JPH0634755B2 - Calcium ion quantification reagent - Google Patents
Calcium ion quantification reagentInfo
- Publication number
- JPH0634755B2 JPH0634755B2 JP61036123A JP3612386A JPH0634755B2 JP H0634755 B2 JPH0634755 B2 JP H0634755B2 JP 61036123 A JP61036123 A JP 61036123A JP 3612386 A JP3612386 A JP 3612386A JP H0634755 B2 JPH0634755 B2 JP H0634755B2
- Authority
- JP
- Japan
- Prior art keywords
- reagent
- calcium ion
- present
- phospholipase
- quantification reagent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は，カルシウムイオン定量用試薬に関するもので
ある。TECHNICAL FIELD The present invention relates to a reagent for quantifying calcium ions.
（従来の技術） 現在，臨床検査の分野で，副甲状腺の疾患，閉塞性黄疸
等の診断の目的で血中のカルシウムイオン濃度が定量さ
れている。その定量は大きく２つに分けられる。１つは
原子吸光法，電極法に代表される機器分析法による定量
であり，さらに１つは，オルトクレゾールフタレインコ
ンプレキソン法（以後OCPC法と略記する。）などの化学
的手法による定量である。(Prior Art) Currently, in the field of clinical examination, the calcium ion concentration in blood is quantified for the purpose of diagnosing diseases of the parathyroid gland, obstructive jaundice, and the like. The quantification can be roughly divided into two. One is quantification by instrumental analysis method represented by atomic absorption method and electrode method, and the other is quantification by chemical method such as orthocresolphthalein complexone method (hereinafter abbreviated as OCPC method). is there.
一方，特開昭57-58893号公報には，コリンオキシダーゼ
を含有する分析用組成物が提案されている。しかしなが
ら，この組成物はコリン又はベタインアルデヒドを定量
するものであって，カルシウムイオンを定量するもので
はない。On the other hand, JP-A-57-58893 discloses a composition for analysis containing choline oxidase. However, this composition quantifies choline or betaine aldehyde, not calcium ion.
（発明が解決しようとする問題点） 前記の機器分析法による定量では比較的正確な結果が得
られるが，高価な機器を特別に必要とし，操作も繁雑で
ある。(Problems to be Solved by the Invention) Although quantitative results by the above-mentioned instrumental analysis method can obtain relatively accurate results, they require special expensive instruments and their operations are complicated.
一方，前記したOCPC法などによる定量では，特別な分析
装置を必要とせず，簡単な比色計で定量可能であるとい
う理由から普及しているが，発色の度合いが，気温やpH
などによって変化する他にカルシウムイオンに対する特
異性に欠けるため，共存する物質の影響が避けられず
に，大きな誤差要因を含んだ結果しか得られないという
問題点があった。On the other hand, the quantification by the above-mentioned OCPC method is popular because it can be quantified with a simple colorimeter without requiring a special analyzer, but the degree of color development depends on the temperature and pH.
In addition to changes due to factors such as the above, the specificity for calcium ions is lacking, so the effect of coexisting substances is unavoidable, and there is the problem that only results containing large error factors can be obtained.
（問題点を解決するための手段） そこで本発明者らは，このような点に鑑み，簡単に，か
つ正確にカルシウムイオンを定量することができる試薬
を提供することを目的として鋭意研究した結果，レシチ
ン，ホスホリパーゼＤ及びコリンオキシダーゼからなる
試薬が試料中のカルシウムイオン濃度に比例して発現
し，正確なカルシウムイオンが定量できることを見い出
し，本発明を完成した。(Means for Solving Problems) Therefore, in view of the above points, the present inventors have earnestly studied for the purpose of providing a reagent that can easily and accurately quantify calcium ions. The present inventors have completed the present invention by discovering that a reagent consisting of lecithin, phospholipase D and choline oxidase is expressed in proportion to the calcium ion concentration in a sample, and accurate calcium ion can be quantified.
すなわち，本発明はレシチン，ホスホリパーゼＤ及びコ
リンオキシダーゼからなるカルシウムイオン定量用試薬
を要旨とするものである。That is, the gist of the present invention is a reagent for quantifying calcium ions, which comprises lecithin, phospholipase D and choline oxidase.
本発明の試薬はレシチン，ホスホリパーゼＤ及びコリン
オキシダーゼからなり，その他通常の賦活剤などの添加
物及び緩衝液が含有されている。The reagent of the present invention is composed of lecithin, phospholipase D and choline oxidase, and also contains additives such as ordinary activators and a buffer solution.
本発明にいうレシチンとは，別名1,2−ジアシル−sn−
グリセロ−(3)−ホスホコリンと言われる〔1979年発行
の『生化学データブックＩ』第８３１頁（東京化学同
人）参照〕。Lecithin as used in the present invention is also known as 1,2-diacyl-sn-
It is said to be glycero- (3) -phosphocholine [see Biochemistry Data Book I, 1979, page 831 (Tokyo Kagaku Dojin)].
本発明に用いられるホスホリパーゼＤとしては，例え
ば，キヤベツ，ニンジン，ブタ膵などの動植物由来，ス
トレプトマイセス・クロモフアカス（Streptomyces Cho
romofuscus）などの微生物由来のものがあげられ，ま
た，コリンオキシダーゼとしては，例えば，アースロバ
クター・グラビホーミス（Arthrobacter globiformi
s），アルカリゲネス・スピーシーズ（Alcaligenes S
P.）などの微生物由来のものがあげられる。Examples of the phospholipase D used in the present invention include those derived from plants and animals such as cabbages, carrots, pig pancreas, and Streptomyces Chomofacus (Streptomyces Cho
romofuscus) and the like, and examples of choline oxidase include Arthrobacter globiformi.
s), Alcaligenes S
P.) and the like derived from microorganisms.
本発明の試薬の各成分の濃度としては，例えば，レシチ
ンを0.2〜10.0mg/ml，ホスホリパーゼＤを0.01〜1.0U/m
l，コリンオキシダーゼを0.1〜10.0U/ml使用すればよ
い。特にレシチンを0.4〜2.0mg/ml，ホスホリパーゼＤ
を0.05〜0.5U/ml，コリンオキシダーゼを0.5〜2.0U/ml
使用することが好ましい。The concentration of each component of the reagent of the present invention is, for example, 0.2 to 10.0 mg / ml of lecithin and 0.01 to 1.0 U / m of phospholipase D.
l, Choline oxidase may be used at 0.1-10.0 U / ml. Lecithin 0.4-2.0 mg / ml, phospholipase D
0.05-0.5U / ml, choline oxidase 0.5-2.0U / ml
Preference is given to using.
本発明の試薬の測定原理を次に示す。The measurement principle of the reagent of the present invention is shown below.
反応式におけるホスホリパーゼＤの活性発現にはカル
シウムイオンの存在が必須であり，ホスホリパーゼＤの
活性はカルシウムイオンの濃度に比例する。最終的に反
応式において生成したH2O2を過酸化水素電極で定量す
るか，またはある色原体の存在下ペルオキシダーゼを作
用させ，色調の変化を比色定量すればカルシウムイオン
の定量は完了する。特に後者が特別な装置を必要としな
いという点で日常の検査には適している。その原理を下
に示す。 The presence of calcium ions is essential for the expression of phospholipase D activity in the reaction formula, and the activity of phospholipase D is proportional to the concentration of calcium ions. Finally, the H 2 O 2 generated in the reaction formula is quantified with a hydrogen peroxide electrode, or peroxidase is allowed to act in the presence of a chromogen to perform colorimetric quantification of the change in color to complete the quantification of calcium ions. To do. In particular, the latter is suitable for daily inspection in that no special device is required. The principle is shown below.
上記で用いられるペルオキシダーゼとしては，例えば西
洋ワサビ由来のものがあげられ，その濃度としては，例
えば10〜300U/dl，好ましくは100〜250U/dlである。 Examples of the peroxidase used above include those derived from horseradish, and the concentration thereof is, for example, 10 to 300 U / dl, preferably 100 to 250 U / dl.
本発明の試薬を用いてカルシウムイオンを定量するに
は，例えば，まず，試薬を約３分間保温（25℃，30℃，
37℃のいずれの温度でもよい。）したのち，サンプルを
加えて約１０分間反応させる。次に反応停止液を添加し
た後，生成したH2O2を上記方法により定量すればよい。
色調の変化を比色定量してH2O2を定量する場合，例え
ば，上記した色原体の存在下ペルオキシダーゼを作用さ
せ，500nmにおける吸光度の上昇を測定すればよい。To quantify calcium ions using the reagent of the present invention, for example, first, the reagent is kept warm for about 3 minutes (25 ° C., 30 ° C.,
Any temperature of 37 ° C may be used. After that, the sample is added and reacted for about 10 minutes. Next, after adding the reaction stop solution, the produced H 2 O 2 may be quantified by the above method.
When H 2 O 2 is quantified by colorimetrically quantifying the change in color tone, for example, peroxidase is allowed to act in the presence of the above-mentioned chromogen and the increase in absorbance at 500 nm may be measured.
（実施例） 次に本発明を実施例によって具体的に説明する。(Examples) Next, the present invention will be specifically described with reference to Examples.
実施例１ NaCl120mM，キヤベツ由来のホスホリパーゼＤ（ベーリ
ンガーマンハイム山ノ内株式会社より購入）0.15U/ml，
アルカリゲネス・スピーシーズ由来のコリンオキシダー
ゼ（東洋紡績株式会社より購入）1.35U/ml，西洋ワサビ
由来のペルオキシダーゼ（東洋紡績株式会社より購入）
200U/dl，レシチン0.8mg/ml，４−アミノアンチピリン
７mg/dl，フエノール40mg/dl,50mMトリス緩衝液（pH7.
4）から成る試薬を調製した。Example 1 NaCl 120 mM, phospholipase D derived from cabbage (purchased from Boehringer Mannheim Yamanouchi Co., Ltd.) 0.15 U / ml,
Choline oxidase derived from Alcaligenes species (purchased from Toyobo Co., Ltd.) 1.35U / ml, peroxidase derived from horseradish (purchased from Toyobo Co., Ltd.)
200 U / dl, lecithin 0.8 mg / ml, 4-aminoantipyrine 7 mg / dl, phenol 40 mg / dl, 50 mM Tris buffer (pH 7.
A reagent consisting of 4) was prepared.
次いであらかじめ３０℃に保温した上記試薬0.45mlとサ
ンプル0.015ml（既知濃度のカルシウム標準液）を混和
し，３０℃で１０分間反応させた。その後，0.2MのEDTA
0.10mlを添加して反応を停止し，500nmにおける吸光度
を測定した。Next, 0.45 ml of the above reagent preliminarily kept warm at 30 ° C. and 0.015 ml of a sample (calcium standard solution of known concentration) were mixed and reacted at 30 ° C. for 10 minutes. Then 0.2M EDTA
The reaction was stopped by adding 0.10 ml, and the absorbance at 500 nm was measured.
その結果を第１図に示す。第１図はカルシウム濃度と吸
光度との相関関係を示すもので，10mg/dlまでの良好な
直線性を有することが判明した。The results are shown in FIG. Fig. 1 shows the correlation between the calcium concentration and the absorbance, and it was found to have good linearity up to 10 mg / dl.
次に上記と同一のカルシウムイオン標準液をカルシウム
イオン電極法を用いて測定し，その相関関係を調べた。Next, the same calcium ion standard solution as above was measured using the calcium ion electrode method, and the correlation was investigated.
その結果を第２図に示す。The results are shown in FIG.
第２図により，本発明の試薬によって得られた値とカル
シウムイオン電極法によって得られた値とは良好な相関
関係を示していることが明らかである。From FIG. 2, it is clear that the values obtained by the reagent of the present invention and the values obtained by the calcium ion electrode method show a good correlation.
（発明の効果） 本発明の試薬は，簡便で，かつ正確にカルシウムイオン
を定量することができ，特に副甲状腺の疾患，閉塞性黄
疸等の診断に適用できる。(Effect of the Invention) The reagent of the present invention can easily and accurately quantify calcium ions, and is particularly applicable to diagnosis of diseases of the parathyroid gland, obstructive jaundice and the like.
第１図は，本発明の試薬におけるカルシウムイオン標準
液と500nmにおける１０分間の吸光度変化量との関係を
示す図で，第２図は，カルシウムイオン電極法における
測定値と本発明の試薬における測定値との関係を示す図
である。FIG. 1 is a diagram showing the relationship between the calcium ion standard solution in the reagent of the present invention and the change in absorbance at 500 nm for 10 minutes, and FIG. 2 is the measured value in the calcium ion electrode method and the measurement in the reagent of the present invention. It is a figure which shows the relationship with a value.
Claims (1)
キシダーゼからなるカルシウムイオン定量用試薬。1. A calcium ion quantification reagent comprising lecithin, phospholipase D and choline oxidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61036123A JPH0634755B2 (en) | 1986-02-19 | 1986-02-19 | Calcium ion quantification reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61036123A JPH0634755B2 (en) | 1986-02-19 | 1986-02-19 | Calcium ion quantification reagent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62195297A JPS62195297A (en) | 1987-08-28 |
| JPH0634755B2 true JPH0634755B2 (en) | 1994-05-11 |
Family
ID=12461003
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61036123A Expired - Lifetime JPH0634755B2 (en) | 1986-02-19 | 1986-02-19 | Calcium ion quantification reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0634755B2 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2533236B2 (en) * | 1990-11-20 | 1996-09-11 | ユニチカ株式会社 | Reagent for calcium ion measurement |
| US5460972A (en) * | 1991-04-08 | 1995-10-24 | Research Foundation Of The State University Of New York | Ionized magnesium2+ concentrations in biological samples |
| JPH06335400A (en) * | 1993-05-31 | 1994-12-06 | Kyowa Medex Co Ltd | Method for measuring ionized calcium |
| US6387646B1 (en) | 1998-12-11 | 2002-05-14 | Toyo Boseki Kabushiki Kaisha | Reagent compositions for measuring electrolyte |
| CN100354630C (en) * | 2002-10-31 | 2007-12-12 | 世诺临床诊断制品株式会社 | Reagent for testing calcium in sample and test method thereof |
| CN100434913C (en) * | 2006-07-25 | 2008-11-19 | 暨南大学 | Lithangiuria intelligent diagnosing instrument based on five-electrode method |
-
1986
- 1986-02-19 JP JP61036123A patent/JPH0634755B2/en not_active Expired - Lifetime
Non-Patent Citations (2)
| Title |
|---|
| BIOCHEMICHAL JOURNAL=1967 * |
| JOURNAL OF BIOCHEMISTRY=1978 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62195297A (en) | 1987-08-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3080770B2 (en) | Method for measuring ions in liquid and composition therefor | |
| US5516700A (en) | Automated urinalysis method | |
| CS250227B2 (en) | Testing system for substances' determination liquids | |
| JPH0634755B2 (en) | Calcium ion quantification reagent | |
| Bissbort et al. | Novel test and its automation for the determination of erythrocyte acetylcholinesterase and its application to organophosphate exposure | |
| JPH08507206A (en) | Compositions useful for anaerobic determination of analytes | |
| Schwartz et al. | Serum creatine kinase isoenzyme BB is a poor index to the size of various brain lesions. | |
| Kayamori et al. | Enzymatic method for assaying calcium in serum and urine with porcine pancreatic alpha-amylase | |
| Perlstein et al. | Spectrophotometric study of bilirubin and hemoglobin interactions in several hydrogen peroxide generating procedures | |
| US4141792A (en) | Composition and method for the quantitative determination of phospholipids | |
| JPH0698031B2 (en) | LD-1 isozyme assay and reagents | |
| Purcell et al. | Evaluation of the BMC glucose oxidase/peroxidase-4-aminophenazone-phenol procedure for glucose as adapted to the Technicon SMAC. | |
| US5902730A (en) | Reagent for calcium ion level determination | |
| JP3217180B2 (en) | Substance measurement method | |
| Levinson | Use of an enzymatic kit method for cholesterol, designed for continuous-flow instrumentation, with discrete bichromatic and centrifugal analyzers. | |
| Kazmierczak et al. | Measuring carboxypeptidase A activity with a centrifugal analyzer: analytical and clinical considerations. | |
| CA2334276C (en) | Liquid reagent set for l-lactate determination | |
| JPH1118798A (en) | Determination of cholesterol and reagent therefor | |
| US6627153B1 (en) | Integrated assay for organ function, drugs and/or one or more metabolites | |
| Kusu et al. | Fluorometric determination of pseudocholinesterase activity in postmortem blood samples | |
| JPH0731498A (en) | Determination kit for 1,5-anhydroglucitol and determination method using the kit | |
| JP4073527B2 (en) | Isozyme activity assay | |
| CA2255851A1 (en) | Process to eliminate haemoglobin errors when analysing medical samples | |
| JPH09168397A (en) | Determination of sulfuric acid included bile acid and its kit | |
| RU2081411C1 (en) | Composition for detecting sodium ions |