JP5982608B2 - Reagent for measuring creatine kinase activity - Google Patents

Description

  The present invention relates to a reagent for measuring creatine kinase activity, which is stable and accurate for a long period of time. The present invention is suitable for measurement of creatine kinase activity in a biological sample particularly important in the field of clinical examination.

クレアチンキナーゼは、骨格筋、心筋及び脳等に分布し、エネルギー代謝上重要な役割を果たしている酵素である。
そして、このクレアチンキナーゼは、二量体酵素であり、Ｍ型（筋型）とＢ型（脳型）のサブユニットの組み合わせにより、ＭＭ型（ＣＫ−３）、ＭＢ型（ＣＫ−２）及びＢＢ型（ＣＫ−１）の３種のアイソザイムが存在し、生体組織中では骨格筋にＭＭ型が、心筋にＭＭ型とＭＢ型が、脳にＢＢ型が多く存在する。
生体由来試料中のクレアチンキナーゼ活性は、急性心筋梗塞等の虚血性心疾患、多発性筋炎又は進行性筋ジストロフィー症等の原発性筋疾患、及び甲状腺機能異常等の疾病において高値となるため、生体由来試料中のクレアチンキナーゼ活性の測定はこれらの疾病の診断にとって大変有用なものである。
また、心筋に多く存在しているクレアチンキナーゼのＭＢ型アイソザイムの測定は、心筋梗塞に特異性の高い検査として重要視されている。
クレアチンキナーゼ活性の測定は、主として、下記の反応式で示されるクレアチンキナーゼ活性の測定反応、すなわち、「クレアチンキナーゼが触媒として働く、クレアチンリン酸又はこの塩とＡＤＰから、クレアチンとＡＴＰを生成する反応」、「ヘキソキナーゼ又はグルコキナーゼが触媒として働く、グルコースとＡＴＰから、グルコース−６−リン酸とＡＤＰを生成する反応」、及び「グルコース−６−リン酸デヒドロゲナーゼが触媒として働く、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から、６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）を生成する反応」、により生成するβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）の生成速度を３４０ｎｍにおける吸光度の増加速度より算出する方法により行われている。〔以下、β−ニコチンアミドアデニンジヌクレオチド（酸化型）又はこの塩を「ＮＡＤ^＋」ということがあり、β−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）又はこの塩を「ＮＡＤＰ^＋」ということがあり、この「ＮＡＤ^＋」と「ＮＡＤＰ^＋」を総称して「ＮＡＤ（Ｐ）^＋」ということがあり、そして、β−ニコチンアミドアデニンジヌクレオチド（還元型）又はこの塩を「ＮＡＤＨ」ということがあり、β−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）又はこの塩を「ＮＡＤＰＨ」ということがあり、この「ＮＡＤＨ」と「ＮＡＤＰＨ」を総称して「ＮＡＤ（Ｐ）Ｈ」ということがある。〕

ところで、赤血球、筋肉又は肝臓等に存在するアデニル酸キナーゼ（ミオキナーゼ）は、下記の反応を触媒する。

よって、溶血した試料等の試料中にアデニル酸キナーゼが存在すると、クレアチンキナーゼ活性測定試薬中に含まれるＡＤＰ２分子よりＡＴＰとアデノシン５’−一リン酸〔以下、アデノシン５’−一リン酸又はこの塩を「ＡＭＰ」ということがある。〕が生成し、この生じたＡＴＰよりヘキソキナーゼ（又はグルコキナーゼ）及びグルコース−６−リン酸デヒドロゲナーゼ（グルコース−６−リン酸脱水素酵素）の触媒する反応によりＮＡＤ（Ｐ）Ｈが生成する。
従って、試料中にアデニル酸キナーゼが存在すると、本来はクレアチンキナーゼの存在により生成するＮＡＤ（Ｐ）Ｈが、アデニル酸キナーゼの存在により生成してしまい、試料中のクレアチンキナーゼ活性測定値に正の誤差を与えてしまうことになる。

これに対して、第１試薬と試料の混合液に第２試薬を混合した最終反応液より得られるシグナルの量又はシグナルの変化量から、第１試薬を試料と混合した混合液より得られるシグナルの量又はシグナルの変化量を差し引き、この差の値より試料中のクレアチンキナーゼ活性値を求めることにより、例え、試料中にアデニル酸キナーゼが存在しても、正確なクレアチンキナーゼ活性値が得られる、いわゆるダブルカイネティック法が考えられ、広く使用されるようになった。
これは、試料が溶血等によりアデニル酸キナーゼを含むものであっても、第１試薬と試料の混合後の測定により得られるアデニル酸キナーゼの反応に由来する測定値（吸光度変化量）を、第１試薬と試料の混合液に第２試薬を混合した後の測定により得られるアデニル酸キナーゼの反応に由来する吸光度変化量とクレアチンキナーゼの反応に由来する吸光度変化量の両方を含む測定値（吸光度変化量）から差し引くことにより、アデニル酸キナーゼの存在により生じる正誤差が差し引かれた、正確なクレアチンキナーゼの活性値が得られる方法である。
ところで、クレアチンキナーゼは、血清等の生体由来試料中で、温度依存性の不可逆的失活と活性中心のＳＨ基がブロックされることによる可逆的失活により速やかに不活性化されるので、活性測定時にチオール化合物を存在させ、予備加温（予備インキュベーション）を行うことにより、クレアチンキナーゼを活性化してその活性値を測定する必要がある。
このクレアチンキナーゼの活性化剤であるチオール化合物としては、種々のものが使用されてきたが、近年は、無臭で取り扱い易く安価なことからＮ−アセチル−Ｌ−システイン（ＮＡＣ）が広く用いられてきた（特許文献１参照。）。
ところが、Ｎ−アセチル−Ｌ−システインは溶液中では不安定であり、そのＳＨ基が少しずつ酸化されることによりクレアチンキナーゼを活性化する能力が低下してしまい、クレアチンキナーゼ活性の測定値も負の誤差を生じるようになる。
更に、Ｎ−アセチル−Ｌ−システインの酸化物は、クレアチンキナーゼの活性を阻害するとの報告もある（非特許文献１参照。）。
また、クレアチンキナーゼの活性化剤として用いられる還元型グルタチオン及びジチオスレイトールなどの他のチオール化合物も溶液中で更に不安定であり、測定値に誤差を生じさせるようになるものであった。
このように、チオール化合物を含むクレアチンキナーゼ活性測定試薬及びチオール化合物を存在させるクレアチンキナーゼ活性測定方法は不安定なものであり、長期間測定に使用することができないものであった。
チオール化合物であるＮ−アセチル−Ｌ−システインを安定化することができ、３７℃での保存で最長１４日間使用することができる測定試薬として、ジチオスレイトール、メルカプトエタノール又はチオグリセロールなどのＮ−アセチル−Ｌ−システイン以外のチオール化合物（ＳＨ基含有化合物）とＮ−アセチル−Ｌ−システインとを含有するクレアチンキナーゼ活性測定試薬が開示されている（特許文献２参照。）。
しかしこれは、ジチオスレイトールが高価であること、メルカプトエタノールは悪臭を放つこと、そしてチオグリセロールは溶液状のため凍結乾燥に適さないことなどの難点を有するものであった。
また、やはりチオール化合物であるＮ−アセチル−Ｌ−システインを安定化することができ、４℃での保存で２８日間使用することが可能な測定試薬として、少なくともＮ−アセチル−Ｌ−システイン及びエチレンジアミン四酢酸塩を含む試薬群とマグネシウムを含む試薬群に分けて構成若しくは調製するクレアチンキナーゼ活性測定試薬が開示されている（特許文献３参照。）。
しかしこれは、第１試薬中にマグネシウムが存在せずヘキソキナーゼ（又はグルコキナーゼ）及びグルコース−６−リン酸デヒドロゲナーゼによる反応が進行しないため、前記のダブルカイネティック法による測定を行うことができないものであった。
長期間正確にクレアチンキナーゼ活性の測定を行うことができるという効果を有する測定試薬として、チオール化合物を含むクレアチンキナーゼ活性測定試薬において、亜硫酸等の硫黄を中心原子とするオキソ酸若しくはこのチオ酸又はこれらの塩を含有するクレアチンキナーゼ活性測定試薬が発明された（特許文献４参照。）。
また、顕著な機能低下なしに２℃〜８℃で１２ヵ月まで安定な液状の測定試薬として、グルコースとは異なり、酵素的にリン酸化され得るヘキソースをグルコースに加え、又はこれの代わりにヘキソキナーゼの基質として使用し、ヘキソキナーゼ反応で生成するグルコース−６−リン酸と異なるヘキソース−６−リン酸を、グルコース−６−リン酸デヒドロゲナーゼと異なるヘキソース−６−リン酸デヒドロゲナーゼを使用して直接測定するか、又は酵素的異性体化の後にグルコース−６−リン酸デヒドロゲナーゼによってグルコ−ス−６−リン酸として測定するクレアチンキナーゼ活性測定方法、及びこの測定方法のためのクレアチンキナーゼ活性測定試薬が開示されている（特許文献５参照。）。
そして、長期間安定な液状の測定試薬として、２つの別個の試薬成分に分割されており、検出のために試薬成分を相互に混合する形式のものにおいて、Ｇ６ＰＤＨ（グルコース−６−リン酸デヒドロゲナーゼ）が１つの成分中に含まれており、チオグリセロールが、少なくとも１つの他の物質とともに他の成分に含まれているクレアチンキナーゼ活性測定試薬が開示されている（特許文献６参照。）。
更に、２〜８℃で長期貯蔵した後の非常にすぐれた安定性を特徴とする測定試薬として、亜硫酸等の有機又は無機イオウ化合物又はイオウ塩をクレアチンキナーゼ活性化物質に対してサブモル量で含むクレアチンキナーゼ活性測定試薬が開示されている（特許文献７参照。）。
また、長期間安定な測定試薬として、第１試薬中のグルコース濃度が０．０５〜０．４ｍＭであり、かつ第２試薬中のグルコース濃度が第１試薬と第２試薬とを混合した時のグルコース終濃度で１６〜２５ｍＭになるように調整されているクレアチンキナーゼ活性測定試薬が開示されている（特許文献８参照。）。
更に、長期保存が可能な測定試薬として、Ｎ−アセチル−Ｌ−システイン等のクレアチンキナーゼ活性化剤と、アミノメタンスルホン酸とを含有するクレアチンキナーゼ活性測定試薬が開示されている（特許文献９参照。）。
しかしながら、近年の液状の活性測定試薬の普及に伴い、クレアチンキナーゼ活性測定試薬の製造、流通、保管及び使用の各段階を通じての更なる安定性の向上が望まれていた。Creatine kinase (CK, creatine phosphokinase, or CPK) [hereinafter sometimes referred to as “CK”. [E. C. 2.7.3.2] is adenosine 5′-triphosphate [hereinafter adenosine 5′-triphosphate or a salt thereof is sometimes referred to as “ATP”. ] Or adenosine 5′-diphosphate [hereinafter, adenosine 5′-diphosphate or a salt thereof may be referred to as “ADP”. ] Is a enzyme that catalyzes the following reaction.

Creatine kinase is an enzyme that is distributed in skeletal muscle, heart muscle, brain and the like and plays an important role in energy metabolism.
And this creatine kinase is a dimer enzyme, MM type (CK-3), MB type (CK-2) and MB type (CK-2) by combination of M type (muscle type) and B type (brain type) subunits. There are three types of isozymes of type BB (CK-1). In living tissue, MM type exists in skeletal muscle, MM type and MB type exist in myocardium, and BB type exists in brain.
Creatine kinase activity in biological samples is high in ischemic heart diseases such as acute myocardial infarction, primary myopathy such as polymyositis or progressive muscular dystrophy, and diseases such as thyroid dysfunction. Measurement of creatine kinase activity in a sample is very useful for the diagnosis of these diseases.
In addition, the measurement of MB type isozyme of creatine kinase, which is present abundantly in the myocardium, is regarded as an important test for myocardial infarction.
The measurement of creatine kinase activity is mainly performed by the measurement reaction of creatine kinase activity represented by the following reaction formula, that is, “a reaction in which creatine kinase acts as a catalyst to produce creatine and ATP from creatine phosphate or a salt thereof and ADP. , “A reaction in which glucose or ATP produces glucose-6-phosphate and ADP from hexokinase or glucokinase as a catalyst”, and “glucose-6-phosphate in which glucose-6-phosphate dehydrogenase acts as a catalyst” 6-phosphogluconic acid and β-nicotinamide adenine dinucleotide (reduced form) or β-nicotine from acid and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) Amidoadenine dinucleotide phosphorus (Reaction that produces (reduced form)) ”, the production rate of β-nicotinamide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) produced by calculation is calculated from the rate of increase in absorbance at 340 nm It is done by the method. [Hereinafter, β-nicotinamide adenine dinucleotide (oxidized form) or a salt thereof may be referred to as “NAD ⁺ ”, and β-nicotinamide adenine dinucleotide phosphate (oxidized form) or a salt thereof may be referred to as “NADP ⁺ ”. These “NAD ⁺ ” and “NADP ⁺ ” may be collectively referred to as “NAD (P) ⁺ ”, and β-nicotinamide adenine dinucleotide (reduced form) or a salt thereof may be referred to as “NADH”. In some cases, β-nicotinamide adenine dinucleotide phosphate (reduced form) or a salt thereof is sometimes referred to as “NADPH”, and “NADH” and “NADPH” are collectively referred to as “NAD (P) H”. There is. ]

By the way, adenylate kinase (myokinase) present in erythrocytes, muscles or liver catalyzes the following reaction.

Therefore, if adenylate kinase is present in a sample such as a hemolyzed sample, ATP and adenosine 5′-monophosphate [hereinafter referred to as adenosine 5′-monophosphate or The salt is sometimes referred to as “AMP”. NAD (P) H is produced from the resulting ATP by a reaction catalyzed by hexokinase (or glucokinase) and glucose-6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase).
Therefore, if adenylate kinase is present in the sample, NAD (P) H originally produced by the presence of creatine kinase is produced by the presence of adenylate kinase, which is positive for the measured value of creatine kinase activity in the sample. An error will be given.

On the other hand, the signal obtained from the mixed solution obtained by mixing the first reagent with the sample from the amount of signal obtained from the final reaction solution obtained by mixing the second reagent in the mixed solution of the first reagent and the sample or the amount of change in the signal. The creatine kinase activity value in the sample is obtained from the difference value by subtracting the amount of change or signal change, for example, even if adenylate kinase is present in the sample, an accurate creatine kinase activity value can be obtained. The so-called double kinetic method was conceived and widely used.
This is because even if the sample contains adenylate kinase due to hemolysis or the like, the measured value (absorbance change amount) derived from the reaction of adenylate kinase obtained by the measurement after mixing the first reagent and the sample Measurement value (absorbance) including both the amount of change in absorbance derived from the reaction of adenylate kinase and the amount of change in absorbance derived from the reaction of creatine kinase obtained by measurement after mixing the second reagent with the mixture of one reagent and sample By subtracting from (variation amount), a correct creatine kinase activity value is obtained by subtracting the positive error caused by the presence of adenylate kinase.
By the way, creatine kinase is rapidly inactivated by temperature-dependent irreversible deactivation and reversible deactivation by blocking the active group SH group in biological samples such as serum. It is necessary to activate creatine kinase and measure the activity value by making a thiol compound exist at the time of measurement and performing preheating (preincubation).
Various thiol compounds as activators of creatine kinase have been used, but in recent years N-acetyl-L-cysteine (NAC) has been widely used because it is odorless and easy to handle. (See Patent Document 1).
However, N-acetyl-L-cysteine is unstable in solution, and its ability to activate creatine kinase is reduced by the gradual oxidation of its SH group, and the measured value of creatine kinase activity is negative. This causes an error.
Furthermore, there is a report that the oxide of N-acetyl-L-cysteine inhibits the activity of creatine kinase (see Non-Patent Document 1).
In addition, other thiol compounds such as reduced glutathione and dithiothreitol used as activators of creatine kinase are also more unstable in the solution and cause errors in the measured values.
Thus, the creatine kinase activity measuring reagent containing a thiol compound and the creatine kinase activity measuring method in which a thiol compound is present are unstable and cannot be used for long-term measurement.
As a measuring reagent that can stabilize the thiol compound N-acetyl-L-cysteine and can be used at storage at 37 ° C. for a maximum of 14 days, N-acetylamine such as dithiothreitol, mercaptoethanol, or thioglycerol can be used. A creatine kinase activity measurement reagent containing a thiol compound (SH group-containing compound) other than acetyl-L-cysteine and N-acetyl-L-cysteine is disclosed (see Patent Document 2).
However, this has the disadvantages that dithiothreitol is expensive, mercaptoethanol gives off a bad odor, and thioglycerol is not suitable for lyophilization because it is in solution.
In addition, at least N-acetyl-L-cysteine and ethylenediamine can be used as measurement reagents that can stabilize N-acetyl-L-cysteine, which is also a thiol compound, and can be used for 28 days when stored at 4 ° C. A reagent for measuring creatine kinase activity, which is composed or prepared by dividing into a reagent group containing tetraacetate and a reagent group containing magnesium, is disclosed (see Patent Document 3).
However, since magnesium does not exist in the first reagent and the reaction by hexokinase (or glucokinase) and glucose-6-phosphate dehydrogenase does not proceed, the measurement by the double kinetic method cannot be performed. there were.
As a measuring reagent having the effect of being able to accurately measure creatine kinase activity for a long period of time, in a creatine kinase activity measuring reagent containing a thiol compound, an oxo acid having sulfur as a central atom such as sulfite or the thioacid or these Invented a reagent for measuring creatine kinase activity containing a salt of (see Patent Document 4).
Also, as a liquid measurement reagent that is stable for up to 12 months at 2 ° C. to 8 ° C. without significant functional deterioration, hexose that can be enzymatically phosphorylated, unlike glucose, is added to glucose or instead of hexokinase. Whether hexose-6-phosphate, which is used as a substrate and is different from glucose-6-phosphate produced by a hexokinase reaction, is directly measured using hexose-6-phosphate dehydrogenase, which is different from glucose-6-phosphate dehydrogenase Or a method for measuring creatine kinase activity which is measured as glucose-6-phosphate by glucose-6-phosphate dehydrogenase after enzymatic isomerization, and a reagent for measuring creatine kinase activity for this measurement method. (See Patent Document 5).
Then, as a liquid measurement reagent that is stable for a long period of time, it is divided into two separate reagent components, and G6PDH (glucose-6-phosphate dehydrogenase) Is contained in one component, and a reagent for measuring creatine kinase activity is disclosed in which thioglycerol is contained in another component together with at least one other substance (see Patent Document 6).
Furthermore, as a measuring reagent characterized by very good stability after long-term storage at 2-8 ° C., it contains organic or inorganic sulfur compounds such as sulfite or sulfur salts in submolar amounts relative to the creatine kinase activator. A reagent for measuring creatine kinase activity has been disclosed (see Patent Document 7).
In addition, as a measurement reagent that is stable for a long time, the glucose concentration in the first reagent is 0.05 to 0.4 mM, and the glucose concentration in the second reagent is a mixture of the first reagent and the second reagent. A reagent for measuring creatine kinase activity that is adjusted to 16 to 25 mM at the final glucose concentration is disclosed (see Patent Document 8).
Furthermore, a creatine kinase activity measuring reagent containing a creatine kinase activator such as N-acetyl-L-cysteine and aminomethanesulfonic acid is disclosed as a measuring reagent capable of long-term storage (see Patent Document 9). .)
However, with the spread of liquid activity measuring reagents in recent years, it has been desired to further improve the stability throughout the production, distribution, storage and use stages of the creatine kinase activity measuring reagent.

JP-A 49-106397 JP-A 61-289900 JP 62-104598 A JP-A-9-70298 JP 11-196899 A JP 2000-189195 A JP-T-2003-517264 JP 2005-130773 A JP 2007-49931 A

Gerhardt et al., Scand. J. et al. Clin. Lab. Invest. 39, 737-742, 1979

As described above, the conventional creatine kinase activity measuring reagent and creatine kinase activity measuring method are unstable, difficult to store and use for a long period of time, and further improvement in stability has been demanded.
As a result of intensive studies aimed at solving the problems of the conventional creatine kinase activity measuring reagent and creatine kinase activity measuring method, the present inventors have found that in the creatine kinase activity measuring reagent comprising the first reagent and the second reagent. The first reagent contains glucose at a concentration of 0.5 mM to 10 mM, and the second reagent contains no glucose or glucose at a concentration of 10 mM or less, thereby measuring creatine kinase activity stably and accurately over a long period of time. As a result, the present invention has been completed.

The present invention includes the following inventions.
(1) A creatine kinase activity measurement reagent comprising a first reagent and a second reagent, wherein the first reagent contains glucose at a concentration of 0.5 mM to 10 mM, and the second reagent does not contain glucose or is 10 mM or less A reagent for measuring creatine kinase activity, comprising glucose at a concentration of
(2) A creatine kinase activity measuring reagent comprising a first reagent and a second reagent, wherein the first reagent generates ADP and glucose-6-phosphate from adenosine 5′-diphosphate or a salt thereof, ATP and glucose 6-phosphogluconic acid and β-nicotinamide from glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) Enzyme that catalyzes a reaction to produce H ⁺ with adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form), β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine Dinucleotide phosphate (oxidized form) or a salt thereof, a thiol compound, and glucose And the concentration of the glucose is 0.5 mM to 10 mM, the second reagent contains creatine phosphate or a salt thereof, and does not contain glucose or contains 10 mM or less of glucose. The reagent for measuring creatine kinase activity according to (1) above.
(3) The reagent for measuring creatine kinase activity according to (1) or (2) above, wherein the glucose concentration of the first reagent is 1.25 mM to 5 mM.
(4) The reagent for measuring creatine kinase activity according to any one of (1) to (3), wherein the glucose concentration of the first reagent is 2 mM to 5 mM.
(5) The reagent for measuring creatine kinase activity according to any one of (1) to (4), wherein the second reagent does not contain glucose or contains glucose at a concentration of 5 mM or less.
(6) The reagent for measuring creatine kinase activity according to any one of (1) to (5), wherein the second reagent does not contain glucose.
(7) The second reagent is glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine. Any one of said (1)-(6) containing the enzyme which catalyzes reaction which produces | generates H ⁺ with amide adenine dinucleotide (reduced type) or (beta) -nicotinamide adenine dinucleotide phosphate (reduced type). The reagent for measuring creatine kinase activity as described.
(8) The first reagent and the second reagent are changed from glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid. And β-nicotinamide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and an enzyme that catalyzes the reaction to produce H ⁺ , of (1) to (7) above The reagent for measuring creatine kinase activity according to any one of the above.
(9) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine The activity value of an enzyme that catalyzes a reaction that generates H ⁺ with amide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) is 200 Unit / L to 5,000 Unit / L. The reagent for measuring creatine kinase activity according to any one of (1) to (8).
(10) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine The activity value of an enzyme that catalyzes a reaction that generates H ⁺ with amide adenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) is 500 Unit / L to 2,000 Unit / L. The reagent for measuring creatine kinase activity according to any one of (1) to (9).
(11) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine The activity value of the enzyme that catalyzes the reaction of producing amide adenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H ⁺ is 500 Unit / L to 1,000 Unit / L. The creatine kinase activity measuring reagent according to any one of (1) to (10).
(12) Glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) of the second reagent, 6-phosphogluconic acid and β-nicotine Amide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and the activity value of the enzyme that catalyzes the reaction that produces H ⁺ are mixed with the sample, the first reagent, and the second reagent. The reagent for measuring creatine kinase activity according to any one of (1) to (11), wherein the reagent is adjusted to be 500 Unit / L or more in the final reaction solution.
(13) From the second reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and the activity value of the enzyme that catalyzes the reaction that produces H ⁺ are mixed with the sample, the first reagent, and the second reagent. The reagent for measuring creatine kinase activity according to any one of (1) to (12), wherein the reagent is adjusted to be 800 Unit / L or more in the final reaction solution.
(14) From the second reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and the activity value of the enzyme that catalyzes the reaction that produces H ⁺ are mixed with the sample, the first reagent, and the second reagent. The reagent for measuring creatine kinase activity according to any one of (1) to (13), which is adjusted to be 1,600 Unit / L or more in the final reaction solution.
(15) The creatine kinase activity according to any one of (1) to (14) above, wherein the enzyme that catalyzes a reaction for producing ADP and glucose-6-phosphate from ATP and glucose is hexokinase or glucokinase. Measuring reagent.
(16) Glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotinamide adenine dinucleotide ( Any of the above (1) to (15), wherein the enzyme that catalyzes the reaction that produces (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and H ⁺ is glucose-6-phosphate dehydrogenase The reagent for measuring creatine kinase activity according to any one of the above.
(17) The reagent for measuring creatine kinase activity according to any one of (1) to (16), wherein the first reagent and the second reagent are liquid reagents.
(18) β-nicotinamide adenine dinucleotide (reduced form) or β-thionicotinamide adenine dinucleotide (reduced form) or β-thionicotinamide adenine dinucleotide (oxidized form) or salt thereof instead of β-nicotinamide adenine dinucleotide (oxidized form) or salt thereof Β-thionicotinamide adenine dinucleotide (reduced form) or salt thereof instead of this salt, β-nicotinamide adenine dinucleotide phosphate (oxidized form) or this salt instead of β-thionicotinamide adenine dinucleotide Phosphoric acid (oxidized form) or a salt thereof is used, or β-thionicotinamide adenine dinucleotide phosphoric acid (reduced form) or a salt thereof is used instead of β-nicotinamide adenine dinucleotide phosphoric acid (reduced form) or a salt thereof The use according to any one of (1) to (17). Rare Chin kinase activity measuring reagent.

The reagent for measuring creatine kinase activity of the present invention has the effect that it can be kept stable for a long period of time and can accurately measure creatine kinase activity for a long period of time.
Further, the creatine kinase activity measuring reagent of the present invention has an effect that it can maintain linearity in a high value range for a long period of time.
Furthermore, the reagent for measuring creatine kinase activity of the present invention is applicable to a double kinetic method capable of correcting a positive error caused by adenylate kinase in a hemolyzed sample or the like, and exhibits the above-described effect. It is.

（５） なお、本発明においては、ＮＡＤ^＋に替えてチオＮＡＤ^＋を用いてもよく、ＮＡＤＨに替えてチオＮＡＤＨを用いてもよく、ＮＡＤＰ^＋に替えてチオＮＡＤＰ^＋を用いてもよく、又はＮＡＤＰＨに替えてチオＮＡＤＰＨを用いてもよい。
そして、本発明においては、「グルコース−６−リン酸とＮＡＤ（Ｐ）^＋から６−ホスホグルコン酸とＮＡＤ（Ｐ）ＨとＨ^＋を生成する反応を触媒する酵素」及び６−ホスホグルコン酸脱水素酵素のいずれにおいても、ＮＡＤ^＋又はＮＡＤＰ^＋に替えてチオＮＡＤ^＋又はチオＮＡＤＰ^＋をそれぞれ用いることができ、この場合、ＮＡＤＨ又はＮＡＤＰＨに替わって、これらの酵素が触媒する反応によりチオＮＡＤＨ又はチオＮＡＤＰＨがそれぞれ生成する。
４．本発明のクレアチンキナーゼ活性測定方法の効果
本発明のクレアチンキナーゼ活性測定方法によれば、長期間正確にクレアチンキナーゼ活性の測定を行うことができる。すなわち、正確なクレアチンキナーゼ活性測定値を、長期間に亘り提供することができる。
また、長期間、高値域における直線性を維持することができる。
更に、溶血した試料等におけるアデニル酸キナーゼによる正の誤差を補正することができるダブルカイネティック法を適用することが可能なものである。
ＩＩＩ．クレアチンキナーゼ活性測定試薬の安定化方法
本発明のクレアチンキナーゼ活性測定試薬（ＣＫ活性測定試薬）の安定化方法であるが、前記の本発明のクレアチンキナーゼ活性測定試薬の構成により当該測定試薬の安定化を図ることができるものである。
以下、詳細に説明を行う。
１．本発明のクレアチンキナーゼ活性測定試薬の安定化方法
本発明のクレアチンキナーゼ活性測定試薬の安定化方法は、以下の発明を包含するものである。
（１） 第１試薬及び第２試薬よりなるクレアチンキナーゼ活性測定試薬の安定化方法であって、第１試薬が０．５ｍＭ〜１０ｍＭの濃度のグルコースを含み、かつ第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことを特徴とする、クレアチンキナーゼ活性測定試薬の安定化方法。
（２） 第１試薬及び第２試薬よりなるクレアチンキナーゼ活性測定試薬の安定化方法であって、第１試薬がアデノシン５’−二リン酸又はこの塩、ＡＴＰとグルコースからＡＤＰとグルコース−６−リン酸を生成する反応を触媒する酵素、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素、β−ニコチンアミドアデニンジヌクレオチド（酸化型）若しくはβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）又はこれらの塩、チオール化合物、及びグルコースを含み、かつ当該グルコースの濃度を０．５ｍＭ〜１０ｍＭとし、第２試薬がクレアチンリン酸又はこの塩を含み、かつグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことを特徴とする、前記（１）記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（３） 第１試薬のグルコースの濃度が１．２５ｍＭ〜５ｍＭである、前記（１）又は（２）記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（４） 第１試薬のグルコースの濃度が２ｍＭ〜５ｍＭである、前記（１）〜（３）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（５） 第２試薬がグルコースを含まないか又は５ｍＭ以下の濃度のグルコースを含むものである、前記（１）〜（４）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（６） 第２試薬がグルコースを含まないものである、前記（１）〜（５）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（７） 第２試薬が、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素を含むものである、前記（１）〜（６）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（８） 第１試薬及び第２試薬が、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素を含むものである、前記（１）〜（７）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（９） 第１試薬の、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素の活性値が、２００Ｕｎｉｔ／Ｌ〜５，０００Ｕｎｉｔ／Ｌである、前記（１）〜（８）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１０） 第１試薬の、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素の活性値が、５００Ｕｎｉｔ／Ｌ〜２，０００Ｕｎｉｔ／Ｌである、前記（１）〜（９）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１１） 第１試薬の、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素の活性値が、５００Ｕｎｉｔ／Ｌ〜１，０００Ｕｎｉｔ／Ｌである、前記（１）〜（１０）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１２） 第２試薬の、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素の活性値が、試料と第１試薬と第２試薬が混合された最終反応液において５００Ｕｎｉｔ／Ｌ以上となるように調整されている、前記（１）〜（１１）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１３） 第２試薬の、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素の活性値が、試料と第１試薬と第２試薬が混合された最終反応液において８００Ｕｎｉｔ／Ｌ以上となるように調整されている、前記（１）〜（１２）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１４） 第２試薬の、グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素の活性値が、試料と第１試薬と第２試薬が混合された最終反応液において１，６００Ｕｎｉｔ／Ｌ以上となるように調整されている、前記（１）〜（１３）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１５） ＡＴＰとグルコースからＡＤＰとグルコース−６−リン酸を生成する反応を触媒する酵素が、ヘキソキナーゼ又はグルコキナーゼである、前記（１）〜（１４）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１６） グルコース−６−リン酸とβ−ニコチンアミドアデニンジヌクレオチド（酸化型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）から６−ホスホグルコン酸とβ−ニコチンアミドアデニンジヌクレオチド（還元型）又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）とＨ^＋を生成する反応を触媒する酵素が、グルコース−６−リン酸デヒドロゲナーゼである、前記（１）〜（１５）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１７） 第１試薬及び第２試薬が液状試薬である、前記（１）〜（１６）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
（１８） β−ニコチンアミドアデニンジヌクレオチド（酸化型）若しくはこの塩に替えてβ−チオニコチンアミドアデニンジヌクレオチド（酸化型）若しくはこの塩を用い、β−ニコチンアミドアデニンジヌクレオチド（還元型）若しくはこの塩に替えてβ−チオニコチンアミドアデニンジヌクレオチド（還元型）若しくはこの塩を用い、β−ニコチンアミドアデニンジヌクレオチドリン酸（酸化型）若しくはこの塩に替えてβ−チオニコチンアミドアデニンジヌクレオチドリン酸（酸化型）若しくはこの塩を用い、又はβ−ニコチンアミドアデニンジヌクレオチドリン酸（還元型）若しくはこの塩に替えてβ−チオニコチンアミドアデニンジヌクレオチドリン酸（還元型）若しくはこの塩を用いる、前記（１）〜（１７）のいずれか一項記載のクレアチンキナーゼ活性測定試薬の安定化方法。
前記の通り、第１試薬及び第２試薬よりなるクレアチンキナーゼ活性測定試薬において、第１試薬が０．５ｍＭ〜１０ｍＭの濃度のグルコースを含み、かつ第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことにより、クレアチンキナーゼ活性測定試薬を安定化することができる。
すなわち、本発明のクレアチンキナーゼ活性測定試薬の安定化方法は、本発明のクレアチンキナーゼ活性測定試薬により達成することができる。
よって、本発明のクレアチンキナーゼ活性測定試薬の安定化方法の詳細は、前記の「Ｉ．クレアチンキナーゼ活性測定試薬」に記載した通りである。
２．本発明のクレアチンキナーゼ活性測定試薬の安定化方法の効果
本発明のクレアチンキナーゼ活性測定試薬の安定化方法によれば、クレアチンキナーゼ活性測定試薬を長期間安定に保つことができる。これにより、長期間正確にクレアチンキナーゼ活性の測定を行うことができる。すなわち、正確なクレアチンキナーゼ活性測定値を、長期間に亘り提供することができる。
また、クレアチンキナーゼ活性測定試薬において、長期間、高値域における直線性を維持することができる。 I. Reagent for measuring creatine kinase activity
  In the present invention, the creatine kinase activity measuring reagent (CK activity measuring reagent) is composed of a first reagent and a second reagent. That is, it is a measuring reagent kit comprising a first reagent and a second reagent.
  Details will be described below.
1. glucose
(1) First reagent
  In the present invention, the first reagent contains glucose at a concentration of 0.5 mM or more.
  When the concentration of glucose contained in the first reagent is less than 0.5 mM, it is derived from adenylate kinase contained in the sample in the mixed solution after mixing the first reagent and the sample in the double kinetic method. The series of reactions to be performed is not sufficiently performed, and the amount of signal (or the amount of change in signal) derived from this reaction by adenylate kinase cannot be accurately measured.
  In the present invention, since the second reagent does not contain glucose or contains glucose at a concentration of 10 mM or less, if the concentration of glucose contained in the first reagent is less than 0.5 mM, the first reagent and the sample In the final reaction mixture after mixing the second reagent with the mixture of, the reaction with creatine kinase (and adenylate kinase) is not sufficiently performed, and the amount of signal (or the amount of change in signal) derived from this reaction is accurately determined. Cannot be measured.
  In addition, since it becomes possible to respond | correspond to the measurement of creatine kinase of a higher activity value, it is preferable that the density | concentration of the glucose which a 1st reagent contains is 1.25 mM or more, and it is more preferable that it is 2 mM or more.
  In the present invention, the concentration of glucose contained in the first reagent needs to be 10 mM or less.
  When the first reagent contains glucose at a concentration of more than 10 mM, the effect of the present invention that “the reagent can be kept stable for a long period of time and creatine kinase activity can be accurately measured for a long period of time” You can't get enough.
  In addition, since the effect of stabilizing the reagent in the present invention can be further enhanced, the concentration of glucose contained in the first reagent is preferably 5 mM or less.
  For the reasons described above, in the present invention, the first reagent contains glucose at a concentration of 0.5 mM to 10 mM. The concentration of glucose contained in the first reagent is preferably 1.25 mM to 5 mM, and more preferably 2 mM to 5 mM.
(2) Second reagent
  In the present invention, the second reagent does not contain glucose or contains glucose at a concentration of 10 mM or less.
  When the second reagent contains glucose at a concentration exceeding 10 mM, the effect of the present invention that “the reagent can be kept stable for a long period of time and creatine kinase activity can be accurately measured for a long period of time” is sufficiently obtained. You will not be able to get to.
  In addition, since the effect of stabilizing the reagent in the present invention can be further enhanced, the concentration of glucose contained in the second reagent is preferably 5 mM or less, and the second reagent preferably does not contain glucose. preferable.
2. Adenosine 5'-diphosphate or a salt thereof
  In the present invention, the first reagent includes “adenosine 5′-diphosphate or a salt thereof” (ADP).
  The second reagent may also contain this “adenosine 5′-diphosphate or salt thereof” (ADP), but this is not essential.
  This “adenosine 5′-diphosphate or a salt thereof” (ADP) is not particularly limited, and examples thereof include an alkali metal salt such as lithium, sodium or potassium, or an alkaline earth such as magnesium or calcium. And the like.
  The concentration of the “adenosine 5′-diphosphate or salt thereof” (ADP) is preferably 0.5 mM or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed.
  There is no particular upper limit to the concentration in the final reaction solution, but considering the cost, up to 50 mM is sufficient.
  The concentration in the final reaction solution is more preferably 0.5 mM to 10 mM, and particularly preferably 1 mM to 5 mM.
  For example, the concentration of “adenosine 5′-diphosphate or a salt thereof” (ADP) contained in the first reagent is preferably 0.625 mM to 62.5 mM, and preferably 0.625 mM to 12.5 mM. More preferably, it is particularly preferably 1.25 mM to 6.25 mM.
  For example, when “adenosine 5′-diphosphate or a salt thereof” (ADP) is included in both the first reagent and the second reagent, “adenosine 5′-diphosphate or this The concentration of “salt” (ADP) is preferably 0.415 mM to 41.5 mM, more preferably 0.415 mM to 8.3 mM, and particularly preferably 0.83 mM to 4.15 mM.
  In this case, the concentration of “adenosine 5′-diphosphate or a salt thereof” (ADP) contained in the second reagent is preferably 0.835 mM to 83.5 mM, and 0.835 mM to 16.7 mM. More preferably, it is particularly preferably 1.67 mM to 8.35 mM.
3. Enzyme that catalyzes the reaction of producing ADP and glucose-6-phosphate from ATP and glucose
  In the present invention, the first reagent includes “an enzyme that catalyzes a reaction for producing ADP and glucose-6-phosphate from ATP and glucose”.
  The second reagent may also contain this “enzyme that catalyzes the reaction for producing ADP and glucose-6-phosphate from ATP and glucose”, but this is not essential.
  This “enzyme that catalyzes the reaction of producing ADP and glucose-6-phosphate from ATP and glucose” is not particularly limited, and catalyzes the reaction of producing ADP and glucose-6-phosphate from ATP and glucose. There is no limitation on the origin of the enzyme, and examples thereof include those derived from microorganisms such as Yeast and Leuconostoc mesenteroides, animals or plants, and those prepared by gene recombination techniques. .
  Examples of the “enzyme that catalyzes the reaction for producing ADP and glucose-6-phosphate from ATP and glucose” include hexokinase and glucokinase.
  The activity value of the “enzyme that catalyzes the reaction for producing ADP and glucose-6-phosphate from ATP and glucose” is 300 activity / unit in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed. It is preferable that it is more than L (unit / L).
  There is no particular upper limit to the activity value in this final reaction solution, but considering the cost, 6,000 Unit / L is sufficient.
  The activity value in this final reaction solution is more preferably 500 Unit / L to 5,000 Unit / L, and particularly preferably 700 Unit / L to 3,500 Unit / L.
  The activity value of the “enzyme that catalyzes the reaction of producing ADP and glucose-6-phosphate from ATP and glucose” contained in the first reagent is the adenylate kinase contained in the sample when the double kinetic method is applied. It is preferable that the activity value can be measured, for example, preferably 375 Unit / L to 7,500 Unit / L, more preferably 625 Unit / L to 6,250 Unit / L, and 875 Unit. / L to 4,375 Unit / L is particularly preferable.
  The activity value of the “enzyme that catalyzes the reaction for producing ADP and glucose-6-phosphate from ATP and glucose” described above is obtained according to various conditions of the enzyme activity measurement method. It is known that activity values are different.
  The activity value of the “enzyme that catalyzes the reaction of producing ADP and glucose-6-phosphate from ATP and glucose” described above or below is determined according to “hexokinase [HK] in the catalog of Oriental Yeast Co., Ltd. (revised in June 2006). ] (GRADE-S) from Yeast (page 29), the activity value determined by the enzyme activity measurement method described in “Analysis method”.
  That is, 2.40 mL of 0.1 M triethanolamine-hydrochloric acid-sodium hydroxide buffer (pH 7.5) containing 50 mg / mL glucose, 0.30 mL of 0.1 M magnesium chloride solution, 10 mM ATP solution 0.15 mL, 10 mM NADP⁺0.15 mL of the solution and 0.01 mL of the 500 Unit / mL glucose-6-phosphate dehydrogenase solution were sequentially added and mixed, and the prepared reaction solution was adjusted to about 3 Unit / mL with “ATP and glucose 0.02 mL of a solution of “enzyme that catalyzes the reaction to produce ADP and glucose-6-phosphate” is added, the reaction is performed at 25 ° C., and the absorbance is measured at a wavelength of 340 nm and an optical path length of 1 cm.
  Then, the activity value of the “enzyme that catalyzes the reaction of producing ADP and glucose-6-phosphate from ATP and glucose” is calculated by the following calculation formula.
  Activity value [Unit / mL] = (ΔA / min × V × D) ÷ (6.2 × d × v)
  Here, “ΔA / min” is the amount of change in absorbance per minute at 340 nm obtained by measurement, and is “an enzyme that catalyzes the reaction of producing ADP and glucose-6-phosphate from ATP and glucose. "V" is the final solution volume (3.03 mL), "D" is the enzyme dilution, and "6.2" is NADPH at 340 nm. Millimolar extinction coefficient (L × mmol)^-1× cm^-1), “D” represents the optical path length (1 cm), and “v” represents the amount of the enzyme solution (0.02 mL).
4). Glucose-6-phosphate and NAD⁺Or NADP⁺To 6-phosphogluconic acid and NADH or NADPH and H⁺Enzymes that catalyze reactions that produce
  In the present invention, the first reagent is “glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) [NAD⁺] Or β-nicotinamide adenine dinucleotide phosphate (oxidized form) [NADP⁺] To 6-phosphogluconic acid and β-nicotinamide adenine dinucleotide (reduced) [NADH] or β-nicotinamide adenine dinucleotide phosphate (reduced) [NADPH] and H⁺An enzyme that catalyzes the reaction to produce ".
  This “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺The enzyme that catalyzes the reaction that produces "may be included in both the first reagent and the second reagent. Inclusion in both the first reagent and the second reagent is preferable because the stability of the reagent is further improved.
  This “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺The enzyme that catalyzes the reaction that produces bismuth is not particularly limited, and glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺Any enzyme can be used as long as it can catalyze the reaction to produce the enzyme, and its origin is not limited, for example, those derived from microorganisms such as Yeast and Leuconostoc mesenteroides, animals or plants, or prepared by gene recombination techniques Can be mentioned.
  This “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺Examples of the “enzyme that catalyzes the reaction to produce” include glucose-6-phosphate dehydrogenase.
  This “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺In the final reaction solution in which the sample, the first reagent and the second reagent are mixed, it is preferably 160 Unit / L or more.
  There is no particular upper limit to the activity value in this final reaction solution, but considering the cost, 4,000 Unit / L is sufficient.
  The activity value in the final reaction solution is more preferably 400 Unit / L to 2,400 Unit / L, and particularly preferably 800 Unit / L to 1,600 Unit / L.
  “Glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺In the case where the enzyme that catalyzes the reaction that produces “is included only in the first reagent, the activity value of the enzyme in the first reagent is determined by the adenylate kinase contained in the sample when the double kinetic method is applied. It is preferable that the activity value can be measured, for example, 200 Unit / L to 5,000 Unit / L, more preferably 500 Unit / L to 3,000 Unit / L, It is especially preferable that it is 000Unit / L-2,000Unit / L.
  In addition, “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺The stability of the reagent is further improved by including “an enzyme that catalyzes the reaction that produces” in both the first reagent and the second reagent. In this case, “glucose-6” Phosphoric acid and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺The activity value of the “enzyme that catalyzes the reaction to produce a” is preferably 200 Unit / L to 5,000 Unit / L, more preferably 500 Unit / L to 3,000 Unit / L, and 1,000 Unit / L. It is particularly preferred to be ˜2,000 Unit / L.
  In this case, the second reagent contains “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺The activity value of the “enzyme that catalyzes the reaction to produce” is preferably 500 Unit / L or more, more preferably 800 Unit / L or more, and particularly preferably 1,600 Unit / L or more. The upper limit of the activity value of the second reagent is not particularly limited, but up to 12,000 Unit / L is sufficient considering the cost.
  In addition, the aforementioned “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺It is known that the activity value of the enzyme differs depending on various conditions of the enzyme activity measurement method.
  "Glucose-6-phosphate and NAD (P)" above or below⁺To 6-phosphogluconic acid and NAD (P) H and H⁺The activity value of the “enzyme that catalyzes the reaction to produce a glucose” is “glucose-6-phosphate dehydrogenase [G-6-PDH] from Yeast” (No. 24) in the catalog of Oriental Yeast Co., Ltd. (revised in June 2006). The activity value obtained by the enzyme activity measuring method described in “Analytical method” on page).
  That is, 2.50 mL of 0.1 M glycylglycine buffer (pH 8.5), 0.30 mL of 0.2 M magnesium chloride solution, 0.15 mL of 10 mM glucose-6-phosphate, and 10 mM NADP.⁺0.15 mL of the solution was sequentially added, mixed, and the prepared reaction solution was adjusted to about 3 Unit / mL with “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺0.02 mL of the solution of “enzyme that catalyzes the reaction to produce” is added, the reaction is performed at 25 ° C., and the absorbance is measured at a wavelength of 340 nm and an optical path length of 1 cm.
  And according to the following calculation formula, this “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺The activity value of the “enzyme that catalyzes the reaction that produces“ is calculated.
  Activity value [Unit / mL] = (ΔA / min × V × D) ÷ (6.2 × d × v)
  Here, “ΔA / min” is the amount of change in absorbance per minute at 340 nm obtained by measurement, and “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺In the blank reaction without the presence of “enzyme that catalyzes the reaction to generate a reaction”, the amount of change in absorbance was subtracted and corrected, “V” is the final liquid volume (3.12 mL), “D” is the enzyme dilution rate, and “6. The value of “2” is the molar molecular extinction coefficient of NADPH at 340 nm (L × mmol^-1× cm^-1), “D” represents the optical path length (1 cm), and “v” represents the amount of the enzyme solution (0.02 mL).
5). NAD⁺Or NADP⁺
  In the present invention, the first reagent is “β-nicotinamide adenine dinucleotide (oxidized form) [NAD⁺] Or β-nicotinamide adenine dinucleotide phosphate (oxidized form) [NADP⁺Or a salt thereof.
  The second reagent also contains this “NAD”.⁺Or NADP⁺Or “salts thereof” may be included, but this is not essential.
  This "NAD⁺Or NADP⁺“These salts” of “or these salts” are not particularly limited, and examples thereof include alkali metal salts such as lithium, sodium and potassium, or alkaline earth metal salts such as magnesium and calcium.
  This "NAD⁺Or NADP⁺Or the concentration of these salts ”is preferably 0.9 mM or more in the final reaction solution in which the sample, the first reagent and the second reagent are mixed.
  There is no particular upper limit to the concentration in the final reaction solution, but up to 15 mM is sufficient considering the cost.
  Further, the concentration in the final reaction solution is more preferably 1.4 mM to 10 mM, and particularly preferably 2 mM to 4 mM.
  For example, “NAD included in the first reagent⁺Or NADP⁺Or the concentration of these salts ”is preferably 1.13 mM to 18.75 mM, more preferably 1.75 mM to 12.5 mM, and particularly preferably 2.5 mM to 5 mM.
  In the present invention, NAD⁺Β-thionicotinamide adenine dinucleotide (oxidized form) or a salt thereof may be used instead of β, and β-thionicotinamide adenine dinucleotide (reduced form) or a salt thereof may be used instead of NADH.⁺Β-thionicotinamide adenine dinucleotide phosphate (oxidized form) or a salt thereof may be used instead of β, or β-thionicotinamide adenine dinucleotide phosphate (reduced form) or a salt thereof may be used instead of NADPH May be. [Hereinafter, β-thionicotinamide adenine dinucleotide (oxidized form) or a salt thereof is referred to as “thioNAD”.⁺Β-thionicotinamide adenine dinucleotide phosphate (oxidized form) or a salt thereof is referred to as “thio-NADP”.⁺"Thio NAD⁺"And" thio NADP⁺"Thio NAD (P)"⁺And β-thionicotinamide adenine dinucleotide (reduced form) or a salt thereof may be referred to as “thioNADH”, and β-thionicotinamide adenine dinucleotide phosphate (reduced form) or a salt thereof. Is sometimes referred to as “thioNADPH”, and “thioNADH” and “thioNADPH” may be collectively referred to as “thioNADP (P) H”. ]
  In the present invention, “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺NAD in both “enzymes that catalyze reactions that produce” and 6-phosphogluconate dehydrogenase⁺Or NADP⁺Instead of Thio NAD⁺Or Thio NADP⁺In this case, in place of NADH or NADPH, thioNADH or thioNADPH is produced by a reaction catalyzed by these enzymes, respectively.
6). Creatine phosphate or its salt
  In the present invention, the second reagent includes “creatine phosphate or a salt thereof”.
  This “creatine phosphate or salt thereof” is not particularly limited, and examples thereof include alkali metal salts such as lithium, sodium or potassium, or alkaline earth metal salts such as magnesium or calcium. Can do.
  The concentration of this “creatine phosphate or salt thereof” is preferably 10 mM or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed.
  In addition, although there is no upper limit in particular in the density | concentration in this final reaction liquid, when considering the cost, up to 100 mM is sufficient.
  Further, the concentration in the final reaction solution is more preferably 15 mM to 75 mM, and particularly preferably 25 mM to 50 mM.
  For example, when “creatine phosphate or a salt thereof” is included only in the second reagent, the concentration in the second reagent is preferably 50 mM to 500 mM, more preferably 75 mM to 375 mM, and 125 mM to 250 mM. It is particularly preferred that
7). Thiol compound
  In the present invention, the first reagent contains a thiol compound.
  The second reagent may also contain this thiol compound, but this is not essential.
  The thiol compound may be any compound having an SH group, and is not particularly limited. For example, N-acetyl-L-cysteine, thioglycerol, dithiothreitol, reduced glutathione, cysteine, dithioerythritol, odor 2-aminoethylisothiouronium, 2-thioglucose, thioglycolic acid, 2-mercaptoethanol, N-guanyl-L-cysteine, mercaptoacetic acid, mercaptosuccinic acid, 2-mercaptoethanesulfonic acid, cysteamine, etc. be able to.
  As this thiol compound, N-acetyl-L-cysteine is preferable.
  The concentration of the thiol compound is preferably 1.6 mM or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed.
  There is no particular upper limit to the concentration in this final reaction solution, but up to 80 mM is sufficient considering the cost.
  The concentration in the final reaction solution is more preferably 4 mM to 64 mM, and particularly preferably 8 mM to 48 mM.
  For example, when the thiol compound is included only in the first reagent, the concentration in the first reagent is preferably 2 mM to 100 mM, more preferably 5 mM to 80 mM, and particularly preferably 10 mM to 60 mM. .
8). Reducing substance
  In the present invention, it is preferable to include the reducing substance in the first reagent, the second reagent, or both the first reagent and the second reagent.
  There is a report that the thiol compound is easily oxidized and unstable, and the oxide of the thiol compound inhibits the activity of creatine kinase. It is necessary to keep the thiol compound coexisting (contacted) with this in a reduced state.
  On the other hand, it is preferable to bring the reducing substance into contact with the thiol compound because the thiol compound can be prevented from being oxidized and kept in the reduced state, or the oxidized thiol compound can be returned to the reduced state.
  The reducing substance is contained in the same reagent as the thiol compound in the first reagent and the second reagent, and the reduced state of the thiol compound is maintained by contacting the thiol compound and the reducing substance in the reagent. Anyway.
  Further, this reducing substance is contained in a reagent different from the thiol compound among the first reagent and the second reagent, and reduced with the thiol compound in the final reaction liquid (mixed liquid of the sample, the first reagent and the second reagent) A substance may be contacted so that the thiol compound oxidized during storage returns to a reduced state.
  The reducing substance is not particularly limited as long as it can maintain the reduced state of the thiol compound by bringing it into contact with the thiol compound or return the oxidized thiol compound to the reduced state. Can do.
  More specifically, examples of the reducing substance include “an oxo acid having sulfur as a central atom, or a thioacid thereof, or a salt thereof”.
  Examples of the “oxo acid having sulfur as a central atom or this thioacid” include sulfoxylic acid [H₂SO₂], Sulfurous acid [H₂SO₃], Thiosulfite [H₂S₂O₂], Thiosulfuric acid [H₂S₂O₃], Dithionite [H₂S₂O₄], Disulfite [H₂S₂O₅], Dithionic acid [H₂S₂O₆], Disulfuric acid [H₂S₂O₇] Or polythionic acid [H₂SxO₆(X = 3, 4...)] And the like.
  The “sulfur-centered oxoacid or this thioacid or salt thereof” is dissociated from the “sulfur-centered oxoacid or this thioacid” as a proton. This is a compound in which part or all of hydrogen that can be substituted is replaced with an alkali metal, an alkaline earth metal, another metal, or a basic group.
  For example, examples of the alkali metal include lithium, sodium, potassium, rubidium, and cesium, and examples of the alkaline earth metal include beryllium, magnesium, calcium, strontium, and barium. Examples of the group include an ammonium group.
  The concentration of the reducing substance is preferably 0.0008% (w / v) or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed.
  There is no particular upper limit to the concentration in the final reaction solution, but considering the cost, 0.8% (w / v) is sufficient.
  The concentration in the final reaction solution is more preferably 0.004% (w / v) to 0.4% (w / v), and particularly preferably 0.008% (w / v) to 0.00. 16% (w / v).
  For example, when the reducing substance is contained only in the first reagent, the concentration in the first reagent is preferably 0.001% (w / v) to 1% (w / v), 0.005% ( W / v) to 0.5% (w / v) is more preferable, and 0.01% (w / v) to 0.2% (w / v) is particularly preferable.
  For example, when the reducing substance is included in both the first reagent and the second reagent, the concentration of the reducing substance contained in the first reagent is 0.0008% (w / v) to 0.8% (w / v). ), Preferably 0.004% (w / v) to 0.4% (w / v), 0.008% (w / v) to 0.16% (w / v) It is particularly preferred that v).
  In this case, the concentration of the reducing substance contained in the second reagent is preferably 0.0008% (w / v) to 0.8% (w / v), and 0.004% (w / v). More preferably, it is -0.4% (w / v), and it is especially preferable that it is 0.008% (w / v) -0.16% (w / v).
  For example, when the reducing substance is included only in the second reagent, the concentration in the second reagent is preferably 0.004% (w / v) to 4% (w / v), and 0.02 % (W / v) to 2% (w / v) is more preferable, and 0.04% (w / v) to 0.8% (w / v) is particularly preferable.
9. Magnesium ion
  In the present invention, magnesium ions can be contained in the first reagent, the second reagent, or both the first reagent and the second reagent.
  Magnesium ions are preferably included for the reaction of the “enzyme that catalyzes the reaction for producing ADP and glucose-6-phosphate from ATP and glucose”.
  The magnesium ion is more preferably contained in the first reagent or both the first reagent and the second reagent, and particularly preferably contained in the first reagent.
  There is no limitation in particular as this magnesium ion, For example, the salt etc. which contain magnesium ion can be mentioned.
  The salt containing magnesium ion is not particularly limited as long as it is a compound composed of an acid group such as a halogen ion or an acid group of an organic acid and magnesium ion.
  Examples of the salt containing magnesium ions include magnesium chloride, magnesium acetate, and magnesium sulfate.
  As the salt containing magnesium ions, magnesium acetate is preferable.
  The concentration of magnesium ion (or a salt containing magnesium ion) is preferably 0.8 mM or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed.
  In addition, although there is no upper limit in particular in the density | concentration in this final reaction liquid, when considering the cost, up to 40 mM is sufficient.
  Further, the concentration in this final reaction solution is more preferably 4 mM to 24 mM, and particularly preferably 8 mM to 16 mM.
  For example, when magnesium ion (or a salt containing magnesium ion) is contained only in the first reagent, the concentration in the first reagent is preferably 1 mM to 50 mM, more preferably 5 mM to 30 mM, and 10 mM. Particularly preferred is ˜20 mM.
10. Chelating agent
  In the present invention, the “chelating agent or salt thereof” can be contained in the first reagent, the second reagent, or both the first reagent and the second reagent.
  Ca²⁺, Fe³⁺Or Cu²⁺In contrast to this, metal ions such as creatine inhibit competitively the creatine kinase activity, but by inhibiting the inhibition of creatine kinase activity by the metal ion by the presence of “chelating agent or salt thereof”, Since it is possible to stabilize the thiol compound, it is preferable to include “a chelating agent or a salt thereof”.
  The “chelating agent or salt thereof” is more preferably contained in the first reagent, or both the first reagent and the second reagent, and particularly preferably contained in the first reagent.
  The “chelating agent or salt thereof” is not particularly limited as long as it can bind to a metal ion.
  Examples of the “chelating agent or salt thereof” include ethylenediamine-N, N, N ′, N′-tetraacetic acid [EDTA], O, O′-bis (2-aminophenyl) ethylene glycol-N, N, N ′, N′-tetraacetic acid [BAPTA], N, N-bis (2-hydroxyethyl) glycine [Bicine], trans-1,2-diaminocyclohexane-N, N, N ′, N′-tetraacetic acid [ CyDTA], diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid [DTPA], ethylenediamine-N, N′-dipropionic acid [EDDP], N- (2-hydroxyethyl) ethylenediamine— N, N ′, N ″ -triacetic acid [EDTA-OH], O, O′-bis (2-aminoethyl) ethylene glycol-N, N, N ′, N′-tetraacetic acid [GEDTA (EGT )], N- (2-hydroxyethyl) iminodiacetic acid [HIDA], iminodiacetic acid [IDA], nitrilotriacetic acid [NTA], nitrilotri (methylphosphonic acid) [NTPO], N, N, N ′, N′— Tetrakis (2-pyridylmethyl) ethylenediamine [TPEN], or triethylenetetramine-N, N, N ′, N ″, N ′ ″, N ′ ″-hexaacetic acid [TTHA], or a salt thereof Can be mentioned.
  As the “chelating agent or salt thereof”, EDTA or EDTA disodium salt is preferable.
  The concentration of the “chelating agent or salt thereof” is preferably 0.08 mM or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed.
  There is no particular upper limit to the concentration in the final reaction solution, but considering the cost, up to 8 mM is sufficient.
  The concentration in the final reaction solution is more preferably 0.4 mM to 3.2 mM, and particularly preferably 0.8 mM to 2.4 mM.
  For example, when the “chelating agent or salt thereof” is included only in the first reagent, the concentration in the first reagent is preferably 0.1 mM to 10 mM, more preferably 0.5 mM to 4 mM, Particularly preferred is 1 mM to 3 mM.
11. Adenylate kinase inhibitor
  In the present invention, an inhibitor of adenylate kinase can be contained in the first reagent, the second reagent, or both the first reagent and the second reagent.
  As described above, the presence of adenylate kinase in the sample causes a positive error in the measured value of creatine kinase activity. However, due to the presence of an inhibitor of adenylate kinase, the positive error derived from adenylate kinase in the measurement of creatine kinase activity. Therefore, it is preferable to include an inhibitor of adenylate kinase in the reagent.
  The inhibitor of adenylate kinase is more preferably contained in the first reagent or both the first reagent and the second reagent, and particularly preferably contained in the first reagent.
  The inhibitor of adenylate kinase is not particularly limited as long as it has an ability to inhibit the activity of adenylate kinase.
  Examples of inhibitors of this adenylate kinase include adenosine 5'-monophosphate or a salt thereof (AMP), P¹, P⁵-Diadenosine-5'-pentaphosphoric acid (AP₅A), diadenosine-5'-tetraphosphate (AP₄A) or diadenosine-5'-hexaphosphate (AP₆Examples thereof include diadenosine polyphosphoric acid such as A) or a salt thereof, or fluorine ions or fluorides.
  The concentration of this adenylate kinase inhibitor is preferably 1 μM to 100 μM in the first reagent.
  In addition, the concentration of the inhibitor of adenylate kinase in the second reagent may be a concentration that can effectively correct an error based on the activity of adenylate kinase when measured by the double kinetic method. .
12 pH
(1) pH of the final reaction solution
  In the present invention, the pH of the final reaction solution in which the sample, the first reagent, and the second reagent are mixed is preferably pH 6.0 (30 ° C.) to pH 7.2 (30 ° C.), and pH 6.4 (30 ° C.) to pH 6.8 (30 ° C.), more preferably pH 6.6 (30 ° C.) to pH 6.8 (30 ° C.).
(2) pH of the first reagent
  In the present invention, the pH of the first reagent is preferably from pH 5.6 (30 ° C.) to pH 6.6 (30 ° C.), and preferably from pH 6.0 (30 ° C.) to pH 6.6 (30 ° C.). Is more preferable, and it is especially preferable that it exists in pH6.3 (30 degreeC)-pH6.6 (30 degreeC).
(3) pH of the second reagent
  In the present invention, the pH of the second reagent may be such that when the sample, the first reagent, and the second reagent are mixed, the pH described in “Final reaction solution pH” in (1) above is obtained. It ’s fine.
  In consideration of the stability of creatine phosphate or a salt thereof contained in the second reagent, the pH of the second reagent is preferably pH 7.0 (30 ° C.) to pH 11.0 (30 ° C.), pH 7 More preferably, it is in the range of 0.5 (30 ° C) to 10.0 (30 ° C), and particularly preferably in the range of 8.0 (30 ° C) to 9.0 (30 ° C).
13. Buffer
  In the present invention, the buffer can be contained in the first reagent, the second reagent, or both the first reagent and the second reagent.
  In order to allow the final reaction solution, the first reagent, and the second reagent to be set to the pH described in “12. The first reagent and the second reagent are preferably included in a concentration having
  Examples of such a buffer include MES, Bis-Tris, Bis-Tris propane, ADA, PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, and Bicine. , TAPS, CHES, phosphoric acid, phosphate, boric acid, borate, glycine, glycylglycine, imidazole, or tris (hydroxymethyl) aminomethane [Tris].
  As this buffer, imidazole is preferred.
14 Other ingredients
  The reagent for measuring creatine kinase activity of the present invention may contain the following components in addition to the above components.
(1) 6-phosphogluconolactonase
  In the present invention, 6-phosphogluconolactonase can be included in the first reagent, the second reagent, or both the first reagent and the second reagent.
  The inclusion of 6-phosphogluconolactonase is preferable because the measurement range can be expanded and the linearity can be improved.
  The 6-phosphogluconolactonase is more preferably contained in the first reagent or both the first reagent and the second reagent, and particularly preferably contained in the first reagent.
  The 6-phosphogluconolactonase is not particularly limited as long as it is an enzyme that can catalyze the reaction of hydrolyzing 6-phosphogluconolactone to 6-phosphogluconic acid, and its origin is also limited. For example, those derived from microorganisms such as Yeast and Leuconostoc mesenteroides, animals or plants, or those prepared by genetic recombination techniques can be used.
  The activity value of 6-phosphogluconolactonase is preferably 8 Unit / L or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed.
  There is no particular upper limit to the activity value in this final reaction solution, but considering the cost, 4,000 Unit / L is sufficient.
  The activity value in this final reaction solution is more preferably 320 Unit / L to 2,000 Unit / L, and particularly preferably 480 Unit / L to 800 Unit / L.
  For example, the activity value of 6-phosphogluconolactonase contained in the first reagent is preferably 10 Unit / L to 5,000 Unit / L, more preferably 400 Unit / L to 2,500 Unit / L, It is particularly preferably 600 Unit / L to 1,000 Unit / L.
  The activity value of 6-phosphogluconolactonase described above is known to vary depending on various conditions of the enzyme activity measurement method.
  The activity value of 6-phosphogluconolactonase described above or below is an activity value obtained by an enzyme activity measurement method defined by Roche Diagnostics.
  Specifically, for sample measurement, 0.855 mL of 0.1 M MES buffer (pH 6.5) containing 30 mM sodium chloride and 2 mM magnesium chloride, 0.050 mL of 12.7 mM NADP aqueous solution, 500 Units. 0.020 mL of / mL glucose-6-phosphate dehydrogenase aqueous solution and 0.010 mL of 200 Unit / mL 6-phosphogluconate dehydrogenase aqueous solution were sequentially added and mixed, and the prepared mixture was at 25 ° C. After allowing to stand, 0.015 mL of a 10 mM glucose-6-phosphate aqueous solution was added to the mixed solution, mixed, and the prepared reaction solution was allowed to stand at 25 ° C. for exactly 2 minutes, and then added to the reaction solution. 0.050 of the sample diluted so that the activity value of the 6-phosphogluconolactonase is about 80 to 200 mUnit / mL. L is added, mixed, reacted at 25 ° C., the change in absorbance is measured for about 10 minutes at a wavelength of 340 nm (or wavelength of 365 nm), an optical path length of 1 cm, and the change in absorbance per minute [ΔA / min (Sample)].
  In addition, for reagent blind measurement (reagent blank) measurement, 0.905 mL of 0.1 M MES buffer (pH 6.5) containing 30 mM sodium chloride and 2 mM magnesium chloride, 0 of 12.7 mM NADP aqueous solution. 0.050 mL, 0.020 mL of 500 Unit / mL glucose-6-phosphate dehydrogenase aqueous solution, and 0.010 mL of 200 Unit / mL 6-phosphogluconate dehydrogenase aqueous solution were sequentially added, mixed, and prepared mixed solution Was allowed to stand at 25 ° C., 0.015 mL of 10 mM glucose-6-phosphate aqueous solution was added to the mixture, mixed, and the prepared reaction solution was allowed to stand at 25 ° C. for exactly 2 minutes. This reaction solution was measured at 25 ° C. with a wavelength of 340 nm (or a wavelength of 365 nm), an optical path length of 1 cm, and a change in absorbance was measured for about 10 minutes. Performed, determine the absorbance change per minute [ΔA / min (blank)].
  Next, the activity value of this 6-phosphogluconolactonase is calculated by the following calculation formula.
  Activity value [Unit / mL] = 1.0 ÷ (ε × 0.05 × 1.0) × ΔA / min
  Here, “ΔA / min” is obtained by subtracting “ΔA / min (blank)” from “ΔA / min (sample)” obtained in the above measurement (ΔA / min (sample) −ΔA / min ( blank)), and “ε” is “ε” at a wavelength of 340 nm.₃₄₀= 6.3 (L · mM^-1・ Cm^-1) ”And“ ε ”at a wavelength of 365 nm.₃₆₅= 3.5 (L · mM^-1・ Cm^-1) ”.
(2) 6-phosphogluconate dehydrogenase
  In the present invention, 6-phosphogluconate dehydrogenase can be included in the first reagent, the second reagent, or both the first reagent and the second reagent.
  By adding 6-phosphogluconate dehydrogenase, the aforementioned “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺In addition to NAD (P) H produced by the reaction of “enzyme that catalyzes the reaction to produce NAD (P) H, NAD (P) H can also be produced from the reaction by 6-phosphogluconate dehydrogenase, and thus creatine Two molecules of NAD (P) H can be obtained from a single reaction of producing creatine and ATP from creatine phosphate and ADP by a kinase, and as a result, the creatine kinase activity value can be measured with high sensitivity. preferable.
  The 6-phosphogluconate dehydrogenase is not particularly limited, and 6-phosphogluconate and NAD (P)⁺To D-ribulose-5-phosphate, NAD (P) H and H⁺Any enzyme can be used as long as it can catalyze the reaction to produce the enzyme, and its origin is not limited, and examples thereof include those derived from microorganisms, animals, plants, etc., or those prepared by genetic recombination techniques, etc. Can do.
(3) Anti-CK-M subunit antibody, anti-CK-B subunit antibody
  In the present invention, the anti-CK-M subunit antibody which is an antibody against the M-type subunit of creatine kinase or the anti-CK-B subunit antibody which is an antibody against the B-type subunit of creatine kinase is used as the first reagent, When included in two reagents, or both the first reagent and the second reagent, differential measurement of MB type isozyme of creatine kinase, differential measurement of MM type isozyme of creatine kinase, or differential measurement of BB type isozyme of creatine kinase It can be performed.
  The anti-CK-M subunit antibody is not particularly limited as long as it can bind to the M-type subunit of creatine kinase and inhibit the activity of the M-type subunit.
  The anti-CK-B subunit antibody is not particularly limited as long as it can bind to the B-type subunit of creatine kinase and inhibit the activity of this B-type subunit.
  Each of these anti-CK-M subunit antibody and anti-CK-B subunit antibody may be a polyclonal antibody, a monoclonal antibody, or an antiserum, and the origin thereof is not particularly limited.
  Each of these antibodies is an antibody fragment [Fab or F (ab ')₂Etc.], or a single chain antibody (scFv), or an amino acid of an animal species different from an animal immunized with an immunogen (CK-M subunit or CK-B subunit) by a genetic recombination technique or the like An antibody (such as a chimeric antibody, a humanized antibody, or a fully humanized antibody) changed to a sequence may be used.
(4) Other ingredients
  In the present invention, further, other enzymes; substrates of other enzymes; other coenzymes; other ions or salts containing them; proteins such as albumin or casein; nonionic surfactants, anionic surfactants Surfactant, amphoteric surfactant or cationic surfactant; antiseptics such as sodium azide, antibiotics or synthetic antibacterial agents; stabilizers such as other sugars or polymer compounds; activation Agents: Substances related to elimination of measurement interfering substances contained in a sample, such as ascorbate oxidase, or suppression of effects; or other reagent components, etc., as necessary, as necessary, first reagent, second reagent, or first It can be included in the reagent and the second reagent.
  In addition, the density | concentration at the time of including these is although it does not specifically limit, 0.001 to 10% (w / v) is preferable, and 0.01 to 5% (w / v) is especially preferable.
  Specific examples of the surfactants include, for example, sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxy Nonionic surfactants such as ethylene alkyl ether, polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin An amphoteric surfactant such as betaine acetate; or a polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ester; It can be cited anionic surfactants such as ether acetates.
15. Liquid reagent
  In the present invention, it is suitable when the first reagent, the second reagent, or both the first reagent and the second reagent are reagents composed of solutions, that is, liquid reagents.
  It is particularly suitable when the first reagent and the second reagent are liquid reagents.
16.3 Reagents for measuring creatine kinase activity comprising at least 3 reagents
  The creatine kinase activity measuring reagent comprising the first reagent and the second reagent has been described above. However, the creatine kinase activity measuring reagent of the present invention includes the first reagent, the second reagent, and other reagents (one Or two or more reagents). That is, it may be a measurement reagent kit comprising these reagents.
  That is, the reagent for measuring creatine kinase activity of the present invention includes “a first reagent containing glucose at a concentration of 0.5 mM to 10 mM”, “a second reagent not containing glucose or containing glucose at a concentration of 10 mM or less”, and It may consist of “one or more other reagents”.
  And in the creatine kinase activity measuring reagent of this invention, each said item of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 13 and (1)-(4) of 14 The reagent components described in the respective sections of the above, the "first reagent containing glucose at a concentration of 0.5 mM to 10 mM", "second reagent containing no glucose or a glucose of 10 mM or less", And / or may be included in “one or more other reagents”.
17. Combination with other reagents
  In the present invention, the creatine kinase activity measuring reagent can be sold alone or used for measuring the creatine kinase activity value in a sample.
  In addition, the creatine kinase activity measuring reagent of the present invention can be sold in combination with other reagents other than the reagents described above, or used for measuring the creatine kinase activity value in a sample.
  Other reagents other than the above-described reagents include, for example, a buffer solution, a sample diluent, a reagent diluent, a reagent containing a labeling substance, a reagent containing a substance for performing calibration (calibration), or a quality control. The reagent etc. which contain the substance for performing can be mentioned.
II. Method for measuring creatine kinase activity
  In the present invention, the creatine kinase activity measurement method (CK activity measurement method) can be measured using the creatine kinase activity measurement reagent described in the above-mentioned “I. Creatine kinase activity measurement reagent”.
  Details will be described below.
1. Method for measuring creatine kinase activity of the present invention
  The method for measuring creatine kinase activity of the present invention includes the following inventions.
(1) In a creatine kinase activity measurement method in which a first reagent is mixed with a sample, and then a second reagent is mixed with the sample, and the amount of the obtained signal or the amount of change in the signal is measured. A method for measuring creatine kinase activity, comprising glucose at a concentration of 10 mM and the second reagent not containing glucose or containing glucose at a concentration of 10 mM or less.
(2) Adenosine 5′-diphosphate or a salt thereof, an enzyme that catalyzes a reaction for producing ADP and glucose-6-phosphate from ATP and glucose, glucose-6-phosphate and β-nicotinamide adenine dinucleotide ( Oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotinamide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) H⁺Containing β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) or a salt thereof, a thiol compound, and glucose, A first reagent having a concentration of 0.5 mM to 10 mM is mixed with the sample, and then a second reagent containing creatine phosphate or a salt thereof and not containing glucose or containing glucose at a concentration of 10 mM or less. The method for measuring creatine kinase activity according to (1) above, wherein the reagent is mixed, and the amount of signal obtained or the amount of change in signal is measured.
(3) Obtained from the reaction in the mixed solution in which the first reagent is mixed with the sample from the amount of signal obtained from the reaction in the final reaction solution in which the second reagent is mixed with the mixed solution of the first reagent and the sample or the amount of change in the signal. The method for measuring creatine kinase activity according to (1) or (2) above, wherein a creatine kinase activity value in a sample is determined from the difference value by subtracting the amount of signal or the amount of change in signal.
(4) The method for measuring creatine kinase activity according to any one of (1) to (3) above, wherein the glucose concentration of the first reagent is 1.25 mM to 5 mM.
(5) The method for measuring creatine kinase activity according to any one of (1) to (4), wherein the glucose concentration of the first reagent is 2 mM to 5 mM.
(6) The method for measuring creatine kinase activity according to any one of (1) to (5), wherein the second reagent does not contain glucose or contains glucose at a concentration of 5 mM or less.
(7) The method for measuring creatine kinase activity according to any one of (1) to (6), wherein the second reagent does not contain glucose.
(8) The second reagent is glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine. Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The method for measuring creatine kinase activity according to any one of the above (1) to (7), which comprises an enzyme that catalyzes a reaction that produces creatine.
(9) Glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) is converted to 6-phosphogluconic acid And β-nicotinamide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and H⁺The method for measuring creatine kinase activity according to any one of the above (1) to (8), which comprises an enzyme that catalyzes a reaction that produces creatine.
(10) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The method for measuring creatine kinase activity according to any one of (1) to (9) above, wherein the activity value of an enzyme that catalyzes a reaction that produces 199 is 200 Unit / L to 5,000 Unit / L.
(11) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The creatine kinase activity measuring method according to any one of (1) to (10) above, wherein an activity value of an enzyme that catalyzes a reaction that generates lactic acid is 500 Unit / L to 2,000 Unit / L.
(12) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The creatine kinase activity measuring method according to any one of the above (1) to (11), wherein an activity value of an enzyme that catalyzes a reaction that generates γ is 500 Unit / L to 1,000 Unit / L.
(13) From the second reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺(1) to (12), wherein the activity value of the enzyme that catalyzes the reaction that produces the above is adjusted to be 500 Unit / L or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed. ) The creatine kinase activity measuring method according to any one of the above.
(14) From the second reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺(1) to (13), wherein the activity value of the enzyme that catalyzes the reaction that produces the reaction is adjusted to be 800 Unit / L or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed. ) The creatine kinase activity measuring method according to any one of the above.
(15) Glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) of the second reagent, 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The activity value of the enzyme that catalyzes the reaction that produces the above is adjusted so as to be 1,600 Unit / L or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed. The method for measuring creatine kinase activity according to any one of (14).
(16) The creatine kinase activity according to any one of (1) to (15) above, wherein the enzyme that catalyzes a reaction for producing ADP and glucose-6-phosphate from ATP and glucose is hexokinase or glucokinase. Measuring method.
(17) Glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotinamide adenine dinucleotide ( Reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and H⁺The method for measuring creatine kinase activity according to any one of (1) to (16) above, wherein the enzyme that catalyzes a reaction that produces glucose-6-phosphate dehydrogenase.
(18) The method for measuring creatine kinase activity according to any one of (1) to (17), wherein the first reagent and the second reagent are liquid reagents.
(19) β-nicotinamide adenine dinucleotide (oxidized form) or a salt thereof is used instead of β-thionicotinamide adenine dinucleotide (oxidized form) or a salt thereof, and β-nicotinamide adenine dinucleotide (reduced form) or Β-thionicotinamide adenine dinucleotide (reduced form) or salt thereof instead of this salt, β-nicotinamide adenine dinucleotide phosphate (oxidized form) or this salt instead of β-thionicotinamide adenine dinucleotide Phosphoric acid (oxidized form) or a salt thereof is used, or β-thionicotinamide adenine dinucleotide phosphoric acid (reduced form) or a salt thereof is used instead of β-nicotinamide adenine dinucleotide phosphoric acid (reduced form) or a salt thereof The use according to any one of (1) to (18). Rare Chin kinase activity measurement method.
2. sample
  The sample in the creatine kinase activity measurement method of the present invention is not particularly limited as long as it is a sample that may contain creatine kinase (CK).
  Examples of the sample include a sample derived from a human or an animal.
  The sample derived from a human or animal is not particularly limited, for example, body fluid such as blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, or amniotic fluid of human or animal; Excrements such as; organs such as brain, heart, kidney, or liver; tissues such as hair, skin, nails, muscles, or nerves; or cells.
  In addition, since the sample used for measurement in the present invention needs to be a liquid, if the sample is not a liquid, pretreatment such as extraction or solubilization may be performed according to a known method to obtain a liquid sample. Good.
  Further, the sample may be diluted or concentrated as necessary.
3. Specific method for measuring creatine kinase activity
  The method for measuring creatine kinase activity in the present invention will be described more specifically below.
(1) Stage 1
  First, the first reagent and the sample are mixed to prepare a mixed solution.
  The details of the first reagent are as described in the above-mentioned “I. Creatine kinase activity measuring reagent”.
  The amount of the first reagent and the sample to be mixed is the amount of the second reagent, the activity value of creatine kinase contained in the sample, the sensitivity of the signal derived from the color developing substance or the ultraviolet absorbing substance, and the specification of the analyzer to be used. Etc., or may be determined as appropriate according to other conditions.
  In general, the sample may be used in the range of 0.5 to 50 μL, and the first reagent may be used in the range of 20 to 600 μL.
  Incubation is performed after preparation of this mixture.
  The incubation time is not particularly limited, but is usually preferably within 20 minutes, more preferably within 10 minutes, and particularly preferably within 5 minutes.
  Moreover, the temperature at the time of incubation should just be the temperature above the temperature which the said liquid mixture freezes.
  In general, the higher the temperature during the reaction, the higher the reaction rate.
  However, if the temperature is too high, the enzyme involved in the reaction will be denatured and inactivated. Therefore, the incubation temperature should be lower than the temperature at which the enzyme involved in the measurement reaction of the present invention is denatured and deactivated. There is.
  The temperature during the incubation is usually 2 to 70 ° C, preferably 20 to 37 ° C, and more preferably 30 to 37 ° C. If the enzyme involved in the measurement reaction of the present invention is a thermostable enzyme, the temperature may be higher.
  By preparing and incubating the mixed solution of the first reagent and the sample, the creatine kinase contained in the sample comes into contact with the reagent component contained in the first reagent, and the reaction by these components starts.
  When measurement is performed by the double kinetic method described above, when adenylate kinase is present in the sample, this incubation causes a reaction catalyzed by adenylate kinase contained in the sample, and this adenylate kinase is promoted. A signal [NAD (P) H etc.] in an amount corresponding to the activity value of is generated.
  For example, adenylate kinase, “an enzyme that catalyzes the reaction of producing ADP and glucose-6-phosphate from ATP and glucose”, and “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺NAD (P) H is produced by a series of reactions by the “enzyme that catalyzes the reaction that produces”.
  The amount of the generated signal or the amount of change in the signal is measured, and the amount of signal derived from the activity value of adenylate kinase contained in the sample or the amount of change in signal is measured.
  The amount of signal or the amount of change in signal may be measured by measuring the increase or decrease in absorbance during the reaction, that is, by the reaction rate method (rate method) for measuring the reaction rate, or after the reaction has stopped. Alternatively, it may be performed by an end point method (end point method) in which an increase or decrease in absorbance or the like is measured after stopping the reaction.
  When the substance that generates this signal is NAD (P) H [that is, NADH or NADPH], it has absorbance absorption at 340 nm and in the vicinity thereof, so the amount of change per unit time (for example, 1 minute) of absorbance at 340 nm That is, what is necessary is just to obtain | require the change rate (increase rate) of an absorbance.
(2) Stage 2
  Next, the second reagent is mixed with the mixed solution of the first reagent and the sample prepared in Step 1 above. This is the final reaction solution.
  The details of the second reagent are as described in "I. Creatine kinase activity measuring reagent" above.
  The amount of the second reagent to be mixed is the amount of the sample and the first reagent, the activity value of creatine kinase contained in the sample, the sensitivity of the signal derived from the color developing substance or the ultraviolet absorbing substance, and the specification of the analyzer to be used. Etc., or may be determined as appropriate according to other conditions.
  In general, the second reagent may be used in the range of 10 to 400 μL.
  Incubation is performed after preparation of this final reaction solution.
  The incubation time is not particularly limited, but is usually preferably within 20 minutes, more preferably within 10 minutes, and particularly preferably within 5 minutes.
  Moreover, the temperature at the time of incubation should just be the temperature above the temperature which freezes the said last reaction liquid.
  In general, the higher the temperature during the reaction, the higher the reaction rate.
  However, if the temperature is too high, the enzyme involved in the reaction will be denatured and inactivated. Therefore, the incubation temperature should be lower than the temperature at which the enzyme involved in the measurement reaction of the present invention is denatured and deactivated. There is.
  The temperature during the incubation is usually 2 to 70 ° C, preferably 20 to 37 ° C, and more preferably 30 to 37 ° C. If the enzyme involved in the measurement reaction of the present invention is a thermostable enzyme, the temperature may be higher.
  By preparing and incubating the final reaction solution, the reaction in Step 2 is started following the reaction in Step 1, and the reaction for measuring creatine kinase activity is advanced.
  Then, the amount of signal obtained by the progress of this reaction or the amount of change of the signal, that is, the amount of signal corresponding to the activity value of creatine kinase contained in the sample or the amount of change of signal is measured.
  When the substance that generates this signal is NAD (P) H [that is, NADH or NADPH], it has absorbance absorption at 340 nm and in the vicinity thereof, so the amount of change per unit time (for example, 1 minute) of absorbance at 340 nm That is, what is necessary is just to obtain | require the change rate (increase rate) of an absorbance.
  In addition, calculating the activity value of creatine kinase contained in a sample from the amount of signal measured or the amount of change in signal is calculated from the measured absorbance based on the molar extinction coefficient of the substance that generates the signal. Or a method of calculating by comparing with the absorbance of a standard substance whose activity value or concentration is known.
  Note that the amount of signal obtained by the reaction in Step 2 or the amount of change in signal is the amount of signal derived from the activity value of creatine kinase contained in the sample or the amount of change in signal, and included in the sample. This is a combination of the amount of signal derived from the adenylate kinase activity value or the amount of change in signal.
  On the other hand, when the measurement is performed by the double kinetic method, the amount of signal obtained by the reaction in Step 1 or the amount of change in the signal is equal to the activity value of adenylate kinase contained in the sample. The amount of signal derived or the amount of change in signal.
  Therefore, when performing measurement by this double kinetic method, the amount of signal or the amount of change in signal obtained in Step 1 is subtracted from the amount of signal or the amount of change in signal obtained in Step 2. From the difference value, the activity value of creatine kinase contained in the sample is determined.
  As a result, it is possible to obtain an accurate activity value of creatine kinase from which a positive error caused by the presence of adenylate kinase in the sample is subtracted.
(3) Note that the measurement operation in each of the above steps 1 and 2 may be performed by the measurer himself or herself, or may be performed using an apparatus such as an automatic analyzer.
  In addition, as described in “Creatin kinase activity measurement reagent comprising 16.3 reagents or more” in “I. Creatine kinase activity measurement reagent”, the creatine kinase activity measurement reagent comprises a first reagent, a second reagent, and When it is composed of other reagents (one or two or more reagents), that is, when it is composed of three or more reagents, the number of steps necessary to perform the measurement using these reagents (two steps if necessary) Alternatively, the measurement reaction may be performed through three or more stages) to measure the creatine kinase activity in the sample.
(4) Specific examples of the reaction in Step 1 and the reaction in Step 2 are shown below.

(5) In the present invention, NAD⁺Instead of Thio NAD⁺May be used, thio-NADH may be used instead of NADH, and NADP⁺Instead of thio NADP⁺Or thio-NADPH may be used instead of NADPH.
  In the present invention, “glucose-6-phosphate and NAD (P)⁺To 6-phosphogluconic acid and NAD (P) H and H⁺NAD in both “enzymes that catalyze reactions that produce” and 6-phosphogluconate dehydrogenase⁺Or NADP⁺Instead of Thio NAD⁺Or Thio NADP⁺In this case, in place of NADH or NADPH, thioNADH or thioNADPH is produced by a reaction catalyzed by these enzymes, respectively.
4). Effect of the method for measuring creatine kinase activity of the present invention
  According to the method for measuring creatine kinase activity of the present invention, creatine kinase activity can be accurately measured for a long period of time. That is, an accurate measured value of creatine kinase activity can be provided over a long period of time.
  In addition, the linearity in the high value range can be maintained for a long time.
  Furthermore, it is possible to apply a double kinetic method capable of correcting a positive error caused by adenylate kinase in a hemolyzed sample or the like.
III. Stabilization method of creatine kinase activity measuring reagent
  This is a method for stabilizing the creatine kinase activity measuring reagent (CK activity measuring reagent) of the present invention, and the measurement reagent can be stabilized by the configuration of the creatine kinase activity measuring reagent of the present invention.
  Details will be described below.
1. Method for stabilizing reagent for measuring creatine kinase activity of the present invention
  The method for stabilizing the reagent for measuring creatine kinase activity of the present invention includes the following inventions.
(1) A method for stabilizing a creatine kinase activity measuring reagent comprising a first reagent and a second reagent, wherein the first reagent contains glucose at a concentration of 0.5 mM to 10 mM, and the second reagent does not contain glucose Or a method for stabilizing a reagent for measuring creatine kinase activity, comprising glucose at a concentration of 10 mM or less.
(2) A method for stabilizing a creatine kinase activity measuring reagent comprising a first reagent and a second reagent, wherein the first reagent is adenosine 5′-diphosphate or a salt thereof, ATP and glucose to ADP and glucose-6 Enzymes that catalyze the reaction to produce phosphate, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid β-nicotinamide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and H⁺Containing β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) or a salt thereof, a thiol compound, and glucose, The concentration of is 0.5 mM to 10 mM, the second reagent contains creatine phosphate or a salt thereof, and does not contain glucose or contains glucose at a concentration of 10 mM or less. A method for stabilizing a reagent for measuring creatine kinase activity.
(3) The method for stabilizing a creatine kinase activity measuring reagent according to (1) or (2) above, wherein the glucose concentration of the first reagent is 1.25 mM to 5 mM.
(4) The method for stabilizing a creatine kinase activity measuring reagent according to any one of (1) to (3), wherein the glucose concentration of the first reagent is 2 mM to 5 mM.
(5) The method for stabilizing a creatine kinase activity measuring reagent according to any one of (1) to (4), wherein the second reagent does not contain glucose or contains glucose at a concentration of 5 mM or less.
(6) The method for stabilizing a creatine kinase activity measuring reagent according to any one of (1) to (5), wherein the second reagent does not contain glucose.
(7) The second reagent is glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine. Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The method for stabilizing a reagent for measuring creatine kinase activity according to any one of the above (1) to (6), which comprises an enzyme that catalyzes a reaction that produces creatine.
(8) The first reagent and the second reagent are changed from glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid. And β-nicotinamide adenine dinucleotide (reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and H⁺The method for stabilizing a reagent for measuring creatine kinase activity according to any one of the above (1) to (7), which comprises an enzyme that catalyzes a reaction that produces creatine.
(9) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The method for stabilizing a reagent for measuring creatine kinase activity according to any one of (1) to (8) above, wherein the activity value of an enzyme that catalyzes a reaction that generates 199 is 200 Unit / L to 5,000 Unit / L.
(10) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The method for stabilizing a reagent for measuring creatine kinase activity according to any one of (1) to (9) above, wherein an activity value of an enzyme that catalyzes a reaction that generates γ is 500 Unit / L to 2,000 Unit / L.
(11) From the first reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The method for stabilizing a reagent for measuring creatine kinase activity according to any one of (1) to (10) above, wherein the activity value of an enzyme that catalyzes a reaction that produces γ is 500 Unit / L to 1,000 Unit / L.
(12) Glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) of the second reagent, 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺(1) to (11), wherein the activity value of the enzyme that catalyzes the reaction that produces the reaction is adjusted to be 500 Unit / L or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed The method for stabilizing a creatine kinase activity measuring reagent according to any one of the above.
(13) From the second reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺(1) to (12), wherein the activity value of the enzyme that catalyzes the reaction that produces the above is adjusted to be 800 Unit / L or more in the final reaction liquid in which the sample, the first reagent, and the second reagent are mixed The method for stabilizing a creatine kinase activity measuring reagent according to any one of the above.
(14) From the second reagent, glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotine Amidoadenine dinucleotide (reduced) or β-nicotinamide adenine dinucleotide phosphate (reduced) and H⁺The activity value of the enzyme that catalyzes the reaction that produces the above is adjusted so as to be 1,600 Unit / L or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed. (13) The method for stabilizing a creatine kinase activity measuring reagent according to any one of (13).
(15) The creatine kinase activity according to any one of (1) to (14) above, wherein the enzyme that catalyzes a reaction for producing ADP and glucose-6-phosphate from ATP and glucose is hexokinase or glucokinase. Stabilization method of measurement reagent.
(16) Glucose-6-phosphate and β-nicotinamide adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) to 6-phosphogluconic acid and β-nicotinamide adenine dinucleotide ( Reduced form) or β-nicotinamide adenine dinucleotide phosphate (reduced form) and H⁺The method for stabilizing a reagent for measuring creatine kinase activity according to any one of (1) to (15), wherein the enzyme that catalyzes a reaction that produces glucose-6-phosphate dehydrogenase.
(17) The method for stabilizing a creatine kinase activity measuring reagent according to any one of (1) to (16), wherein the first reagent and the second reagent are liquid reagents.
(18) β-nicotinamide adenine dinucleotide (reduced form) or β-thionicotinamide adenine dinucleotide (reduced form) or β-thionicotinamide adenine dinucleotide (oxidized form) or salt thereof instead of β-nicotinamide adenine dinucleotide (oxidized form) or salt thereof Β-thionicotinamide adenine dinucleotide (reduced form) or salt thereof instead of this salt, β-nicotinamide adenine dinucleotide phosphate (oxidized form) or this salt instead of β-thionicotinamide adenine dinucleotide Phosphoric acid (oxidized form) or a salt thereof is used, or β-thionicotinamide adenine dinucleotide phosphoric acid (reduced form) or a salt thereof is used instead of β-nicotinamide adenine dinucleotide phosphoric acid (reduced form) or a salt thereof The use according to any one of (1) to (17). Method for stabilizing a rare Chin kinase activity measuring reagent.
  As described above, in the creatine kinase activity measurement reagent comprising the first reagent and the second reagent, the first reagent contains glucose at a concentration of 0.5 mM to 10 mM, and the second reagent does not contain glucose or is 10 mM or less. By containing a concentration of glucose, the creatine kinase activity measuring reagent can be stabilized.
  That is, the method for stabilizing the creatine kinase activity measuring reagent of the present invention can be achieved by the creatine kinase activity measuring reagent of the present invention.
  Therefore, the details of the method for stabilizing the creatine kinase activity measuring reagent of the present invention are as described in the above-mentioned “I. Creatine kinase activity measuring reagent”.
2. Effect of the method for stabilizing a creatine kinase activity measuring reagent of the present invention
  According to the method for stabilizing a creatine kinase activity measuring reagent of the present invention, a creatine kinase activity measuring reagent can be kept stable for a long period of time. Thereby, creatine kinase activity can be accurately measured for a long period of time. That is, an accurate measured value of creatine kinase activity can be provided over a long period of time.
  Moreover, in the reagent for measuring creatine kinase activity, linearity in a high value range can be maintained for a long time.

６．考察
（１） 表１において、試料がコントロール血清−１、又はコントロール血清−２であるとき、第１試薬が３７℃で７日間保存した第１試薬〔従来発明（２５ｍＭ）〕である場合には、５℃で７日間保存した第１試薬〔対照試薬〕の場合に比べて、測定値が３．１％〜３．４％低い値となっている。
これに対して、第１試薬が３７℃で７日間保存した第１試薬〔本発明（１ｍＭ）〕、第１試薬〔本発明（５ｍＭ）〕、又は第１試薬〔本発明（１０ｍＭ）〕である場合には、５℃で７日間保存した第１試薬〔対照試薬〕の場合に比べて、測定値は２．１％〜２．６％低い値となっている。
この第１試薬を３７℃又は５℃で７日間保存した結果より、第１試薬のグルコース濃度が１ｍＭ、５ｍＭ、又は１０ｍＭである場合は、第１試薬のグルコース濃度が２５ｍＭである場合に比較して、測定試薬保存時の測定値の低下度が小さく、安定化されていることが分かる。
（２） 表２において、試料が試料−１〜試料−１０であるとき、第１試薬が３７℃で７日間保存した第１試薬〔従来発明（２５ｍＭ）〕である場合には、５℃で７日間保存した第１試薬〔対照試薬〕の場合に比べて、測定値が３．３％〜４５．３％低い値となっている。
特に、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きく、例えば、試料−８においてはその測定値は３５．０％低く、試料−９においてはその測定値は４０．２％低く、そして試料−１０においてはその測定値は４５．３％低いものとなっている。
これに対して、第１試薬が３７℃で７日間保存した第１試薬〔本発明（１ｍＭ）〕、第１試薬〔本発明（５ｍＭ）〕、又は第１試薬〔本発明（１０ｍＭ）〕である場合には、５℃で７日間保存した第１試薬〔対照試薬〕の場合に比べて、測定値は１．３％〜１７．７％低い値となっている。
この場合も、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きいが、その測定値の低下度は、例えば、試料−８では８．５％〜１２．９％であり、試料−９では９．７％〜１５．１％であり、そして試料−１０では１１．６％〜１７．７％である。
この第１試薬を３７℃又は５℃で７日間保存した結果より、第１試薬のグルコース濃度が１ｍＭ、５ｍＭ、又は１０ｍＭである場合は、第１試薬のグルコース濃度が２５ｍＭである場合に比較して、測定試薬保存時の測定値の低下度には大きな差異があり、測定値の低下が抑制され、安定化されていることが分かる。
（３） 以上のことより、第１試薬が０．５ｍＭ〜１０ｍＭの濃度のグルコースを含み、かつ第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことを特徴とする本発明のクレアチンキナーゼ活性測定試薬は、従来のクレアチンキナーゼ活性測定試薬に比べ、３７℃という非常に過酷な条件での保存時においても、その測定値の低下が抑制され、安定化されたものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間安定に保つことができ、かつ長期間正確にクレアチンキナーゼ活性の測定を行うことができるという効果を有するものであることが確かめられた。
また、本発明のクレアチンキナーゼ活性測定試薬は、従来のクレアチンキナーゼ活性測定試薬に比べ、特にクレアチンキナーゼ活性値が高い高値域における測定値の低下の抑制に効果があるものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間、高値域における直線性を維持することができるという効果を有するものであることが確かめられた。
〔実施例２〕（本発明の保存時の安定化の効果の確認−２）
本発明のクレアチンキナーゼ活性測定試薬における保存時の安定化効果を確認した。
具体的には、本発明のクレアチンキナーゼ活性測定試薬の第１試薬を、５℃、３７℃それぞれで７日間保存した際の安定化の効果を確認した。
１．クレアチンキナーゼ活性測定試薬
（１）第１試薬
（ａ）第１試薬〔従来発明（０ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ６．５（２０℃）に調整して、グルコースを含まない（つまり０ｍＭである）、第１試薬〔従来発明（０ｍＭ）〕を調製した。
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 １，０００Ｕｎｉｔ／Ｌ
ヘキソキナーゼ〔ロシュ・ダイアグノスティックス社、Ｙｅａｓｔ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ３，０００Ｕｎｉｔ／Ｌ
イミダゾール １００ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
Ｎ−アセチル−Ｌ−システイン〔ロシュ・ダイアグノスティックス社〕 ２７ｍＭ
ＮＡＤＰ（酸化型）二ナトリウム塩 ２．７ｍＭ
ＡＭＰ二ナトリウム塩 ６．７ｍＭ
ジアデノシンペンタリン酸三リチウム塩 １３μＭ
二亜硫酸カリウム〔ナカライテスク社〕 １．４ｍＭ
（ｂ）第１試薬〔従来発明（０．０７８１ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ６．５（２０℃）に調整して、０．０７８１ｍＭの濃度のグルコースを含む、第１試薬〔従来発明（０．０７８１ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ０．０７８１ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 １，０００Ｕｎｉｔ／Ｌ
ヘキソキナーゼ〔ロシュ・ダイアグノスティックス社、Ｙｅａｓｔ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ３，０００Ｕｎｉｔ／Ｌ
イミダゾール １００ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
Ｎ−アセチル−Ｌ−システイン〔ロシュ・ダイアグノスティックス社〕 ２７ｍＭ
ＮＡＤＰ（酸化型）二ナトリウム塩 ２．７ｍＭ
ＡＭＰ二ナトリウム塩 ６．７ｍＭ
ジアデノシンペンタリン酸三リチウム塩 １３μＭ
二亜硫酸カリウム〔ナカライテスク社〕 １．４ｍＭ
（ｃ）第１試薬〔従来発明（０．１５６ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ６．５（２０℃）に調整して、０．１５６ｍＭの濃度のグルコースを含む、第１試薬〔従来発明（０．１５６ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ０．１５６ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 １，０００Ｕｎｉｔ／Ｌ
ヘキソキナーゼ〔ロシュ・ダイアグノスティックス社、Ｙｅａｓｔ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ３，０００Ｕｎｉｔ／Ｌ
イミダゾール １００ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
Ｎ−アセチル−Ｌ−システイン〔ロシュ・ダイアグノスティックス社〕 ２７ｍＭ
ＮＡＤＰ（酸化型）二ナトリウム塩 ２．７ｍＭ
ＡＭＰ二ナトリウム塩 ６．７ｍＭ
ジアデノシンペンタリン酸三リチウム塩 １３μＭ
二亜硫酸カリウム〔ナカライテスク社〕 １．４ｍＭ
（ｄ）第１試薬〔従来発明（０．３１３ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ６．５（２０℃）に調整して、０．３１３ｍＭの濃度のグルコースを含む、第１試薬〔従来発明（０．３１３ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ０．３１３ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 １，０００Ｕｎｉｔ／Ｌ
ヘキソキナーゼ〔ロシュ・ダイアグノスティックス社、Ｙｅａｓｔ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ３，０００Ｕｎｉｔ／Ｌ
イミダゾール １００ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
Ｎ−アセチル−Ｌ−システイン〔ロシュ・ダイアグノスティックス社〕 ２７ｍＭ
ＮＡＤＰ（酸化型）二ナトリウム塩 ２．７ｍＭ
ＡＭＰ二ナトリウム塩 ６．７ｍＭ
ジアデノシンペンタリン酸三リチウム塩 １３μＭ
二亜硫酸カリウム〔ナカライテスク社〕 １．４ｍＭ
（ｅ）第１試薬〔本発明（０．６２５ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ６．５（２０℃）に調整して、０．６２５ｍＭの濃度のグルコースを含む、第１試薬〔本発明（０．６２５ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ０．６２５ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 １，０００Ｕｎｉｔ／Ｌ
ヘキソキナーゼ〔ロシュ・ダイアグノスティックス社、Ｙｅａｓｔ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ３，０００Ｕｎｉｔ／Ｌ
イミダゾール １００ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
Ｎ−アセチル−Ｌ−システイン〔ロシュ・ダイアグノスティックス社〕 ２７ｍＭ
ＮＡＤＰ（酸化型）二ナトリウム塩 ２．７ｍＭ
ＡＭＰ二ナトリウム塩 ６．７ｍＭ
ジアデノシンペンタリン酸三リチウム塩 １３μＭ
二亜硫酸カリウム〔ナカライテスク社〕 １．４ｍＭ
（ｆ）第１試薬〔本発明（１．２５ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ６．５（２０℃）に調整して、１．２５ｍＭの濃度のグルコースを含む、第１試薬〔本発明（１．２５ｍＭ）〕を調製した。
Ｄ−グルコース（無水） １．２５ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 １，０００Ｕｎｉｔ／Ｌ
ヘキソキナーゼ〔ロシュ・ダイアグノスティックス社、Ｙｅａｓｔ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ３，０００Ｕｎｉｔ／Ｌ
イミダゾール １００ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
Ｎ−アセチル−Ｌ−システイン〔ロシュ・ダイアグノスティックス社〕 ２７ｍＭ
ＮＡＤＰ（酸化型）二ナトリウム塩 ２．７ｍＭ
ＡＭＰ二ナトリウム塩 ６．７ｍＭ
ジアデノシンペンタリン酸三リチウム塩 １３μＭ
二亜硫酸カリウム〔ナカライテスク社〕 １．４ｍＭ
（ｇ）第１試薬〔本発明（２．５ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ６．５（２０℃）に調整して、２．５ｍＭの濃度のグルコースを含む、第１試薬〔本発明（２．５ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ２．５０ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 １，０００Ｕｎｉｔ／Ｌ
ヘキソキナーゼ〔ロシュ・ダイアグノスティックス社、Ｙｅａｓｔ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ３，０００Ｕｎｉｔ／Ｌ
イミダゾール １００ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
Ｎ−アセチル−Ｌ−システイン〔ロシュ・ダイアグノスティックス社〕 ２７ｍＭ
ＮＡＤＰ（酸化型）二ナトリウム塩 ２．７ｍＭ
ＡＭＰ二ナトリウム塩 ６．７ｍＭ
ジアデノシンペンタリン酸三リチウム塩 １３μＭ
二亜硫酸カリウム〔ナカライテスク社〕 １．４ｍＭ
（ｈ）第１試薬〔本発明（５ｍＭ）〕
前記の実施例１の１の（１）の（ｂ）の「第１試薬〔本発明５ｍＭ〕」を、「第１試薬〔本発明（５ｍＭ）〕」として用いた。
（ｉ）第１試薬〔本発明（１０ｍＭ）〕
前記の実施例１の１の（１）の（ｃ）の「第１試薬〔本発明１０ｍＭ〕」を、「第１試薬〔本発明（１０ｍＭ）〕」として用いた。
（ｊ）第１試薬〔対照試薬〕
市販のクレアチンキナーゼ活性測定試薬「アキュラスオート ＣＫ−ＪＳ」の第１試薬〔製造販売元：シノテスト社、製造番号：Ｅ９１３〕を、第１試薬〔対照試薬〕とした。
（２）第２試薬
（ａ）第２試薬〔本発明（０ｍＭ）〕
前記の実施例１の１の（２）の（ａ）の「第２試薬〔本発明０ｍＭ〕」を、「第２試薬〔本発明（０ｍＭ）〕」として用いた。
（ｂ）第２試薬〔対照試薬〕
市販のクレアチンキナーゼ活性測定試薬「アキュラスオート ＣＫ−ＪＳ」の第２試薬〔製造販売元：シノテスト社、製造番号：Ｅ９２３〕を、第２試薬〔対照試薬〕とした。
２．試料
（１）希釈試料
ヒト血清試料にラビットのクレアチンキナーゼＭＭアイソザイムを添加したものを調製し（添加血清試料）、この添加血清試料を、「試料希釈液」（５．０ｍＭコハク酸緩衝液〔ｐＨ７．０（２０℃）〕）により、それぞれ１／１０、２／１０、３／１０、４／１０、５／１０、６／１０、７／１０、８／１０、及び９／１０となるように希釈し、下記の「試料−ａ」〜「試料−ｉ」を調製した。
また、前記の添加血清試料を、「試料−ｊ」（１０／１０）とした。
（ａ）「試料−ａ」： 添加血清試料を１／１０希釈
（ｂ）「試料−ｂ」： 添加血清試料を２／１０希釈
（ｃ）「試料−ｃ」： 添加血清試料を３／１０希釈
（ｄ）「試料−ｄ」： 添加血清試料を４／１０希釈
（ｅ）「試料−ｅ」： 添加血清試料を５／１０希釈
（ｆ）「試料−ｆ」： 添加血清試料を６／１０希釈
（ｇ）「試料−ｇ」： 添加血清試料を７／１０希釈
（ｈ）「試料−ｈ」： 添加血清試料を８／１０希釈
（ｉ）「試料−ｉ」： 添加血清試料を９／１０希釈
（ｊ）「試料−ｊ」： 添加血清試料
（２）不含試料
前記（１）における「試料希釈液」を、クレアチンキナーゼを含まない、クレアチンキナーゼ活性値が０Ｕｎｉｔ／Ｌ（単位／Ｌ）の試料、すなわち「不含試料」とした。
３．試薬の保存
（１）３７℃での保存
前記１の（１）の（ａ）の第１試薬〔本発明（１ｍＭ）〕、（ｂ）の第１試薬〔本発明（５ｍＭ）〕、（ｃ）の第１試薬〔本発明（１０ｍＭ）〕、及び（ｄ）の第１試薬〔従来発明（２５ｍＭ）〕については、それぞれ３７℃で暗所に７日間保存した。
（２）５℃での保存
前記１の（１）の（ａ）の第１試薬〔本発明（１ｍＭ）〕、（ｂ）の第１試薬〔本発明（５ｍＭ）〕、（ｃ）の第１試薬〔本発明（１０ｍＭ）〕、及び（ｄ）の第１試薬〔従来発明（２５ｍＭ）〕については、５℃においても、それぞれ暗所に７日間保存した。
また、前記１の（１）の（ｅ）の第１試薬〔対照試薬〕、前記１の（２）の（ａ）の第２試薬〔本発明（０ｍＭ）〕、及び（ｂ）の第２試薬〔対照試薬〕についても、それぞれ５℃で暗所に７日間保存した。
４．試料のクレアチンキナーゼ活性値の測定
前記２の各試料のクレアチンキナーゼ活性値の測定は、東芝メディカルシステムズ社の自動分析装置ＴＢＡ−１２０ＦＲを使用して、ダブルカイネティック法により行った。
（１） 前記２の（１）〜（２）のそれぞれの試料５．６μＬに、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ａ）の第１試薬〔従来発明（０ｍＭ）〕の１６０μＬを添加し、３７℃で反応させた。
（２） 次に、８ポイント（第１試薬添加後１３６．１８秒）から１６ポイント（第１試薬添加後２８０．４６秒）に掛けての吸光度変化量を主波長３４０ｎｍ、副波長４０５ｎｍにて測定した〔「測光１」〕。（試料中に含まれていたアデニル酸キナーゼの活性値に応じた吸光度変化量の測定）
（３） 次に、１６ポイント（第１試薬添加後２８０．４６秒）から１７ポイント（第１試薬添加後２９８．４９秒）の間に、前記３の（２）で５℃にて７日間保存した前記１の（２）の（ａ）の第２試薬〔本発明（０ｍＭ）〕の４０μＬを添加し、３７℃で反応させた。
（４） 次に、２４ポイント（第１試薬添加後４２４．７４秒）から３３ポイント（第１試薬添加後５８７．０５秒）に掛けての吸光度変化量を主波長３４０ｎｍ、副波長４０５ｎｍにて測定した〔「測光２」〕。（試料中に含まれていたクレアチンキナーゼの活性値に応じた吸光度変化量と、試料中に含まれていたアデニル酸キナーゼの活性値に応じた吸光度変化量が合わさった吸光度変化量の測定）
（５） 次に、試料に含まれていたクレアチンキナーゼの活性値を、下記の計算式により求めた。
クレアチンキナーゼ活性値（Ｕｎｉｔ／Ｌ）＝｛（ΔＡ_２−ΔＲＢ_２）−ｃ×（ΔＡ_１−ΔＲＢ_１）｝×（Ｓ＋Ｖ_１＋Ｖ_２）×１０^６÷（Ｋ×ｄ×Ｓ）＝｛（ΔＡ_２−ΔＲＢ_２）−０．８０５×（ΔＡ_１−ΔＲＢ_１）｝×５．８３×１０^３
なお、この計算式において、ΔＡ_１は「測光１」において測定した１分間当りの吸光度変化量（Ａｂｓ／ｍｉｎ）を、ΔＡ_２は「測光２」において測定した１分間当りの吸光度変化量（Ａｂｓ／ｍｉｎ）を、ΔＲＢ_１は試料が前記の「不含試料」であるときの「測光１」において測定した１分間当りの吸光度変化量〔試薬盲検値〕（Ａｂｓ／ｍｉｎ）を、ΔＲＢ_２は試料が前記の「不含試料」であるときの「測光２」において測定した１分間当りの吸光度変化量〔試薬盲検値〕（Ａｂｓ／ｍｉｎ）を、Ｓは試料の量〔５．６μＬ〕を、Ｖ_１は第１試薬の量〔１６０μＬ〕を、Ｖ_２は第２試薬の量〔４０μＬ〕を、ｃは液量補正係数〔（Ｓ＋Ｖ_１）÷（Ｓ＋Ｖ_１＋Ｖ_２）＝０．８０５〕を、ＫはＮＡＤＰＨの理論モル吸光係数〔６．３０×１０^３Ｌ・ｍｏｌｅ^−１・ｃｍ^−１（主波長：３４０ｎｍ、副波長：４０５ｎｍ）〕、及びｄはセルの光路長〔１．００ｃｍ〕を表す。
（６） また、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（２）で５℃にて７日間保存した前記１の（１）の（ａ）の第１試薬〔従来発明（０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第１試薬として前記の第１試薬〔従来発明（０ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（７） 更に、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ｂ）の第１試薬〔従来発明（０．０７８１ｍＭ）〕を用いること、又は前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｂ）の第１試薬〔従来発明（０．０７８１ｍＭ）〕を用いること以外は、前記（１）〜（６）の通りに操作を行い、第１試薬として前記の第１試薬〔従来発明（０．０７８１ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（８） また、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ｃ）の第１試薬〔従来発明（０．１５６ｍＭ）〕を用いること、又は前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｃ）の第１試薬〔従来発明（０．１５６ｍＭ）〕を用いること以外は、前記（１）〜（６）の通りに操作を行い、第１試薬として前記の第１試薬〔従来発明（０．１５６ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（９） 更に、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ｄ）の第１試薬〔従来発明（０．３１３ｍＭ）〕を用いること、又は前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｄ）の第１試薬〔従来発明（０．３１３ｍＭ）〕を用いること以外は、前記（１）〜（６）の通りに操作を行い、第１試薬として前記の第１試薬〔従来発明（０．３１３ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１０） また、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ｅ）の第１試薬〔本発明（０．６２５ｍＭ）〕を用いること、又は前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｅ）の第１試薬〔本発明（０．６２５ｍＭ）〕を用いること以外は、前記（１）〜（６）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（０．６２５ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１１） 更に、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ｆ）の第１試薬〔本発明（１．２５ｍＭ）〕を用いること、又は前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｆ）の第１試薬〔本発明（１．２５ｍＭ）〕を用いること以外は、前記（１）〜（６）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（１．２５ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１２） また、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ｇ）の第１試薬〔本発明（２．５ｍＭ）〕を用いること、又は前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｇ）の第１試薬〔本発明（２．５ｍＭ）〕を用いること以外は、前記（１）〜（６）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（２．５ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１３） 更に、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ｈ）の第１試薬〔本発明（５ｍＭ）〕を用いること、又は前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｈ）の第１試薬〔本発明（５ｍＭ）〕を用いること以外は、前記（１）〜（６）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（５ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１４） また、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（１）の（ｉ）の第１試薬〔本発明（１０ｍＭ）〕を用いること、又は前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｉ）の第１試薬〔本発明（１０ｍＭ）〕を用いること以外は、前記（１）〜（６）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（１０ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１５） 更に、前記（１）において、前記３の（１）で３７℃にて７日間保存した第１試薬〔従来発明（０ｍＭ）〕に替えて、前記３の（２）で５℃にて７日間保存した前記１の（１）の（ｊ）の第１試薬〔対照試薬〕を用いること、及び、前記（３）において、前記３の（２）で５℃にて７日間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（２）で５℃にて７日間保存した前記１の（２）の（ｂ）の第２試薬〔対照試薬〕を用いること、以外は、前記（１）〜（５）の通りに操作を行い、第１試薬として前記の第１試薬〔対照試薬〕を用い、そして第２試薬として前記の第２試薬〔対照試薬〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
５．測定結果
前記４において各試料のクレアチンキナーゼ活性値の測定を行って得られた結果のうち、前記の第１試薬〔従来発明（０ｍＭ）〕〜第１試薬〔本発明（１０ｍＭ）〕の保存温度が５℃の場合のものを表３に示し、そして、前記の第１試薬〔従来発明（０ｍＭ）〕〜第１試薬〔本発明（１０ｍＭ）〕の保存温度が３７℃の場合のものを表４に示した。
なお、この表３及び表４において、「〔１〕活性値」の欄に示した値は各試料のクレアチンキナーゼの活性値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔２〕活性値差」の欄に示した値は、各試料における「〔１〕活性値」の欄の値から、同じ試料の第１試薬及び第２試薬ともが「対照試薬」である場合の「〔１〕活性値」の欄の値を差し引いた差の値〔Ｕｎｉｔ／Ｌ〕を示す。
そして、「〔３〕相対比率」の欄に示した値は、各試料における「〔１〕活性値」の欄の値を、同じ試料の第１試薬及び第２試薬ともが「対照試薬」である場合の「〔１〕活性値」の欄の値で除した値をパーセント表示で示す。

６．考察
（１） 表３より、前記の第１試薬〔従来発明（０ｍＭ）〕〜第１試薬〔本発明（１０ｍＭ）〕の保存温度が５℃であるとき、第１試薬が５℃で７日間保存した第１試薬〔従来発明（０ｍＭ）〕〜第１試薬〔従来発明（０．３１３ｍＭ）〕である場合には、その測定値は本来のクレアチンキナーゼ活性値よりも大幅に低いものであり、試料中のクレアチンキナーゼ活性値の測定を定量的に行うことができないものであることが分かる。
これに対して、第１試薬が５℃で７日間保存した第１試薬〔本発明（０．６２５ｍＭ）〕〜第１試薬〔本発明（１０ｍＭ）〕である場合には、高値域まで（９／１０希釈又は１０／１０の試料まで）試料中のクレアチンキナーゼ活性値の測定を定量的に行うことができるものであることが分かる。
この第１試薬を３７℃又は５℃で７日間保存した結果より、第１試薬が０．５ｍＭ〜１０ｍＭの濃度のグルコースを含み、かつ第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことを特徴とする本発明のクレアチンキナーゼ活性測定試薬においては、試料中のクレアチンキナーゼ活性値の測定を高値域まで定量的に行うことができるが、第１試薬がグルコースを含まないか又はそのグルコース濃度が１０ｍＭ未満の場合には、試料中のクレアチンキナーゼ活性値の測定を定量的に行うことができないことが確かめられた。
（２） また、表４より、前記の第１試薬〔従来発明（０ｍＭ）〕〜第１試薬〔本発明（１０ｍＭ）〕の保存温度が３７℃であるとき、第１試薬が３７℃で７日間保存した第１試薬〔従来発明（０ｍＭ）〕〜第１試薬〔従来発明（０．３１３ｍＭ）〕である場合には、その測定値はやはり本来のクレアチンキナーゼ活性値よりも大幅に低いものであり、試料中のクレアチンキナーゼ活性値の測定を定量的に行うことができないものであることが分かる。
これに対して、第１試薬が３７℃で７日間保存した第１試薬〔本発明（０．６２５ｍＭ）〕〜第１試薬〔本発明（１０ｍＭ）〕である場合には、高値域まで試料中のクレアチンキナーゼ活性値の測定を定量的に行うことができるものであることが分かる。
また、第１試薬が５℃で７日間保存した第１試薬〔対照試薬〕である場合の測定値に対する、第１試薬が３７℃で７日間保存した第１試薬〔本発明（０．６２５ｍＭ）〕〜第１試薬〔本発明（１０ｍＭ）〕である場合の測定値の相対比率は、試料−ｊにおいて、第１試薬〔本発明（０．６２５ｍＭ）〕では７２．２％であり、第１試薬〔本発明（１．２５ｍＭ）〕では９４．１％であり、第１試薬〔本発明（２．５ｍＭ）〕では９５．３％であり、第１試薬〔本発明（５ｍＭ）〕では９５．１％であり、第１試薬〔本発明（１０ｍＭ）〕では９４．３％である。
この第１試薬を３７℃又は５℃で７日間保存した結果より、第１試薬が第１試薬〔本発明（０．６２５ｍＭ）〕〜第１試薬〔本発明（１０ｍＭ）〕である場合には、測定試薬保存時の測定値の低下が抑制され、安定化されていることが分かる。
すなわち、第１試薬が０．５ｍＭ〜１０ｍＭの濃度のグルコースを含み、かつ第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことを特徴とする本発明のクレアチンキナーゼ活性測定試薬は、第１試薬を３７℃で７日間保存した場合においても、試料中のクレアチンキナーゼ活性値の測定を高値域まで定量的に行うことができるが、第１試薬がグルコースを含まないか又はそのグルコース濃度が１０ｍＭ未満の場合には、試料中のクレアチンキナーゼ活性値の測定を定量的に行うことができないことが確かめられた。
また、本実施例により、本発明のクレアチンキナーゼ活性測定試薬が、３７℃という非常に過酷な条件での保存時においても、その測定値の低下が抑制され、安定化されたものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間安定に保つことができ、かつ長期間正確にクレアチンキナーゼ活性の測定を行うことができるという効果を有するものであることが改めて確かめられた。
更に、本実施例により、本発明のクレアチンキナーゼ活性測定試薬が、特にクレアチンキナーゼ活性値が高い高値域における測定値の低下の抑制に効果があるものであり、長期間、高値域における直線性を維持することができるという効果を有するものであることが改めて確かめられた。
〔実施例３〕（本発明の保存時の安定化の効果の確認−３）
本発明のクレアチンキナーゼ活性測定試薬における保存時の安定化効果を確認した。
具体的には、本発明のクレアチンキナーゼ活性測定試薬の第２試薬を、３７℃で７日間保存した際の安定化の効果を確認した。
１．クレアチンキナーゼ活性測定試薬
（１）第１試薬
・第１試薬〔本発明（５ｍＭ）〕
前記の実施例１の１の（１）の（ｂ）の「第１試薬〔本発明５ｍＭ〕」を、「第１試薬〔本発明（５ｍＭ）〕」として用いた。
（２）第２試薬
（ａ）第２試薬〔本発明（０ｍＭ）〕
前記の実施例１の１の（２）の（ａ）の「第２試薬〔本発明０ｍＭ〕」を、「第２試薬〔本発明（０ｍＭ）〕」として用いた。
（ｂ）第２試薬〔本発明（１０ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ８．０（２０℃）に調整して、１０ｍＭの濃度のグルコースを含む、第２試薬〔本発明（１０ｍＭ）〕を調製した。
Ｄ−グルコース（無水） １０．０ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ４，０００Ｕｎｉｔ／Ｌ
イミダゾール １２０ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
ジアデノシンペンタリン酸三リチウム塩 ２６μＭ
クレアチンリン酸二ナトリウム塩 １２０ｍＭ
（ｃ）第２試薬〔従来発明（３０ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ８．０（２０℃）に調整して、３０ｍＭの濃度のグルコースを含む、第２試薬〔従来発明（３０ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ３０．０ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ４，０００Ｕｎｉｔ／Ｌ
イミダゾール １２０ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
ジアデノシンペンタリン酸三リチウム塩 ２６μＭ
クレアチンリン酸二ナトリウム塩 １２０ｍＭ
（ｄ）第２試薬〔従来発明（４０ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ８．０（２０℃）に調整して、４０ｍＭの濃度のグルコースを含む、第２試薬〔従来発明（４０ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ４０．０ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ４，０００Ｕｎｉｔ／Ｌ
イミダゾール １２０ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
ジアデノシンペンタリン酸三リチウム塩 ２６μＭ
クレアチンリン酸二ナトリウム塩 １２０ｍＭ
（ｅ）第２試薬〔従来発明（５０ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ８．０（２０℃）に調整して、５０ｍＭの濃度のグルコースを含む、第２試薬〔従来発明（５０ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ５０．０ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ４，０００Ｕｎｉｔ／Ｌ
イミダゾール １２０ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
ジアデノシンペンタリン酸三リチウム塩 ２６μＭ
クレアチンリン酸二ナトリウム塩 １２０ｍＭ
（ｆ）第２試薬〔従来発明（６０ｍＭ）〕の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、０．１Ｎ酢酸でｐＨをｐＨ８．０（２０℃）に調整して、６０ｍＭの濃度のグルコースを含む、第２試薬〔従来発明（６０ｍＭ）〕を調製した。
Ｄ−グルコース（無水） ６０．０ｍＭ
グルコース−６−リン酸デヒドロゲナーゼ〔ロシュ・ダイアグノスティックス社、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ，ｒｅｃｏｍｂｉｎａｎｔ由来〕 ４，０００Ｕｎｉｔ／Ｌ
イミダゾール １２０ｍＭ
エチレンジアミン四酢酸二ナトリウム塩 ２ｍＭ
アジ化ナトリウム ７．７ｍＭ
酢酸マグネシウム（４水和物） １０ｍＭ
ＡＤＰ一カリウム塩 ２ｍＭ
ジアデノシンペンタリン酸三リチウム塩 ２６μＭ
クレアチンリン酸二ナトリウム塩 １２０ｍＭ
２．試料
（１）希釈試料
ヒト血清試料にラビットのクレアチンキナーゼＭＭアイソザイムを添加したものを調製し（添加血清試料）、この添加血清試料を、「試料希釈液」（５．０ｍＭコハク酸緩衝液〔ｐＨ７．０（２０℃）〕）により、それぞれ１／１０、２／１０、３／１０、４／１０、５／１０、６／１０、７／１０、８／１０、及び９／１０となるように希釈し、下記の「試料−Ａ」〜「試料−Ｉ」を調製した。
また、前記の添加血清試料を、「試料−Ｊ」（１０／１０）とした。
（ａ）「試料−Ａ」： 添加血清試料を１／１０希釈
（ｂ）「試料−Ｂ」： 添加血清試料を２／１０希釈
（ｃ）「試料−Ｃ」： 添加血清試料を３／１０希釈
（ｄ）「試料−Ｄ」： 添加血清試料を４／１０希釈
（ｅ）「試料−Ｅ」： 添加血清試料を５／１０希釈
（ｆ）「試料−Ｆ」： 添加血清試料を６／１０希釈
（ｇ）「試料−Ｇ」： 添加血清試料を７／１０希釈
（ｈ）「試料−Ｈ」： 添加血清試料を８／１０希釈
（ｉ）「試料−Ｉ」： 添加血清試料を９／１０希釈
（ｊ）「試料−Ｊ」： 添加血清試料
（２）不含試料
前記（１）における「試料希釈液」を、クレアチンキナーゼを含まない、クレアチンキナーゼ活性値が０Ｕｎｉｔ／Ｌ（単位／Ｌ）の試料、すなわち「不含試料」とした。
３．試薬の保存
（１）３７℃での保存
前記１の（２）の（ａ）の第２試薬〔本発明（０ｍＭ）〕、（ｂ）の第２試薬〔本発明（１０ｍＭ）〕、（ｃ）の第２試薬〔従来発明（３０ｍＭ）〕、（ｄ）の第２試薬〔従来発明（４０ｍＭ）〕、（ｅ）の第２試薬〔従来発明（５０ｍＭ）〕、及び（ｆ）の第２試薬〔従来発明（６０ｍＭ）〕については、それぞれ３７℃で暗所に７日間保存した。
（２）５℃での保存
前記１の（１）の第１試薬〔本発明（５ｍＭ）〕については、５℃で暗所に７日間保存した。
４．試料のクレアチンキナーゼ活性値の測定
前記２の各試料のクレアチンキナーゼ活性値の測定は、東芝メディカルシステムズ社の自動分析装置ＴＢＡ−１２０ＦＲを使用して、ダブルカイネティック法により行った。
（１） 前記２の（１）〜（２）のそれぞれの試料５．６μＬに、前記３の（２）で５℃にて７日間保存した前記１の（１）の第１試薬〔本発明（５ｍＭ）〕の１６０μＬを添加し、３７℃で反応させた。
（２） 次に、８ポイント（第１試薬添加後１３６．１８秒）から１６ポイント（第１試薬添加後２８０．４６秒）に掛けての吸光度変化量を主波長３４０ｎｍ、副波長４０５ｎｍにて測定した〔「測光１」〕。（試料中に含まれていたアデニル酸キナーゼの活性値に応じた吸光度変化量の測定）
（３） 次に、１６ポイント（第１試薬添加後２８０．４６秒）から１７ポイント（第１試薬添加後２９８．４９秒）の間に、前記３の（１）で３７℃にて７日間保存した前記１の（２）の（ａ）の第２試薬〔本発明（０ｍＭ）〕の４０μＬを添加し、３７℃で反応させた。
（４） 次に、２４ポイント（第１試薬添加後４２４．７４秒）から３３ポイント（第１試薬添加後５８７．０５秒）に掛けての吸光度変化量を主波長３４０ｎｍ、副波長４０５ｎｍにて測定した〔「測光２」〕。（試料中に含まれていたクレアチンキナーゼの活性値に応じた吸光度変化量と、試料中に含まれていたアデニル酸キナーゼの活性値に応じた吸光度変化量が合わさった吸光度変化量の測定）
（５） 次に、試料に含まれていたクレアチンキナーゼの活性値を、下記の計算式により求めた。
クレアチンキナーゼ活性値（Ｕｎｉｔ／Ｌ）＝｛（ΔＡ_２−ΔＲＢ_２）−ｃ×（ΔＡ_１−ΔＲＢ_１）｝×（Ｓ＋Ｖ_１＋Ｖ_２）×１０^６÷（Ｋ×ｄ×Ｓ）＝｛（ΔＡ_２−ΔＲＢ_２）−０．８０５×（ΔＡ_１−ΔＲＢ_１）｝×５．８３×１０^３
なお、この計算式において、ΔＡ_１は「測光１」において測定した１分間当りの吸光度変化量（Ａｂｓ／ｍｉｎ）を、ΔＡ_２は「測光２」において測定した１分間当りの吸光度変化量（Ａｂｓ／ｍｉｎ）を、ΔＲＢ_１は試料が前記の「不含試料」であるときの「測光１」において測定した１分間当りの吸光度変化量〔試薬盲検値〕（Ａｂｓ／ｍｉｎ）を、ΔＲＢ_２は試料が前記の「不含試料」であるときの「測光２」において測定した１分間当りの吸光度変化量〔試薬盲検値〕（Ａｂｓ／ｍｉｎ）を、Ｓは試料の量〔５．６μＬ〕を、Ｖ_１は第１試薬の量〔１６０μＬ〕を、Ｖ_２は第２試薬の量〔４０μＬ〕を、ｃは液量補正係数〔（Ｓ＋Ｖ_１）÷（Ｓ＋Ｖ_１＋Ｖ_２）＝０．８０５〕を、ＫはＮＡＤＰＨの理論モル吸光係数〔６．３０×１０^３Ｌ・ｍｏｌｅ^−１・ｃｍ^−１（主波長：３４０ｎｍ、副波長：４０５ｎｍ）〕、及びｄはセルの光路長〔１．００ｃｍ〕を表す。
（６） また、前記（３）において、前記３の（１）で３７℃にて７日間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（２）の（ｂ）の第２試薬〔本発明（１０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第２試薬として前記の第２試薬〔本発明（１０ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（７） 更に、前記（３）において、前記３の（１）で３７℃にて７日間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（２）の（ｃ）の第２試薬〔従来発明（３０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第２試薬として前記の第２試薬〔従来発明（３０ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（８） また、前記（３）において、前記３の（１）で３７℃にて７日間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（２）の（ｄ）の第２試薬〔従来発明（４０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第２試薬として前記の第２試薬〔従来発明（４０ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（９） 更に、前記（３）において、前記３の（１）で３７℃にて７日間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（２）の（ｅ）の第２試薬〔従来発明（５０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第２試薬として前記の第２試薬〔従来発明（５０ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１０） また、前記（３）において、前記３の（１）で３７℃にて７日間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で３７℃にて７日間保存した前記１の（２）の（ｆ）の第２試薬〔従来発明（６０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第２試薬として前記の第２試薬〔従来発明（６０ｍＭ）〕を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
５．測定結果
前記４において各試料のクレアチンキナーゼ活性値の測定を行って得られた結果を、表５に示した。
なお、この表５において、「〔１〕活性値」の欄に示した値は各試料のクレアチンキナーゼの活性値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔２〕活性値差」の欄に示した値は、各試料における「〔１〕活性値」の欄の値から、同じ試料の、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・７日間保存＞であり、そして第２試薬が第２試薬〔本発明（０ｍＭ）〕＜３７℃・暗所・７日間保存＞である場合の「〔１〕活性値」の欄の値を差し引いた差の値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔３〕相対比率」の欄に示した値は、各試料における「〔１〕活性値」の欄の値を、同じ試料の、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・７日間保存＞であり、そして第２試薬が第２試薬〔本発明（０ｍＭ）〕＜３７℃・暗所・７日間保存＞である場合の「〔１〕活性値」の欄の値で除した値をパーセント表示で示す。

６．考察
（１） 表５において、第２試薬が３７℃で７日間保存した第２試薬〔本発明（１０ｍＭ）〕である場合の測定値は、やはり３７℃で７日間保存した第２試薬〔本発明（０ｍＭ）〕の場合の測定値と、ほぼ同一の値となっている。
これに対して、第２試薬が３７℃で７日間保存した第２試薬〔従来発明（３０ｍＭ）〕、第２試薬〔従来発明（４０ｍＭ）〕、第２試薬〔従来発明（５０ｍＭ）〕、又は第２試薬〔従来発明（６０ｍＭ）〕である場合には、３７℃で７日間保存した第２試薬〔本発明（０ｍＭ）〕の場合に比べて、その測定値は低い値となっている。
この場合、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きい傾向にあり、その測定値の低下度（表５の「〔２〕活性値差」の値）は、例えば、試料が「試料−Ｈ」〜「試料−Ｊ」においては、第２試薬が第２試薬〔従来発明（３０ｍＭ）〕の場合には−３Ｕｎｉｔ／Ｌ〜−１９Ｕｎｉｔ／Ｌであり、第２試薬〔従来発明（４０ｍＭ）〕の場合には−２９Ｕｎｉｔ／Ｌ〜−３９Ｕｎｉｔ／Ｌであり、第２試薬〔従来発明（５０ｍＭ）〕の場合には−４４Ｕｎｉｔ／Ｌ〜−５３Ｕｎｉｔ／Ｌであり、そして、第２試薬〔従来発明（６０ｍＭ）〕の場合には−７０Ｕｎｉｔ／Ｌ〜−８６Ｕｎｉｔ／Ｌである。
この第２試薬を３７℃で７日間保存した結果より、第２試薬のグルコース濃度が、３０ｍＭ、４０ｍＭ、５０ｍＭ、又は６０ｍＭである場合には、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合に比較して、測定試薬保存時の測定値の低下度には大きな差異があり、測定値が低下してしまっていることが分かる。
そして、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合には、測定値の低下が抑制され、安定化されていることが分かる。
（２） 以上のことより、第１試薬が０．５ｍＭ〜１０ｍＭの濃度のグルコースを含み、かつ第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことを特徴とする本発明のクレアチンキナーゼ活性測定試薬は、従来のクレアチンキナーゼ活性測定試薬に比べ、３７℃という非常に過酷な条件での保存時においても、その測定値の低下が抑制され、安定化されたものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間安定に保つことができ、かつ長期間正確にクレアチンキナーゼ活性の測定を行うことができるという効果を有するものであることが改めて確かめられた。
また、本発明のクレアチンキナーゼ活性測定試薬は、従来のクレアチンキナーゼ活性測定試薬に比べ、特にクレアチンキナーゼ活性値が高い高値域における測定値の低下の抑制に効果があるものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間、高値域における直線性を維持することができるという効果を有するものであることが改めて確かめられた。
〔実施例４〕（本発明の保存時の安定化の効果の確認−４）
本発明のクレアチンキナーゼ活性測定試薬における保存時の安定化効果を確認した。
具体的には、本発明のクレアチンキナーゼ活性測定試薬において、第１試薬は測定時に調製したもの又は５℃で１０ヶ月間保存したものを使用し、第２試薬は５℃で１０ヶ月間保存したもの（又は測定時に調製したもの）を使用し、安定化の効果を確認した。
１．クレアチンキナーゼ活性測定試薬
（１）第１試薬
（ａ）第１試薬〔本発明（５ｍＭ）〕
前記の実施例１の１の（１）の（ｂ）の「第１試薬〔本発明５ｍＭ〕」を、「第１試薬〔本発明（５ｍＭ）〕」として用いた。
（ｂ）第１試薬〔本発明（１０ｍＭ）〕
前記の実施例１の１の（１）の（ｃ）の「第１試薬〔本発明１０ｍＭ〕」を、「第１試薬〔本発明（１０ｍＭ）〕」として用いた。
（２）第２試薬
（ａ）第２試薬〔本発明（０ｍＭ）〕
前記の実施例１の１の（２）の（ａ）の「第２試薬〔本発明０ｍＭ〕」を、「第２試薬〔本発明（０ｍＭ）〕」として用いた。
（ｂ）第２試薬〔本発明（１０ｍＭ）〕
前記の実施例３の１の（２）の（ｂ）の「第２試薬〔本発明１０ｍＭ〕」を、「第２試薬〔本発明（１０ｍＭ）〕」として用いた。
（ｃ）第２試薬〔従来発明（６０ｍＭ）〕
前記の実施例３の１の（２）の（ｆ）の「第２試薬〔従来発明６０ｍＭ〕」を、「第２試薬〔従来発明（６０ｍＭ）〕」として用いた。
２．試料
（１）希釈試料
ヒト血清試料にヒトのクレアチンキナーゼＢＢアイソザイムを添加したものを調製し（添加血清試料）、この添加血清試料を「試料希釈液」（５．０ｍＭコハク酸緩衝液〔ｐＨ７．０（２０℃）〕）により、それぞれ１／１０、２／１０、３／１０、４／１０、５／１０、６／１０、７／１０、８／１０、及び９／１０となるように希釈し、下記の「試料−（１）」〜「試料−（９）」を調製した。
また、前記の添加血清試料を「試料−（１０）」（１０／１０）とした。
（ａ）「試料−（１）」： 添加血清試料を１／１０希釈
（ｂ）「試料−（２）」： 添加血清試料を２／１０希釈
（ｃ）「試料−（３）」： 添加血清試料を３／１０希釈
（ｄ）「試料−（４）」： 添加血清試料を４／１０希釈
（ｅ）「試料−（５）」： 添加血清試料を５／１０希釈
（ｆ）「試料−（６）」： 添加血清試料を６／１０希釈
（ｇ）「試料−（７）」： 添加血清試料を７／１０希釈
（ｈ）「試料−（８）」： 添加血清試料を８／１０希釈
（ｉ）「試料−（９）」： 添加血清試料を９／１０希釈
（ｊ）「試料−（１０）」： 添加血清試料
（２）不含試料
前記（１）における「試料希釈液」を、クレアチンキナーゼを含まない、クレアチンキナーゼ活性値が０Ｕｎｉｔ／Ｌ（単位／Ｌ）の試料、すなわち「不含試料」とした。
３．試薬の保存又は測定時の調製
（１）５℃での保存
前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕、及び（ｂ）の第１試薬〔本発明（１０ｍＭ）〕については、それぞれ５℃で暗所に１０ヶ月間保存した。
また、前記１の（２）の（ａ）の第２試薬〔本発明（０ｍＭ）〕、（ｂ）の第２試薬〔本発明（１０ｍＭ）〕、及び（ｃ）の第２試薬〔従来発明（６０ｍＭ）〕についても、それぞれ５℃で暗所に１０ヶ月間保存した。
（２）測定時の調製
前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕、及び（ｂ）の第１試薬〔本発明（１０ｍＭ）〕については、下記４の試料のクレアチンキナーゼ活性値の測定時にも調製を行い、測定に使用した。
また、前記１の（２）の（ｃ）の第２試薬〔従来発明（６０ｍＭ）〕については、これも下記４の試料のクレアチンキナーゼ活性値の測定時にも調製を行い、測定に使用した。
４．試料のクレアチンキナーゼ活性値の測定
前記２の各試料のクレアチンキナーゼ活性値の測定は、東芝メディカルシステムズ社の自動分析装置ＴＢＡ−１２０ＦＲを使用して、ダブルカイネティック法により行った。
（１） 前記２の（１）〜（２）のそれぞれの試料５．６μＬに、前記３の（２）の通りこの測定時に調製した前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕の１６０μＬを添加し、３７℃で反応させた。
（２） 次に、８ポイント（第１試薬添加後１３６．１８秒）から１６ポイント（第１試薬添加後２８０．４６秒）に掛けての吸光度変化量を主波長３４０ｎｍ、副波長４０５ｎｍにて測定した〔「測光１」〕。（試料中に含まれていたアデニル酸キナーゼの活性値に応じた吸光度変化量の測定）
（３） 次に、１６ポイント（第１試薬添加後２８０．４６秒）から１７ポイント（第１試薬添加後２９８．４９秒）の間に、前記３の（１）で５℃にて１０ヶ月間保存した前記１の（２）の（ａ）の第２試薬〔本発明（０ｍＭ）〕の４０μＬを添加し、３７℃で反応させた。
（４） 次に、２４ポイント（第１試薬添加後４２４．７４秒）から３３ポイント（第１試薬添加後５８７．０５秒）に掛けての吸光度変化量を主波長３４０ｎｍ、副波長４０５ｎｍにて測定した〔「測光２」〕。（試料中に含まれていたクレアチンキナーゼの活性値に応じた吸光度変化量と、試料中に含まれていたアデニル酸キナーゼの活性値に応じた吸光度変化量が合わさった吸光度変化量の測定）
（５） 次に、試料に含まれていたクレアチンキナーゼの活性値を、下記の計算式により求めた。
クレアチンキナーゼ活性値（Ｕｎｉｔ／Ｌ）＝｛（ΔＡ_２−ΔＲＢ_２）−ｃ×（ΔＡ_１−ΔＲＢ_１）｝×（Ｓ＋Ｖ_１＋Ｖ_２）×１０^６÷（Ｋ×ｄ×Ｓ）＝｛（ΔＡ_２−ΔＲＢ_２）−０．８０５×（ΔＡ_１−ΔＲＢ_１）｝×５．８３×１０^３
なお、この計算式において、ΔＡ_１は「測光１」において測定した１分間当りの吸光度変化量（Ａｂｓ／ｍｉｎ）を、ΔＡ_２は「測光２」において測定した１分間当りの吸光度変化量（Ａｂｓ／ｍｉｎ）を、ΔＲＢ_１は試料が前記の「不含試料」であるときの「測光１」において測定した１分間当りの吸光度変化量〔試薬盲検値〕（Ａｂｓ／ｍｉｎ）を、ΔＲＢ_２は試料が前記の「不含試料」であるときの「測光２」において測定した１分間当りの吸光度変化量〔試薬盲検値〕（Ａｂｓ／ｍｉｎ）を、Ｓは試料の量〔５．６μＬ〕を、Ｖ_１は第１試薬の量〔１６０μＬ〕を、Ｖ_２は第２試薬の量〔４０μＬ〕を、ｃは液量補正係数〔（Ｓ＋Ｖ_１）÷（Ｓ＋Ｖ_１＋Ｖ_２）＝０．８０５〕を、ＫはＮＡＤＰＨの理論モル吸光係数〔６．３０×１０^３Ｌ・ｍｏｌｅ^−１・ｃｍ^−１（主波長：３４０ｎｍ、副波長：４０５ｎｍ）〕、及びｄはセルの光路長〔１．００ｃｍ〕を表す。
（６） また、前記（３）において、前記３の（１）で５℃にて１０ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で５℃にて１０ヶ月間保存した前記１の（２）の（ｂ）の第２試薬〔本発明（１０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第２試薬として前記の第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（７） 更に、前記（３）において、前記３の（１）で５℃にて１０ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で５℃にて１０ヶ月間保存した前記１の（２）の（ｃ）の第２試薬〔従来発明（６０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第２試薬として前記の第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（８） また、前記（３）において、前記３の（１）で５℃にて１０ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（２）の通りこの測定時に調製した前記１の（２）の（ｃ）の第２試薬〔従来発明（６０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第２試薬として前記の第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（９） 更に、前記（１）において、前記３の（２）の通りこの測定時に調製した前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕に替えて、前記３の（２）の通りこの測定時に調製した前記１の（１）の（ｂ）の第１試薬〔本発明（１０ｍＭ）〕を用いること以外は、前記（１）〜（８）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（１０ｍＭ）〕＜測定時調製＞を用い、第２試薬として前記の第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、又は第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞をそれぞれ用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１０） また、前記（１）において、前記３の（２）の通りこの測定時に調製した前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕に替えて、前記３の（１）で５℃にて１０ヶ月間保存した前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕を用いること以外は、前記（１）〜（８）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞を用い、第２試薬として前記の第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、又は第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞をそれぞれ用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１１） 更に、前記（１）において、前記３の（２）の通りこの測定時に調製した前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕に替えて、前記３の（１）で５℃にて１０ヶ月間保存した前記１の（１）の（ｂ）の第１試薬〔本発明（１０ｍＭ）〕を用いること以外は、前記（１）〜（８）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞を用い、第２試薬として前記の第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、又は第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞をそれぞれ用いた場合の各試料のクレアチンキナーゼの活性値を得た。
５．測定結果
（１）第１試薬〔本発明（５ｍＭ）〕＜測定時調製＞使用時
前記４の（１）〜（８）における、第１試薬として前記の第１試薬〔本発明（５ｍＭ）〕＜測定時調製＞を用い、第２試薬として前記の第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、又は第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞をそれぞれ用いた場合の各試料のクレアチンキナーゼの活性値の測定結果を、表６に示した。
なお、この表６において、「〔１〕活性値」の欄に示した値は各試料のクレアチンキナーゼの活性値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔２〕活性値差」の欄に示した値は、各試料における「〔１〕活性値」の欄の値から、同じ試料の、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜測定時調製＞であり、そして第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞である場合の「〔１〕活性値」の欄の値を差し引いた差の値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔３〕相対比率」の欄に示した値は、各試料における「〔１〕活性値」の欄の値を、同じ試料の、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜測定時調製＞であり、そして第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞である場合の「〔１〕活性値」の欄の値で除した値をパーセント表示で示す。
（２）第１試薬〔本発明（１０ｍＭ）〕＜測定時調製＞使用時
前記４の（９）における、第１試薬として前記の第１試薬〔本発明（１０ｍＭ）〕＜測定時調製＞を用い、第２試薬として前記の第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、又は第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞をそれぞれ用いた場合の各試料のクレアチンキナーゼの活性値の測定結果を、表７に示した。
なお、この表７において、「〔１〕活性値」の欄に示した値は各試料のクレアチンキナーゼの活性値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔２〕活性値差」の欄に示した値は、各試料における「〔１〕活性値」の欄の値から、同じ試料の、第１試薬が第１試薬〔本発明（１０ｍＭ）〕＜測定時調製＞であり、そして第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞である場合の「〔１〕活性値」の欄の値を差し引いた差の値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔３〕相対比率」の欄に示した値は、各試料における「〔１〕活性値」の欄の値を、同じ試料の、第１試薬が第１試薬〔本発明（１０ｍＭ）〕＜測定時調製＞であり、そして第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞である場合の「〔１〕活性値」の欄の値で除した値をパーセント表示で示す。
（３）第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞使用時
前記４の（１０）における、第１試薬として前記の第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞を用い、第２試薬として前記の第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、又は第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞をそれぞれ用いた場合の各試料のクレアチンキナーゼの活性値の測定結果を、表８に示した。
なお、この表８において、「〔１〕活性値」の欄に示した値は各試料のクレアチンキナーゼの活性値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔２〕活性値差」の欄に示した値は、各試料における「〔１〕活性値」の欄の値から、同じ試料の、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞であり、そして第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞である場合の「〔１〕活性値」の欄の値を差し引いた差の値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔３〕相対比率」の欄に示した値は、各試料における「〔１〕活性値」の欄の値を、同じ試料の、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞であり、そして第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞である場合の「〔１〕活性値」の欄の値で除した値をパーセント表示で示す。
（４）第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞使用時
前記４の（１１）における、第１試薬として前記の第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞を用い、第２試薬として前記の第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞、又は第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞をそれぞれ用いた場合の各試料のクレアチンキナーゼの活性値の測定結果を、表９に示した。
なお、この表９において、「〔１〕活性値」の欄に示した値は各試料のクレアチンキナーゼの活性値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔２〕活性値差」の欄に示した値は、各試料における「〔１〕活性値」の欄の値から、同じ試料の、第１試薬が第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞であり、そして第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞である場合の「〔１〕活性値」の欄の値を差し引いた差の値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔３〕相対比率」の欄に示した値は、各試料における「〔１〕活性値」の欄の値を、同じ試料の、第１試薬が第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞であり、そして第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜測定時調製＞である場合の「〔１〕活性値」の欄の値で除した値をパーセント表示で示す。

６．考察
（１）第１試薬〔本発明（５ｍＭ）〕＜測定時調製＞使用時
第１試薬が第１試薬〔本発明（５ｍＭ）〕＜測定時調製＞であるときの測定結果を示した表６において、第２試薬が５℃で１０ヶ月間保存した第２試薬〔従来発明（６０ｍＭ）〕である場合の測定値は、やはり５℃で１０ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕や第２試薬〔本発明（１０ｍＭ）〕の場合の測定値に比べ、低い値となっている。
この場合、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きい傾向にあり、その測定値の低下度（表６の「〔２〕活性値差」の値）は、例えば、試料が「試料−（８）」〜「試料−（１０）」においては、第２試薬が第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−２９１Ｕｎｉｔ／Ｌ〜−３６５Ｕｎｉｔ／Ｌであり、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−２７４Ｕｎｉｔ／Ｌ〜−２９０Ｕｎｉｔ／Ｌであり、そして、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−３９０Ｕｎｉｔ／Ｌ〜−４５８Ｕｎｉｔ／Ｌである。
この第２試薬を５℃で１０ヶ月間保存（又は測定時調製）した結果より、第２試薬のグルコース濃度が６０ｍＭである場合には、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合に比較して、測定試薬保存時の測定値の低下度には大きな差異があり、測定値が低下してしまっていることが分かる。
そして、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合には、測定値の低下が抑制され、安定化されていることが分かる。
（２）第１試薬〔本発明（１０ｍＭ）〕＜測定時調製＞使用時
第１試薬が第１試薬〔本発明（１０ｍＭ）〕＜測定時調製＞であるときの測定結果を示した表７において、第２試薬が５℃で１０ヶ月間保存した第２試薬〔従来発明（６０ｍＭ）〕である場合の測定値は、やはり５℃で１０ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕や第２試薬〔本発明（１０ｍＭ）〕の場合の測定値に比べ、低い値となっている。
この場合も、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きい傾向にあり、その測定値の低下度（表７の「〔２〕活性値差」の値）は、例えば、試料が「試料−（８）」〜「試料−（１０）」においては、第２試薬が第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−２９４Ｕｎｉｔ／Ｌ〜−３４１Ｕｎｉｔ／Ｌであり、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−２７８Ｕｎｉｔ／Ｌ〜−２８５Ｕｎｉｔ／Ｌであり、そして、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−３７９Ｕｎｉｔ／Ｌ〜−４３９Ｕｎｉｔ／Ｌである。
この第２試薬を５℃で１０ヶ月間保存（又は測定時調製）した結果より、第２試薬のグルコース濃度が６０ｍＭである場合には、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合に比較して、測定試薬保存時の測定値の低下度には大きな差異があり、測定値が低下してしまっていることが分かる。
そして、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合には、測定値の低下が抑制され、安定化されていることが分かる。
（３）第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞使用時
第１試薬が第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞であるときの測定結果を示した表８において、第２試薬が５℃で１０ヶ月間保存した第２試薬〔従来発明（６０ｍＭ）〕である場合の測定値は、やはり５℃で１０ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕や第２試薬〔本発明（１０ｍＭ）〕の場合の測定値に比べ、低い値となっている。
この場合も、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きい傾向にあり、その測定値の低下度（表８の「〔２〕活性値差」の値）は、例えば、試料が「試料−（８）」〜「試料−（１０）」においては、第２試薬が第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−５９８Ｕｎｉｔ／Ｌ〜−７０４Ｕｎｉｔ／Ｌであり、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−５６０Ｕｎｉｔ／Ｌ〜−６７４Ｕｎｉｔ／Ｌであり、そして、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−８５１Ｕｎｉｔ／Ｌ〜−１，１２２Ｕｎｉｔ／Ｌである。
この第２試薬を５℃で１０ヶ月間保存（又は測定時調製）した結果より、第２試薬のグルコース濃度が６０ｍＭである場合には、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合に比較して、測定試薬保存時の測定値の低下度には大きな差異があり、測定値が低下してしまっていることが分かる。
更に、この第２試薬のグルコース濃度が６０ｍＭである場合の測定値の低下度は、このように第１試薬を５℃にて１０ヶ月間保存したときは、前記（１）及び（２）のように第１試薬が測定時に調製されたものであるときに比べ、より大きいものであることが分かる。
そして、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合には、測定値の低下が抑制され、安定化されていることが分かる。
（４）第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞使用時
第１試薬が第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞であるときの測定結果を示した表９において、第２試薬が５℃で１０ヶ月間保存した第２試薬〔従来発明（６０ｍＭ）〕である場合の測定値は、やはり５℃で１０ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕や第２試薬〔本発明（１０ｍＭ）〕の場合の測定値に比べ、低い値となっている。
この場合も、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きい傾向にあり、その測定値の低下度（表９の「〔２〕活性値差」の値）は、例えば、試料が「試料−（８）」〜「試料−（１０）」においては、第２試薬が第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−６５２Ｕｎｉｔ／Ｌ〜−７６９Ｕｎｉｔ／Ｌであり、第２試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−６１７Ｕｎｉｔ／Ｌ〜−７５１Ｕｎｉｔ／Ｌであり、そして、第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１０ヶ月間保存＞の場合には−１，０５７Ｕｎｉｔ／Ｌ〜−１，２７９Ｕｎｉｔ／Ｌである。
この第２試薬を５℃で１０ヶ月間保存（又は測定時調製）した結果より、第２試薬のグルコース濃度が６０ｍＭである場合には、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合に比較して、測定試薬保存時の測定値の低下度には大きな差異があり、測定値が低下してしまっていることが分かる。
更に、この第２試薬のグルコース濃度が６０ｍＭである場合の測定値の低下度は、このように第１試薬を５℃にて１０ヶ月間保存したときは、前記（１）及び（２）のように第１試薬が測定時に調製されたものであるときに比べ、より大きいものであることが分かる。
そして、第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含む場合には、測定値の低下が抑制され、安定化されていることが分かる。
（５）まとめ
以上のことより、第１試薬が０．５ｍＭ〜１０ｍＭの濃度のグルコースを含み、かつ第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことを特徴とする本発明のクレアチンキナーゼ活性測定試薬は、従来のクレアチンキナーゼ活性測定試薬に比べ、５℃にて１０ヶ月間という長期間の保存時においても、その測定値の低下が抑制され、安定化されたものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間安定に保つことができ、かつ長期間正確にクレアチンキナーゼ活性の測定を行うことができるという効果を有するものであることが改めて確かめられた。
また、本発明のクレアチンキナーゼ活性測定試薬は、従来のクレアチンキナーゼ活性測定試薬に比べ、特にクレアチンキナーゼ活性値が高い高値域における測定値の低下の抑制に効果があるものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間、高値域における直線性を維持することができるという効果を有するものであることが改めて確かめられた。
〔実施例５〕（本発明の保存時の安定化の効果の確認−５）
本発明のクレアチンキナーゼ活性測定試薬における保存時の安定化効果を確認した。
具体的には、本発明のクレアチンキナーゼ活性測定試薬において、第１試薬は５℃で１２ヶ月間保存したもの（又は測定時に調製したもの）を使用し、第２試薬は５℃で１２ヶ月間保存したもの（又は測定時に調製したもの）を使用し、安定化の効果を確認した。
１．クレアチンキナーゼ活性測定試薬
（１）第１試薬
（ａ）第１試薬〔本発明（５ｍＭ）〕
前記の実施例１の１の（１）の（ｂ）の「第１試薬〔本発明５ｍＭ〕」を、「第１試薬〔本発明（５ｍＭ）〕」として用いた。
（ｂ）第１試薬〔本発明（１０ｍＭ）〕
前記の実施例１の１の（１）の（ｃ）の「第１試薬〔本発明１０ｍＭ〕」を、「第１試薬〔本発明（１０ｍＭ）〕」として用いた。
（ｃ）第１試薬〔従来発明（２５ｍＭ）〕
前記の実施例１の１の（１）の（ｄ）の「第１試薬〔従来発明２５ｍＭ〕」を、「第１試薬〔従来発明（２５ｍＭ）〕」として用いた。
（２）第２試薬
（ａ）第２試薬〔本発明（０ｍＭ）〕
前記の実施例１の１の（２）の（ａ）の「第２試薬〔本発明０ｍＭ〕」を、「第２試薬〔本発明（０ｍＭ）〕」として用いた。
（ｂ）第２試薬〔従来発明（３０ｍＭ）〕
前記の実施例３の１の（２）の（ｃ）の「第２試薬〔従来発明３０ｍＭ〕」を、「第２試薬〔従来発明（３０ｍＭ）〕」として用いた。
（ｃ）第２試薬〔従来発明（６０ｍＭ）〕
前記の実施例３の１の（２）の（ｆ）の「第２試薬〔従来発明６０ｍＭ〕」を、「第２試薬〔従来発明（６０ｍＭ）〕」として用いた。
２．試料
（１）希釈試料
ヒト血清試料にヒトのクレアチンキナーゼＢＢアイソザイムを添加したものを調製し（添加血清試料）、この添加血清試料を「試料希釈液」（５．０ｍＭコハク酸緩衝液〔ｐＨ７．０（２０℃）〕）により、それぞれ１／１０、２／１０、３／１０、４／１０、５／１０、６／１０、７／１０、８／１０、及び９／１０となるように希釈し、下記の「試料−〔１〕」〜「試料−〔９〕」を調製した。
また、前記の添加血清試料を「試料−〔１０〕」（１０／１０）とした。
（ａ）「試料−〔１〕」： 添加血清試料を１／１０希釈
（ｂ）「試料−〔２〕」： 添加血清試料を２／１０希釈
（ｃ）「試料−〔３〕」： 添加血清試料を３／１０希釈
（ｄ）「試料−〔４〕」： 添加血清試料を４／１０希釈
（ｅ）「試料−〔５〕」： 添加血清試料を５／１０希釈
（ｆ）「試料−〔６〕」： 添加血清試料を６／１０希釈
（ｇ）「試料−〔７〕」： 添加血清試料を７／１０希釈
（ｈ）「試料−〔８〕」： 添加血清試料を８／１０希釈
（ｉ）「試料−〔９〕」： 添加血清試料を９／１０希釈
（ｊ）「試料−〔１０〕」： 添加血清試料
（２）不含試料
前記（１）における「試料希釈液」を、クレアチンキナーゼを含まない、クレアチンキナーゼ活性値が０Ｕｎｉｔ／Ｌ（単位／Ｌ）の試料、すなわち「不含試料」とした。
３．試薬の保存又は測定時の調製
（１）５℃での保存
前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕、（ｂ）の第１試薬〔本発明（１０ｍＭ）〕、及び（ｃ）第１試薬〔従来発明（２５ｍＭ）〕については、それぞれ５℃で暗所に１２ヶ月間保存した。
また、前記１の（２）の（ａ）の第２試薬〔本発明（０ｍＭ）〕、（ｂ）の第２試薬〔従来発明（３０ｍＭ）〕、及び（ｃ）の第２試薬〔従来発明（６０ｍＭ）〕についても、それぞれ５℃で暗所に１２ヶ月間保存した。
（２）測定時の調製
前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕については、下記４の試料のクレアチンキナーゼ活性値の測定時にも調製を行い、第１試薬の対照試薬として測定に使用した。
また、前記１の（２）の（ｃ）の第２試薬〔従来発明（６０ｍＭ）〕については、これも下記４の試料のクレアチンキナーゼ活性値の測定時にも調製を行い、第２試薬の対照試薬として測定に使用した。
４．試料のクレアチンキナーゼ活性値の測定
前記２の各試料のクレアチンキナーゼ活性値の測定は、東芝メディカルシステムズ社の自動分析装置ＴＢＡ−１２０ＦＲを使用して、ダブルカイネティック法により行った。
（１） 前記２の（１）〜（２）のそれぞれの試料５．６μＬに、前記３の（１）で５℃にて１２ヶ月保存した前記１の（１）の（ａ）の第１試薬〔本発明（５ｍＭ）〕の１６０μＬを添加し、３７℃で反応させた。
（２） 次に、８ポイント（第１試薬添加後１３６．１８秒）から１６ポイント（第１試薬添加後２８０．４６秒）に掛けての吸光度変化量を主波長３４０ｎｍ、副波長４０５ｎｍにて測定した〔「測光１」〕。（試料中に含まれていたアデニル酸キナーゼの活性値に応じた吸光度変化量の測定）
（３） 次に、１６ポイント（第１試薬添加後２８０．４６秒）から１７ポイント（第１試薬添加後２９８．４９秒）の間に、前記３の（１）で５℃にて１２ヶ月間保存した前記１の（２）の（ａ）の第２試薬〔本発明（０ｍＭ）〕の４０μＬを添加し、３７℃で反応させた。
（４） 次に、２４ポイント（第１試薬添加後４２４．７４秒）から３３ポイント（第１試薬添加後５８７．０５秒）に掛けての吸光度変化量を主波長３４０ｎｍ、副波長４０５ｎｍにて測定した〔「測光２」〕。（試料中に含まれていたクレアチンキナーゼの活性値に応じた吸光度変化量と、試料中に含まれていたアデニル酸キナーゼの活性値に応じた吸光度変化量が合わさった吸光度変化量の測定）
（５） 次に、試料に含まれていたクレアチンキナーゼの活性値を、下記の計算式により求めた。
クレアチンキナーゼ活性値（Ｕｎｉｔ／Ｌ）＝｛（ΔＡ_２−ΔＲＢ_２）−ｃ×（ΔＡ_１−ΔＲＢ_１）｝×（Ｓ＋Ｖ_１＋Ｖ_２）×１０^６÷（Ｋ×ｄ×Ｓ）＝｛（ΔＡ_２−ΔＲＢ_２）−０．８０５×（ΔＡ_１−ΔＲＢ_１）｝×５．８３×１０^３
なお、この計算式において、ΔＡ_１は「測光１」において測定した１分間当りの吸光度変化量（Ａｂｓ／ｍｉｎ）を、ΔＡ_２は「測光２」において測定した１分間当りの吸光度変化量（Ａｂｓ／ｍｉｎ）を、ΔＲＢ_１は試料が前記の「不含試料」であるときの「測光１」において測定した１分間当りの吸光度変化量〔試薬盲検値〕（Ａｂｓ／ｍｉｎ）を、ΔＲＢ_２は試料が前記の「不含試料」であるときの「測光２」において測定した１分間当りの吸光度変化量〔試薬盲検値〕（Ａｂｓ／ｍｉｎ）を、Ｓは試料の量〔５．６μＬ〕を、Ｖ_１は第１試薬の量〔１６０μＬ〕を、Ｖ_２は第２試薬の量〔４０μＬ〕を、ｃは液量補正係数〔（Ｓ＋Ｖ_１）÷（Ｓ＋Ｖ_１＋Ｖ_２）＝０．８０５〕を、ＫはＮＡＤＰＨの理論モル吸光係数〔６．３０×１０^３Ｌ・ｍｏｌｅ^−１・ｃｍ^−１（主波長：３４０ｎｍ、副波長：４０５ｎｍ）〕、及びｄはセルの光路長〔１．００ｃｍ〕を表す。
（６） また、前記（１）において、前記３の（１）で５℃にて１２ヶ月間保存した第１試薬〔本発明（５ｍＭ）〕に替えて、前記３の（１）で５℃にて１２ヶ月間保存した前記１の（１）の（ｂ）の第１試薬〔本発明（１０ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（７） 更に、前記（１）において、前記３の（１）で５℃にて１２ヶ月間保存した第１試薬〔本発明（５ｍＭ）〕に替えて、前記３の（１）で５℃にて１２ヶ月間保存した前記１の（１）の（ｃ）の第１試薬〔従来発明（２５ｍＭ）〕を用いること以外は、前記（１）〜（５）の通りに操作を行い、第１試薬として前記の第１試薬〔従来発明（２５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（８） また、前記（３）において、前記３の（１）で５℃にて１２ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で５℃にて１２ヶ月間保存した前記１の（２）の（ｂ）の第２試薬〔従来発明（３０ｍＭ）〕を用いること以外は、前記（１）〜（７）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞、第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞、又は第１試薬〔従来発明（２５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞をそれぞれ用い、第２試薬として前記の第２試薬〔従来発明（３０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（９） 更に、前記（３）において、前記３の（１）で５℃にて１２ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（１）で５℃にて１２ヶ月間保存した前記１の（２）の（ｃ）の第２試薬〔従来発明（６０ｍＭ）〕を用いること以外は、前記（１）〜（７）の通りに操作を行い、第１試薬として前記の第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞、第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞、又は第１試薬〔従来発明（２５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞をそれぞれ用い、第２試薬として前記の第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
（１０） また、〔Ａ〕前記（１）において、前記３の（１）で５℃にて１２ヶ月間保存した第１試薬〔本発明（５ｍＭ）〕に替えて、前記３の（２）の通りこの測定時に調製した前記の第１試薬の対照試薬を用いること、及び、〔Ｂ〕前記（３）において、前記３の（１）で５℃にて１２ヶ月間保存した第２試薬〔本発明（０ｍＭ）〕に替えて、前記３の（２）の通りこの測定時に調製した前記の第２試薬の対照試薬を用いることの、これら〔Ａ〕と〔Ｂ〕以外のことは、前記（１）〜（５）の通りに操作を行い、前記の第１試薬の対照試薬、及び前記の第２試薬の対照試薬を用いた場合の各試料のクレアチンキナーゼの活性値を得た。
５．測定結果
前記４における各試料のクレアチンキナーゼの活性値の測定結果を、表１０に示した。
なお、この表１０において、「〔１〕活性値」の欄に示した値は各試料のクレアチンキナーゼの活性値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔２〕活性値差」の欄に示した値は、各試料における「〔１〕活性値」の欄の値から、同じ試料の、第１試薬が前記の第１試薬の対照試薬であり、そして第２試薬が前記の第２試薬の対照試薬である場合の「〔１〕活性値」の欄の値を差し引いた差の値〔Ｕｎｉｔ／Ｌ〕を示す。
また、「〔３〕相対比率」の欄に示した値は、各試料における「〔１〕活性値」の欄の値を、同じ試料の、第１試薬が前記の第１試薬の対照試薬であり、そして第２試薬が前記の第２試薬の対照試薬である場合の「〔１〕活性値」の欄の値で除した値をパーセント表示で示す。

６．考察
（１）第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞使用時
表１０で、第２試薬が第２試薬〔本発明（０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞であるときの測定結果において、第１試薬が５℃で１２ヶ月間保存した第１試薬〔従来発明（２５ｍＭ）〕である場合の測定値は、５℃で１２ヶ月間保存した第１試薬〔本発明（５ｍＭ）〕や第１試薬〔本発明（１０ｍＭ）〕の場合の測定値に比べ、低くなる傾向にある。
この場合、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きい傾向にあり、その測定値の低下度（表１０の「〔２〕活性値差」の値）は、例えば、試料が「試料−〔８〕」〜「試料−〔１０〕」においては、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には−３２Ｕｎｉｔ／Ｌ〜−１１７Ｕｎｉｔ／Ｌであり、第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には−８８Ｕｎｉｔ／Ｌ〜−４８７Ｕｎｉｔ／Ｌであり、そして、第１試薬〔従来発明（２５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には−１５７Ｕｎｉｔ／Ｌ〜−６６０Ｕｎｉｔ／Ｌである。
（２）第２試薬〔従来発明（３０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞使用時
表１０で、第２試薬が第２試薬〔従来発明（３０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞であるときの測定結果においても、第１試薬が５℃で１２ヶ月間保存した第１試薬〔従来発明（２５ｍＭ）〕である場合の測定値は、５℃で１２ヶ月間保存した第１試薬〔本発明（５ｍＭ）〕や第１試薬〔本発明（１０ｍＭ）〕の場合の測定値に比べ、低くなる傾向にある。
この場合においても、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きい傾向にあり、その測定値の低下度（表１０の「〔２〕活性値差」の値）は、例えば、試料が「試料−〔８〕」〜「試料−〔１０〕」においては、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には６４Ｕｎｉｔ／Ｌ〜−４５７Ｕｎｉｔ／Ｌであり、第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には−３６９Ｕｎｉｔ／Ｌ〜−４７２Ｕｎｉｔ／Ｌであり、そして、第１試薬〔従来発明（２５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には−４０３Ｕｎｉｔ／Ｌ〜−５００Ｕｎｉｔ／Ｌである。
（３）第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞使用時
表１０で、第２試薬が第２試薬〔従来発明（６０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞であるときの測定結果においても、第１試薬が５℃で１２ヶ月間保存した第１試薬〔従来発明（２５ｍＭ）〕である場合の測定値は、５℃で１２ヶ月間保存した第１試薬〔本発明（５ｍＭ）〕や第１試薬〔本発明（１０ｍＭ）〕の場合の測定値に比べ、低くなる傾向にある。
この場合においても、そのクレアチンキナーゼ活性値が高い試料ほど測定試薬保存による測定値の低下度は大きい傾向にあり、その測定値の低下度（表１０の「〔２〕活性値差」の値）は、例えば、試料が「試料−〔８〕」〜「試料−〔１０〕」においては、第１試薬が第１試薬〔本発明（５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には−１８８Ｕｎｉｔ／Ｌ〜−６８８Ｕｎｉｔ／Ｌであり、第１試薬〔本発明（１０ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には−３００Ｕｎｉｔ／Ｌ〜−７０２Ｕｎｉｔ／Ｌであり、そして、第１試薬〔従来発明（２５ｍＭ）〕＜５℃・暗所・１２ヶ月間保存＞の場合には−３２０Ｕｎｉｔ／Ｌ〜−８０３Ｕｎｉｔ／Ｌである。
（４）第１試薬についてのまとめ
前記の通り、第１試薬を５℃で１２ヶ月間保存（又は測定時調製）した結果より、第１試薬のグルコース濃度が２５ｍＭである場合には、第１試薬のグルコース濃度が５ｍＭ又は１０ｍＭである場合に比較して、測定試薬保存時の測定値の低下度には差異があり、測定値が低下する傾向にあることが分かる。
そして、第１試薬のグルコース濃度が５ｍＭ又は１０ｍＭである場合には、いずれの場合においても、測定値の低下が抑制され、安定化されていることが分かる。
（５）第２試薬についてのまとめ
前記の通り、第２試薬を５℃で１２ヶ月間保存（又は測定時調製）した結果より、第２試薬のグルコース濃度が３０ｍＭ又は６０ｍＭである場合には、第２試薬のグルコース濃度が０ｍＭである場合（すなわち、第２試薬がグルコースを含まない場合）に比較して、測定試薬保存時の測定値の低下度には差異があり、測定値が低下する傾向にあることが分かる。
そして、第２試薬がグルコースを含まない場合（０ｍＭの場合）には、測定値の低下が抑制され、安定化されていることが分かる。
（６）総まとめ
以上のことより、第１試薬が０．５ｍＭ〜１０ｍＭの濃度のグルコースを含み、かつ第２試薬がグルコースを含まないか又は１０ｍＭ以下の濃度のグルコースを含むことを特徴とする本発明のクレアチンキナーゼ活性測定試薬は、従来のクレアチンキナーゼ活性測定試薬に比べ、５℃にて１２ヶ月間という更なる長期間の保存時においても、その測定値の低下が抑制され、安定化されたものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間安定に保つことができ、かつ長期間正確にクレアチンキナーゼ活性の測定を行うことができるという効果を有するものであることが改めて確かめられた。
また、本発明のクレアチンキナーゼ活性測定試薬は、従来のクレアチンキナーゼ活性測定試薬に比べ、特にクレアチンキナーゼ活性値が高い高値域における測定値の低下の抑制に効果があるものであり、すなわち、本発明のクレアチンキナーゼ活性測定試薬は、長期間、高値域における直線性を維持することができるという効果を有するものであることが改めて確かめられた。   EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by description of this Example.
[Example 1] (Confirmation of stabilizing effect of the present invention at the time of storage-1)
  The stabilizing effect at the time of storage in the reagent for measuring creatine kinase activity of the present invention was confirmed.
  Specifically, the effect of stabilization when the first reagent of the creatine kinase activity measuring reagent of the present invention was stored at 37 ° C. for 7 days was confirmed.
1. Reagent for measuring creatine kinase activity
(1) First reagent
(A) Preparation of first reagent [present invention (1 mM)]
  The following reagent components were dissolved in pure water to the respective concentrations described above, adjusted to pH 6.5 (20 ° C.) with 0.1N acetic acid, and containing 1 mM glucose, the first reagent [ The present invention (1 mM)] was prepared.
  D-glucose (anhydrous) 1.00 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(B) Preparation of first reagent [present invention (5 mM)]
  The following reagent components were dissolved in pure water to the respective concentrations described above, adjusted to pH 6.5 (20 ° C.) with 0.1N acetic acid, and containing 5 mM glucose, the first reagent [ The present invention (5 mM)] was prepared.
  D-glucose (anhydrous) 5.00 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(C) Preparation of first reagent [present invention (10 mM)]
  The following reagent components are dissolved in pure water to the respective concentrations described above, adjusted to pH 6.5 (20 ° C.) with 0.1 N acetic acid, and containing 10 mM glucose, the first reagent [ The present invention (10 mM)] was prepared.
  D-glucose (anhydrous) 10.0 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(D) Preparation of first reagent [conventional invention (25 mM)]
  The following reagent components were dissolved in pure water to the respective concentrations described above, adjusted to pH 6.5 (20 ° C.) with 0.1N acetic acid, and the first reagent containing 25 mM glucose [ Conventional invention (25 mM)] was prepared.
  D-glucose (anhydrous) 25.0 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(E) First reagent [control reagent]
  The first reagent [manufacturer / seller: Sinotest Co., production number: A921] of a commercially available creatine kinase activity measuring reagent “Aculus Auto CK-JS” was used as the first reagent [control reagent].
(2) Second reagent
(A) Preparation of second reagent [present invention (0 mM)]
  The following reagent components are dissolved in pure water to the respective concentrations described above, adjusted to pH 8.0 (20 ° C.) with 0.1 N acetic acid, and do not contain glucose (that is, 0 mM). Two reagents [invention (0 mM)] were prepared.
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 4,000 Unit / L
  Imidazole 120 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  Diadenosine pentaphosphate trilithium salt 26μM
  Creatine phosphate disodium salt 120 mM
(B) Second reagent [control reagent]
  A second reagent [manufacturer / distributor: Sinotest, production number: A912] of a commercially available creatine kinase activity measuring reagent “Aculus Auto CK-JS” was used as the second reagent [control reagent].
2. sample
(1) Control serum-1
  A commercially available control serum for quality control, “Aalto Control I Y” (import / release source: Sinotest) was used as “control serum-1”.
(2) Control serum-2
  “Aalto Control CRPII U” (import / release source: Sinotest), which is a commercially available control serum for quality control, was used as “control serum-2”.
(3) Diluted sample
  A human serum sample prepared by adding rabbit creatine kinase MM isozyme was prepared (added serum sample), and this added serum sample was prepared as “sample diluent” (5.0 mM succinate buffer [pH 7.0 (20 ° C.) ]) To be 1/10, 2/10, 3/10, 4/10, 5/10, 6/10, 7/10, 8/10, and 9/10, respectively. “Sample-1” to “Sample-9” were prepared.
  The added serum sample was designated as “Sample-10” (10/10).
  (A) "Sample-1": 1/10 dilution of added serum sample
  (B) “Sample-2”: 2/10 dilution of added serum sample
  (C) “Sample-3”: 3/10 dilution of added serum sample
  (D) “Sample-4”: dilution of added serum sample by 4/10
  (E) “Sample-5”: 5/10 dilution of added serum sample
  (F) “Sample-6”: dilution of added serum sample by 6/10
  (G) “Sample-7”: Diluted added serum sample by 7/10
  (H) "Sample-8": diluted added serum sample 8/10
  (I) “Sample-9”: The added serum sample was diluted by 9/10
  (J) “Sample-10”: added serum sample
(4) Free sample
  The “sample diluent” in (3) above was a sample containing no creatine kinase and having a creatine kinase activity value of 0 Unit / L (unit / L), that is, a “free sample”.
3. Reagent storage
(1) Storage at 37 ° C
  (1) (a) first reagent [present invention (1 mM)], (b) first reagent [present invention (5 mM)], (c) first reagent [present invention (10 mM) And the first reagent (conventional invention (25 mM)) of (d) were stored at 37 ° C. in the dark for 7 days.
(2) Storage at 5 ° C
  (1) (e) first reagent [control reagent], (1) (2) (a) second reagent [present invention (0 mM)], and (b) second reagent [1] Control reagents] were each stored at 5 ° C. in the dark for 7 days.
4). Measurement of creatine kinase activity in samples
  The measurement of the creatine kinase activity value of each sample in the above 2 was performed by the double kinetic method using an automatic analyzer TBA-120FR manufactured by Toshiba Medical Systems.
(1) The first of (1) (a) of (1) above, which was stored in 5.6 μL of each sample of (2) (1) to (4) at 37 ° C. for 7 days in (3) of (3) above 160 μL of a reagent [the present invention (1 mM)] was added and reacted at 37 ° C.
  [0192]
(2) Next, the amount of change in absorbance from 8 points (136.18 seconds after the addition of the first reagent) to 16 points (280.46 seconds after the addition of the first reagent) is measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 1”]. (Measurement of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(3) Next, between 16 points (280.46 seconds after addition of the first reagent) and 17 points (298.49 seconds after addition of the first reagent), 7 days at 5 ° C. in (3) above 40 μL of the stored second reagent (a) of the above (2) (the present invention (0 mM)) was added and reacted at 37 ° C.
(4) Next, the change in absorbance from 24 points (424.74 seconds after the addition of the first reagent) to 33 points (587.005 seconds after the addition of the first reagent) was measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 2”]. (Measurement of change in absorbance by combining the amount of change in absorbance according to the activity value of creatine kinase contained in the sample and the amount of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(5) Next, the activity value of creatine kinase contained in the sample was determined by the following calculation formula.
  Creatine kinase activity value (Unit / L) = {(ΔA₂-ΔRB₂) −c × (ΔA₁-ΔRB₁)} × (S + V₁+ V₂) × 10⁶÷ (K × d × S) = {(ΔA₂-ΔRB₂) −0.805 × (ΔA₁-ΔRB₁)} × 5.83 × 10³
  In this calculation formula, ΔA₁Is the change in absorbance per minute (Abs / min) measured in “Photometry 1”, ΔA₂Is the change in absorbance per minute (Abs / min) measured in “Photometry 2”, ΔRB₁Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 1” when the sample is the above “free sample”, ΔRB₂Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 2” when the sample is the above “free sample”, and S is the amount of sample [5.6 μL ], V₁Is the amount of the first reagent [160 μL], V₂Is the amount of the second reagent [40 μL], c is the liquid amount correction coefficient [(S + V₁) ÷ (S + V₁+ V₂) = 0.805], K is the theoretical molar extinction coefficient of NADPH [6.30 × 10³L. mole^-1・ Cm^-1(Main wavelength: 340 nm, sub wavelength: 405 nm)] and d represent the optical path length [1.00 cm] of the cell.
(6) Further, in the above (1), instead of the first reagent [the present invention (1 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). Except for using the first reagent (1) of (1) (b) [the present invention (5 mM)], which was stored for 7 days, the operation was performed as described in (1) to (5) above. As a result, the activity value of creatine kinase was obtained for each sample when the first reagent [the present invention (5 mM)] was used.
(7) Furthermore, in (1) above, instead of the first reagent [this invention (1 mM)] stored at 37 ° C. for 7 days in (3) above, the temperature of the first reagent (37) in 3 above (1) is changed to 37 ° C. Except that the first reagent (1) of (1) (c) [present invention (10 mM)] stored for 7 days is used, and the operation is performed as described in (1) to (5) above. As a result, the activity value of creatine kinase of each sample when the first reagent [the present invention (10 mM)] was used was obtained.
(8) Further, in the above (1), instead of the first reagent (the present invention (1 mM)) stored at 37 ° C. for 7 days in the above (1), the temperature is increased to 37 ° C. in the above (1). Except that the first reagent (1) of (1) (d) [conventional invention (25 mM)] stored for 7 days is used, and the operation is performed as described in (1) to (5) above. As a result, the activity value of creatine kinase was obtained for each sample when the first reagent [conventional invention (25 mM)] was used.
(9) Further, in the above (1), in place of the first reagent (the present invention (1 mM)) stored at 37 ° C. for 7 days in the above (1), the temperature is increased to 5 ° C. in the above (2). And using the first reagent (control reagent) of (1) (1) of (1) above stored for 7 days, and storing for 7 days at 5 ° C in (2) of (3) above in (3) Instead of the second reagent [present invention (0 mM)], use the second reagent (control reagent) of (1) (2) (b) stored for 7 days at 5 ° C. in (3) of the above 3. Except for the above, the operations are carried out as in the above (1) to (5), the first reagent [control reagent] is used as the first reagent, and the second reagent [control reagent] is used as the second reagent. When used, the creatine kinase activity value of each sample was obtained.
5). Measurement result
  Among the results obtained by measuring the creatine kinase activity value of each sample in 4 above, the results when the sample is the control serum-1 or the control serum-2 are shown in Table 1, and the sample is the sample. The results for -1 to Sample-10 are shown in Table 2.
  In Tables 1 and 2, the value shown in the column “[1] Activity value” indicates the activity value [Unit / L] of creatine kinase of each sample.
  In addition, the values shown in the column “[2] Activity value difference” are the values in the column “[1] Activity value” of each sample, and both the first reagent and the second reagent of the same sample are “control reagents”. The difference value [Unit / L] obtained by subtracting the value in the “[1] Activity value” column is shown.
  The values shown in the column “[3] Relative ratio” are the values in the column “[1] Activity value” for each sample, and the first and second reagents of the same sample are “control reagents”. The value divided by the value in the “[1] Activity value” column in a certain case is shown as a percentage.

6). Consideration
(1) In Table 1, when the sample is control serum-1 or control serum-2, when the first reagent is the first reagent [conventional invention (25 mM)] stored at 37 ° C. for 7 days, Compared with the case of the first reagent [control reagent] stored at 5 ° C. for 7 days, the measured value is 3.1% to 3.4% lower.
  In contrast, the first reagent stored in the first reagent for 7 days at 37 ° C. [the present invention (1 mM)], the first reagent [the present invention (5 mM)], or the first reagent [the present invention (10 mM)]. In some cases, the measured value is 2.1% to 2.6% lower than that of the first reagent [control reagent] stored at 5 ° C. for 7 days.
  From the result of storing this first reagent at 37 ° C. or 5 ° C. for 7 days, when the glucose concentration of the first reagent is 1 mM, 5 mM, or 10 mM, compared with the case where the glucose concentration of the first reagent is 25 mM. Thus, it can be seen that the degree of decrease in the measured value when the measurement reagent is stored is small and stabilized.
(2) In Table 2, when the samples are Sample-1 to Sample-10, when the first reagent is the first reagent [conventional invention (25 mM)] stored at 37 ° C. for 7 days, the temperature is 5 ° C. Compared with the case of the first reagent [control reagent] stored for 7 days, the measured value is 3.3% to 45.3% lower.
  In particular, the higher the creatine kinase activity value, the greater the degree of decrease in the measured value due to storage of the measurement reagent. For example, the measured value is 35.0% lower in Sample-8, and the measured value is 40% in Sample-9. .2% lower, and in Sample-10, the measured value is 45.3% lower.
  In contrast, the first reagent stored in the first reagent for 7 days at 37 ° C. [the present invention (1 mM)], the first reagent [the present invention (5 mM)], or the first reagent [the present invention (10 mM)]. In some cases, the measured value is 1.3% to 17.7% lower than that of the first reagent [control reagent] stored at 5 ° C. for 7 days.
  In this case as well, the higher the creatine kinase activity value, the greater the degree of decrease in the measured value due to storage of the measurement reagent, but the degree of decrease in the measured value is, for example, 8.5% to 12.9% in Sample-8. Yes, from 9.7% to 15.1% for Sample-9 and from 11.6% to 17.7% for Sample-10.
  From the result of storing this first reagent at 37 ° C. or 5 ° C. for 7 days, when the glucose concentration of the first reagent is 1 mM, 5 mM, or 10 mM, compared with the case where the glucose concentration of the first reagent is 25 mM. Thus, it can be seen that there is a large difference in the degree of decrease in the measured value when the measurement reagent is stored, and the decrease in the measured value is suppressed and stabilized.
(3) From the above, the present invention is characterized in that the first reagent contains glucose at a concentration of 0.5 mM to 10 mM, and the second reagent does not contain glucose or contains glucose at a concentration of 10 mM or less. The reagent for measuring creatine kinase activity of the present invention is stabilized by suppressing a decrease in the measured value even when stored under extremely severe conditions of 37 ° C. compared to the conventional reagent for measuring creatine kinase activity, That is, it was confirmed that the creatine kinase activity measuring reagent of the present invention has an effect that it can be kept stable for a long period of time and can accurately measure creatine kinase activity for a long period of time.
  Further, the creatine kinase activity measuring reagent of the present invention is more effective than the conventional creatine kinase activity measuring reagent in suppressing the decrease in the measured value particularly in the high range where the creatine kinase activity value is high. It was confirmed that the reagent for measuring creatine kinase activity has an effect of maintaining linearity in a high value range for a long period of time.
[Example 2] (Confirmation of stabilizing effect during storage of the present invention-2)
  The stabilizing effect at the time of storage in the reagent for measuring creatine kinase activity of the present invention was confirmed.
  Specifically, the effect of stabilization when the first reagent of the creatine kinase activity measuring reagent of the present invention was stored at 5 ° C. and 37 ° C. for 7 days was confirmed.
1. Reagent for measuring creatine kinase activity
(1) First reagent
(A) Preparation of first reagent [conventional invention (0 mM)]
  The following reagent components are dissolved in pure water to the respective concentrations described above, adjusted to pH 6.5 (20 ° C.) with 0.1N acetic acid, and do not contain glucose (that is, 0 mM). One reagent [conventional invention (0 mM)] was prepared.
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(B) Preparation of first reagent [conventional invention (0.0781 mM)]
  Each of the following reagent components is dissolved in pure water so as to have the stated concentration, adjusted to pH 6.5 (20 ° C.) with 0.1N acetic acid, and contains glucose at a concentration of 0.0781 mM. A reagent [conventional invention (0.0781 mM)] was prepared.
  D-glucose (anhydrous) 0.0781 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(C) Preparation of first reagent [conventional invention (0.156 mM)]
  Each of the following reagent components is dissolved in pure water so as to have the stated concentration, adjusted to pH 6.5 (20 ° C.) with 0.1N acetic acid, and contains glucose at a concentration of 0.156 mM. A reagent [conventional invention (0.156 mM)] was prepared.
  D-glucose (anhydrous) 0.156 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(D) Preparation of first reagent [conventional invention (0.313 mM)]
  Each of the following reagent components is dissolved in pure water so as to have the stated concentration, adjusted to pH 6.5 (20 ° C.) with 0.1 N acetic acid, and contains glucose at a concentration of 0.313 mM. A reagent [conventional invention (0.313 mM)] was prepared.
  D-glucose (anhydrous) 0.313 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(E) Preparation of first reagent [present invention (0.625 mM)]
  Each of the following reagent components is dissolved in pure water so as to have the stated concentration, adjusted to pH 6.5 (20 ° C.) with 0.1 N acetic acid, and contains glucose at a concentration of 0.625 mM. A reagent [present invention (0.625 mM)] was prepared.
  D-glucose (anhydrous) 0.625 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(F) Preparation of first reagent [present invention (1.25 mM)]
  Each of the following reagent components is dissolved in pure water so as to have the stated concentration, adjusted to pH 6.5 (20 ° C.) with 0.1 N acetic acid, and contains glucose at a concentration of 1.25 mM, A reagent [the present invention (1.25 mM)] was prepared.
  D-glucose (anhydrous) 1.25 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(G) Preparation of first reagent [present invention (2.5 mM)]
  The following reagent components are dissolved in pure water so as to have the stated concentrations, adjusted to pH 6.5 (20 ° C.) with 0.1 N acetic acid, and contain 2.5 mM glucose. A reagent [present invention (2.5 mM)] was prepared.
  D-glucose (anhydrous) 2.50 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 1,000 Unit / L
  Hexokinase (from Roche Diagnostics, Yeast, recombinant) 3,000Unit / L
  Imidazole 100 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  N-acetyl-L-cysteine (Roche Diagnostics) 27 mM
  NADP (oxidized form) disodium salt 2.7 mM
  AMP disodium salt 6.7 mM
  Diadenosine pentaphosphate trilithium salt 13μM
  Potassium disulfite (Nacalai Tesque) 1.4 mM
(H) First reagent [the present invention (5 mM)]
  The “first reagent [present invention (5 mM)]” in (1) (b) of Example 1 was used as the “first reagent [present invention (5 mM)]”.
(I) First reagent [the present invention (10 mM)]
  The “first reagent [present invention (10 mM)]” in (1) (1) of Example 1 was used as the “first reagent [present invention (10 mM)]”.
(J) First reagent [control reagent]
  The first reagent [manufacturer / seller: Shinotest, serial number: E913] of a commercially available creatine kinase activity measuring reagent “Aculus Auto CK-JS” was used as the first reagent [control reagent].
(2) Second reagent
(A) Second reagent [the present invention (0 mM)]
  The “second reagent [present invention (0 mM)]” in (2) (a) of Example 1 was used as the “second reagent [present invention (0 mM)]”.
(B) Second reagent [control reagent]
  The second reagent [manufacturer / seller: Shinotest, serial number: E923] of a commercially available creatine kinase activity measuring reagent “Aculus Auto CK-JS” was used as the second reagent [control reagent].
2. sample
(1) Diluted sample
  A human serum sample prepared by adding rabbit creatine kinase MM isozyme was prepared (added serum sample), and this added serum sample was prepared as “sample diluent” (5.0 mM succinate buffer [pH 7.0 (20 ° C.)]. ]) To be 1/10, 2/10, 3/10, 4/10, 5/10, 6/10, 7/10, 8/10, and 9/10, respectively. “Sample-a” to “sample-i” were prepared.
  The added serum sample was designated as “Sample-j” (10/10).
  (A) “Sample-a”: 1/10 dilution of added serum sample
  (B) “Sample-b”: 2/10 dilution of added serum sample
  (C) “Sample-c”: 3/10 dilution of added serum sample
  (D) “Sample-d”: 4/10 dilution of added serum sample
  (E) “Sample-e”: 5/10 dilution of added serum sample
  (F) “Sample-f”: 6/10 dilution of added serum sample
  (G) “Sample-g”: 7/10 dilution of added serum sample
  (H) “Sample-h”: diluted added serum sample 8/10
  (I) "Sample-i": 9/10 dilution of added serum sample
  (J) “Sample-j”: added serum sample
(2) Non-containing sample
  The “sample dilution liquid” in the above (1) was a sample not containing creatine kinase and having a creatine kinase activity value of 0 Unit / L (unit / L), that is, a “free sample”.
3. Reagent storage
(1) Storage at 37 ° C
  (1) (a) first reagent [present invention (1 mM)], (b) first reagent [present invention (5 mM)], (c) first reagent [present invention (10 mM) And the first reagent (conventional invention (25 mM)) of (d) were stored at 37 ° C. in the dark for 7 days.
(2) Storage at 5 ° C
  (1) (a) first reagent [present invention (1 mM)], (b) first reagent [present invention (5 mM)], (c) first reagent [present invention (10 mM) And the first reagent (conventional invention (25 mM)) of (d), each was stored in a dark place for 7 days even at 5 ° C.
  In addition, the first reagent (control reagent) of (1) (e) of (1), the second reagent of (a) of (1) of (1) [the present invention (0 mM)], and the second reagent of (b) of (1). Reagents (control reagents) were each stored at 5 ° C. in the dark for 7 days.
4). Measurement of creatine kinase activity in samples
  The measurement of the creatine kinase activity value of each sample in the above 2 was performed by the double kinetic method using an automatic analyzer TBA-120FR manufactured by Toshiba Medical Systems.
(1) The first of (1) (a) of (1) above, which was stored in 5.6 μL of each sample of (2) (1) to (2) above at (3) (7) at 37 ° C. for 7 days. 160 μL of a reagent [conventional invention (0 mM)] was added and reacted at 37 ° C.
(2) Next, the amount of change in absorbance from 8 points (136.18 seconds after the addition of the first reagent) to 16 points (280.46 seconds after the addition of the first reagent) is measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 1”]. (Measurement of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(3) Next, between 16 points (280.46 seconds after addition of the first reagent) and 17 points (298.49 seconds after addition of the first reagent), 7 days at 5 ° C. in (3) above 40 μL of the stored second reagent (a) of the above (2) (the present invention (0 mM)) was added and reacted at 37 ° C.
(4) Next, the change in absorbance from 24 points (424.74 seconds after the addition of the first reagent) to 33 points (587.005 seconds after the addition of the first reagent) was measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 2”]. (Measurement of change in absorbance by combining the amount of change in absorbance according to the activity value of creatine kinase contained in the sample and the amount of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(5) Next, the activity value of creatine kinase contained in the sample was determined by the following calculation formula.
  Creatine kinase activity value (Unit / L) = {(ΔA₂-ΔRB₂) −c × (ΔA₁-ΔRB₁)} × (S + V₁+ V₂) × 10⁶÷ (K × d × S) = {(ΔA₂-ΔRB₂) −0.805 × (ΔA₁-ΔRB₁)} × 5.83 × 10³
  In this calculation formula, ΔA₁Is the change in absorbance per minute (Abs / min) measured in “Photometry 1”, ΔA₂Is the change in absorbance per minute (Abs / min) measured in “Photometry 2”, ΔRB₁Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 1” when the sample is the above “free sample”, ΔRB₂Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 2” when the sample is the above “free sample”, and S is the amount of sample [5.6 μL ], V₁Is the amount of the first reagent [160 μL], V₂Is the amount of the second reagent [40 μL], c is the liquid amount correction coefficient [(S + V₁) ÷ (S + V₁+ V₂) = 0.805], K is the theoretical molar extinction coefficient of NADPH [6.30 × 10³L. mole^-1・ Cm^-1(Main wavelength: 340 nm, sub wavelength: 405 nm)] and d represent the optical path length [1.00 cm] of the cell.
(6) Further, in the above (1), instead of the first reagent [conventional invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is increased to 5 ° C. in the above (2). Except that the first reagent of (1) (a) of [1] (conventional invention (0 mM)) stored for 7 days is used, and the operation is performed as in (1) to (5) above. As a result, the activity value of creatine kinase was obtained for each sample when the first reagent [conventional invention (0 mM)] was used.
(7) Further, in the above (1), instead of the first reagent [conventional invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). The first reagent (1) of (1) (b) [conventional invention (0.0781 mM)] stored for 7 days, or the above 1 (1) stored for 7 days at 5 ° C. in (3) above (1) (b) except that the first reagent [conventional invention (0.0781 mM)] is used, the operation is performed as described in (1) to (6) above, and the first reagent is used as the first reagent. The activity value of creatine kinase was obtained for each sample when the reagent [conventional invention (0.0781 mM)] was used.
(8) Further, in the above (1), instead of the first reagent [conventional invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). The first reagent of (1) (c) of the above (1) (conventional invention (0.156 mM)) stored for 7 days is used, or the 1 of the above (1) stored for 7 days at 5 ° C. (1) except that the first reagent of (c) [conventional invention (0.156 mM)] is used, the operation is performed as described in the above (1) to (6), and the first reagent is used as the first reagent. The activity value of creatine kinase of each sample when the reagent [conventional invention (0.156 mM)] was used was obtained.
(9) Further, in the above (1), instead of the first reagent [conventional invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). The first reagent (1) of (1) (d) [conventional invention (0.313 mM)] stored for 7 days is used, or the above 1 (1) stored for 7 days at 5 ° C. in (3) above (1) except that the first reagent (d) of [1] (conventional invention (0.313 mM)) is used, the operation is carried out as in the above (1) to (6), and the first reagent is used as the first reagent. The activity value of creatine kinase of each sample when the reagent [conventional invention (0.313 mM)] was used was obtained.
(10) Further, in the above (1), instead of the first reagent [conventional invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). The first reagent (1) (e) of the above (1) (this invention (0.625 mM)) stored for 7 days is used, or the above 1 (1) stored for 7 days at 5 ° C. in (3) above (1) except that the first reagent (e) of the present invention (the present invention (0.625 mM)) is used, the operation is performed as in the above (1) to (6), and the first reagent is used as the first reagent. The activity value of creatine kinase of each sample when the reagent [present invention (0.625 mM)] was used was obtained.
(11) Further, in (1) above, instead of the first reagent (conventional invention (0 mM)) stored at 37 ° C. for 7 days in (3) above, the temperature is increased to 37 ° C. in (1) above. The first reagent (1) of (1) (f) [this invention (1.25 mM)] stored for 7 days is used, or the above 1 (1) stored for 7 days at 5 ° C. in (3) above (1) except that the first reagent (f) of the present invention (the present invention (1.25 mM)) is used, the operation is carried out as in the above (1) to (6), and the first reagent is used as the first reagent. The activity value of creatine kinase of each sample when the reagent [present invention (1.25 mM)] was used was obtained.
(12) Also, in the above (1), instead of the first reagent (conventional invention (0 mM)) stored at 37 ° C. for 7 days in the above (1), the temperature is increased to 37 ° C. in the above (1). The first reagent (1) (g) of the above (1) (this invention (2.5 mM)) stored for 7 days is used, or the above 1 (1) stored for 7 days at 5 ° C. in (3) above (1) except that the first reagent (g) of the present invention (the present invention (2.5 mM)) is used, the operation is performed as in the above (1) to (6), and the first reagent is used as the first reagent. The activity value of creatine kinase of each sample when the reagent [present invention (2.5 mM)] was used was obtained.
(13) Further, in the above (1), instead of the first reagent [conventional invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). The first reagent (1) of (1) (h) [invention (5 mM)] stored for 7 days is used, or the (1) of (1) stored for 7 days at 5 ° C. in (3) above. 1) (h) except that the first reagent [the present invention (5 mM)] is used, the operation is performed as in the above (1) to (6), and the first reagent [the present invention] is used as the first reagent. (5 mM)] was used to obtain the creatine kinase activity value of each sample.
(14) Further, in the above (1), instead of the first reagent (conventional invention (0 mM)) stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). The first reagent (1) (i) of the above (1) (this invention (10 mM)) stored for 7 days is used, or the 1 (1) stored for 7 days at 5 ° C. in (3) above. 1) (i) The first reagent [the present invention (10 mM)] is used except that the first reagent [the present invention] is used as the first reagent. (10 mM)] was used to obtain the activity value of creatine kinase in each sample.
(15) Further, in the above (1), in place of the first reagent (conventional invention (0 mM)) stored at 37 ° C. for 7 days in the above (1), the temperature is increased to 5 ° C. in the above (2). Using the first reagent (control reagent) of (1) (1) of (1) above stored for 7 days, and storing for 7 days at 5 ° C in (2) of (3) above in (3) Instead of the second reagent [present invention (0 mM)], use the second reagent (control reagent) of (1) (2) (b) stored for 7 days at 5 ° C. in (3) of the above 3. Except for the above, the operations are carried out as in the above (1) to (5), the first reagent [control reagent] is used as the first reagent, and the second reagent [control reagent] is used as the second reagent. When used, the creatine kinase activity value of each sample was obtained.
5). Measurement result
  Among the results obtained by measuring the creatine kinase activity value of each sample in 4 above, the storage temperature of the first reagent [conventional invention (0 mM)] to the first reagent [present invention (10 mM)] is 5 Table 3 shows the results at the temperature of ° C., and Table 4 shows the results when the storage temperature of the first reagent [conventional invention (0 mM)] to the first reagent [present invention (10 mM)] is 37 ° C. Indicated.
  In Tables 3 and 4, the value shown in the column “[1] Activity value” indicates the activity value [Unit / L] of creatine kinase of each sample.
  In addition, the values shown in the column “[2] Activity value difference” are the values in the column “[1] Activity value” of each sample, and both the first reagent and the second reagent of the same sample are “control reagents”. The difference value [Unit / L] obtained by subtracting the value in the “[1] Activity value” column is shown.
  The values shown in the column “[3] Relative ratio” are the values in the column “[1] Activity value” for each sample, and the first and second reagents of the same sample are “control reagents”. The value divided by the value in the “[1] Activity value” column in a certain case is shown as a percentage.

6). Consideration
(1) From Table 3, when the storage temperature of the first reagent [conventional invention (0 mM)] to the first reagent [present invention (10 mM)] is 5 ° C., the first reagent is stored at 5 ° C. for 7 days. In the case of the first reagent [conventional invention (0 mM)] to the first reagent [conventional invention (0.313 mM)], the measured value is significantly lower than the original creatine kinase activity value. It turns out that the measurement of the creatine kinase activity value in it cannot be performed quantitatively.
  On the other hand, when the first reagent is the first reagent [the present invention (0.625 mM)] to the first reagent [the present invention (10 mM)] stored for 7 days at 5 ° C. It can be seen that the creatine kinase activity value in the sample can be measured quantitatively (up to / 10 dilution or 10/10 sample).
  From the result of storing this first reagent at 37 ° C. or 5 ° C. for 7 days, the first reagent contains glucose at a concentration of 0.5 mM to 10 mM, and the second reagent does not contain glucose or has a concentration of 10 mM or less. In the creatine kinase activity measuring reagent of the present invention characterized by containing glucose, the creatine kinase activity value in the sample can be quantitatively measured up to a high value range, but the first reagent does not contain glucose. Alternatively, when the glucose concentration was less than 10 mM, it was confirmed that the creatine kinase activity value in the sample could not be quantitatively measured.
(2) From Table 4, when the storage temperature of the first reagent [conventional invention (0 mM)] to the first reagent [present invention (10 mM)] is 37 ° C., the first reagent is 7 ° C. at 7 ° C. In the case of the first reagent [conventional invention (0 mM)] to the first reagent [conventional invention (0.313 mM)] stored for a day, the measured value is still significantly lower than the original creatine kinase activity value. It can be seen that the creatine kinase activity value in the sample cannot be quantitatively measured.
  On the other hand, when the first reagent is the first reagent [the present invention (0.625 mM)] to the first reagent [the present invention (10 mM)] stored at 37 ° C. for 7 days, It can be seen that the creatine kinase activity value of can be quantitatively measured.
  In addition, the first reagent was stored for 7 days at 37 ° C. [the present invention (0.625 mM) relative to the measured value when the first reagent was the first reagent [control reagent] stored for 7 days at 5 ° C. ] To 1st reagent [invention (10 mM)], the relative ratio of the measured values in sample-j is 72.2% in the first reagent [invention (0.625 mM)], The reagent [present invention (1.25 mM)] is 94.1%, the first reagent [present invention (2.5 mM)] is 95.3%, and the first reagent [present invention (5 mM)] is 95%. 1%, and 94.3% for the first reagent [the present invention (10 mM)].
  From the result of storing the first reagent at 37 ° C. or 5 ° C. for 7 days, when the first reagent is the first reagent [the present invention (0.625 mM)] to the first reagent [the present invention (10 mM)] It can be seen that a decrease in the measured value during storage of the measurement reagent is suppressed and stabilized.
  That is, the reagent for measuring creatine kinase activity of the present invention, wherein the first reagent contains glucose at a concentration of 0.5 mM to 10 mM, and the second reagent contains no glucose or glucose at a concentration of 10 mM or less. Even if the first reagent is stored at 37 ° C. for 7 days, the creatine kinase activity value in the sample can be quantitatively measured up to a high value range, but the first reagent does not contain glucose or its When the glucose concentration was less than 10 mM, it was confirmed that the creatine kinase activity value in the sample could not be quantitatively measured.
  In addition, according to this example, the creatine kinase activity measuring reagent of the present invention was stabilized by suppressing the decrease in measured value even when stored under extremely severe conditions of 37 ° C., that is, It was again confirmed that the creatine kinase activity measuring reagent of the present invention has an effect that it can be kept stable for a long period of time and can accurately measure creatine kinase activity for a long period of time.
  Furthermore, according to this example, the creatine kinase activity measuring reagent of the present invention is effective in suppressing the decrease in the measured value particularly in the high value range where the creatine kinase activity value is high. It was confirmed once again that it has the effect that it can be maintained.
[Example 3] (Confirmation of stabilizing effect of the present invention at the time of storage-3)
  The stabilizing effect at the time of storage in the reagent for measuring creatine kinase activity of the present invention was confirmed.
  Specifically, the effect of stabilization when the second reagent of the creatine kinase activity measuring reagent of the present invention was stored at 37 ° C. for 7 days was confirmed.
1. Reagent for measuring creatine kinase activity
(1) First reagent
First reagent [invention (5 mM)]
  The “first reagent [present invention (5 mM)]” in (1) (b) of Example 1 was used as the “first reagent [present invention (5 mM)]”.
(2) Second reagent
(A) Second reagent [the present invention (0 mM)]
  The “second reagent [present invention (0 mM)]” in (2) (a) of Example 1 was used as the “second reagent [present invention (0 mM)]”.
(B) Preparation of second reagent [present invention (10 mM)]
  The following reagent components were dissolved in pure water to the respective concentrations described above, adjusted to pH 8.0 (20 ° C.) with 0.1N acetic acid, and containing a 10 mM glucose, a second reagent [ The present invention (10 mM)] was prepared.
  D-glucose (anhydrous) 10.0 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 4,000 Unit / L
  Imidazole 120 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  Diadenosine pentaphosphate trilithium salt 26μM
  Creatine phosphate disodium salt 120 mM
(C) Preparation of second reagent [conventional invention (30 mM)]
  The following reagent components were dissolved in pure water so as to have the respective concentrations described above, adjusted to pH 8.0 (20 ° C.) with 0.1 N acetic acid, and containing a 30 mM glucose, a second reagent [ Conventional invention (30 mM)] was prepared.
  D-glucose (anhydrous) 30.0 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 4,000 Unit / L
  Imidazole 120 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  Diadenosine pentaphosphate trilithium salt 26μM
  Creatine phosphate disodium salt 120 mM
(D) Preparation of second reagent [conventional invention (40 mM)]
  The following reagent components were dissolved in pure water so as to have the respective concentrations described above, adjusted to pH 8.0 (20 ° C.) with 0.1N acetic acid, and a second reagent containing 40 mM glucose [ Conventional invention (40 mM)] was prepared.
  D-glucose (anhydrous) 40.0 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 4,000 Unit / L
  Imidazole 120 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  Diadenosine pentaphosphate trilithium salt 26μM
  Creatine phosphate disodium salt 120 mM
(E) Preparation of second reagent [conventional invention (50 mM)]
  The following reagent components were dissolved in pure water to the respective concentrations described above, adjusted to pH 8.0 (20 ° C.) with 0.1N acetic acid, and containing a 50 mM glucose, a second reagent [ Conventional invention (50 mM)] was prepared.
  D-glucose (anhydrous) 50.0 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 4,000 Unit / L
  Imidazole 120 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  Diadenosine pentaphosphate trilithium salt 26μM
  Creatine phosphate disodium salt 120 mM
(F) Preparation of second reagent [conventional invention (60 mM)]
  Each of the following reagent components was dissolved in pure water so as to have the stated concentration, adjusted to pH 8.0 (20 ° C.) with 0.1N acetic acid, and contained a 60 mM concentration of second reagent [ Conventional invention (60 mM)] was prepared.
  D-glucose (anhydrous) 60.0 mM
  Glucose-6-phosphate dehydrogenase [Roche Diagnostics, Leuconostoc mesenteroides, derived from recombinant] 4,000 Unit / L
  Imidazole 120 mM
  Ethylenediaminetetraacetic acid disodium salt 2mM
  Sodium azide 7.7 mM
  Magnesium acetate (tetrahydrate) 10 mM
  ADP monopotassium salt 2 mM
  Diadenosine pentaphosphate trilithium salt 26μM
  Creatine phosphate disodium salt 120 mM
2. sample
(1) Diluted sample
  A human serum sample prepared by adding rabbit creatine kinase MM isozyme was prepared (added serum sample), and this added serum sample was prepared as “sample diluent” (5.0 mM succinate buffer [pH 7.0 (20 ° C.)]. ]) To be 1/10, 2/10, 3/10, 4/10, 5/10, 6/10, 7/10, 8/10, and 9/10, respectively. “Sample-A” to “Sample-I” were prepared.
  The added serum sample was designated as “Sample-J” (10/10).
  (A) “Sample-A”: 1/10 dilution of added serum sample
  (B) “Sample-B”: 2/10 dilution of added serum sample
  (C) “Sample-C”: diluted serum sample by 3/10
  (D) “Sample-D”: 4/10 dilution of added serum sample
  (E) “Sample-E”: diluted serum sample 5/10
  (F) “Sample-F”: 6/10 dilution of added serum sample
  (G) “Sample-G”: dilution of added serum sample by 7/10
  (H) “Sample-H”: diluted added serum sample 8/10
  (I) “Sample-I”: added serum sample diluted 9/10
  (J) “Sample-J”: added serum sample
(2) Non-containing sample
  The “sample dilution liquid” in the above (1) was a sample not containing creatine kinase and having a creatine kinase activity value of 0 Unit / L (unit / L), that is, a “free sample”.
3. Reagent storage
(1) Storage at 37 ° C
  (2) (a) second reagent [present invention (0 mM)], (b) second reagent [present invention (10 mM)], (c) second reagent [conventional invention (30 mM) ], (D) second reagent [conventional invention (40 mM)], (e) second reagent [conventional invention (50 mM)], and (f) second reagent [conventional invention (60 mM)], Each was stored at 37 ° C. in the dark for 7 days.
(2) Storage at 5 ° C
  The first reagent (1) of the first aspect (the present invention (5 mM)) was stored at 5 ° C. in a dark place for 7 days.
4). Measurement of creatine kinase activity in samples
  The measurement of the creatine kinase activity value of each sample in the above 2 was performed by the double kinetic method using an automatic analyzer TBA-120FR manufactured by Toshiba Medical Systems.
(1) The first reagent of (1) of (1) above, which is stored in 5.6 μL of each of the samples of (2) (1) to (2) at 5 ° C. for 7 days in (2) of the above [the present invention (5 mM)] was added and reacted at 37 ° C.
(2) Next, the amount of change in absorbance from 8 points (136.18 seconds after the addition of the first reagent) to 16 points (280.46 seconds after the addition of the first reagent) is measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 1”]. (Measurement of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(3) Next, between 16 points (280.46 seconds after addition of the first reagent) and 17 points (298.49 seconds after addition of the first reagent), 7 days at 37 ° C. in (1) above 40 μL of the stored second reagent (a) of the above (2) (the present invention (0 mM)) was added and reacted at 37 ° C.
(4) Next, the change in absorbance from 24 points (424.74 seconds after the addition of the first reagent) to 33 points (587.005 seconds after the addition of the first reagent) was measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 2”]. (Measurement of change in absorbance by combining the amount of change in absorbance according to the activity value of creatine kinase contained in the sample and the amount of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(5) Next, the activity value of creatine kinase contained in the sample was determined by the following calculation formula.
  Creatine kinase activity value (Unit / L) = {(ΔA₂-ΔRB₂) −c × (ΔA₁-ΔRB₁)} × (S + V₁+ V₂) × 10⁶÷ (K × d × S) = {(ΔA₂-ΔRB₂) −0.805 × (ΔA₁-ΔRB₁)} × 5.83 × 10³
  In this calculation formula, ΔA₁Is the change in absorbance per minute (Abs / min) measured in “Photometry 1”, ΔA₂Is the change in absorbance per minute (Abs / min) measured in “Photometry 2”, ΔRB₁Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 1” when the sample is the above “free sample”, ΔRB₂Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 2” when the sample is the above “free sample”, and S is the amount of sample [5.6 μL ], V₁Is the amount of the first reagent [160 μL], V₂Is the amount of the second reagent [40 μL], c is the liquid amount correction coefficient [(S + V₁) ÷ (S + V₁+ V₂) = 0.805], K is the theoretical molar extinction coefficient of NADPH [6.30 × 10³L. mole^-1・ Cm^-1(Main wavelength: 340 nm, sub wavelength: 405 nm)] and d represent the optical path length [1.00 cm] of the cell.
(6) Further, in the above (3), instead of the second reagent [the present invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). Except that the second reagent of (1) (2) (b) [present invention (10 mM)] stored for 7 days is used, and the operation is performed as in (1) to (5) above. As a result, the creatine kinase activity value of each sample was obtained when the second reagent [the present invention (10 mM)] was used.
(7) Further, in the above (3), instead of the second reagent [the present invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is increased to 37 ° C. in the above (1). Except that the second reagent (c) of 1) (2) (conventional invention (30 mM)) stored for 7 days is used, and the operation is performed as in (1) to (5) above. As a result, the activity value of creatine kinase of each sample when the second reagent [conventional invention (30 mM)] was used was obtained.
(8) Further, in the above (3), instead of the second reagent [the present invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is changed to 37 ° C. in the above (1). Except that the second reagent (d) of (1) (2) (conventional invention (40 mM)) stored for 7 days is used, and the operation is performed as in (1) to (5) above. As a result, the creatine kinase activity value of each sample was obtained when the second reagent [conventional invention (40 mM)] was used.
(9) Further, in (3) above, instead of the second reagent [invention (0 mM)] stored at 37 ° C. for 7 days in (3) above, the temperature is increased to 37 ° C. in (1) above. Except that the second reagent (e) of (1) (2) (conventional invention (50 mM)) stored for 7 days is used, and the operation is performed as in (1) to (5) above. As a result, the activity value of creatine kinase was obtained for each sample when the second reagent [conventional invention (50 mM)] was used.
(10) Further, in the above (3), instead of the second reagent [the present invention (0 mM)] stored at 37 ° C. for 7 days in the above (1), the temperature is adjusted to 37 ° C. in the above (1). Except that the second reagent (2) of (1) (2) (conventional invention (60 mM)) stored for 7 days is used, and the procedure is performed as in (1) to (5) above. As a result, the creatine kinase activity value of each sample was obtained when the second reagent [conventional invention (60 mM)] was used.
5). Measurement result
  Table 5 shows the results obtained by measuring the creatine kinase activity value of each sample in 4 above.
  In Table 5, the value shown in the column “[1] Activity value” indicates the activity value [Unit / L] of creatine kinase of each sample.
  In addition, the value shown in the column of “[2] Activity value difference” indicates that the first reagent of the same sample is the first reagent [present invention (5 mM) from the value of the “[1] activity value” column in each sample. )] <5 ° C., dark place, stored for 7 days> and the second reagent is the second reagent [invention (0 mM)] <37 ° C., dark place, stored for 7 days> The difference value [Unit / L] obtained by subtracting the value in the “activity value” column is shown.
  The value shown in the column “[3] Relative ratio” is the same as the value in the column “[1] Activity value” in each sample, and the first reagent of the same sample is the first reagent [present invention (5 mM) [<1] activity when the second reagent is the second reagent [the present invention (0 mM)] <37 ° C, dark place, preserved for 7 days> The value divided by the value in the “value” column is shown as a percentage.

6). Consideration
(1) In Table 5, when the second reagent is the second reagent stored at 37 ° C. for 7 days [the present invention (10 mM)], the measured value is also the second reagent stored at 37 ° C. for 7 days [the present invention (0 mM)] is almost the same as the measured value.
  In contrast, the second reagent stored at 37 ° C. for 7 days [conventional invention (30 mM)], the second reagent [conventional invention (40 mM)], the second reagent [conventional invention (50 mM)], or In the case of the second reagent [conventional invention (60 mM)], the measured value is lower than that of the second reagent [present invention (0 mM)] stored at 37 ° C. for 7 days.
  In this case, the higher the creatine kinase activity value, the greater the degree of decrease in the measured value due to storage of the measurement reagent. The degree of decrease in the measured value (the value of “[2] Activity value difference” in Table 5) is For example, when the sample is “Sample-H” to “Sample-J”, the second reagent is −3 Unit / L to −19 Unit / L when the second reagent is the second reagent [conventional invention (30 mM)], In the case of the reagent [conventional invention (40 mM)], it is -29 Unit / L to -39 Unit / L, and in the case of the second reagent [conventional invention (50 mM)], it is -44 Unit / L to -53 Unit / L. In the case of the second reagent [conventional invention (60 mM)], it is -70 Unit / L to -86 Unit / L.
  From the result of storing this second reagent at 37 ° C. for 7 days, when the glucose concentration of the second reagent is 30 mM, 40 mM, 50 mM, or 60 mM, the second reagent does not contain glucose or is 10 mM or less. It can be seen that there is a large difference in the degree of decrease in the measured value when the measurement reagent is stored compared to the case where the concentration of glucose is included, and the measured value has decreased.
  And when a 2nd reagent does not contain glucose or contains glucose with a density | concentration of 10 mM or less, it turns out that the fall of a measured value is suppressed and stabilized.
(2) From the above, the present invention is characterized in that the first reagent contains glucose at a concentration of 0.5 mM to 10 mM, and the second reagent does not contain glucose or contains glucose at a concentration of 10 mM or less. The reagent for measuring creatine kinase activity of the present invention is stabilized by suppressing a decrease in the measured value even when stored under extremely severe conditions of 37 ° C. compared to the conventional reagent for measuring creatine kinase activity, That is, it was reconfirmed that the creatine kinase activity measuring reagent of the present invention has an effect that it can be kept stable for a long period of time and can accurately measure creatine kinase activity for a long period of time.
  Further, the creatine kinase activity measuring reagent of the present invention is more effective than the conventional creatine kinase activity measuring reagent in suppressing the decrease in the measured value particularly in the high range where the creatine kinase activity value is high. It was reconfirmed that the reagent for measuring creatine kinase activity has the effect of maintaining linearity in the high value range for a long period of time.
[Example 4] (Confirmation of stabilizing effect of the present invention upon storage-4)
  The stabilizing effect at the time of storage in the reagent for measuring creatine kinase activity of the present invention was confirmed.
  Specifically, in the reagent for measuring creatine kinase activity of the present invention, the first reagent was prepared at the time of measurement or stored at 5 ° C. for 10 months, and the second reagent was stored at 5 ° C. for 10 months. The thing (or the thing prepared at the time of measurement) was used, and the effect of stabilization was confirmed.
1. Reagent for measuring creatine kinase activity
(1) First reagent
(A) First reagent [the present invention (5 mM)]
  The “first reagent [present invention (5 mM)]” in (1) (b) of Example 1 was used as the “first reagent [present invention (5 mM)]”.
(B) First reagent [the present invention (10 mM)]
  The “first reagent [present invention (10 mM)]” in (1) (1) of Example 1 was used as the “first reagent [present invention (10 mM)]”.
(2) Second reagent
(A) Second reagent [the present invention (0 mM)]
  The “second reagent [present invention (0 mM)]” in (2) (a) of Example 1 was used as the “second reagent [present invention (0 mM)]”.
(B) Second reagent [the present invention (10 mM)]
  The “second reagent [the present invention (10 mM)]” in (2) and (b) of Example 3 was used as the “second reagent [the present invention (10 mM)]”.
(C) Second reagent [conventional invention (60 mM)]
  The “second reagent [conventional invention 60 mM]” of (2), (f) of Example 3 was used as the “second reagent [conventional invention (60 mM)]”.
2. sample
(1) Diluted sample
  A human serum sample prepared by adding human creatine kinase BB isozyme was prepared (added serum sample), and this added serum sample was designated as “sample diluent” (5.0 mM succinate buffer (pH 7.0 (20 ° C.)). ) Are diluted to 1/10, 2/10, 3/10, 4/10, 5/10, 6/10, 7/10, 8/10, and 9/10, respectively. Sample- (1) ”to“ Sample- (9) ”were prepared.
  The added serum sample was designated as “Sample- (10)” (10/10).
  (A) “Sample- (1)”: 1/10 dilution of added serum sample
  (B) “Sample— (2)”: 2/10 dilution of added serum sample
  (C) “Sample- (3)”: diluted serum sample by 3/10
  (D) "Sample- (4)": Addition serum sample diluted 4/10
  (E) "Sample- (5)": Addition serum sample diluted 5/10
  (F) “Sample- (6)”: 6/10 dilution of added serum sample
  (G) "Sample- (7)": Addition serum sample diluted 7/10
  (H) "Sample- (8)": Addition serum sample diluted 8/10
  (I) "Sample- (9)": Addition serum sample diluted 9/10
  (J) “Sample- (10)”: added serum sample
(2) Non-containing sample
  The “sample dilution liquid” in the above (1) was a sample not containing creatine kinase and having a creatine kinase activity value of 0 Unit / L (unit / L), that is, a “free sample”.
3. Reagent storage or measurement preparation
(1) Storage at 5 ° C
  The first reagent (1) (a) of the present invention (1) (the present invention (5 mM)) and the first reagent (the present invention (10 mM)) of (1) are each at 5 ° C. for 10 months in the dark. saved.
  In addition, the second reagent of the above (2) (a) [the present invention (0 mM)], the second reagent of the (b) [present invention (10 mM)], and the second reagent of the (c) [conventional invention] (60 mM)] were also stored at 5 ° C. in the dark for 10 months.
(2) Preparation during measurement
  For the first reagent (1) (a) of the present invention (1) (the present invention (5 mM)) and the first reagent (the present invention (10 mM)) of (b), the creatine kinase activity value of the following 4 samples Preparation was also performed at the time of measurement and used for measurement.
  In addition, the second reagent (conventional invention (60 mM)) of (1) (2) (1) above was also prepared and used for measurement when measuring the creatine kinase activity value of the following 4 samples.
4). Measurement of creatine kinase activity in samples
  The measurement of the creatine kinase activity value of each sample in the above 2 was performed by the double kinetic method using an automatic analyzer TBA-120FR manufactured by Toshiba Medical Systems.
(1) The first reagent of (a) in (1) (a) prepared at the time of this measurement as described in (2) above in 5.6 μL of each of the samples in (2) (1) to (2) above [2] 160 μL of the present invention (5 mM)] was added and reacted at 37 ° C.
(2) Next, the amount of change in absorbance from 8 points (136.18 seconds after the addition of the first reagent) to 16 points (280.46 seconds after the addition of the first reagent) is measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 1”]. (Measurement of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(3) Next, between 16 points (280.46 seconds after addition of the first reagent) and 17 points (298.49 seconds after addition of the first reagent), 10 months at 5 ° C. in (1) above 40 μL of the second reagent (a) (1) (the present invention (0 mM)) stored in (1) above was added and reacted at 37 ° C.
(4) Next, the change in absorbance from 24 points (424.74 seconds after the addition of the first reagent) to 33 points (587.005 seconds after the addition of the first reagent) was measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 2”]. (Measurement of change in absorbance by combining the amount of change in absorbance according to the activity value of creatine kinase contained in the sample and the amount of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(5) Next, the activity value of creatine kinase contained in the sample was determined by the following calculation formula.
  Creatine kinase activity value (Unit / L) = {(ΔA₂-ΔRB₂) −c × (ΔA₁-ΔRB₁)} × (S + V₁+ V₂) × 10⁶÷ (K × d × S) = {(ΔA₂-ΔRB₂) −0.805 × (ΔA₁-ΔRB₁)} × 5.83 × 10³
  In this calculation formula, ΔA₁Is the change in absorbance per minute (Abs / min) measured in “Photometry 1”, ΔA₂Is the change in absorbance per minute (Abs / min) measured in “Photometry 2”, ΔRB₁Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 1” when the sample is the above “free sample”, ΔRB₂Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 2” when the sample is the above “free sample”, and S is the amount of sample [5.6 μL ], V₁Is the amount of the first reagent [160 μL], V₂Is the amount of the second reagent [40 μL], c is the liquid amount correction coefficient [(S + V₁) ÷ (S + V₁+ V₂) = 0.805], K is the theoretical molar extinction coefficient of NADPH [6.30 × 10³L. mole^-1・ Cm^-1(Main wavelength: 340 nm, sub wavelength: 405 nm)] and d represent the optical path length [1.00 cm] of the cell.
(6) Further, in the above (3), in place of the second reagent [the present invention (0 mM)] stored at 5 ° C. for 10 months in the above (1), 5 ° C. in the above (3). Except that the second reagent (b) of (1) (2) (this invention (10 mM)) stored for 10 months is used as in (1) to (5). The creatine kinase activity value of each sample was obtained when the second reagent [invention (10 mM)] <5 ° C., dark place, stored for 10 months> was used as two reagents.
(7) Further, in the above (3), in place of the second reagent [the present invention (0 mM)] stored at 5 ° C. for 10 months in the above (1), 5 ° C. in the above (3). (1) to (5), except that the second reagent of (1) (2) (c) [conventional invention (60 mM)] stored for 10 months is used. The creatine kinase activity value of each sample was obtained when the second reagent [conventional invention (60 mM)] <5 ° C., dark place, stored for 10 months> was used as two reagents.
(8) Also, in the above (3), instead of the second reagent [the present invention (0 mM)] stored at 5 ° C. for 10 months in the above (1), this is the same as (3) in (3) above. Except for using the second reagent of (1) (2) (c) [conventional invention (60 mM)] prepared at the time of measurement, the operation was carried out as in the above (1) to (5) as the second reagent. The activity value of creatine kinase was obtained for each sample when the second reagent [conventional invention (60 mM)] <preparation during measurement> was used.
(9) Further, in the above (1), instead of the first reagent (1) (a) of the above (1) (1) [this invention (5 mM)] prepared at the time of this measurement as in (2) of the above 3, As described in (1) to (8) above, except that the first reagent (1) (b) of the above 1 (1) (this invention (10 mM)) prepared at the time of this measurement is used as described in 3 (2). The first reagent [present invention (10 mM)] <preparation during measurement> was used as the first reagent, and the second reagent [present invention (0 mM)] <5 ° C. in the dark.・ Storage for 10 months>, second reagent [invention (10 mM)] <5 ° C., dark place, storage for 10 months>, second reagent [conventional invention (60 mM)] <5 ° C., dark place, for 10 months Preservation> or the second reagent [conventional invention (60 mM)] <preparation during measurement> for each sample of creatine kinase To obtain a sexual value.
(10) Further, in the above (1), instead of the first reagent (1) (a) of the above (1) (1) [this invention (5 mM)] prepared at the time of this measurement as in (2) of the above 3, (1) to (8) above, except that the first reagent (the present invention (5 mM)) of (1) of (1) of the above (1) stored at 5 ° C. for 10 months in (1) of 3 is used. The above-mentioned first reagent (the present invention (5 mM)) <5 ° C., dark place, stored for 10 months> is used as the first reagent, and the second reagent [the present invention is used as the second reagent] (0 mM)] <5 ° C., dark place, stored for 10 months>, second reagent [present invention (10 mM)] <5 ° C., dark place, stored for 10 months>, second reagent [conventional invention (60 mM)] <5 ° C, dark place, storage for 10 months> or the second reagent [conventional invention (60 mM)] <preparation during measurement> Obtain an active value of Chin kinase.
(11) Furthermore, in the above (1), instead of the first reagent (1) (a) of the above (1) (1) [this invention (5 mM)] prepared at the time of this measurement as in (2) of the above 3, (1) to (8) above, except that the first reagent (the present invention (10 mM)) of (1) (b) of 1 above (1) stored at 5 ° C. for 10 months in 3 (1) is used. The above-mentioned first reagent (the present invention (10 mM)) <5 ° C., dark place, stored for 10 months> is used as the first reagent, and the second reagent [the present invention is used as the second reagent. (0 mM)] <5 ° C., dark place, stored for 10 months>, second reagent [present invention (10 mM)] <5 ° C., dark place, stored for 10 months>, second reagent [conventional invention (60 mM)] <5 ° C., dark place, storage for 10 months>, or second sample [conventional invention (60 mM)] <preparation during measurement> Obtain an active value of rare Chin kinase.
5). Measurement result
(1) First reagent [invention (5 mM)] <Preparation during measurement>
  In the above (1) to (8), the first reagent [present invention (5 mM)] <preparation during measurement> is used as the first reagent, and the second reagent [present invention (0 mM) is used as the second reagent. )] <5 ° C./dark place / 10 months storage>, second reagent [present invention (10 mM)] <5 ° C./dark place / 10 months storage>, second reagent [conventional invention (60 mM)] <5 Table 6 shows the measurement results of the activity value of creatine kinase of each sample when the second reagent [conventional invention (60 mM)] <preparation at the time of measurement> is used, respectively. It was.
  In Table 6, the value shown in the column “[1] Activity value” indicates the activity value [Unit / L] of creatine kinase of each sample.
  In addition, the value shown in the column of “[2] Activity value difference” indicates that the first reagent of the same sample is the first reagent [present invention (5 mM) from the value of the “[1] activity value” column in each sample. )] <Preparation upon measurement> and the difference between the second reagent [conventional invention (60 mM)] and <preparation upon measurement> minus the value in the column "[1] Activity value" Indicates the value [Unit / L].
  The value shown in the column “[3] Relative ratio” is the same as the value in the column “[1] Activity value” in each sample, and the first reagent of the same sample is the first reagent [present invention (5 mM) ] The value obtained by dividing by the value in the column of [1] Activity Value when the second reagent is the second reagent [conventional invention (60 mM)] <preparation at measurement>. Shown in the display.
(2) First reagent [invention (10 mM)] <Preparation during measurement> During use
  In the above (9), the first reagent [the present invention (10 mM)] <preparation during measurement> is used as the first reagent, and the second reagent [the present invention (0 mM)] <5 as the second reagent. C, dark place, storage for 10 months>, second reagent [invention (10 mM)] <5 ° C., dark place, stored for 10 months>, second reagent [conventional invention (60 mM)] <5 ° C., dark place Table 7 shows the measurement results of the activity value of creatine kinase of each sample when the storage was performed for 10 months> or the second reagent [conventional invention (60 mM)] <preparation during measurement> was used.
  In Table 7, the value shown in the column “[1] Activity value” indicates the activity value [Unit / L] of creatine kinase of each sample.
  In addition, the value shown in the column “[2] Activity value difference” indicates that the value of the column “[1] Activity value” in each sample is the same as that of the first reagent [the present invention (10 mM) of the same sample. )] <Preparation upon measurement> and the difference between the second reagent [conventional invention (60 mM)] and <preparation upon measurement> minus the value in the column "[1] Activity value" Indicates the value [Unit / L].
  In addition, the value shown in the column “[3] Relative ratio” is the same as the value in the column “[1] Activity value” in each sample, and the first reagent of the same sample is the first reagent [invention (10 mM) ] The value obtained by dividing by the value in the column of [1] Activity Value when the second reagent is the second reagent [conventional invention (60 mM)] <preparation at measurement>. Shown in the display.
(3) First reagent [invention (5 mM)] <5 ° C., dark place, stored for 10 months>
  In the above (10), the first reagent [the present invention (5 mM)] <5 ° C., dark place, stored for 10 months> is used as the first reagent, and the second reagent [the present reagent is used as the second reagent. Invention (0 mM)] <5 ° C./dark place / 10 months storage>, second reagent [present invention (10 mM)] <5 ° C./dark place / 10 months storage>, second reagent [conventional invention (60 mM) ] <5 ° C., dark place, storage for 10 months>, or the measurement result of the activity value of creatine kinase of each sample when using the second reagent [conventional invention (60 mM)] <preparation at measurement> This is shown in FIG.
  In Table 8, the value shown in the column of “[1] Activity value” indicates the activity value [Unit / L] of creatine kinase of each sample.
  In addition, the value shown in the column of “[2] Activity value difference” indicates that the first reagent of the same sample is the first reagent [present invention (5 mM) from the value of the “[1] activity value” column in each sample. )] <5 [deg.] C., dark place, storage for 10 months> and the second reagent is the second reagent [conventional invention (60 mM)] <preparation upon measurement> The difference value [Unit / L] obtained by subtracting the value is shown.
  The value shown in the column “[3] Relative ratio” is the same as the value in the column “[1] Activity value” in each sample, and the first reagent of the same sample is the first reagent [present invention (5 mM) ] <5 ° C, dark place, storage for 10 months>, and the second reagent is the second reagent [conventional invention (60 mM)] <preparation during measurement> in the column of [1] Activity Value The value divided by the value is shown as a percentage.
(4) First reagent [invention (10 mM)] <5 ° C./dark place / store for 10 months>
  In the above (11), the first reagent [the present invention (10 mM)] <5 ° C., dark place, stored for 10 months> is used as the first reagent, and the second reagent [the present one is used as the second reagent. Invention (0 mM)] <5 ° C./dark place / 10 months storage>, second reagent [present invention (10 mM)] <5 ° C./dark place / 10 months storage>, second reagent [conventional invention (60 mM) ] <5 ° C., dark place, storage for 10 months>, or the measurement result of the activity value of creatine kinase of each sample when using the second reagent [conventional invention (60 mM)] <preparation at measurement> 9 shows.
  In Table 9, the value shown in the column “[1] Activity value” indicates the activity value [Unit / L] of creatine kinase of each sample.
  In addition, the value shown in the column “[2] Activity value difference” indicates that the value of the column “[1] Activity value” in each sample is the same as that of the first reagent [the present invention (10 mM) of the same sample. )] <5 [deg.] C., dark place, storage for 10 months> and the second reagent is the second reagent [conventional invention (60 mM)] <preparation upon measurement> The difference value [Unit / L] obtained by subtracting the value is shown.
  In addition, the value shown in the column “[3] Relative ratio” is the same as the value in the column “[1] Activity value” in each sample, and the first reagent of the same sample is the first reagent [invention (10 mM) ] <5 ° C, dark place, storage for 10 months>, and the second reagent is the second reagent [conventional invention (60 mM)] <preparation during measurement> in the column of [1] Activity Value The value divided by the value is shown as a percentage.

6). Consideration
(1) First reagent [invention (5 mM)] <Preparation during measurement>
  In Table 6 showing the measurement results when the first reagent is the first reagent [present invention (5 mM)] <Preparation at measurement>, the second reagent was stored for 10 months at 5 ° C. [Conventional invention] (60 mM)], the measured value was compared with the measured values of the second reagent [the present invention (0 mM)] and the second reagent [the present invention (10 mM)], which were also stored at 5 ° C. for 10 months, The value is low.
  In this case, the higher the creatine kinase activity value, the greater the degree of decrease in the measured value due to storage of the measurement reagent. The degree of decrease in the measured value (the value of “[2] Activity value difference” in Table 6) is For example, when the sample is “Sample- (8)” to “Sample- (10)”, the second reagent is the second reagent [the present invention (0 mM)] <5 ° C., dark place, stored for 10 months> Is -291 Unit / L to -365 Unit / L, and in the case of the second reagent [the present invention (10 mM)] <5 ° C., dark place and stored for 10 months>, it is −274 Unit / L to −290 Unit / L. Yes, and in the case of the second reagent [conventional invention (60 mM)] <5 ° C., dark place, storage for 10 months>, it is -390 Unit / L to -458 Unit / L.
  From the result of storing this second reagent at 5 ° C. for 10 months (or preparation at the time of measurement), when the glucose concentration of the second reagent is 60 mM, the second reagent does not contain glucose or has a concentration of 10 mM or less. It can be seen that there is a large difference in the degree of decrease in the measured value when the measurement reagent is stored compared to the case of containing glucose, and the measured value has decreased.
  And when a 2nd reagent does not contain glucose or contains glucose with a density | concentration of 10 mM or less, it turns out that the fall of a measured value is suppressed and stabilized.
(2) First reagent [invention (10 mM)] <Preparation during measurement> During use
  In Table 7 showing the measurement results when the first reagent is the first reagent [present invention (10 mM)] <preparation during measurement> the second reagent stored for 10 months at 5 ° C. [conventional invention] (60 mM)], the measured value was compared with the measured values of the second reagent [the present invention (0 mM)] and the second reagent [the present invention (10 mM)], which were also stored at 5 ° C. for 10 months, The value is low.
  In this case as well, the higher the creatine kinase activity value, the greater the degree of decrease in the measured value due to storage of the measurement reagent. The degree of decrease in the measured value (the value of “[2] Activity value difference” in Table 7) is For example, in the case of samples “Sample- (8)” to “Sample- (10)”, the second reagent is the second reagent [invention (0 mM)] <5 ° C., dark place, stored for 10 months> In the case of -294 Unit / L to -341 Unit / L, and in the case of the second reagent [the present invention (10 mM)] <5 ° C., dark place and stored for 10 months> -278 Unit / L to -285 Unit / L In the case of the second reagent [conventional invention (60 mM)] <5 ° C., dark place, preserved for 10 months>, it is −379 Unit / L to −439 Unit / L.
  From the result of storing this second reagent at 5 ° C. for 10 months (or preparation at the time of measurement), when the glucose concentration of the second reagent is 60 mM, the second reagent does not contain glucose or has a concentration of 10 mM or less. It can be seen that there is a large difference in the degree of decrease in the measured value when the measurement reagent is stored compared to the case of containing glucose, and the measured value has decreased.
  And when a 2nd reagent does not contain glucose or contains glucose with a density | concentration of 10 mM or less, it turns out that the fall of a measured value is suppressed and stabilized.
(3) First reagent [invention (5 mM)] <5 ° C., dark place, stored for 10 months>
  In Table 8 showing the measurement results when the first reagent is the first reagent [the present invention (5 mM)] <5 ° C., dark place, stored for 10 months>, the second reagent is stored for 10 months at 5 ° C. The measured value in the case of the second reagent [conventional invention (60 mM)] is the same as that of the second reagent [present invention (0 mM)] or the second reagent [present invention (10 mM)] stored for 10 months at 5 ° C. It is a low value compared with the measured value in the case.
  In this case as well, the higher the creatine kinase activity value, the greater the degree of decrease in the measured value due to storage of the measurement reagent. The degree of decrease in the measured value (the value of “[2] Activity value difference” in Table 8) For example, in the case of samples “Sample- (8)” to “Sample- (10)”, the second reagent is the second reagent [invention (0 mM)] <5 ° C., dark place, stored for 10 months> In the case of -598 Unit / L to -704 Unit / L, and in the case of the second reagent [the present invention (10 mM)] <5 ° C., dark place and stored for 10 months>, −560 Unit / L to −674 Unit / L In the case of the second reagent [conventional invention (60 mM)] <5 ° C., dark place, preserved for 10 months>, it is −851 Unit / L to −1,122 Unit / L.
  From the result of storing this second reagent at 5 ° C. for 10 months (or preparation at the time of measurement), when the glucose concentration of the second reagent is 60 mM, the second reagent does not contain glucose or has a concentration of 10 mM or less. It can be seen that there is a large difference in the degree of decrease in the measured value when the measurement reagent is stored compared to the case of containing glucose, and the measured value has decreased.
  Furthermore, when the glucose concentration of the second reagent is 60 mM, the degree of decrease in the measured value is as described in (1) and (2) above when the first reagent is stored at 5 ° C. for 10 months. Thus, it can be seen that the first reagent is larger than the one prepared at the time of measurement.
  And when a 2nd reagent does not contain glucose or contains glucose with a density | concentration of 10 mM or less, it turns out that the fall of a measured value is suppressed and stabilized.
(4) First reagent [invention (10 mM)] <5 ° C./dark place / store for 10 months>
  In Table 9 showing the measurement results when the first reagent is the first reagent [invention (10 mM)] <5 ° C., dark place, stored for 10 months>, the second reagent is stored for 10 months at 5 ° C. The measured value in the case of the second reagent [conventional invention (60 mM)] is the same as that of the second reagent [present invention (0 mM)] or the second reagent [present invention (10 mM)] stored for 10 months at 5 ° C. It is a low value compared with the measured value in the case.
  In this case as well, the higher the creatine kinase activity value, the greater the decrease in the measured value due to the storage of the measurement reagent. The degree of decrease in the measured value (the value of “[2] Activity value difference” in Table 9) is For example, in the case of samples “Sample- (8)” to “Sample- (10)”, the second reagent is the second reagent [invention (0 mM)] <5 ° C., dark place, stored for 10 months> In the case of −652 Unit / L to −769 Unit / L, and in the case of the second reagent [the present invention (10 mM)] <5 ° C., dark place and stored for 10 months>, −617 Unit / L to −751 Unit / L In the case of the second reagent [conventional invention (60 mM)] <5 ° C., dark place, storage for 10 months>, it is from −1,057 Unit / L to −1,279 Unit / L.
  From the result of storing this second reagent at 5 ° C. for 10 months (or preparation at the time of measurement), when the glucose concentration of the second reagent is 60 mM, the second reagent does not contain glucose or has a concentration of 10 mM or less. It can be seen that there is a large difference in the degree of decrease in the measured value when the measurement reagent is stored compared to the case of containing glucose, and the measured value has decreased.
  Furthermore, when the glucose concentration of the second reagent is 60 mM, the degree of decrease in the measured value is as described in (1) and (2) above when the first reagent is stored at 5 ° C. for 10 months. Thus, it can be seen that the first reagent is larger than the one prepared at the time of measurement.
  And when a 2nd reagent does not contain glucose or contains glucose with a density | concentration of 10 mM or less, it turns out that the fall of a measured value is suppressed and stabilized.
(5) Summary
  From the above, the creatine kinase of the present invention is characterized in that the first reagent contains glucose at a concentration of 0.5 mM to 10 mM and the second reagent contains no glucose or contains glucose at a concentration of 10 mM or less. The activity measuring reagent is stabilized and prevented from lowering its measured value even when stored for a long period of 10 months at 5 ° C., compared to a conventional creatine kinase activity measuring reagent, It was reconfirmed that the reagent for measuring creatine kinase activity of the present invention has an effect that it can be kept stable for a long period of time and can accurately measure creatine kinase activity for a long period of time.
  Further, the creatine kinase activity measuring reagent of the present invention is more effective than the conventional creatine kinase activity measuring reagent in suppressing the decrease in the measured value particularly in the high range where the creatine kinase activity value is high. It was reconfirmed that the reagent for measuring creatine kinase activity has the effect of maintaining linearity in the high value range for a long period of time.
[Example 5] (Confirmation of stabilizing effect of the present invention upon storage-5)
  The stabilizing effect at the time of storage in the reagent for measuring creatine kinase activity of the present invention was confirmed.
  Specifically, in the reagent for measuring creatine kinase activity of the present invention, the first reagent used is one stored at 5 ° C. for 12 months (or prepared at the time of measurement), and the second reagent is used at 5 ° C. for 12 months. Using the stored one (or the one prepared at the time of measurement), the effect of stabilization was confirmed.
1. Reagent for measuring creatine kinase activity
(1) First reagent
(A) First reagent [the present invention (5 mM)]
  The “first reagent [present invention (5 mM)]” in (1) (b) of Example 1 was used as the “first reagent [present invention (5 mM)]”.
(B) First reagent [the present invention (10 mM)]
  The “first reagent [present invention (10 mM)]” in (1) (1) of Example 1 was used as the “first reagent [present invention (10 mM)]”.
(C) First reagent [conventional invention (25 mM)]
  The “first reagent [conventional invention (25 mM)]” of (1) (1) in Example 1 was used as the “first reagent [conventional invention (25 mM)]”.
(2) Second reagent
(A) Second reagent [the present invention (0 mM)]
  The “second reagent [present invention (0 mM)]” in (2) (a) of Example 1 was used as the “second reagent [present invention (0 mM)]”.
(B) Second reagent [conventional invention (30 mM)]
  The “second reagent [conventional invention 30 mM]” in (2) (c) of Example 3 was used as the “second reagent [conventional invention (30 mM)]”.
(C) Second reagent [conventional invention (60 mM)]
  The “second reagent [conventional invention 60 mM]” of (2), (f) of Example 3 was used as the “second reagent [conventional invention (60 mM)]”.
2. sample
(1) Diluted sample
  A human serum sample prepared by adding human creatine kinase BB isozyme was prepared (added serum sample), and this added serum sample was designated as “sample diluent” (5.0 mM succinate buffer (pH 7.0 (20 ° C.)). ) Are diluted to 1/10, 2/10, 3/10, 4/10, 5/10, 6/10, 7/10, 8/10, and 9/10, respectively. Sample- [1] ”to“ Sample- [9] ”were prepared.
  The added serum sample was designated as “Sample- [10]” (10/10).
  (A) “Sample- [1]”: 1/10 dilution of added serum sample
  (B) "Sample- [2]": Addition serum sample diluted 2/10
  (C) “Sample- [3]”: 3/10 dilution of added serum sample
  (D) "Sample- [4]": The added serum sample was diluted 4/10
  (E) "Sample- [5]": Addition serum sample diluted 5/10
  (F) "Sample- [6]": Addition serum sample diluted 6/10
  (G) “Sample- [7]”: Diluted added serum sample by 7/10
  (H) "Sample- [8]": Addition serum sample diluted 8/10
  (I) "Sample- [9]": Addition serum sample diluted 9/10
  (J) "Sample- [10]": added serum sample
(2) Non-containing sample
  The “sample dilution liquid” in the above (1) was a sample not containing creatine kinase and having a creatine kinase activity value of 0 Unit / L (unit / L), that is, a “free sample”.
3. Reagent storage or measurement preparation
(1) Storage at 5 ° C
  (1) (a) first reagent [present invention (5 mM)], (b) first reagent [present invention (10 mM)], and (c) first reagent [conventional invention (25 mM)] ] Were each stored at 5 ° C. in the dark for 12 months.
  In addition, the second reagent (a) of the above (2) (the present invention (0 mM)), the second reagent of the (b) [conventional invention (30 mM)], and the second reagent of the (c) [conventional invention] (60 mM)] was also stored at 5 ° C. in the dark for 12 months.
(2) Preparation during measurement
  The first reagent (1) of (1) (a) [invention (5 mM)] is also prepared at the time of measuring the creatine kinase activity value of the following four samples, and is used as a control reagent for the first reagent. used.
  The second reagent (1) of (2) (c) above (conventional invention (60 mM)) was also prepared at the time of measuring the creatine kinase activity value of the following four samples, and the control of the second reagent Used as a reagent for the measurement.
4). Measurement of creatine kinase activity in samples
  The measurement of the creatine kinase activity value of each sample in the above 2 was performed by the double kinetic method using an automatic analyzer TBA-120FR manufactured by Toshiba Medical Systems.
(1) First of (1) (a) of (1) above, which was stored in 5.6 μL of each sample of (2) (1) to (2) above at (5) at 12 ° C. for 12 months. 160 μL of a reagent [the present invention (5 mM)] was added and reacted at 37 ° C.
(2) Next, the amount of change in absorbance from 8 points (136.18 seconds after the addition of the first reagent) to 16 points (280.46 seconds after the addition of the first reagent) is measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 1”]. (Measurement of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(3) Next, between 16 points (280.46 seconds after addition of the first reagent) and 17 points (298.49 seconds after addition of the first reagent), 12 months at 5 ° C. in (1) above 40 μL of the second reagent (a) (1) (the present invention (0 mM)) stored in (1) above was added and reacted at 37 ° C.
(4) Next, the change in absorbance from 24 points (424.74 seconds after the addition of the first reagent) to 33 points (587.005 seconds after the addition of the first reagent) was measured at the main wavelength of 340 nm and the sub wavelength of 405 nm. Measured [“photometry 2”]. (Measurement of change in absorbance by combining the amount of change in absorbance according to the activity value of creatine kinase contained in the sample and the amount of change in absorbance according to the activity value of adenylate kinase contained in the sample)
(5) Next, the activity value of creatine kinase contained in the sample was determined by the following calculation formula.
  Creatine kinase activity value (Unit / L) = {(ΔA₂-ΔRB₂) −c × (ΔA₁-ΔRB₁)} × (S + V₁+ V₂) × 10⁶÷ (K × d × S) = {(ΔA₂-ΔRB₂) −0.805 × (ΔA₁-ΔRB₁)} × 5.83 × 10³
  In this calculation formula, ΔA₁Is the change in absorbance per minute (Abs / min) measured in “Photometry 1”, ΔA₂Is the change in absorbance per minute (Abs / min) measured in “Photometry 2”, ΔRB₁Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 1” when the sample is the above “free sample”, ΔRB₂Is the absorbance change amount per minute (reagent blind value) (Abs / min) measured in “photometry 2” when the sample is the above “free sample”, and S is the amount of sample [5.6 μL ], V₁Is the amount of the first reagent [160 μL], V₂Is the amount of the second reagent [40 μL], c is the liquid amount correction coefficient [(S + V₁) ÷ (S + V₁+ V₂) = 0.805], K is the theoretical molar extinction coefficient of NADPH [6.30 × 10³L. mole^-1・ Cm^-1(Main wavelength: 340 nm, sub wavelength: 405 nm)] and d represent the optical path length [1.00 cm] of the cell.
(6) Further, in the above (1), in place of the first reagent [the present invention (5 mM)] stored at 5 ° C. for 12 months in the above (1), 5 ° C. in the above (1) Except that the first reagent (1) of (1) (b) [this invention (10 mM)] stored for 12 months is used as in (1) to (5), The activity value of creatine kinase of each sample was obtained when the above-mentioned first reagent [the present invention (10 mM)] <5 ° C., dark place, stored for 12 months> was used as one reagent.
(7) Further, in the above (1), in place of the first reagent [the present invention (5 mM)] stored at 5 ° C. for 12 months in the above (1), the above (1), 5 ° C. Except that the first reagent (1) of (1) (c) [conventional invention (25 mM)] stored for 12 months is used as in (1) to (5). The creatine kinase activity value of each sample was obtained when the above-mentioned first reagent [conventional invention (25 mM)] <5 ° C., dark place, stored for 12 months> was used as one reagent.
(8) Further, in the above (3), in place of the second reagent [the present invention (0 mM)] stored at 5 ° C. for 12 months in 3 (1) above, 5 ° C. in (1) above. (1) to (7), except that the second reagent of (1) (2) (b) [conventional invention (30 mM)] stored for 12 months was used. As the first reagent, the first reagent [invention (5 mM)] <5 ° C., dark place, stored for 12 months>, the first reagent [invention (10 mM)] <5 ° C., dark place, stored for 12 months> Or the first reagent [conventional invention (25 mM)] <5 ° C., dark place, preserved for 12 months>, and the second reagent as the second reagent [conventional invention (30 mM)] <5 ° C., dark place -The activity value of creatine kinase was obtained for each sample when the storage for 12 months was used.
(9) Further, in the above (3), in place of the second reagent [the present invention (0 mM)] stored at 5 ° C. for 12 months in the above (1), 5 ° C. in the above (3) (1) to (7) except that the second reagent (c) of (1) (2) (conventional invention (60 mM)) stored for 12 months was used. As the first reagent, the first reagent [invention (5 mM)] <5 ° C., dark place, stored for 12 months>, the first reagent [invention (10 mM)] <5 ° C., dark place, stored for 12 months> Or the first reagent [conventional invention (25 mM)] <5 ° C./dark place / stored for 12 months>, and the second reagent [conventional invention (60 mM)] <5 ° C./dark place -The activity value of creatine kinase was obtained for each sample when the storage for 12 months was used.
(10) Further, [A] In the above (1), instead of the first reagent [this invention (5 mM)] stored at 5 ° C. for 12 months in the above (1), the above (2) Using the control reagent of the first reagent prepared at the time of the measurement as described above, and [B] in the above (3), the second reagent stored at 5 ° C. for 12 months in (3) above [3] In place of the present invention (0 mM)], using the control reagent of the second reagent prepared at the time of measurement as described in (2) of 3 above, except for these [A] and [B], The operation was carried out as in (1) to (5), and the activity value of creatine kinase in each sample was obtained when the control reagent of the first reagent and the control reagent of the second reagent were used.
5). Measurement result
  Table 10 shows the measurement results of the creatine kinase activity value of each sample in 4.
  In Table 10, the value shown in the column “[1] Activity value” indicates the activity value [Unit / L] of creatine kinase of each sample.
  The value shown in the column “[2] Activity value difference” is the same as that of the first reagent in the same sample from the value in the column “[1] Activity value” for each sample. And the difference value [Unit / L] obtained by subtracting the value in the “[1] Activity value” column when the second reagent is the control reagent of the second reagent.
  The value shown in the column “[3] Relative ratio” is the same as the value in the column “[1] Activity value” for each sample, and the first reagent of the same sample is the control reagent of the first reagent. Yes, and the value divided by the value in the column “[1] Activity value” when the second reagent is the control reagent of the second reagent is shown as a percentage.

6). Consideration
(1) Second reagent [invention (0 mM)] <5 ° C., dark place, stored for 12 months>
  In Table 10, in the measurement results when the second reagent is the second reagent [the present invention (0 mM)] <5 ° C., dark place, stored for 12 months>, the first reagent was stored at 5 ° C. for 12 months. The measured value in the case of the first reagent [conventional invention (25 mM)] is the value of the first reagent [the present invention (5 mM)] and the first reagent [the present invention (10 mM)] stored at 5 ° C. for 12 months. It tends to be lower than the measured value.
  In this case, the higher the creatine kinase activity value, the greater the degree of decrease in the measured value due to storage of the measurement reagent. The degree of decrease in the measured value (the value of “[2] Activity value difference” in Table 10) is For example, in the case of “sample- [8]” to “sample- [10]”, the first reagent is the first reagent [the present invention (5 mM)] <5 ° C., dark place, stored for 12 months> -32 Unit / L to -117 Unit / L, and in the case of the first reagent [the present invention (10 mM)] <5 ° C., dark place, stored for 12 months>, from −88 Unit / L to −487 Unit / L In the case of the first reagent [conventional invention (25 mM)] <5 ° C., dark place, preserved for 12 months>, it is -157 Unit / L to -660 Unit / L.
(2) Second reagent [conventional invention (30 mM)] <5 ° C., dark place, stored for 12 months>
  In Table 10, even when the second reagent is the second reagent [conventional invention (30 mM)] <5 ° C., dark place, stored for 12 months>, the first reagent is stored at 5 ° C. for 12 months. The measured value in the case of the first reagent [conventional invention (25 mM)] is the case of the first reagent [present invention (5 mM)] and the first reagent [present invention (10 mM)] stored for 12 months at 5 ° C. It tends to be lower than the measured value.
  Even in this case, the sample having a higher creatine kinase activity value tends to have a greater degree of decrease in the measured value due to storage of the measurement reagent, and the degree of decrease in the measured value (value of “[2] difference in activity value” in Table 10). For example, in the case of samples “sample- [8]” to “sample- [10]”, the first reagent is the first reagent [invention (5 mM)] <5 ° C., dark place, stored for 12 months> Is 64 Unit / L to -457 Unit / L, and in the case of the first reagent [the present invention (10 mM)] <5 ° C, dark place and stored for 12 months>, -369 Unit / L to -472 Unit / L In the case of the first reagent [conventional invention (25 mM)] <5 ° C., dark place, preserved for 12 months>, it is -403 Unit / L to -500 Unit / L.
(3) Second reagent [conventional invention (60 mM)] <5 ° C., dark place, stored for 12 months>
  In Table 10, even when the second reagent is the second reagent [conventional invention (60 mM)] <5 ° C., dark place, stored for 12 months>, the first reagent is stored at 5 ° C. for 12 months. The measured value in the case of the first reagent [conventional invention (25 mM)] is the case of the first reagent [present invention (5 mM)] and the first reagent [present invention (10 mM)] stored for 12 months at 5 ° C. It tends to be lower than the measured value.
  Even in this case, the sample having a higher creatine kinase activity value tends to have a greater degree of decrease in the measured value due to storage of the measurement reagent, and the degree of decrease in the measured value (value of “[2] difference in activity value” in Table 10). For example, in the case of samples “sample- [8]” to “sample- [10]”, the first reagent is the first reagent [invention (5 mM)] <5 ° C., dark place, stored for 12 months> Is -188 Unit / L to -688 Unit / L, and in the case of the first reagent [invention (10 mM)] <5 ° C, dark place, stored for 12 months>, -300 Unit / L to -702 Unit / L In the case of the first reagent [conventional invention (25 mM)] <5 ° C., dark place, preserved for 12 months>, it is -320 Unit / L to -803 Unit / L.
(4) Summary of the first reagent
  As described above, from the result of storing the first reagent at 5 ° C. for 12 months (or preparation during measurement), when the glucose concentration of the first reagent is 25 mM, the glucose concentration of the first reagent is 5 mM or 10 mM. Compared to a certain case, it can be seen that there is a difference in the degree of decrease in the measured value when the measurement reagent is stored, and the measured value tends to decrease.
  And when the glucose concentration of a 1st reagent is 5 mM or 10 mM, it turns out that the fall of a measured value is suppressed and stabilized in any case.
(5) Summary of the second reagent
  As described above, when the glucose concentration of the second reagent is 30 mM or 60 mM based on the result of storing the second reagent at 5 ° C. for 12 months (or preparation during measurement), the glucose concentration of the second reagent is 0 mM. It can be seen that there is a difference in the degree of decrease in the measured value when the measurement reagent is stored compared to a certain case (that is, when the second reagent does not contain glucose), and the measured value tends to decrease.
  And when a 2nd reagent does not contain glucose (in the case of 0 mM), the fall of a measured value is suppressed and it turns out that it is stabilized.
(6) Summary
  From the above, the creatine kinase of the present invention is characterized in that the first reagent contains glucose at a concentration of 0.5 mM to 10 mM and the second reagent contains no glucose or contains glucose at a concentration of 10 mM or less. The activity measuring reagent is stabilized by suppressing a decrease in the measured value even when stored for a further long period of 12 months at 5 ° C., compared to a conventional creatine kinase activity measuring reagent, That is, it was reconfirmed that the creatine kinase activity measuring reagent of the present invention has an effect that it can be kept stable for a long period of time and can accurately measure creatine kinase activity for a long period of time.
Further, the creatine kinase activity measuring reagent of the present invention is more effective than the conventional creatine kinase activity measuring reagent in suppressing the decrease in the measured value particularly in the high range where the creatine kinase activity value is high. It was reconfirmed that the reagent for measuring creatine kinase activity has the effect of maintaining linearity in the high value range for a long period of time.

Claims (9)

A reagent for measuring creatine kinase activity comprising a first reagent and a second reagent, wherein the first reagent is adenosine 5′-diphosphate or a salt thereof, hexokinase or glucokinase , glucose-6-phosphate dehydrogenase , β-nicotinamide Adenine dinucleotide (oxidized form) or β-nicotinamide adenine dinucleotide phosphate (oxidized form) or a salt thereof, a thiol compound, and glucose, and the concentration of the glucose is 0.5 mM to 10 mM, wherein the reagent comprises a creatine phosphate or a salt and glucose-6-phosphate dehydrogenase, and is intended to include glucose or 10mM following concentrations without glucose, creatine kinase activity measuring reagent. The concentration of glucose in the first reagent is 1.25MM～5mM, claim 1 Symbol placement of creatine kinase activity measuring reagent. The creatine kinase activity measuring reagent according to any one of claims 1 to 2 , wherein the glucose concentration of the first reagent is 2 mM to 5 mM. The reagent for measuring creatine kinase activity according to any one of claims 1 to 3 , wherein the second reagent does not contain glucose or contains glucose at a concentration of 5 mM or less. The reagent for measuring creatine kinase activity according to any one of claims 1 to 4 , wherein the second reagent does not contain glucose. The creatine kinase activity measuring reagent according to any one of claims 1 to 5 , wherein the activity value of glucose-6-phosphate dehydrogenase of the first reagent is 200 Unit / L to 5,000 Unit / L. The activity value of glucose-6-phosphate dehydrogenase of the second reagent is adjusted so as to be 500 Unit / L or more in the final reaction solution in which the sample, the first reagent, and the second reagent are mixed. The creatine kinase activity measuring reagent according to any one of claims 6 to 6 . The creatine kinase activity measuring reagent according to any one of claims 1 to 7 , wherein the first reagent and the second reagent are liquid reagents. In place of β-nicotinamide adenine dinucleotide (oxidized form) or salt thereof, β-thionicotinamide adenine dinucleotide (oxidized form) or salt thereof is used, and β-nicotinamide adenine dinucleotide (reduced form) or salt thereof is used. In place of β-thionicotinamide adenine dinucleotide (reduced form) or a salt thereof, β-nicotinamide adenine dinucleotide phosphate (oxidized form) or in place of this salt, β-thionicotinamide adenine dinucleotide phosphate ( Oxidized form) or a salt thereof, or β-thionicotinamide adenine dinucleotide phosphate (reduced form) or a salt thereof instead of β-nicotinamide adenine dinucleotide phosphate (reduced form) or a salt thereof, The creatine tincture according to any one of claims 1 to 8. Reagent activity measurement reagent.