WO1993013219A1 - Calcium ion determining composition - Google Patents

Calcium ion determining composition Download PDF

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Publication number
WO1993013219A1
WO1993013219A1 PCT/JP1992/001680 JP9201680W WO9313219A1 WO 1993013219 A1 WO1993013219 A1 WO 1993013219A1 JP 9201680 W JP9201680 W JP 9201680W WO 9313219 A1 WO9313219 A1 WO 9313219A1
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Prior art keywords
reagent
measuring
composition
calcium
lysolecithin
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PCT/JP1992/001680
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French (fr)
Japanese (ja)
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Shigeyuki Imamura
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Asahi Kasei Kogyo Kabushiki Kaisha
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides

Definitions

  • the present invention relates to a composition for measuring calcium ions. More particularly calcium I O emissions measured composition having Complex Li phospholipids, phospholipase A 2 and lysolecithin measuring reagent.
  • Conventional technology having Complex Li phospholipids, phospholipase A 2 and lysolecithin measuring reagent.
  • Measurement of calcium in body fluids provides useful information on various physiological functions such as nerve and muscle excitability, blood coagulation reaction, cell membrane permeability, and parathyroid function, and has high clinical significance.
  • methods such as an electrode method using an ion-selective electrode have been put to practical use and generally used for measuring calcium ions in body fluids.
  • this method requires a dedicated device, and therefore requires attention to the maintenance and management of the device, and has problems such as poor sample analysis processing efficiency.
  • An object of the present invention is to provide a simple calcium ion measuring reagent which does not require maintenance and management of a special measuring device, has excellent measurement accuracy, and gives accurate analysis results even with a high-value calcium sample.
  • the present invention is a phospholipid, calcium I O emissions measured composition characterized by containing the phospholipase A 2 and 1 / Zoreshichin measuring reagent.
  • the phospholipid used in the present invention is represented by the following general formula.
  • Rz represents an acyl group having 6 to 22 carbon atoms
  • X represents collin or ethanolamine.
  • this phospholipid examples include natural lecithins such as egg yolk lecithin and soy lecithin, giorail lecithin, dipalmitoyl lecithin, distearoyl lecithin, zimiris toy / lelecithin, zirino leis relecithin, zirino leioinolecithin, I—Palmi Toinole 1—Oleyl Lecithin, 1— ⁇ Lumi Toilet 1—Lino Rail Lecithin, 1—Palmi Toileu 2—Lino Leisleine, 1—Stear Mouth 2—Oleino Lecithin, 1—Stearoyl 2—Lino Lele Lecithin And synthetic lecithin.
  • natural lecithins such as egg yolk lecithin and soy lecithin, giorail lecithin, dipalmitoyl lecithin, distearoyl lecithin, zimiris toy /
  • the reagent for measuring lysolecithin is composed of a combination of enzymes and reagents for detecting lysolecithin, which is a reaction product of phospholipase and the phospholipid represented by the above general formula. That is, at least lysophospholipase, glycerylphosphorylcholine phosphogen
  • the amount of hydrogen peroxide produced by the reaction using a lysolecithin measurement reagent consisting of a sterase and glycerophosphate oxidase or choline oxidase'- or ethanolamine oxidase can be determined.
  • glycerylphosphorylcholine phosphodiesterase is used as a substrate when X is ethanolamine in the above general formula, so that the reaction proceeds even when glyceryl phosphorylethanolamine is used.
  • the bi venom, bee venom forces the enzyme of origin such as the present the animal ⁇ it is known maximum activation with calcium I O emissions lightly-doped region It is known to happen.
  • phosphorylase Horipaze eight 2 from scan Torebutomaisesu is activated in proportion to the concentration to a relatively high concentration of calcium I-one as shown in FIG. 1 can be seen.
  • Reputomaisesu derived phospholipase eight 2 (Toyo Jozo Co., catalog T one 3 1) is advantageous, also Re Yu calcium I O emissions measured composition of the present invention this point It is a point.
  • the source of lysophospholipase as a reagent for measuring lysolecithin is not particularly limited, but an enzyme obtained from animals, plants, and microorganisms and which does not act on lecithin is used.
  • Suitable enzymes derived from microorganisms include enzymes derived from the genus Vibrio (Catalog T-32, manufactured by Toyo Brewing Co., Ltd.). Glyceryl phosphorylco
  • the origin of the phosphodiesterase is not particularly limited, and enzymes derived from, animals and microorganisms can be used. Similarly, glycerophosphate
  • the origin of the enzyme, choline oxidase and ethanolamine oxidase is not limited at all, and enzymes derived from microorganisms can be preferably used.For these various enzymes, commercially available enzymes are used. That It is simple.
  • any buffer solution such as a phosphate buffer, an ethanol-lamine buffer, a borate buffer, and various good buffers can be used in the composition for measuring calcium ion of the present invention. What is the pH of the buffer? ⁇ 8 is preferred.
  • composition for measuring calcium ion of the present invention phospholipids 2 to 5 mM, phospholipase 21 to; L0 UZml, lysophospholipase 0.5 to 5 UZml, glycerylphosphorylcholine phosphodiesterase 0.05 ⁇ 1 U / m 1, glycemic acid phosphate 2 ⁇ 201 1 111, choline oxide 2 ⁇ 20 U / m 1, ethano-lamine oxidase 2 ⁇ 20 U / m ⁇ , Peroxidase 1 to 10 U / m 1, 4-aminoantipyrine 0.02% to 0.2%, phenol or aniline derivative 0.02% to 0.2% .
  • a surfactant for example, a nonionic surfactant such as 0.1 to 0.5% of Triton X—100, P It is preferable to have a preservative, a stabilizing agent, etc., and to easily disperse the phospholipid in the composition for measurement by ultrasonic treatment, or to disperse easily by using a surfactant in combination. I just need.
  • body fluids such as blood, serum, and urine.
  • the measurement of hydrogen peroxide is carried out by a conventional method, which is described in, for example, Japanese Patent Publication No. 2-319600.
  • Reagents may be divided into two or more and appropriately combined. Specifically, calcium ion measurement consisting of the above composition Add a serum sample of 100 to 201 to the reaction solution containing the composition for use, and then react for 5 to 10 minutes at 37 ° C. In the case of a color reaction, the absorbance at 500 to 550 nm may be measured.
  • composition for measuring calcium ion of the present invention has good fractionation efficiency, excellent measurement accuracy, good linearity, and can measure accurately and easily up to a high concentration region.
  • this measurement reagent is applicable to continuous measurement using an automatic analyzer often used in clinical laboratories.
  • Figure 1 shows the results of measurement of enzymes derived from Streptomyces and bee venom.
  • FIG. 2 shows the measurement results using the reagent in Example 1 of the present invention.
  • FIG. 3 shows a measurement result using a reagent in Example 3 of the present invention.
  • FIG. 4 shows the measurement results using the reagent in Example 4 of the present invention.
  • the calcium ion concentration was measured by the following measurement method using the following reagents.
  • Human serum was used as a test solution.
  • the reagents and the measurement method were measured under the same conditions as in Example 1. Incidentally, it issued Ki serum calcium ⁇ beam concentration 1 mMC a CI Z as a standard solution. The results are shown in Table 1.
  • test solution 0.5 mM CaCl 2
  • Reagent 1 having the above composition
  • the increase in absorbance at 0 nm was measured continuously with a spectrophotometer. The results are shown in FIG. Example 4
  • the calcium ion concentration was measured by the following measurement method using the following reagent.
  • composition for measuring calcium ion having the following composition
  • composition for measuring calcium ion having the following composition
  • a composition for measuring calcium ion having the following composition.

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Abstract

A calcium ion determining composition containing a phopholipid, phospholipase A2 and a lysolecithin determining reagent as the components for determining the calcium ion concentration in a biological specimen. The composition facilitates the determination and gives accurate analytical results even with a high-calcium specimen.

Description

明 細 書  Specification
カルシウムィォン測定用組成物 技術分野  Technical field of calcium ion measuring composition
本発明は、 カルシウムイオンの測定用組成物に関する。 更に詳しく はリ ン脂質、 ホスホリパーゼ A 2 およびリゾレシチン測定用試薬を舍 有するカルシウムィォン測定用組成物に関する。 従来の技術 The present invention relates to a composition for measuring calcium ions. More particularly calcium I O emissions measured composition having Complex Li phospholipids, phospholipase A 2 and lysolecithin measuring reagent. Conventional technology
体液中のカルシウムの測定は、 神経、 筋肉の興奮性、 血液凝固反応、 細胞膜の透過性等の種々の生理機能や副甲状腺機能などの有用な情報 を与え、 臨床的意義が高い。 従来、 体液中のカルシウムイオンの測定 には、 イオン選択電極を用いた電極法などの方法が実用化され、 一般 に用いられていた。 ところが、 この方法は専用の装置が必要であり、 従って機器の保守 ·管理に注意を要し、 試料の分析処理効率が悪い等 の問題があった。  Measurement of calcium in body fluids provides useful information on various physiological functions such as nerve and muscle excitability, blood coagulation reaction, cell membrane permeability, and parathyroid function, and has high clinical significance. Conventionally, methods such as an electrode method using an ion-selective electrode have been put to practical use and generally used for measuring calcium ions in body fluids. However, this method requires a dedicated device, and therefore requires attention to the maintenance and management of the device, and has problems such as poor sample analysis processing efficiency.
また、 近年ホスホリパーゼ D反応におけるカルシウムイオンによる 活性化 （カルシウム要求性） を利用したカルシウムイオンの測定方法 が報告されている （ 1 9 9 1年度日本農芸化学会大会要旨、 3 0 9頁） 。 この方法は、 高濃度領域のカルシウムィォンの測定のために反応系に M nイオンを添加する操作が必要であるなどの問題があった。 発明が解決しょうとする課題  In recent years, a method for measuring calcium ion using activation by calcium ion (calcium requirement) in phospholipase D reaction has been reported (Abstract of the Japanese Society of Agricultural Chemistry, 1991, p. 309). This method had problems such as the necessity of adding Mn ions to the reaction system to measure calcium ions in a high concentration region. Problems to be solved by the invention
本発明は、 特殊な測定装置の保守，管理が不要であり、 測定精度に 優れ、 かつカルシゥムの高値検体でも正確な分析結果を与える簡便な カルシウムィォン測定用試薬を提供するものである。 課題を解決するための手段 An object of the present invention is to provide a simple calcium ion measuring reagent which does not require maintenance and management of a special measuring device, has excellent measurement accuracy, and gives accurate analysis results even with a high-value calcium sample. Means for solving the problem
本発明者らは、 上記のごとく目的を達成するために種々銳意検計し た結果、 本発明を完成するに至った。 すなわち、 本発明は、 リン脂質 、 ホスホリパーゼ A2 および1/ゾレシチン測定用試薬を含有すること を特徴とするカルシウムィォン測定用組成物である。 The inventors of the present invention have made various measurements to achieve the object as described above, and as a result, have completed the present invention. That is, the present invention is a phospholipid, calcium I O emissions measured composition characterized by containing the phospholipase A 2 and 1 / Zoreshichin measuring reagent.
本発明に用いられるリン脂質は、 下記一般式で示される。  The phospholipid used in the present invention is represented by the following general formula.
R： 0 C H2 R: 0 CH 2
H C 0 R2 HC 0 R 2
0 H  0 H
H2 C-0-P-0-X H 2 C-0-P-0-X
II  II
0 0
(式中、 、 Rz は炭素原子数 6〜22のァシル基、 Xはコリ ンま たはエタノールアミンを示す。 ） (In the formula, Rz represents an acyl group having 6 to 22 carbon atoms, and X represents collin or ethanolamine.)
また、 このリ ン脂質を例示すれば、 卵黄レシチンや大豆レシチンな どの天然レシチン、 ジォレイルレシチン、 ジパルミ トイルレシチン、 ジステアロイルレシチン、 ジミ リス トイ /レレシチン、 ジリノ レイスレレ シチン、 ジリノ レオイノレレシチン、 I—パルミ トイノレ一 2—ォレイル レシチン、 1一ノ\°ルミ トイゾレ一 2—リノ レイルレシチン、 1一パルミ トィルー 2—リノ レオイスレ、 1ーステア口ィルー 2—ォレイノレレシチ ン、 1—ステアロイルー 2—リノ レイルレシチンなどの合成レシチン が挙げられる。  Examples of this phospholipid include natural lecithins such as egg yolk lecithin and soy lecithin, giorail lecithin, dipalmitoyl lecithin, distearoyl lecithin, zimiris toy / lelecithin, zirino leis relecithin, zirino leioinolecithin, I—Palmi Toinole 1—Oleyl Lecithin, 1— \ Lumi Toilet 1—Lino Rail Lecithin, 1—Palmi Toileu 2—Lino Leisleine, 1—Stear Mouth 2—Oleino Lecithin, 1—Stearoyl 2—Lino Lele Lecithin And synthetic lecithin.
リゾレシチン測定用試薬としては、 ホスホリパーゼ と上記の一 般式で示されるリン脂質との反応'生成物であるリゾレシチンを検出す るための諸酵素や試薬の組み合わせから構成される。 すなわち、 少な く ともリゾホスホリパーゼ、 グリセリルホスホリルコ リ ンホスホジェ ステラーゼおよびグリセロリ ン酸ォキシダーゼまたはコ リ ンォキシタ' —ゼまたはェタノ一ルァミンォキシダーゼから構成されるリゾレシチ ン測定用試薬を用いた反応により生成された過酸化水素を定量すればThe reagent for measuring lysolecithin is composed of a combination of enzymes and reagents for detecting lysolecithin, which is a reaction product of phospholipase and the phospholipid represented by the above general formula. That is, at least lysophospholipase, glycerylphosphorylcholine phosphogen The amount of hydrogen peroxide produced by the reaction using a lysolecithin measurement reagent consisting of a sterase and glycerophosphate oxidase or choline oxidase'- or ethanolamine oxidase can be determined.
* よく、 またペルォキシダ一ゼを用いて分光学的に測定できるものであ * Good and can be measured spectrophotometrically using peroxidase
る。  You.
一般にはグリセリルホスホリルコ リ ンホスホジエステラ一ゼは、 上 記一般式において Xがエタノールァミンの場合も基質として利用する ものであるので、 グリセリルホスホリエタノ一ルァ ミ ンの場合も反応 が進行する。 ' 本発明に用いられれるホスホリパーゼ八2 としては、 へビ毒、 ハチ 毒、 動物薛臓などにその存在が知られている力 これら起源の酵素は 低濃度領域のカルシウムィォンで最大の活性化が起きることが知られ ている。 本発明者により究明された、 ス トレブトマイセス由来のホス ホリパーゼ八2 は、 第 1図に示すように比較的高濃度のカルシウムィ オンまで濃度に比例した活性化がみられる。 In general, glycerylphosphorylcholine phosphodiesterase is used as a substrate when X is ethanolamine in the above general formula, so that the reaction proceeds even when glyceryl phosphorylethanolamine is used. 'As the phospholipase eight 2 to be used in the present invention, the bi venom, bee venom, forces the enzyme of origin such as the present the animal薛臓it is known maximum activation with calcium I O emissions lightly-doped region It is known to happen. Was investigated by the present inventors, phosphorylase Horipaze eight 2 from scan Torebutomaisesu is activated in proportion to the concentration to a relatively high concentration of calcium I-one as shown in FIG. 1 can be seen.
従って、 カルシウムィォン測定の目的にはス ト レプトマイセス由来 のホスホリパーゼ八2 (東洋醸造株式会社製、 カタログ T一 3 1 ) が 好都合であり、 この点も本発明のカルシウムィォン測定用組成物の優 れた点である。 リゾレシチン測定用試薬としてのリゾホスホリパーゼ の起源は特に限定されるものでないが、 動物、 植物、 微生物から得ら れる酵素でレシチンに作用しない酵素が利用される。 Therefore, for the purposes of calcium I O emissions measurements be sampled Reputomaisesu derived phospholipase eight 2 (Toyo Jozo Co., catalog T one 3 1) is advantageous, also Re Yu calcium I O emissions measured composition of the present invention this point It is a point. The source of lysophospholipase as a reagent for measuring lysolecithin is not particularly limited, but an enzyme obtained from animals, plants, and microorganisms and which does not act on lecithin is used.
微生物由来の酵素としては、 ビブリオ属由来の酵素 （東洋醸造株式 会社製、 カタログ T— 3 2 ) が適している。 グリ セリルホスホリルコ Suitable enzymes derived from microorganisms include enzymes derived from the genus Vibrio (Catalog T-32, manufactured by Toyo Brewing Co., Ltd.). Glyceryl phosphorylco
* リ ンホスホジエステラーゼもその起源は特に限定されるものではなく、 , 動物、 微生物由来の酵素が使用できる。 同様にグリセロリ ン酸ォキシ * The origin of the phosphodiesterase is not particularly limited, and enzymes derived from, animals and microorganisms can be used. Similarly, glycerophosphate
ダ一ゼ、 コ リ ンオシダ一ゼやエタノールアミ ンォキシダ一ゼも、 その 起源に何ら限定されるものではなく、 好ましくは微生物由来の酵素が 使用でき、 これら種々の酵素については市販のものを使用することが 簡便である。 The origin of the enzyme, choline oxidase and ethanolamine oxidase is not limited at all, and enzymes derived from microorganisms can be preferably used.For these various enzymes, commercially available enzymes are used. That It is simple.
本発明のカルシウムイオン測定用組成物にはリン酸緩衝液、 ェタノ —ルァミン緩衝液、 ホウ酸緩衝液、 各種グッド緩衝液などあらゆる緩 衝液が使用できる。 緩衝液の P Hは？〜 8が好ましい。  Any buffer solution such as a phosphate buffer, an ethanol-lamine buffer, a borate buffer, and various good buffers can be used in the composition for measuring calcium ion of the present invention. What is the pH of the buffer? ~ 8 is preferred.
本発明のカルシウムィォン測定用組成物について例示すれば、 リン 脂質 2〜5 mM、 ホスホリパーゼ 2 1〜； L 0 UZm l、 リゾホ スホリパーゼ 0. 5〜5 UZm l、 グリセリルホスホリルコリンホス ホジエステラーゼ 0. 0 5〜1 U /m 1、 グリセ口リン酸ォキシダ一 ゼ2〜2 0 1ノ111 1、 コリンォキシダ一ゼ 2〜2 0 U/m 1、 ェタノ —ルァミンォキシダーゼ 2〜 2 0 U/m〗、 ペルォキシダーゼ 1〜 1 0 U/m 1、 4ーァミノアンチピリン 0. 0 2 %〜0. 2 %、 フエノ —ルまたばァニリ ン誘導体 0. 0 2 %〜0 2 %を適宜選択して含有 する。 本 ¾明のカルシウムィォン測定用組成物にば必要に応じて异面 活性剤、 例えば 0. 1〜0. 5 %のトリ トン X— 1 0 0などの非ィォ ン性界面活性剤、 P方腐剤、 安定化剤等を舍有するとよく、 また、 上記 リン脂質ば超音波処理により当該測定用組成物中に分散し易く行うか 、 またば界面活性剤を併用して分散し易くすればよい。 このカルシゥ ムイオン測定用組成物を用いて測定される試料としては、 例えば血液 、 血清や尿などの体液が挙げられる。 As an example of the composition for measuring calcium ion of the present invention, phospholipids 2 to 5 mM, phospholipase 21 to; L0 UZml, lysophospholipase 0.5 to 5 UZml, glycerylphosphorylcholine phosphodiesterase 0.05 ~ 1 U / m 1, glycemic acid phosphate 2 ~ 201 1 111, choline oxide 2 ~ 20 U / m 1, ethano-lamine oxidase 2 ~ 20 U / m〗, Peroxidase 1 to 10 U / m 1, 4-aminoantipyrine 0.02% to 0.2%, phenol or aniline derivative 0.02% to 0.2% . In the composition for measuring calcium ion of the present invention, if necessary, a surfactant, for example, a nonionic surfactant such as 0.1 to 0.5% of Triton X—100, P It is preferable to have a preservative, a stabilizing agent, etc., and to easily disperse the phospholipid in the composition for measurement by ultrasonic treatment, or to disperse easily by using a surfactant in combination. I just need. Examples of the sample measured using the calcium ion measuring composition include body fluids such as blood, serum, and urine.
試料中のカルシウムイオンを測定するには、 緩衝液、 リン脂質、 ホ スホリパーゼ八2 およびリゾレシチン測定用試薬を舍有する試薬に試 料を添加して反応を行わせ、 その結果生成するリゾレシチンをリゾレ シチン測定用試薬処理、 いわゆる酵素法により生成する過酸化水素を 過酸化水素電極またばペルォキシダーゼを用いて分光学的に測定すれ ばよい。 To measure calcium ions in a sample, buffer, phospholipids, was added to specimen a host Suhoripaze eight 2 and lysolecithin measuring reagent in the reagent to舍有to perform the reaction, Rizore lecithin and lysolecithin which result generated Hydrogen peroxide generated by treatment with a measuring reagent, a so-called enzymatic method, may be measured spectrophotometrically using a hydrogen peroxide electrode or peroxidase.
過酸化水素の測定ば常法により行われ、 それは例えば特公平 2— 3 1 9 6 0号に記載されている。 試薬ば 2つ以上に分けて、 適宜組み合 わせてもよい。 具体的には、 上記組成からなるカルシウムイオン測定 用組成物を舍む反応液 5 0 0〜 1 0 0 0 //〗 に 1 0〜 2 0 1 の血清 検体を加え、 37てで 5〜 1 0分間反応を行い、 ペルォキシダ一ゼに よる呈色反応の場合は、 5 0 0〜5 5 0 n mにおける吸光度を測定す ればよい。 The measurement of hydrogen peroxide is carried out by a conventional method, which is described in, for example, Japanese Patent Publication No. 2-319600. Reagents may be divided into two or more and appropriately combined. Specifically, calcium ion measurement consisting of the above composition Add a serum sample of 100 to 201 to the reaction solution containing the composition for use, and then react for 5 to 10 minutes at 37 ° C. In the case of a color reaction, the absorbance at 500 to 550 nm may be measured.
本発明のカルシウムイオン測定用組成物は、 分折効率が良く、 測定 精度に優れ、 良好な直線性をもち、 高濃度領域まで正確かつ簡便に測 定できる。 また、 本測定試薬は、 臨床検査室でもよく使われている自 動分析機を用いての連続測定にも適応可能である。 図面の簡単な説明  The composition for measuring calcium ion of the present invention has good fractionation efficiency, excellent measurement accuracy, good linearity, and can measure accurately and easily up to a high concentration region. In addition, this measurement reagent is applicable to continuous measurement using an automatic analyzer often used in clinical laboratories. BRIEF DESCRIPTION OF THE FIGURES
第 1図はストレブトマイセ.ス由来酵素およびハチ毒由来酵素の測定 結果である。  Figure 1 shows the results of measurement of enzymes derived from Streptomyces and bee venom.
第 2図は本発明の実施例 1における試薬を用いた測定結果である。 第 3図は本発明の実施例 3における試薬を用いた測定結果である。 第 4図は本発明の実施例 4における試薬を用いた測定結果である。 次に、 本発明を実施例により説明する。  FIG. 2 shows the measurement results using the reagent in Example 1 of the present invention. FIG. 3 shows a measurement result using a reagent in Example 3 of the present invention. FIG. 4 shows the measurement results using the reagent in Example 4 of the present invention. Next, the present invention will be described with reference to examples.
実施例 1 Example 1
下記試薬を用いて下記測定法によりカルシウムイオン濃度を測定し た。  The calcium ion concentration was measured by the following measurement method using the following reagents.
ぐ試薬 1 >  Reagent 1>
0. 0 5 M トリス一塩酸緩衝液 （PH7. 5 )  0.05 M Tris monohydrochloride buffer (PH7.5)
1 m M ジォレオイルレシチン  1 mM M Giore oil lecithin
0. 2 % トリ トン X 1 0 0  0.2% Triton X 1 0 0
1 U/ml リ ゾホスホリパ一ゼ  1 U / ml lysophospholipase
0. 1 U/ml グリセリルホスホリルコ リ ンホスホジエステ ラーセ  0.1 U / ml glyceryl phosphorylcholine phosphodiesterase
U/ml グリセロリ ン酸ォキシダ一ゼ 5 U/ml ぺゾレオキシタ "一ゼ U / ml glycerophosphate oxidase 5 U / ml zoleoxyta
0. 0 5 % フエノール  0.0 5% phenol
5 IJ ml ホスホリパーゼ A2 (ス トレプトマイセス属 由来、 東洋醸造社製） 5 IJ ml phospholipase A 2 (from Streptomyces sp., Manufactured by Toyo Brewery)
ぐ試薬 2 >  Reagent 2>
Q . 0 5 M トリス一塩酸緩衝液 （PH7. 5 ) 0. 0 5 % 4ーァミノアンチピリ ン  Q.05 M Tris monohydrochloride buffer (PH7.5) 0.05% 4-aminoantipyridine
ぐ測定方法 > - 塩化カルシウム (C a Clz) または塩化マグネシウム （M g Clz) を舍有せしめた被検液 1 0 l に上記試薬 1を 5 0 0 / 1 加え、 3 7 ΐで 5分間反応した後、 試率 2を加え、 3 7 °Cで 5分間反応させ、 2 %S D S 2 5 0 ii l を加え、 反応を止めた後、 その吸光度を波長 5 0 O nmで測定した。 この結果を、 第 2図に示した。 マグネシウムィォ ンでは殆ど反応性が認められなかつた力 カルシウムイオンに対して 直線性の良好な結果が得られた。 実施例 2 >-Add 50 0/1 of the above reagent 1 to 10 l of test solution containing calcium chloride (Ca Clz) or magnesium chloride (Mg Cl z ), and add 37 3 for 5 minutes. After the reaction, a test rate of 2 was added, the reaction was carried out at 37 ° C for 5 minutes, 2% SDS250iIl was added, the reaction was stopped, and the absorbance was measured at a wavelength of 50 O nm. The results are shown in FIG. Magnesium ion showed almost no reactivity, and good linearity was obtained for calcium ions. Example 2
被検液としてヒ ト血清を用いた。 試薬、 測定方法は実施例 1に同じ 条件で測定を行った。 尚、 1 mMC a CIZを標準液として血清カルシ ゥム濃度を箕出した。 この結果を、 第 1表に示した。 Human serum was used as a test solution. The reagents and the measurement method were measured under the same conditions as in Example 1. Incidentally, it issued Ki serum calcium © beam concentration 1 mMC a CI Z as a standard solution. The results are shown in Table 1.
第 1表 血清 検体 カ ル シ ウ ム 濃 度  Table 1 Serum Specimen Calcium Concentration
A 0. 9 3 mM A 0.93 mM
B 1. 2 5 mM 実施例 3 B 1.2 5 mM Example 3
ぐ試薬 1 >  Reagent 1>
0. 0 5 P I P E S— N a 0 H緩衝液 （pH7. 3 ) 1 mM 卵黄ホスファチジルエタノールァミ ン 0.05 PIPES-NaOH buffer (pH 7.3) 1 mM egg yolk phosphatidylethanolamine
0. 2 % ト リ ト ン X— 1 0 0 0.2% Triton X—1 0 0
1 U/m 1 リゾホスホリパ一ゼ  1 U / m 1 lysophospholipase
1 U/m 1 グリセリルホスホリルコ リ ンホスホジェス ァフ一セ  1 U / m 1 Glycerylphosphorylcholine phosphoges
5 U/m 1 グリセロリ ン酸ォキシダ一ゼ  5 U / m 1 glycerophosphate oxidase
5 U/m 1 ペルォキシダ一ゼ  5 U / m 1 Peroxidase
0. 0 5 % フエノール  0.0 5% phenol
1 0 U/m 1 ホスホリパーゼ 2 (ス ト レブトマイセス属 由来、 東洋醸造社製） 10 U / m 1 Phospholipase 2 (from Streptomyces sp., Manufactured by Toyo Brewery)
く試薬 2 >  Reagent 2>
0. 0 5 M P I P E S -N a O H緩衝液 （ P H 7. 3 ) 0. 0 5 % 4—アミノアンチピリ ン  0.05 M PIPES-NaOH buffer (PH7.3) 0.05% 4-aminoantipyrine
<測定方法 >  <Measurement method>
上記組成の試薬 1の 5 0 0 1 に被検液 （ 0. 5 mMC a Cl2) 1 0 1 を加え、 3 7てで 5分間加温後、 試薬 2を 5 0 0 1 加え、 5 0 0 nmの吸光度の増加を分光光度計で連続的に測定した。 この結果 を、 第 3図に示した。 実施例 4 A test solution (0.5 mM CaCl 2 ) 101 was added to 500 1 of Reagent 1 having the above composition, and the mixture was heated at 37 ° C for 5 minutes. The increase in absorbance at 0 nm was measured continuously with a spectrophotometer. The results are shown in FIG. Example 4
下記試薬を用いて下記測定法によりカルシウムィォン濃度を測定 した。  The calcium ion concentration was measured by the following measurement method using the following reagent.
ぐ試薬 1〉  Reagent 1>
0. 0 5 M トリスー塩酸緩衝液 （ P H 7. 5 ) 0.05 M Tris-HCl buffer (PH 7.5)
1 mM ジォレオイルレシチン 0. 2 % ト！ / トン X— 1 0 0 1 mM dioleoyl lecithin 0.2% G! / Ton X— 1 0 0
1 U m 1 リゾホスホリパ一ゼ  1 U m 1 lysophospholipase
0. 1 U/ml グリセリルホスホリルコリ ンホスホジエステ ラ一セ  0.1 U / ml glyceryl phosphoryl choline phosphodiesterase
5 U/m 1 グリセロリ ン酸ォキシダ一ゼ  5 U / m 1 glycerophosphate oxidase
5 \]/m 1 ペルォキシダーゼ  5 \] / m1 peroxidase
0. 05 % フエノール  0.05% phenol
5 U/m 1 ホスホリパ一ゼ A2 (ス トレブトマイセス属 由来、 東洋醸造社製） 5 U / m 1 Phospholipase A 2 (from Streptomyces sp., Manufactured by Toyo Brewery)
6 υ/Ίιι 1 コリンォキシダ一ゼ  6 υ / Ίιι 1
<試薬 2 > .  <Reagent 2>.
0. 05 M トリスー塩酸緩衝液 （P H 7. 5 ) 0. 05 % 4—ァミノアンチピリ ン  0.05 M Tris-HCl buffer (pH 7.5) 0.05% 4-Aminoantipyridine
ぐ測定方法 >  Measuring method>
塩化力ルシゥムまたは塩化マグネシウムを舍有せしめた被検液 1 0 U 1 に上記試薬 1を 5 00 1 加え、 37てで 5分間反応した後、 試 薬 2を加え、 37°Cで 5分間反応させ、 2%SDS 25 ひ ^】 加え、 反応を止めた後、 その吸光度を波長 5 0 0 nmで測定した。 この結果 を、 第 4図に示した。 マグネシウムイオンでは殆ど反応性が認められ なかったが、 カルシウムィォンでは直線性の良好な結果が得られた。 実施例 5  Add 5001 of the above reagent 1 to 10 U 1 of test solution containing lucidium chloride or magnesium chloride, react at 37 ° C for 5 minutes, add Reagent 2 and react at 37 ° C for 5 minutes. After adding 2% SDS and stopping the reaction, the absorbance was measured at a wavelength of 500 nm. The results are shown in FIG. Almost no reactivity was observed with magnesium ions, but good linearity was obtained with calcium ions. Example 5
下記組成よりなるカルシウムィォン測定用組成物  A composition for measuring calcium ion having the following composition
<試薬 1 >  <Reagent 1>
0. 05 M トリスー塩酸緩衝液 （P H 7. 5 ) 0.05 M Tris-HCl buffer (PH 7.5)
1 mM ジォレオイルレシチン 1 mM dioleoyl lecithin
0. 2 % トリ トン X— 1 0 0  0.2% Triton X—1 0 0
1 U/m 1 リゾホスホリパーゼ 0. 1 U/ml グリセリルホスホリルコ リ ンホスホジエステ ラ— k 1 U / m1 lysophospholipase 0.1 U / ml glyceryl phosphorylcholine phosphodiester k
δ U/m 1 グリセロ リ ン酸ォキシダ一ゼ  δ U / m 1 glycerophosphate oxidase
5 U/m l ペルォキシダ一ゼ  5 U / ml l peroxidase
0. 0 5 % フエノール  0.0 5% phenol
5 U/m ] ホスホリパーゼ A 2 (ス ト レプトマイセス属 由来、 東洋醸造社製） 5 U / m] Phospholipase A 2 (from Streptomyces sp., Manufactured by Toyo Brewery)
く試薬 2 >  Reagent 2>
0. 0 5 M トリスー塩酸緩衝液 （P H 7. 5 ) 0. 0 5 % 4 ーァミノアンチピリ ン 実施例 6  0.05 M Tris-HCl buffer (pH 7.5) 0.05% 4-aminoantipyrine Example 6
下記組成よりなるカルシウムィォン測定用組成物  A composition for measuring calcium ion having the following composition
ぐ試薬 1 >  Reagent 1>
0. 0 5 P I P E S - N a O H緩衝液 （ p H 7. 3 ) 1 mM 卵黄ホスファチジルエタノールァミ ン 0.05 PIPES-NaOH buffer (pH 7.3) 1 mM yolk phosphatidylethanolamine
0. 2 % ト リ トン X - 1 0 0 0.2% Triton X-1 0 0
1 U 1 リゾホスホリパーゼ  1 U 1 lysophospholipase
0. 1 U/ml グリセリルホスホリルコリ ンホスホジエステ ラ一セ  0.1 U / ml glyceryl phosphoryl choline phosphodiesterase
5 U/m 1 グリセ πリ ン酸ォキシダ一ゼ  5 U / m 1 Glyce π-phosphate oxidase
5 U/m ] ペルォキシダーゼ  5 U / m] peroxidase
0. 0 5 % フエノール  0.0 5% phenol
1 0 U/m 1 ホスホリパーゼ A2 (ス ト レプトマイセス属 由来、 東洋醸造社製） 10 U / m 1 Phospholipase A 2 (from Streptomyces sp., Manufactured by Toyo Brewery)
ぐ試薬 2 >  Reagent 2>
0. 0 5 M P I P E S - N a O H緩衝液 （ P H 7. 3 ) 0. 0 5 % 4—ァミノアンチピリ ン 実施例 Ί 0.05 MPIPES-NaOH buffer (PH7.3) 0.05% 4-aminoaminopyridine Example Ί
下記組成よりなるカルシウムィォン測定用組成物。  A composition for measuring calcium ion having the following composition.
<試薬 1 >  <Reagent 1>
0. 0 5 M トリス—塩酸緩衝液 （Ρ Η 7. 5 ) 0.05 M Tris-HCl buffer (Ρ Η 7.5)
1 mM ジォレオイルレシチン 1 mM dioleoyl lecithin
ひ. 2 % トリ トン X - 1 0 0  H. 2% Triton X-1 0 0
1 5/m I リゾホスホリパ一ゼ  1 5 / m I lysophospholipase
0. 1 U ml グリセリフレホスホリルコ リ ンホスホジエステ ラ一ゼ  0.1 U ml glyceryl phosphorylcholine phosphodiesterase
5 Uノ m 1 グリセロリ ン酸ォキシダーゼ  5 U m 1 Glycerolate oxidase
5 U/m 1 ぺ ォキシダ—ゼ  5 U / m 1 キ シ oxidase
0 0 5 % フエノ一ル  0 0 5% phenol
5 U/m 1 ホスホリパ一ゼ Az (ス トレプトマイセス属 由来、 東洋醸造社製） 5 U / m 1 Hosuhoripa Ichize A z (scan Toreputomaisesu genus, manufactured by Toyo Jozo Co.)
6 U/m 1 コリ ンォキシダ一ゼ  6 U / m 1 Coryneoxidase
<薩 2 >  <Satsu 2>
0. 0 5 M 卜リス一塩酸緩衝液 （P H 7. 5 )  0.05 M Tris-hydrochloric acid buffer (pH 7.5)
0. 0 5 % 4ーァミノアンチピリ ン  0.0 5% 4-aminoantipyridine

Claims

請 求 の 範 囲 The scope of the claims
1 . リ ン脂質、 ホスホリパ一ゼ A 2 およびリゾレシチン測定用試薬 を含有することを特徴とするカルシウムィォン測定用組成物。 1. Li phospholipids, calcium I O emissions measured composition characterized by containing Hosuhoripa Ichize A 2 and lysolecithin measuring reagent.
2 . ホスホリパーゼ A z 力く、 ス トレプトマイセス属由来のホスホリ パーゼ A 2 である請求の範囲第 1項記載のカルシウムィォン測定用組 成物。 2. Phospholipase A z Chikaraku, calcium preceding claim claims a Hosuhori lipase A 2 from scan Toreputomaisesu genus I O emissions measurement set Narubutsu.
3 . リゾレシチン測定用試薬が、 リゾホスホリパーゼ、 グリセリル ホスホリルコ リ ンホスホジ^:ステラ一ゼおよびグリセロリ ン酸ォキシ ダーゼを含有してなる試薬である請求の範囲第 1項記載のカルシウム イオン測定用組成物。 3. The composition for measuring calcium ion according to claim 1, wherein the reagent for measuring lysolecithin is a reagent containing lysophospholipase, glyceryl phosphorylcholinephosphodi ^: sterase and glycerophosphate oxidase.
4 . リゾレシチン測定用試薬が、 リゾホスホリパ一ゼ、 グリセリル ホスホリルコ リ ンホスホジエステラーゼおよびコ リ ンォキシダーゼま たはエタノ一ルァミンォキシダーゼを舍有してなる試薬である請求の 範囲第 1項記載の力ルシゥムィォン測定用組成物。 4. The measurement of lysolecithin according to claim 1, wherein the reagent for measuring lysolecithin is a reagent comprising lysophospholipase, glyceryl phosphorylcholine phosphodiesterase and cholinoxidase or ethanolamine oxidase. Composition.
5 . リゾレシチン測定用試薬が、 リゾホスホリパ一ゼ、 グリセリル ホスホリルコリ ンホスホジエステラーゼ、 グリセロリ ン酸ォキシダ一 ゼおよびコ リ ンォキシダ一ゼ、 必要に応じてエタノールアミ ンォキシ ダ一ゼを舍有してなる試薬である請求の範囲第 1項記載のカルシウム イオン測定用組成物。 5. The reagent for measuring lysolecithin is a reagent comprising lysophospholipase, glyceryl phosphorylcholine phosphodiesterase, glycerophosphate oxidase and cholineoxidase, and, if necessary, ethanolamine oxidase. The composition for measuring calcium ions according to claim 1.
6 . 過酸化水素指示薬組成物を含有してなる請求の範囲第 3項ない し第 5項のいずれかの項に記載のカルシウムイオン測定用組成物。 6. The composition for measuring calcium ion according to any one of claims 3 to 5, comprising a hydrogen peroxide indicator composition.
PCT/JP1992/001680 1991-12-24 1992-12-22 Calcium ion determining composition WO1993013219A1 (en)

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