するときは、以下の式を利用
して求めることができる。The substrate for acid phosphatase used in the present invention may be any substrate as long as it reacts with acid phosphatase, particularly acid phosphatase derived from osteoclast, and can measure acid phosphatase activity. Is preferred. As substrates capable of rate assay, α-naphthyl phosphate and salts thereof, 2,6
-Dichloro-4-nitrophenylphosphoric acid and salts thereof,
2-chloro-4-nitrophenylphosphoric acid and salts thereof,
2,6-dichloro-4-acetylphenylphosphoric acid and salts thereof can be exemplified. Among them, 2,6-dichloro-4-acetylphenylphosphoric acid and a salt thereof are preferable because they have high sensitivity and can be measured at a measurement wavelength of 340 nm.
The substrate concentration during the reaction depends on the affinity of each substrate for acid phosphatase, but is preferably 5 to 10 times the Km for acid phosphatase. In the present invention, when measuring the acid phosphatase activity by allowing a substrate to act on acid phosphatase in a sample, the amount of alcohol or phenol produced is measured using a spectrophotometer or an automatic analyzer to determine the change in absorbance at an appropriate wavelength. By measuring, the desired activity can be measured. In this case, when measuring the acid phosphatase activity by the rate assay, it can be obtained by using the following formula.
【００１０】[0010]
【式１】活性＝［（酵素反応中の単位時間当たりの吸光
度変化量−ブランクのときの吸光度変化量）×酵素反応
時の液量］÷［検体量×生成物の吸光分子係数×セル光
路長］ （ただし、「ブランクのときの吸光度変化量」とは、検
体の代わりに生理食塩水または精製水を用いて「酵素反
応中の単位時間当たりの吸光度変化量」と同様な操作を
して求めた値を示す）[Formula 1] Activity = [(Absorbance change per unit time during enzyme reaction-absorbance change in blank) × Liquid volume during enzyme reaction] 検 体 [Sample volume × Absorbent molecular coefficient of product × Cell optical path Length] (However, the “absorbance change in blank” means the same operation as “absorbance change per unit time during enzyme reaction” using physiological saline or purified water instead of sample. Indicates the calculated value)
【００１１】自動分析装置を用いたレートアッセイで
は、約１０分間で測定が終了可能である。本発明では、
酵素反応時の反応液量は、用いた検体量の５倍以上が好
ましく、８〜１００倍がさらに好ましく、１０〜５０倍
が特に好ましい。酵素反応時の反応液量が検体量の５倍
を越えないと、検体中の妨害物質の影響のため破骨細胞
由来酸性ホスファターゼ活性を正確に測定できにくく、
その反応液量が多すぎると測定感度が低くなるため破骨
細胞由来酸性ホスファターゼ活性を測定できない場合が
ある。また、酵素反応の際には、必要に応じて、トリト
ンＸ−１００等の界面活性剤、アルコール類、塩化ナト
リウムなどの塩類、アルブミン等の蛋白質を含んでも構
わない。また、酵素反応の際には、ｐＨ４〜７の酸性域
が好ましく、ｐＨ５．４〜６．８がさらに好ましい。そ
のための緩衝物質として、クエン酸及びその塩、酢酸及
びその塩、ＭＥＳ、Ｂｉｓ−Ｔｒｉｓなどを用いてもよ
い。In a rate assay using an automatic analyzer, measurement can be completed in about 10 minutes. In the present invention,
The amount of the reaction solution at the time of the enzyme reaction is preferably 5 times or more, more preferably 8 to 100 times, particularly preferably 10 to 50 times the amount of the sample used. If the volume of the reaction solution during the enzyme reaction does not exceed 5 times the sample volume, it is difficult to accurately measure osteoclast-derived acid phosphatase activity due to the effects of interfering substances in the sample,
If the amount of the reaction solution is too large, the measurement sensitivity may be low, so that osteoclast-derived acid phosphatase activity may not be measured. In addition, at the time of the enzymatic reaction, a surfactant such as Triton X-100, an alcohol, a salt such as sodium chloride, or a protein such as albumin may be contained as necessary. In the case of an enzymatic reaction, an acidic range of pH 4 to 7 is preferable, and pH 5.4 to 6.8 is more preferable. As a buffer substance therefor, citric acid and its salts, acetic acid and its salts, MES, Bis-Tris and the like may be used.
【００１２】本発明の破骨細胞由来酸性ホスファターゼ
活性の測定方法では、例えば、 ｉ）酒石酸を含む試薬（酒石酸試薬）、 ii) 赤血球および血小板由来酸性ホスファターゼを阻害
しない破骨細胞由来酸性ホスファターゼ阻害剤と酒石酸
とを含む試薬（阻害剤・酒石酸試薬）、及び iii)酸性ホスファターゼ用基質を含む試薬（基質試薬）
を必須構成試薬とする破骨細胞由来酸性ホスファターゼ
活性測定用キットを用いることができる。このキットは
２試薬系の自動分析装置に適用できる。これらの試薬に
は、必要に応じて、トリトンＸ−１００等の界面活性
剤、アルコール類、塩化ナトリウムなどの塩類、アルブ
ミン等の蛋白質を含んでいても構わない。また、緩衝物
質としてクエン酸及びその塩、酢酸及びその塩、ＭＥ
Ｓ、Ｂｉｓ−Ｔｒｉｓなどを含んでいてもよい。In the method for measuring the activity of acid phosphatase derived from osteoclasts of the present invention, for example, i) a reagent containing tartaric acid (tartaric acid reagent); ii) an osteoclast derived acid phosphatase inhibitor which does not inhibit erythrocyte and platelet derived acid phosphatase Reagent containing thiophene and tartaric acid (inhibitor / tartaric acid reagent), and iii) reagent containing a substrate for acid phosphatase (substrate reagent)
A kit for measuring the activity of osteoclast-derived acid phosphatase, which contains as an essential constituent reagent, can be used. This kit can be applied to a two-reagent automatic analyzer. These reagents may contain surfactants such as Triton X-100, alcohols, salts such as sodium chloride, and proteins such as albumin, if necessary. Also, citric acid and its salts, acetic acid and its salts, ME
S, Bis-Tris, etc. may be included.
【００１３】このキットでは、まず、検体と酒石酸試薬
とを混合することにより前立腺由来酸性ホスファターゼ
を阻害し、次いで、その混合液に基質試薬を添加して残
存の酸性ホスファターゼ活性を測定する（測定１）。そ
れとは別に、検体と阻害剤・酒石酸試薬とを混合するこ
とにより、前立腺由来酸性ホスファターゼと破骨細胞由
来酸性ホスファターゼとを阻害し、次いで、その混合液
に基質試薬を添加して残存の酸性ホスファターゼ活性を
測定する（測定２）。得られた測定１での測定値から測
定２で測定値を差し引くことにより、検体中の破骨細胞
由来酸性ホスファターゼ活性を測定することができる。
測定２で用いる阻害剤・酒石酸試薬の液量は、測定１に
用いる酒石酸試薬と同量用いることが、計算上、好まし
い。また、測定の際に用いる酒石酸試薬と基質試薬との
和は、それらの試薬と検体と併せた時、検体量の好まし
くは５倍以上、さらに好ましくは８〜１００倍、特に好
ましくは１０〜５０倍である。上記のキットを用いる
と、本発明の方法を、自動分析装置にも適用でき、簡便
に短時間で数多くの検体の破骨細胞由来酸性ホスファタ
ーゼ活性を測定できる。In this kit, first, a prostate-derived acid phosphatase is inhibited by mixing a specimen with a tartaric acid reagent, and then a substrate reagent is added to the mixture to measure the remaining acid phosphatase activity (measurement 1). ). Separately, prostate-derived acid phosphatase and osteoclast-derived acid phosphatase are inhibited by mixing a specimen with an inhibitor / tartaric acid reagent, and then a substrate reagent is added to the mixture to remove residual acid phosphatase. The activity is measured (measurement 2). The osteoclast-derived acid phosphatase activity in the sample can be measured by subtracting the measured value in Measurement 2 from the measured value in Measurement 1 obtained.
The amount of the inhibitor / tartaric acid reagent used in Measurement 2 is preferably calculated to be the same as that of the tartaric acid reagent used in Measurement 1. The sum of the tartaric acid reagent and the substrate reagent used in the measurement is preferably 5 times or more, more preferably 8 to 100 times, particularly preferably 10 to 50 times the amount of the sample when the reagent and the sample are combined. It is twice. By using the above kit, the method of the present invention can be applied to an automatic analyzer, and the osteoclast-derived acid phosphatase activity of a large number of samples can be measured simply and in a short time.
【００１４】上記のキットの他にも、例えば、ｉ）酒石
酸と酸性ホスファターゼ用基質とを含む試薬、及びii)
赤血球および血小板由来酸性ホスファターゼを阻害しな
い破骨細胞由来酸性ホスファターゼ阻害剤と酒石酸と酸
性ホスファターゼ用基質とを含む試薬を必須構成試薬と
する破骨細胞由来酸性ホスファターゼ活性測定用キット
を用いても本発明の破骨細胞由来酸性ホスファターゼ活
性の測定方法を実施できる。検体と上記試薬ｉ）とを混
合して、測定１の方法で、破骨細胞由来酸性ホスファタ
ーゼ、赤血球由来酸性ホスファターゼおよび血小板由来
酸性ホスファターゼからなる総酒石酸抵抗性酸性ホスフ
ァターゼ活性を求めることができる。また、検体と上記
試薬ii）とを混合して測定２の方法で赤血球由来酸性ホ
スファターゼおよび血小板由来酸性ホスファターゼから
なる酸性ホスファターゼ活性を求めることができる。得
られた測定１のデータから測定２のデータを差し引くこ
とにより、検体中の破骨細胞由来酸性ホスファターゼ活
性を測定することができる。In addition to the above kit, for example, i) a reagent containing tartaric acid and a substrate for acid phosphatase; and ii)
The present invention is also applicable to a kit for measuring osteoclast-derived acid phosphatase activity, which comprises a reagent containing an osteoclast-derived acid phosphatase inhibitor that does not inhibit erythrocyte and platelet-derived acid phosphatase, tartaric acid, and a substrate for acid phosphatase as essential components. For measuring the activity of acid phosphatase derived from osteoclasts. By mixing the sample and the above reagent i), the total tartrate-resistant acid phosphatase activity consisting of osteoclast-derived acid phosphatase, erythrocyte-derived acid phosphatase and platelet-derived acid phosphatase can be determined by the method of Measurement 1. Further, by mixing the sample and the above reagent ii), the acid phosphatase activity composed of red blood cell-derived acid phosphatase and platelet-derived acid phosphatase can be determined by the method of Measurement 2. The osteoclast-derived acid phosphatase activity in the sample can be measured by subtracting the measurement 2 data from the obtained measurement 1 data.
【００１５】[0015]
【実施例】以下、本発明を実施例により詳細に説明する
が、本発明の要旨を越えない限り、本発明は以下の実施
例に限定されるものではない。 実施例１破骨細胞由来酸性ホスファターゼ活性測定法 １）試薬組成 （１）総酒石酸抵抗性酸性ホスファターゼ測定用緩衝液
（酒石酸試薬） １５０ｍＭ ＭＥＳ緩衝液 ｐＨ６．２（２５℃） ６０ｍＭ Ｌ（＋）−酒石酸ナトリウム ０．５％ 牛アルブミン ０．１％ トリトン Ｘ−１００ （２）酒石酸抵抗性フッ化ナトリウム抵抗性酸性ホスフ
ァターゼ測定用緩衝液（阻害剤・酒石酸試薬） １５０ｍＭ ＭＥＳ緩衝液 ｐＨ６．２（２５℃） ６０ｍＭ Ｌ（＋）−酒石酸ナトリウム ５０ｍＭ フッ化ナトリウム ０．５％ 牛アルブミン ０．１％ トリトン Ｘ−１００ （３）基質液（基質試薬） ４５ｍＭ ＤＣＡＰ−Ｐ（２，６−ジクロロ−４−ア
セチルフェニルリン酸） ５０ｍＭ トリスEXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the present invention. Example 1 Osteoclast-derived acid phosphatase activity measuring method 1) Reagent composition (1) Buffer for measuring total tartrate-resistant acid phosphatase (tartaric acid reagent) 150 mM MES buffer pH 6.2 (25 ° C) 60 mM L (+)- Sodium tartrate 0.5% Bovine albumin 0.1% Triton X-100 (2) Buffer for measuring tartrate-resistant sodium fluoride-resistant acid phosphatase (inhibitor / tartaric acid reagent) 150 mM MES buffer pH 6.2 (25 ° C.) ) 60 mM L (+)-sodium tartrate 50 mM sodium fluoride 0.5% bovine albumin 0.1% triton X-100 (3) Substrate solution (substrate reagent) 45 mM DCAP-P (2,6-dichloro-4-acetyl) Phenyl phosphate) 50 mM Tris
【００１６】（４）試料の調整 ａ．牛骨粗精製液（破骨細胞由来酸性ホスファターゼ含
有液） 牛脛骨頭部を約２ｃｍ角に砕き、液体窒素存在下ブレン
ダーにて粗い粉末とする。これに１．５倍量の蛋白分解
酵素阻害剤を含む３Ｍ ＫＣｌ、１％トリトンＸ−１０
０溶液を混和し、ポリトロンホモジナイザーにて懸濁液
とし、２０００ｒｐｍ、２０分遠心し、上清を採取し、
フレオン処理により脂肪分を除き、０．４５μｍのフィ
ルターを通し、主として破骨細胞由来酸性ホスファター
ゼを含む、牛骨粗精製液として使用した。 ｂ．赤血球溶液（赤血球由来酸性ホスファターゼ溶液） ヘパリン血を１０００ｒｐｍ、１０分遠心後の沈殿赤血
球を生理食塩水にて洗浄後 ０．１％トリトンＸ−１０
０を含む生理食塩水にて溶解したものを、主として赤血
球由来酸性ホスファターゼを含む赤血球溶液として使用
した。 ｃ．血小板溶液（血小板由来酸性ホスファターゼ溶液） ヘパリン血を１０００ｒｐｍ、１０分遠心後の上清をさ
らに３０００ｒｐｍ、１０分遠心し、沈殿物を生理食塩
水にて洗浄後、０．１％トリトンＸ−１００を含む生理
食塩水にて溶解したものを、主として血小板由来酸性ホ
スファターゼを含む血小板溶液として使用した。(4) Sample preparation a. Bovine bone crude purified solution (solution containing osteoclast-derived acid phosphatase) The head of a bovine tibia is crushed into approximately 2 cm squares, and a coarse powder is prepared using a blender in the presence of liquid nitrogen. 3M KCl, 1.5% of protease inhibitor, 1% Triton X-10
0 solution was mixed and made into a suspension with a Polytron homogenizer, centrifuged at 2,000 rpm for 20 minutes, and the supernatant was collected.
Fat was removed by Freon treatment, and the solution was passed through a 0.45 μm filter and used as a crude purified bovine bone solution containing mainly acid phosphatase derived from osteoclasts. b. Erythrocyte solution (erythrocyte-derived acid phosphatase solution) Heparin blood is centrifuged at 1000 rpm for 10 minutes, and the precipitated erythrocytes are washed with physiological saline and then 0.1% Triton X-10.
The solution dissolved in physiological saline containing 0 was mainly used as an erythrocyte solution containing erythrocyte-derived acid phosphatase. c. Platelet solution (platelet-derived acid phosphatase solution) The supernatant after centrifugation of heparin blood at 1000 rpm for 10 minutes is further centrifuged at 3000 rpm for 10 minutes, and the precipitate is washed with physiological saline, and 0.1% Triton X-100 is added. The solution dissolved in a physiological saline solution was used as a platelet solution mainly containing platelet-derived acid phosphatase.
【００１７】２）操作方法（汎用の自動分析装置、コバ
スTMファラ（バクスター社）での例） （１）または（２）の緩衝液１５０μｌをキュベットに
添加し、２０秒後に（４）の試薬を１５０μｌ加える。
６０秒間３７℃でインキュベーション後、（３）基質液
を６０μｌ加えて反応液量が試料の１５倍量（＝（１５
０μｌ＋１５μｌ＋６０μｌ）／１５μｌ）で酵素反応
を開始させ、その６０秒後から１８０秒間、３４０ｎｍ
での吸光度変化を測定する。そしてその吸光度変化の直
線部分から１分間あたりの吸光度変化量（酵素反応中の
単位時間当たりの吸光度変化量）を求める。ブランクの
時の吸光度変化は検体の代わりに生理食塩水または精製
水を加え、上記と同様の操作を行って１分間あたりの吸
光度変化量を求める。 ３）計算式 上記操作によって得られた吸光度変化量を下記式に代入
することによって酸性ホスファターゼ活性（Ｕ／ｌ）を
求めることができる。2) Operation method (example of a general-purpose automatic analyzer, Cobas ™ Fara (Baxter)) Add 150 μl of the buffer (1) or (2) to a cuvette, and after 20 seconds, add the reagent of (4) Add 150 μl.
After incubation at 37 ° C. for 60 seconds, (3) 60 μl of the substrate solution was added, and the reaction solution volume was 15 times the sample volume (= (15
0 μl + 15 μl + 60 μl) / 15 μl) to start the enzymatic reaction, 60 seconds after that, 180 seconds at 340 nm
The change in absorbance at is measured. Then, the amount of change in absorbance per minute (the amount of change in absorbance per unit time during the enzyme reaction) is determined from the linear portion of the change in absorbance. For the change in absorbance at the time of blank, physiological saline or purified water is added instead of the sample, and the same operation as described above is performed to determine the amount of change in absorbance per minute. 3) Calculation formula Acid phosphatase activity (U / l) can be determined by substituting the absorbance change amount obtained by the above operation into the following formula.
【００１８】[0018]
【式２】活性（Ｕ／ｌ）＝〔（酵素反応中の単位時間当
たりの吸光度変化量−ブランクの時の吸光度変化量）×
酵素反応時の液量×１０6 〕÷〔検体量×反応生成物の
分子吸光係数×セル光路長〕 △E/min sample：酵素反応中の１分間当たりの吸光度変
化量 △E/min blank ：ブランクの時の１分間当たりの吸光度
変化量 ０．２２５：酵素反応時の液量（ｍｌ） ０．０１５：検体量（ｍｌ） ２１，５００：反応生成物の分子吸光係数（１／ｍｏｌ
・ｃｍ） ０．９：光路長（ｃｍ）## EQU2 ## Activity (U / l) = [(absorbance change per unit time during enzyme reaction−absorbance change in blank) ×
Liquid volume during enzyme reaction × 10 6 ] １０ [sample volume × molecular extinction coefficient of reaction product × cell optical path length] ΔE / min sample: change in absorbance per minute during enzyme reaction ΔE / min blank: change in absorbance per minute when blank 0.225: liquid volume (ml) during enzyme reaction 0.015 : Amount of sample (ml) 21,500: Molecular absorption coefficient of reaction product (1 / mol)
0.9) Optical path length (cm)
【００１９】破骨細胞由来酸性ホスファターゼ活性は、
（１）の総酒石酸抵抗性酸性ホスファターゼ測定用緩衝
液を用いて測定した総酒石酸抵抗性酸性ホスファターゼ
活性（破骨細胞、赤血球および血小板由来酸性ホスファ
ターゼ活性）から（２）の酒石酸抵抗性フッ化ナトリウ
ム抵抗性酸性ホスファターゼ測定用緩衝液を用いて求め
た残存活性（赤血球および血小板由来酸性ホスファター
ゼ活性）を差し引いて求める。なお、１検体の破骨細胞
由来酸性ホスファターゼ活性の測定に要する時間は約１
０分である。また、上記試薬の濃度、組成、液量等は使
用する自動分析装置の機種、適用する測定法により適宜
調節である。 ４）予備測定（フッ化ナトリウム（ＮａＦ）濃度の検
討） 試薬（１）の緩衝液にフッ化ナトリウムを加え、その濃
度０、１０、２０、３０、５０、１００、１５０、２０
０ｍＭに調整した液を用い、試料ａ．牛骨粗精製液、
ｂ．赤血球溶液（ヘモグロビン濃度０．８ｇ／ｄｌ）
ｃ．血小板溶液（血小板数６０００／μｌ）について前
記の（２）操作法に従って各試料の酸性ホスファターゼ
活性を測定した。その結果を表１に示した。The osteoclast-derived acid phosphatase activity is
From the total tartrate-resistant acid phosphatase activity (acid phosphatase activity derived from osteoclast, erythrocyte and platelet) measured using the buffer solution for measuring total tartrate-resistant acid phosphatase of (1), the tartrate-resistant sodium fluoride of (2) It is determined by subtracting the residual activity (acid phosphatase activity derived from red blood cells and platelets) determined using a buffer for measuring resistant acid phosphatase. The time required for measuring the osteoclast-derived acid phosphatase activity of one sample is about 1 hour.
0 minutes. The concentration, composition, liquid volume, etc. of the above reagents are appropriately adjusted depending on the type of automatic analyzer used and the measuring method to be applied. 4) Preliminary measurement (investigation of sodium fluoride (NaF) concentration) Sodium fluoride was added to the buffer of reagent (1), and its concentration was 0, 10, 20, 30, 50, 100, 150, 20.
Using a solution adjusted to 0 mM, sample a. Beef bone crude liquid,
b. Erythrocyte solution (haemoglobin concentration 0.8 g / dl)
c. The acid phosphatase activity of each sample was measured for the platelet solution (platelet count 6000 / μl) according to the above-mentioned (2) operation method. The results are shown in Table 1.
【００２０】[0020]
【表１】 [Table 1]
【００２１】表１の結果から明らかなごとく、赤血球、
血小板溶液はフッ化ナトリウムを加えても得られる活性
測定値に変化はなかった。しかし、牛骨粗精製液ではフ
ッ化ナトリウム添加により得られる活性測定値が低下
し、５０ｍＭでほぼプラトーとなった。この結果より前
記した試薬（２）のフッ化ナトリウム濃度は５０ｍＭと
した。また表１の結果は、酒石酸とフッ化ナトリウムと
が存在すると赤血球及び血小板由来酸性ホスファターゼ
は阻害されないが、破骨細胞由来酸性ホスファターゼは
阻害されたことを示している。なお、フッ化ナトリウム
の代わりに、破骨細胞由来酸性ホスファターゼ阻害剤と
して知られている、モリブデン酸塩を用いると、赤血球
及び血小板由来酸性ホスファターゼも阻害された。As is clear from the results in Table 1, erythrocytes,
The measured platelet activity did not change even when sodium fluoride was added. However, in the crudely purified bovine bone solution, the measured activity value obtained by the addition of sodium fluoride decreased, and the plateau became almost plateau at 50 mM. From this result, the sodium fluoride concentration of the reagent (2) was set to 50 mM. The results in Table 1 show that the presence of tartaric acid and sodium fluoride did not inhibit erythrocyte and platelet-derived acid phosphatase, but inhibited osteoclast-derived acid phosphatase. When molybdate, which is known as an osteoclast-derived acid phosphatase inhibitor, was used instead of sodium fluoride, erythrocyte and platelet-derived acid phosphatase were also inhibited.
【００２２】５）破骨細胞由来酸性ホスファターゼ活性
の測定 血清検体について、前記（１）の緩衝液を用いて２）の
操作に従って総酒石酸抵抗性酸性ホスファターゼ活性を
測定したところ３８．９Ｕ／ｌの測定値が得られた。一
方、前記（２）の緩衝液を用いて２）の操作に従い赤血
球および血小板由来酸性ホスファターゼ活性を測定した
ところ１６．４Ｕ／ｌの測定値が得られた。これらの測
定値から、血清検体中の破骨細胞由来酸性ホスファター
ゼ活性は２２．５Ｕ／ｌ（＝３８．９Ｕ／ｌ−１６．４
Ｕ／ｌ）であることが判った。5) Measurement of osteoclast-derived acid phosphatase activity The total tartrate-resistant acid phosphatase activity of the serum sample was measured using the buffer solution of (1) according to the procedure of 2), and was 38.9 U / l. Measurements were obtained. On the other hand, the erythrocyte- and platelet-derived acid phosphatase activity was measured using the buffer (2) according to the procedure of 2), and a measured value of 16.4 U / l was obtained. From these measured values, the osteoclast-derived acid phosphatase activity in the serum sample was 22.5 U / l (= 38.9 U / l-16.4).
U / l).
【００２３】実施例２添加回収試験 破骨細胞由来酸性ホスファターゼ活性が既知の血清９容
に、破骨細胞由来酸性ホスファターゼ活性が既知の牛骨
粗精製液１容添加したものを検体とし、実施例１の操作
方法に従い検体中の破骨細胞由来酸性ホスファターゼ活
性を測定し添加回収試験を行った。その結果を表２に示
した。Example 2 Addition / recovery test A sample was prepared by adding 9 volumes of serum with known osteoclast-derived acid phosphatase activity to 1 volume of crude bovine bone with known osteoclast-derived acid phosphatase activity. According to the operation method 1, the osteoclast-derived acid phosphatase activity in the sample was measured and an addition recovery test was performed. The results are shown in Table 2.
【００２４】[0024]
【表２】 [Table 2]
【００２５】表２に示すごとく、検討した５種の血清で
測定値は平均で１０４．２％の回収率を示し、ほぼ理論
値に近い値であった。この結果、本発明の方法では正確
に破骨細胞由来酸性ホスファターゼを測定していること
が判明した。As shown in Table 2, the measured value of the five sera examined showed an average recovery of 104.2%, which was almost a theoretical value. As a result, it was found that the method of the present invention accurately measured osteoclast-derived acid phosphatase.
【００２６】実施例３血清の希釈試験 血清を生理食塩水で５段階希釈した検体を実施例１の操
作方法に従い破骨細胞由来酸性ホスファターゼ活性を測
定した。結果を図１に示した。図１から明らかなよう
に、２種の血清でどちらもほぼ原点を通る直線が得られ
た。そのため、本発明の測定法では透析や希釈などの操
作をしないにもかかわらず、血清中の妨害物質の影響を
受けていない事が判明した。EXAMPLE 3 Serum Dilution Test Serum was diluted with physiological saline in five steps, and osteoclast-derived acid phosphatase activity was measured according to the procedure of Example 1. The results are shown in FIG. As is clear from FIG. 1, a straight line almost passing through the origin was obtained for both types of serum. Therefore, it was found that the measuring method of the present invention was not affected by interfering substances in serum, even though dialysis and dilution were not performed.
【００２７】実施例４溶血検体の影響 ヘパリン血漿を溶血前検体として、同一ヘパリン血を攪
拌後２２Ｇの注射針を通して溶血させ、遠心して得た血
漿を溶血後検体として実施例１の操作法に従い、両者の
破骨細胞由来酸性ホスファターゼ活性と溶血後のヘモグ
ロビン（Ｈｂ）濃度を測定した。その結果を表３に示し
た。Example 4 Influence of Hemolyzed Sample Heparin plasma was used as a pre-hemolysis sample, and the same heparin blood was stirred and hemolyzed through a 22G injection needle. Both osteoclast-derived acid phosphatase activities and hemoglobin (Hb) concentration after hemolysis were measured. Table 3 shows the results.
【００２８】[0028]
【表３】 [Table 3]
【００２９】表３から明らかなごとく、溶血前後で破骨
細胞由来酸性ホスファターゼ活性の差はほとんどなく、
本発明の破骨細胞由来酸性ホスファターゼ活性測定法は
溶血の影響を受けないことが判明した。As is clear from Table 3, there is almost no difference in the activity of osteoclast-derived acid phosphatase before and after hemolysis.
It was found that the method for measuring acid phosphatase derived from osteoclasts of the present invention was not affected by hemolysis.
【００３０】実施例５破骨細胞由来酸性ホスファターゼ活性の加齢変化 集団検診で肝機能と腎機能が正常範囲の女性の血清検体
を操作方法に従い破骨細胞由来酸性ホスファターゼ活性
を測定し年齢との関係を検討した。その結果を表４に示
した。Example 5 Age-Related Changes in Osteoclast-Derived Acid Phosphatase Activity Serum samples from women with normal liver function and renal function in a mass screening were assayed for osteoclast-derived acid phosphatase activity in accordance with the operation method to determine the age. Considered the relationship. Table 4 shows the results.
【００３１】[0031]
【表４】 [Table 4]
【００３２】表４の結果から明らかなごとく、得られた
破骨細胞由来酸性ホスファターゼ活性値の平均値は統計
的に有意に４０才代より上昇傾向を示し、閉経による骨
吸収の変化を反映していることを示唆する結果であっ
た。 実施例６干渉物質の影響 実施例１に記載した方法で約２０Ｕ／１の破骨細胞由来
酸性ホスファターゼ活性を有する血清検体に干渉物質を
添加して活性を測定し、干渉物質の影響を検討した。そ
の結果、ヘモグロビンでは０．４ｇ／ｄｌ、乳ビでは３
８００ホルマジン濁度、遊離及び抱合ビリルビンではそ
れぞれ１５ｍｇ／ｄｌまで影響は認められなかった。As is apparent from the results in Table 4, the average value of the obtained osteoclast-derived acid phosphatase activity statistically significantly increased from the age of 40s, reflecting changes in bone resorption due to menopause. The result suggests that Example 6 Effect of Interfering Substance The effect of the interfering substance was examined by adding an interfering substance to a serum sample having an osteoclast-derived acid phosphatase activity of about 20 U / 1 according to the method described in Example 1 and measuring the activity. . As a result, 0.4 g / dl for hemoglobin and 3 g for milk
No effects were observed for 800 formazin turbidity, free and conjugated bilirubin up to 15 mg / dl each.
【００３３】[0033]
【発明の効果】本発明の破骨細胞由来酸性ホスファター
ゼ活性の測定法は、以下のような効果を有する。 １）長時間加熱等の検体の前処理をしなくても正確に破
骨細胞由来酸性ホスファターゼ活性を測定できる。 ２）検体に、試薬を添加するだけで、短時間に簡単に破
骨細胞由来酸性ホスファターゼ活性を測定できる。 ３）検体をあらかじめ希釈することなく、正確に検体中
の破骨細胞由来酸性ホスファターゼ活性を測定できる。 ４）検体として血清を用いても、破骨細胞由来酸性ホス
ファターゼを正確に測定することができる。 ５）汎用型の自動分析装置に適用できるので、多数の検
体中の破骨細胞由来酸性ホスファターゼを短時間で簡単
に測定できる。 ６）検体中のビリルビン等の干渉物質の影響を受けるこ
となく、検体中の破骨細胞由来酸性ホスファターゼ活性
を測定できる。 ７）簡便で正確に測定可能な骨吸収変化の指標として有
用であると示される。 ８）検体中の赤血球由来酸性ホスファターゼ及び血小板
由来酸性ホスファターゼの影響なく、検体中の破骨細胞
由来酸性ホスファターゼ活性を正確に測定できる。The method for measuring osteoclast-derived acid phosphatase activity of the present invention has the following effects. 1) The osteoclast-derived acid phosphatase activity can be accurately measured without pretreatment of the specimen such as heating for a long time. 2) Osteoclast-derived acid phosphatase activity can be easily measured in a short time simply by adding a reagent to a sample. 3) The osteoclast-derived acid phosphatase activity in the sample can be accurately measured without previously diluting the sample. 4) Even if serum is used as a specimen, osteoclast-derived acid phosphatase can be accurately measured. 5) Since it can be applied to a general-purpose automatic analyzer, osteoclast-derived acid phosphatase in a large number of samples can be easily measured in a short time. 6) Osteoclast-derived acid phosphatase activity in a sample can be measured without being affected by an interfering substance such as bilirubin in the sample. 7) It is shown to be useful as a simple and accurate measurable bone resorption index. 8) The osteoclast-derived acid phosphatase activity in the sample can be accurately measured without the influence of red blood cell-derived acid phosphatase and platelet-derived acid phosphatase in the sample.
【図１】本発明の破骨細胞由来酸性ホスファターゼ活性
測定法による血清検体の希釈直線性を示すグラフであ
る。FIG. 1 is a graph showing the dilution linearity of a serum sample according to the method for measuring osteoclast-derived acid phosphatase activity of the present invention.
Claims (9)
石酸の存在下で酸性ホスファターゼ用基質を作用させて
酸性ホスファターゼ活性を測定し、 それとは別に、ii）検体中の酸性ホスファターゼに、赤
血球および血小板由来酸性ホスファターゼを阻害しない
破骨細胞由来酸性ホスファターゼ阻害剤と酒石酸との存
在下で酸性ホスファターゼ用基質を作用させて酸性ホス
ファターゼ活性を測定し、次いで、 iii)上記ｉ）の活性から上記ii）の活性を差し引くこと
により測定することを特徴とする破骨細胞由来酸性ホス
ファターゼ活性の測定方法。1. An acid phosphatase in a sample is reacted with an acid phosphatase substrate in the presence of tartaric acid to measure acid phosphatase activity in the presence of tartaric acid. Separately, ii) an acid phosphatase in a sample is treated with erythrocytes and platelets. Acid phosphatase substrate is allowed to act in the presence of an osteoclast-derived acid phosphatase inhibitor that does not inhibit derived acid phosphatase and tartaric acid, and the acid phosphatase activity is measured. A method for measuring osteoclast-derived acid phosphatase activity, which is performed by subtracting the activity.
ーゼを阻害しない破骨細胞由来酸性ホスファターゼ阻害
剤がフッ化塩である請求項１の測定方法。2. The method according to claim 1, wherein the osteoclast-derived acid phosphatase inhibitor that does not inhibit erythrocyte and platelet-derived acid phosphatase is a fluoride salt.
セイ可能な基質であり、かつ、酸性ホスファターゼ活性
の測定をレートアッセイにより実施する請求項１または
２の測定方法。3. The method according to claim 1, wherein the acid phosphatase substrate is a substrate that can be subjected to a rate assay, and the acid phosphatase activity is measured by a rate assay.
クロロ−４−アセチルフェニルリン酸またはその塩であ
る請求項１〜３のいずれかの測定方法。4. The method according to claim 1, wherein the substrate for acid phosphatase is 2,6-dichloro-4-acetylphenylphosphate or a salt thereof.
スファターゼ用基質を作用させる際、反応液量が用いた
検体の５倍以上である請求項１〜４のいずれかの測定方
法。5. The method according to claim 1, wherein the amount of the reaction solution when the substrate for acid phosphatase is allowed to act on the acid phosphatase in the sample is at least 5 times the amount of the sample used.
しない破骨細胞由来酸性ホスファターゼ阻害剤と酒石酸
とを含む試薬、 及びiii)酸性ホスファターゼ用基質を含む試薬を必須構
成試薬とする破骨細胞由来酸性ホスファターゼ活性測定
用キット。6. A reagent containing tartaric acid, i) a reagent containing tartaric acid, ii) a reagent containing an osteoclast-derived acid phosphatase inhibitor that does not inhibit erythrocyte and platelet-derived acid phosphatase, and iii) a reagent containing a substrate for acid phosphatase. Kit for measuring osteoclast-derived acid phosphatase activity as a constituent reagent.
ーゼを阻害しない破骨細胞由来酸性ホスファターゼ阻害
剤がフッ化塩である請求項６のキット。7. The kit according to claim 6, wherein the osteoclast-derived acid phosphatase inhibitor that does not inhibit erythrocyte and platelet-derived acid phosphatase is a fluoride salt.
セイ可能な基質である請求項６または７のキット。8. The kit according to claim 6, wherein the substrate for acid phosphatase is a substrate capable of performing a rate assay.
クロロ−４−アセチルフェニルリン酸またはその塩であ
る請求項６〜８のいずれかのキット。9. The kit according to claim 6, wherein the substrate for acid phosphatase is 2,6-dichloro-4-acetylphenyl phosphate or a salt thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14961597A JP3713901B2 (en) | 1997-06-06 | 1997-06-06 | Method for measuring osteoclast-derived acid phosphatase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14961597A JP3713901B2 (en) | 1997-06-06 | 1997-06-06 | Method for measuring osteoclast-derived acid phosphatase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH10337198A true JPH10337198A (en) | 1998-12-22 |
| JP3713901B2 JP3713901B2 (en) | 2005-11-09 |
Family
ID=15479092
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14961597A Expired - Lifetime JP3713901B2 (en) | 1997-06-06 | 1997-06-06 | Method for measuring osteoclast-derived acid phosphatase |
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| Country | Link |
|---|---|
| JP (1) | JP3713901B2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7465556B2 (en) | 2003-02-25 | 2008-12-16 | Nitto Boseki Co., Ltd. | Method of immunoassay tartrate resistant acid phosphatase 5b and kit to be used therein |
| US7611855B2 (en) | 2002-12-26 | 2009-11-03 | Nitto Boseki Co., Ltd. | Immunoassay method and kit to be used therein |
| US10214592B2 (en) | 2014-08-22 | 2019-02-26 | Nitto Boseki Co., Ltd. | Protein assay method specific to TRACP-5b (tartrate resistant acid phosphatase 5b) |
| CN114457141A (en) * | 2022-02-16 | 2022-05-10 | 北京九强生物技术股份有限公司 | Quantitative detection kit for prostatic acid phosphatase |
-
1997
- 1997-06-06 JP JP14961597A patent/JP3713901B2/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7611855B2 (en) | 2002-12-26 | 2009-11-03 | Nitto Boseki Co., Ltd. | Immunoassay method and kit to be used therein |
| US7465556B2 (en) | 2003-02-25 | 2008-12-16 | Nitto Boseki Co., Ltd. | Method of immunoassay tartrate resistant acid phosphatase 5b and kit to be used therein |
| US10214592B2 (en) | 2014-08-22 | 2019-02-26 | Nitto Boseki Co., Ltd. | Protein assay method specific to TRACP-5b (tartrate resistant acid phosphatase 5b) |
| CN114457141A (en) * | 2022-02-16 | 2022-05-10 | 北京九强生物技术股份有限公司 | Quantitative detection kit for prostatic acid phosphatase |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3713901B2 (en) | 2005-11-09 |
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