JPH06331624A - Reagent composition for zinc sulfate turbidity test - Google Patents
Reagent composition for zinc sulfate turbidity testInfo
- Publication number
- JPH06331624A JPH06331624A JP13998393A JP13998393A JPH06331624A JP H06331624 A JPH06331624 A JP H06331624A JP 13998393 A JP13998393 A JP 13998393A JP 13998393 A JP13998393 A JP 13998393A JP H06331624 A JPH06331624 A JP H06331624A
- Authority
- JP
- Japan
- Prior art keywords
- reagent
- zinc sulfate
- acid
- standard method
- standard
- 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.)
- Granted
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 119
- 239000000203 mixture Substances 0.000 title claims abstract description 34
- 229960001763 zinc sulfate Drugs 0.000 title claims abstract description 20
- 229910000368 zinc sulfate Inorganic materials 0.000 title claims abstract description 20
- 238000012360 testing method Methods 0.000 title claims abstract description 14
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 title abstract 4
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000000872 buffer Substances 0.000 claims abstract description 16
- 239000002535 acidifier Substances 0.000 claims abstract description 11
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 26
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 239000004327 boric acid Substances 0.000 claims description 6
- OWXMKDGYPWMGEB-UHFFFAOYSA-N HEPPS Chemical compound OCCN1CCN(CCCS(O)(=O)=O)CC1 OWXMKDGYPWMGEB-UHFFFAOYSA-N 0.000 claims description 5
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 239000011701 zinc Substances 0.000 claims description 2
- 229910052725 zinc Inorganic materials 0.000 claims description 2
- 125000005619 boric acid group Chemical group 0.000 claims 2
- GIZQLVPDAOBAFN-UHFFFAOYSA-N HEPPSO Chemical compound OCCN1CCN(CC(O)CS(O)(=O)=O)CC1 GIZQLVPDAOBAFN-UHFFFAOYSA-N 0.000 claims 1
- 239000001384 succinic acid Substances 0.000 claims 1
- 238000010561 standard procedure Methods 0.000 abstract description 45
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 46
- 238000005259 measurement Methods 0.000 description 32
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 26
- 210000002966 serum Anatomy 0.000 description 23
- 238000012937 correction Methods 0.000 description 20
- 238000002835 absorbance Methods 0.000 description 15
- 238000010586 diagram Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 229960002319 barbital Drugs 0.000 description 11
- 239000008213 purified water Substances 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 8
- 238000000691 measurement method Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000007982 barbital buffer Substances 0.000 description 6
- 230000003247 decreasing effect Effects 0.000 description 6
- 238000009472 formulation Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 229960000796 barbital sodium Drugs 0.000 description 4
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- RGHFKWPGWBFQLN-UHFFFAOYSA-M sodium;5,5-diethylpyrimidin-3-ide-2,4,6-trione Chemical compound [Na+].CCC1(CC)C([O-])=NC(=O)NC1=O RGHFKWPGWBFQLN-UHFFFAOYSA-M 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- MGSRCZKZVOBKFT-UHFFFAOYSA-N thymol Chemical compound CC(C)C1=CC=C(C)C=C1O MGSRCZKZVOBKFT-UHFFFAOYSA-N 0.000 description 4
- 238000009835 boiling Methods 0.000 description 3
- 239000006172 buffering agent Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- AJTVSSFTXWNIRG-UHFFFAOYSA-N 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid Chemical compound OCC[NH+](CCO)CCS([O-])(=O)=O AJTVSSFTXWNIRG-UHFFFAOYSA-N 0.000 description 2
- 208000027932 Collagen disease Diseases 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000005844 Thymol Substances 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 108010074605 gamma-Globulins Proteins 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 230000003908 liver function Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 229960000790 thymol Drugs 0.000 description 2
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 description 2
- HYRHLJWTFKJITA-UHFFFAOYSA-N 3-hydroxy-2-(hydroxymethyl)propanamide Chemical compound NC(=O)C(CO)CO HYRHLJWTFKJITA-UHFFFAOYSA-N 0.000 description 1
- GAWIXWVDTYZWAW-UHFFFAOYSA-N C[CH]O Chemical group C[CH]O GAWIXWVDTYZWAW-UHFFFAOYSA-N 0.000 description 1
- VECXFAFHWMJFJG-UHFFFAOYSA-N OCCC(CO)(CO)C(O)=N Chemical compound OCCC(CO)(CO)C(O)=N VECXFAFHWMJFJG-UHFFFAOYSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 229910001626 barium chloride Inorganic materials 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000019788 craving Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【０００１】[0001]
【発明の利用分野】本発明は血清膠質反応を利用した硫
酸亜鉛混濁試験に用いる試薬組成物に関する。FIELD OF THE INVENTION The present invention relates to a reagent composition used in a zinc sulfate turbidity test utilizing a serum oncotic reaction.
【０００２】[0002]
【発明の背景】硫酸亜鉛混濁試験（Zinc sulfate tudid
ity test、以下、ＺＴＴと略記する。）、チモール混濁
試験等の血清膠質反応は、各種肝疾患、慢性感染症、膠
原病、骨髄腫等の診断に於て有効であり、今日でも重要
な検査項目である。特にＺＴＴは、血清と硫酸亜鉛溶液
を混合し、生じた混濁を比濁測定するもので、血清中の
γ−グロブリンの量とよく相関し、臨床的意義も高い。
ＺＴＴについては、1962年に日本消化器病学会肝機能研
究班により標準操作法が提示され（以下、標準法と略記
する。）、現在市販されているＺＴＴ試薬はほぼこの標
準法に基づいて調製されている。この標準法は、例えば
臨床検査法提要、第29版第５刷、710〜711頁、昭和60年11
月20日発行、金原出版株式会社発行、等に記載されている
が、用手法の場合、概略下記の通りである。BACKGROUND OF THE INVENTION Zinc sulfate tudid
City test, hereinafter abbreviated as ZTT. ), The serum colloid reaction such as the thymol opacity test is effective in the diagnosis of various liver diseases, chronic infectious diseases, collagen diseases, myeloma and the like, and is still an important test item even today. In particular, ZTT is a method in which serum and zinc sulfate solution are mixed and turbidimetric measurement of the resulting turbidity is performed, which correlates well with the amount of γ-globulin in serum and has high clinical significance.
Regarding ZTT, the standard operation method was presented by the Japanese Gastroenterological Society Liver Function Research Group in 1962 (hereinafter abbreviated as standard method), and the ZTT reagents currently on the market are prepared based on this standard method. Has been done. This standard method is, for example, the Clinical Laboratory Procedure Recommendation, 29th Edition, 5th edition, pages 710 to 711, 1985.
It is described on the 20th of the month, issued by Kinbara Publishing Co., Ltd., etc., but in the case of the manual method, it is roughly as follows.
【０００３】即ち、まず煮沸してCO2を除去した水にバ
ルビタール302mg、バルビタールナトリウム190mgを溶解
し、これに0.480g/dl硫酸亜鉛・７水和物（ZnSO4・7H
2O）水溶液5.0mlを加え、更に水を加えて全量１リット
ルとする（バルビタール2.6mM）。このとき、試薬のpＨ
は7.60±0.05に厳密に調整されていなくてはならない。
実際の測定は、血清0.1mlと調整した試薬6.0mlを試験管
にとり良く混和し、25±３℃で正確に30分間放置後、66
0nmに於ける吸光度を測定する。一方、塩化バリウムと
硫酸を混合して得られた懸濁液（硫酸バリウム混濁標準
液）を標準液として用い、660nmの吸光度を読み、これ
をクンケル20単位として検量線を作成する。この検量線
から、血清試料のＺＴＴ値を求める。That is, first, 302 mg of barbital and 190 mg of barbital sodium were dissolved in water that had been boiled to remove CO 2 , and 0.480 g / dl zinc sulfate heptahydrate (ZnSO 4 7H) was added to the solution.
2 O) 5.0 ml of aqueous solution is added, and further water is added to make a total volume of 1 liter (barbital 2.6 mM). At this time, the pH of the reagent
Must be strictly adjusted to 7.60 ± 0.05.
For the actual measurement, mix 6.0 ml of reagent adjusted with 0.1 ml of serum in a test tube, mix well, leave it at 25 ± 3 ° C for exactly 30 minutes, and then
Measure the absorbance at 0 nm. On the other hand, a suspension obtained by mixing barium chloride and sulfuric acid (barium sulfate turbidity standard solution) is used as a standard solution, the absorbance at 660 nm is read, and a calibration curve is prepared using this as 20 units of Kunkel. From this calibration curve, the ZTT value of the serum sample is obtained.
【０００４】上記の方法では、試薬及び標準液の調製、
反応条件及び測定方法は厳密に規定されている。特に本
測定法に係る反応はpＨの影響を大きく受け、pＨ7〜8の
間ではpＨが低い方が濁度が高くなるので、試液を調整
する際には厳密なpＨ管理を必要とする。しかしなが
ら、そのために試液のイオン強度をあげると正常値付近
の濁度が高くなり、一方異常域では逆に濁度が低下して
診断効率の低下を招く。そこで標準法では、pＨの調整
に2.6mM バルビタール緩衝液を使用しているが、この緩
衝剤濃度ではpＨ維持能力は極めて低く、開栓状態では
空気中の炭酸ガスを吸収して試薬のpＨが低下してしま
うばかりか、バルビタール自体不安定で、特に硫酸亜鉛
共存下、室温以上で保存すると、経日的にpＨの低下を
招き測定値が高値になってしまう。一方、pＨ安定性を
改善した方法として例えば両性電解質を使用する方法が
提案されている（特開昭62-153759号）。しかし、この
方法も高温下、長期間保存した場合に於ける安定化効果
は未だ十分では無く、従来の標準法と相関性が良好でし
かも経日安定性により優れたＺＴＴ用試薬組成物の開発
が長年渇望されてきた。In the above method, preparation of reagents and standard solutions,
Reaction conditions and measurement methods are strictly specified. In particular, the reaction according to the present measuring method is greatly affected by pH, and the lower the pH becomes, the higher the turbidity becomes between pH 7 and 8. Therefore, strict pH control is required when adjusting the test solution. However, if the ionic strength of the test solution is increased for that reason, the turbidity in the vicinity of the normal value becomes high, while in the abnormal region, the turbidity is decreased and the diagnostic efficiency is decreased. Therefore, in the standard method, 2.6 mM barbital buffer is used to adjust the pH, but at this buffer concentration, the pH maintaining ability is extremely low, and when the cap is opened, the carbon dioxide gas in the air is absorbed and the pH of the reagent becomes In addition to the decrease, the barbital itself is unstable, and especially when it is stored at room temperature or higher in the presence of zinc sulfate, the pH is lowered with the passage of time, and the measured value becomes high. On the other hand, as a method for improving pH stability, for example, a method using an ampholyte has been proposed (JP-A-62-153759). However, this method also does not have a sufficient stabilizing effect when stored at high temperature for a long period of time, and has developed a reagent composition for ZTT which has good correlation with the conventional standard method and is excellent in stability over time. Have been craving for many years.
【０００５】[0005]
【発明の目的】本発明は上記した如き状況に鑑みなされ
たもので、標準法（2.6mM バルビタール緩衝液を用いる
方法）と良好な相関性を維持し、再現性、経日安定性に
より優れたＺＴＴ用試薬組成物を提供することを目的と
する。SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and maintains good correlation with the standard method (method using a 2.6 mM barbital buffer solution) and is excellent in reproducibility and stability over time. It is intended to provide a reagent composition for ZTT.
【０００６】[0006]
【発明の構成】本発明は、硝酸亜鉛と、緩衝剤としての
ビス（2-ヒドロキシエチル）イミノトリス（ヒドロキシ
メチル）メタンと、目的のpＨに調整するための酸性化
剤とを含んで成ることを特徴とするＺＴＴ用試薬組成物
の発明である。即ち、本発明者らは標準法と良く相関し
ながらも、pＨによる影響を受けず、経日安定性に優れ
たＺＴＴ用試薬組成物を得るべく鋭意研究の結果、緩衝
剤としてビス（2-ヒドロキシエチル）イミノトリス（ヒ
ドロキシメチル）メタン（以下、ビストリスと略記す
る。）を用いると、pＨの変動が少なく、しかも経日安
定性に優れたＺＴＴ用試薬組成物が得られることを見い
出し、本発明を完成した。The present invention comprises zinc nitrate, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane as a buffering agent, and an acidifying agent for adjusting the pH to a desired value. It is an invention of a characteristic ZTT reagent composition. That is, the inventors of the present invention have conducted extensive studies to obtain a ZTT reagent composition which is not affected by pH and is excellent in stability over time even though it correlates well with the standard method. The present invention found that the use of (hydroxyethyl) iminotris (hydroxymethyl) methane (hereinafter abbreviated as “bistris”) yields a ZTT reagent composition with little fluctuation in pH and excellent in stability over time. Was completed.
【０００７】本発明に於て用いられるビストリスの使用
濃度は、試薬組成物のpＨを安定化でき、測定値に影響
を与えない濃度であればよいわけであるが、通常は５〜
30mM、好ましくは７〜15mMである。また本測定法に係る
反応はpＨ7.4〜7.7で行うのが最も好ましいが、目的のp
Ｈに調整する為の酸性化剤としては、亜鉛とキレートを
生成しない、例えば塩酸，硫酸，硝酸、硼酸等の無機酸
や、酢酸，琥珀酸，乳酸等の有機酸、或はN-2-ヒドロキ
シエチルピペラジン-N'-2-ヒドロキシプロパン-3-スル
ホン酸（ＨＥＰＰＳＯ），N-2-ヒドロキシエチルピペラ
ジン-N'-3-プロパンスルホン酸（ＥＰＰＳ）等のグット
緩衝剤等が挙げられるが、緩衝力のある硼酸を使用する
とpＨ維持の面から特に有利である。これら酸性化剤の
使用量は、使用する酸性化剤の種類により異なり、一定
ではないが、要は試薬溶液のpＨを7.4〜7.7に調整でき
る量を加えればよい。尚、硫酸亜鉛は通常硫酸亜鉛７水
塩として１リットル当たり20〜30mg程度用いられる。The concentration of bis-tris used in the present invention may be such that it can stabilize the pH of the reagent composition and does not affect the measured value, but it is usually 5 to 5.
It is 30 mM, preferably 7 to 15 mM. It is most preferable to carry out the reaction according to this assay at a pH of 7.4 to 7.7.
As an acidifying agent for adjusting to H, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, boric acid, etc. which do not form a chelate with zinc, organic acids such as acetic acid, succinic acid, lactic acid, or N-2- Examples include Guth's buffer such as hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid (HEPPSO) and N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid (EPPS). The use of buffering boric acid is particularly advantageous in terms of pH maintenance. The amount of the acidifying agent used varies depending on the type of the acidifying agent used and is not constant, but the point is to add an amount that can adjust the pH of the reagent solution to 7.4 to 7.7. Incidentally, zinc sulfate is usually used as zinc sulfate heptahydrate in an amount of about 20 to 30 mg per liter.
【０００８】ＺＴＴは用手法に限らず、自動分析装置に
よる測定も広く行われている。一般にＺＴＴを自動分析
装置を用いて測定する場合、測定条件は装置により異な
るが、例えば検体と試薬組成物を混合後、通常37℃で、
反応時間２〜15分、測定波長600〜660nmで吸光度の測定
を行う。また自動分析装置を用いる場合、予めクンケル
単位の判っている血清を標準として用いるのが一般的で
ある。本発明に係る試薬組成物も、用手法に限らず、自
動分析装置に応用することももちろん可能であり、測定
条件等は従来法に従えばよい。標準法等の従来のＺＴＴ
法では、試薬組成物中のイオン強度が測定値に大きな影
響を与えるため、厳密なpＨ調整が必要であるにもかか
わらず、緩衝剤濃度を増やすと標準法との相関性が悪化
し、正常異常の差が減少して診断効率の低下を招いた。
しかしながら、ビストリスを用いた本発明によれば、緩
衝剤濃度を増加させてもイオン強度の増加は僅かで、そ
のため標準法との相関性を維持しながら緩衝力を増強で
きる。この事実はトリエタノールアミンやトリス（ヒド
ロキシメチル）アミノメタン等ではモル濃度の増加で相
関性が著しく悪化するのと比較すると実に意外であっ
た。以下に実施例を挙げて本発明を更に詳細に説明する
が、本発明はこれら実施例によって何等制約を受けるも
のではない。ZTT is not limited to a manual method, and measurement by an automatic analyzer is widely performed. Generally, when ZTT is measured using an automatic analyzer, the measurement conditions vary depending on the device, but for example, after mixing the sample and the reagent composition, usually at 37 ° C.,
The absorbance is measured at a reaction wavelength of 2 to 15 minutes and a measurement wavelength of 600 to 660 nm. When an automatic analyzer is used, it is common to use serum whose Kunkel unit is known in advance as a standard. The reagent composition according to the present invention can be applied not only to the method used but also to an automatic analyzer, and the measurement conditions and the like may follow conventional methods. Conventional ZTT such as standard method
In the method, the ionic strength in the reagent composition has a large effect on the measured value, so although the pH must be adjusted strictly, the correlation with the standard method deteriorates when the buffer concentration is increased, and the The difference between abnormalities decreased, leading to a decrease in diagnostic efficiency.
However, according to the present invention using bistris, the ionic strength is slightly increased even if the buffer concentration is increased, and therefore the buffering power can be enhanced while maintaining the correlation with the standard method. This fact was quite unexpected when compared with the fact that the correlation is remarkably deteriorated with an increase in the molar concentration of triethanolamine, tris (hydroxymethyl) aminomethane and the like. Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
【０００９】[0009]
【実施例】 実施例１ ［試薬の調製］ 試薬１（ビストリス−硼酸緩衝液） ビストリス 2.092g 硼酸 587mg 硫酸亜鉛７水塩 28mg 煮沸してCO2を除去した精製水１リットルに上記試薬類
を溶解し、pHを7.50に調整した。 試薬２（ビストリス−塩酸緩衝液） ビストリス 2.092g １Ｎ 塩酸 0.9ml 硫酸亜鉛７水塩 24mg 煮沸してCO2を除去した精製水１リットルに上記試薬類
を溶解し、pHを7.50に調整した。 試薬３（ビストリス−ＥＰＰＳ緩衝液） ビストリス 2.092g ＥＰＰＳ 782mg 硫酸亜鉛７水塩 28mg 煮沸してCO2を除去した精製水１リットルに上記試薬類
を溶解し、pHを7.60に調整した。 ［測定方法］日立7050形自動分析装置により、以下の方
法で測定した。ヒト血清10μlを反応セルにとり、調製
した試薬１、試薬２又は試薬３を500μl加えて37℃で10
分間加温した後、主波長600nm、側波長700nmに於ける吸
光度を測定した。16クンケル単位のヒト血清を標準とし
て用い、検体の吸光度をクンケル単位に換算した。該測
定は、各試薬毎に同じ検体について各10回測定を行い、
得られたクンケル単位の最大値及び最小値、測定値の
幅、平均値、標準偏差及び変動係数を求めた。結果を表
１に示す。Examples Example 1 [Preparation of Reagents] Reagent 1 (bistris-borate buffer) Bistris 2.092g Boric acid 587mg Zinc sulfate 7-hydrate 28mg Dissolve the above reagents in 1 liter of purified water after boiling to remove CO 2. Then, the pH was adjusted to 7.50. Reagent 2 (bis-Tris-hydrochloric acid buffer solution) Bis-Tris 2.092 g 1N hydrochloric acid 0.9 ml Zinc sulfate heptahydrate 24 mg The above reagents were dissolved in 1 liter of purified water that had been boiled to remove CO 2 and the pH was adjusted to 7.50. Reagent 3 (bis-Tris-EPPS buffer) Bis-Tris 2.092 g EPPS 782 mg Zinc sulfate 7-hydrate 28 mg The above reagents were dissolved in 1 liter of purified water that had been boiled to remove CO 2 and the pH was adjusted to 7.60. [Measurement method] The measurement was carried out by the following method using a Hitachi 7050 type automatic analyzer. Transfer 10 μl of human serum to a reaction cell, add 500 μl of the prepared reagent 1, reagent 2 or reagent 3 and incubate at 37 ℃.
After heating for a minute, the absorbance at a main wavelength of 600 nm and a side wavelength of 700 nm was measured. Using 16 Kunkel units of human serum as a standard, the absorbance of the sample was converted into Kunkel units. The measurement is performed 10 times for each reagent for the same sample,
The maximum value and the minimum value of the obtained Kunkel unit, the width of the measured value, the average value, the standard deviation and the coefficient of variation were obtained. The results are shown in Table 1.
【００１０】[0010]
【表１】 [Table 1]
【００１１】比較例１（標準法） ［試薬の調製］ 試薬Ａ（標準法処方） バルビタール 302mg バルビタールナトリウム 190mg 硫酸亜鉛７水塩 24mg 煮沸してCO2を除去した精製水１リットルに上記試薬類
を溶解し、pHを7.60に調整した。 ［測定方法］試薬Ａを用い、実施例１と同じヒト血清に
ついて、実施例１と同様の方法でクンケル値測定を10回
行い、得られたクンケル単位の最大値及び最小値、測定
値の幅、平均値、標準偏差及び変動係数を求めた。結果
を表１に併せて示す。表１から明らかなように、ビスト
リスを緩衝剤として用いた本発明による方法は、標準法
と同等の同時再現性を示した。Comparative Example 1 (Standard Method) [Preparation of Reagents] Reagent A (Standard Method Prescription) Barbital 302 mg Barbital Sodium 190 mg Zinc Sulfate 7-hydrate 24 mg The above reagents were added to 1 liter of purified water after boiling to remove CO 2. It was dissolved and the pH was adjusted to 7.60. [Measurement method] Using reagent A, the same human serum as in Example 1 was subjected to the Kunkel value measurement 10 times in the same manner as in Example 1 to obtain the maximum and minimum values in Kunkel units, and the range of the measured values. , Average value, standard deviation and coefficient of variation were determined. The results are also shown in Table 1. As is apparent from Table 1, the method of the present invention using Bis-Tris as a buffer showed the same reproducibility as the standard method.
【００１２】実施例２ 表２に記載のビストリス緩衝剤（試薬１〜８）を用い、
ヒト血清20〜30検体について実施例１と同様にしてクン
ケル値の測定を行った。得られた測定結果［Ｙ］と、標
準法（試薬Ａを用いた方法）［Ｘ］との相関関係を表わ
す回帰式及び相関係数を表２に示す。Example 2 Using the Bis-Tris buffer (reagents 1 to 8) shown in Table 2,
The Kunkel value was measured in the same manner as in Example 1 for 20 to 30 samples of human serum. Table 2 shows the regression equation and the correlation coefficient showing the correlation between the obtained measurement result [Y] and the standard method (method using reagent A) [X].
【００１３】[0013]
【表２】 [Table 2]
【００１４】更に、標準法（試薬Ａを用いた方法）と試
薬１を用いた結果との相関図を図１（300検体）に、標
準法と試薬２を用いた結果との相関図を図２に（300検
体）、標準法と試薬３を用いた結果との相関図を図３に
(300検体）夫々示す。Further, a correlation diagram between the standard method (method using reagent A) and the result using reagent 1 is shown in FIG. 1 (300 samples), and a correlation diagram between the standard method and the result using reagent 2 is shown. 2 (300 specimens), Figure 3 shows the correlation diagram between the standard method and the results using reagent 3.
(300 specimens) shown.
【００１５】比較例２ ［試液の調製］ 試薬Ｂ（高濃度バルビタール緩衝液：15mM） バルビタール 1.17g バルビタールナトリウム 1.11g 硫酸亜鉛７水塩 24mg 煮沸してCO2を除去した精製水１リットルに上記試薬類
を溶解し、pHを7.60に調整した。 試薬Ｃ N,N-ビス(2-ヒドロキシエチル)-2-アミノエタンスルホ
ン酸（ＢＥＳ）213mg トリス（ヒドロキシメチル）アミノメタン 133mg 硫酸亜鉛７水塩 24mg 煮沸してCO2を除去した精製水１リットルに上記試薬類
を溶解し、pＨを7.60に調整した。 ［測定方法］試薬Ｂ、又は試薬Ｃを用い、ヒト血清10検
体について、実施例１と同様の方法でクンケル値の測定
を行った。得られた測定結果［Ｙ］と、標準法（試薬Ａ
を用いた方法）［Ｘ］との相関関係を表わす回帰式及び
相関係数を表２に併せて示す。また、同じく比較例とし
て表２に記載の各緩衝液（試薬Ｄ〜Ｏ）を用いて同様に
測定を行った結果も表２に併せて示す。更に、標準法
（試薬Ａを用いた方法）と試薬Ｂを用いた結果との相関
図を図４に（10検体）、標準法と試薬Ｃを用いた結果と
の相関図を図５に（10検体）夫々示す。Comparative Example 2 [Preparation of Reagent] Reagent B (high-concentration barbital buffer: 15 mM) Barbital 1.17 g Barbital sodium 1.11 g Zinc sulfate heptahydrate 24 mg Boiled to remove CO 2 and purified in 1 liter of the above reagent. The ingredients were dissolved and the pH was adjusted to 7.60. Reagent C N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES) 213 mg Tris (hydroxymethyl) aminomethane 133 mg Zinc sulfate heptahydrate 24 mg Purified water from which CO 2 has been removed by boiling 1 liter The above reagents were dissolved in and the pH was adjusted to 7.60. [Measurement method] Using reagent B or reagent C, the Kunkel value was measured for 10 human serum samples in the same manner as in Example 1. The obtained measurement result [Y] and the standard method (reagent A
Table 2 shows the regression equation and the correlation coefficient showing the correlation with [X]. In addition, similarly, as a comparative example, the results obtained by the same measurement using each of the buffer solutions (reagents D to O) described in Table 2 are also shown in Table 2. Furthermore, the correlation diagram between the standard method (method using reagent A) and the result using reagent B is shown in FIG. 4 (10 samples), and the correlation diagram between the standard method and the result using reagent C is shown in FIG. 10 samples are shown respectively.
【００１６】表２から明らかな如く、本発明に係る試薬
組成物を用いた場合は、何れも標準法処方試薬を用いた
場合と、よい相関を示した。一方、他の緩衝剤を用いた
場合、回帰式から明らかなように、測定値が低くでた
り、高くでたりする傾向が見られ、ＺＴＴ用に好ましく
ないことが判る。また、図１、図２、図３からも、本発
明に係る試薬を用いた場合は、測定値が標準法処方試薬
を用いた場合と高い相関関係にあることが判るが、一
方、図４から明らかな如く、標準法処方試薬に更にバル
ビタールの濃度を上げると、濁度が低くなり、標準法処
方試薬を用いた場合よりも測定値が低くでる傾向が見ら
れる。また、図５から明らかな如く、緩衝剤にＢＥＳを
用いると、濁度が高くなり、測定値が高くでる傾向があ
る。これらのことから、バルビタール，ＢＥＳ共に、Ｚ
ＴＴの測定には好ましくないことが判る。As is clear from Table 2, when the reagent composition according to the present invention was used, each showed a good correlation with the case where the standard method prescription reagent was used. On the other hand, when other buffering agents are used, as is clear from the regression equation, the measured values tend to be low or high, which is not preferable for ZTT. Further, it can be seen from FIGS. 1, 2 and 3 that the measured values have a high correlation with the case where the standard method prescription reagent is used when the reagent according to the present invention is used. As is clear from the above, when the concentration of barbital is further increased in the standard formulation reagent, the turbidity becomes low, and the measured value tends to be lower than when the standard formulation reagent is used. Further, as is clear from FIG. 5, when BES is used as the buffer, the turbidity tends to be high and the measured value tends to be high. From these, both barbital and BES
It turns out that it is not preferable for the measurement of TT.
【００１７】実施例３（経日安定性試験） 実施例１で用いたと同じ試薬１、試薬２、試薬３の夫々
を密封状態で10℃、25℃、40℃で１ヵ月、２ヵ月及び５
ヵ月保存した。一定期間保存後、夫々の試薬のpＨを測
定した。また、一定期間保存後の夫々の試薬を用いて、
実施例１と同様の方法でヒト血清検体について吸光度の
測定を行った。得られたpＨ値、及び吸光度の値を表３
に示す。Example 3 (Stability test over time) The same reagent 1, reagent 2 and reagent 3 used in Example 1 were sealed at 10 ° C., 25 ° C. and 40 ° C. for 1 month, 2 months and 5 respectively.
Stored for a month. After storage for a certain period of time, the pH of each reagent was measured. In addition, using each reagent after storage for a certain period,
The absorbance of human serum samples was measured in the same manner as in Example 1. Table 3 shows the obtained pH value and absorbance value.
Shown in.
【００１８】[0018]
【表３】 [Table 3]
【００１９】比較例３ 比較例１で用いたと同じ試薬Ａ（標準法処方）を密封状
態で、10℃、25℃、40℃で１ヵ月、２ヵ月及び５ヵ月保
存した。一定期間保存後、夫々の試薬のpＨを測定し
た。また、一定期間保存後の夫々の試薬を用いて、実施
例１と同様の方法でヒト血清検体について吸光度の測定
を行った。得られたpＨ値、及び吸光度の値を表３に併
せて示す。表３から明らかなように、標準法処方試薬を
長期保存した場合、経日的にpＨが低下し、吸光度が高
値に出る傾向が顕著であったが、本発明に係る試薬組成
物を用いて測定を行った場合は、pＨ値も吸光度値も変
化がなく、良好な経日安定性を示した。Comparative Example 3 The same reagent A (standard method formulation) used in Comparative Example 1 was sealed and stored at 10 ° C., 25 ° C. and 40 ° C. for 1 month, 2 months and 5 months. After storage for a certain period of time, the pH of each reagent was measured. Further, the absorbance of a human serum sample was measured in the same manner as in Example 1 using the respective reagents stored for a certain period of time. The obtained pH value and absorbance value are also shown in Table 3. As is clear from Table 3, when the standard method prescription reagent was stored for a long period of time, the pH decreased with time and the absorbance tended to increase to a high value. However, using the reagent composition of the present invention, When the measurement was carried out, there was no change in the pH value and the absorbance value, and good day-to-day stability was shown.
【００２０】[0020]
【発明の効果】以上述べた通り、本発明の硫酸亜鉛混濁
試験（ＺＴＴ）用試薬組成物は、経日安定性に著しく優
れ、長期間安定保存が可能であり、40℃の高温下でも５
ヵ月間以上安定に保存し得る点に顕著な効果を奏するも
のであり、斯業に貢献するところ極めて大なる発明であ
る。Industrial Applicability As described above, the reagent composition for zinc sulfate turbidity test (ZTT) of the present invention has extremely excellent stability over time, can be stably stored for a long time, and can be stored at a high temperature of 40 ° C.
It has a remarkable effect in that it can be stably stored for more than one month, and it is an extremely great invention that contributes to the related art.
【００２１】[0021]
【図１】図１は実施例２に於ける相関図(1)を示し、横
軸は標準法処方試薬を用いた測定結果を、縦軸は本発明
の試薬１（ビストリス−硼酸緩衝液）を用いた測定結果
を夫々表わす。FIG. 1 shows a correlation diagram (1) in Example 2, in which the horizontal axis shows the measurement results using a standard method prescription reagent, and the vertical axis shows the reagent 1 of the present invention (bistris-borate buffer solution). The results of measurements using are respectively shown.
【図２】図２は実施例２に於ける相関図(2)を示し、横
軸は標準法処方試薬を用いた測定結果を、縦軸は本発明
の試薬２（ビストリス−塩酸緩衝液）を用いた測定結果
を夫々表わす。FIG. 2 shows a correlation diagram (2) in Example 2, in which the horizontal axis represents the measurement results using the standard method prescription reagent and the vertical axis represents the reagent 2 of the present invention (bistris-hydrochloric acid buffer solution). The results of measurements using are respectively shown.
【図３】図３は実施例２に於ける相関図(3)を示し、横
軸は標準法処方試薬を用いた測定結果を、縦軸は本発明
の試薬３（ビストリス−ＥＰＰＳ緩衝液）を用いた測定
結果を夫々表わす。FIG. 3 shows a correlation diagram (3) in Example 2, in which the horizontal axis represents the measurement results using the standard method prescription reagent and the vertical axis represents the reagent 3 of the present invention (bistris-EPPS buffer solution). The results of measurements using are respectively shown.
【図４】図４は比較例２に於ける相関図(1)を示し、横
軸は標準法処方試薬を用いた測定結果を、縦軸は試薬Ａ
（高濃度バルビタール緩衝液）を用いた測定結果を夫々
表わす。FIG. 4 shows a correlation diagram (1) in Comparative Example 2, in which the horizontal axis shows the measurement results using the standard method prescription reagent and the vertical axis shows the reagent A.
The measurement results using (high-concentration barbital buffer) are shown respectively.
【図５】図５は比較例２に於ける相関図(2)を示し、横
軸は標準法処方試薬を用いた測定結果を、縦軸は試薬Ｃ
（ＢＥＳ−トリス緩衝液）を用いた測定結果を夫々表わ
す。FIG. 5 shows a correlation diagram (2) in Comparative Example 2, in which the horizontal axis represents the measurement result using the standard method prescription reagent and the vertical axis represents the reagent C.
The measurement results using (BES-Tris buffer) are shown respectively.
─────────────────────────────────────────────────────
─────────────────────────────────────────────────── ───
【手続補正書】[Procedure amendment]
【提出日】平成６年６月２７日[Submission date] June 27, 1994
【手続補正２】[Procedure Amendment 2]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００２[Name of item to be corrected] 0002
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【０００２】[0002]
【発明の背景】硫酸亜鉛混濁試験（Ｚｉｎｃ ｓｕｌｆ
ａｔｅ ｔｕｒｂｉｄｉｔｙ ｔｅｓｔ、以下、ＺＴＴ
と略記する。）、チモール混濁試験等の血清膠質反応
は、各種肝疾患、慢性感染症、膠原病、骨髄腫等の診断
に於て有効であり、今日でも重要な検査項目である。特
にＺＴＴは、血清と硫酸亜鉛溶液を混合し、生じた混濁
を比濁測定するもので、血清中のγ−グロブリンの量と
よく相関し、臨床的意義も高い。ＺＴＴについては、１
９６２年に日本消化器病学会肝機能研究班により標準操
作法が提示され（以下、標準法と略記する。）、現在市
販されているＺＴＴ試薬はほぼこの標準法に基づいて調
製されている。この標準法は、例えば臨床検査法提要、
第２９版第５刷、７１０〜７１１頁、昭和６０年１１月
２０日発行、金原出版株式会社発行、等に記載されてい
るが、用手法の場合、概略下記の通りである。BACKGROUND OF THE INVENTION Zinc sulfate turbidity test (Zinc sulf
ate turbine test, hereafter ZTT
Is abbreviated. ), The serum colloid reaction such as the thymol opacity test is effective in the diagnosis of various liver diseases, chronic infectious diseases, collagen diseases, myeloma and the like, and is still an important test item even today. In particular, ZTT is a method in which serum and zinc sulfate solution are mixed and turbidimetric measurement of the resulting turbidity is performed, which correlates well with the amount of γ-globulin in serum and has high clinical significance. 1 for ZTT
The standard operation method was presented by the Japan Gastroenterological Society Society Liver Function Research Group in 962 (hereinafter abbreviated as standard method), and the ZTT reagents currently on the market are prepared based on this standard method. This standard method, for example
29th edition, 5th printing, pages 710 to 711, published on November 20, 1960, published by Kanehara Publishing Co., Ltd., etc., but in the case of a manual method, it is roughly as follows.
【手続補正３】[Procedure 3]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００４[Correction target item name] 0004
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【０００４】上記の方法では、試薬及び標準液の調製、
反応条件及び測定方法は厳密に規定されている。特に本
測定法に係る反応はｐＨの影響を大きく受け、ｐＨ７〜
８の間ではｐＨが低い方が濁度が高くなるので、試液を
調整する際には厳密なｐＨ管理を必要とする。しかしな
がら、そのために試液のイオン強度をあげると正常値付
近の濁度が高くなり、一方異常域では逆に濁度が低下し
て診断効率の低下を招く。そこで標準法では、ｐＨの保
持に２．６ｍＭバルビタール緩衝液を使用しているが、
この緩衝剤濃度ではｐＨ維持能力は極めて低く、開栓状
態では空気中の炭酸ガスを吸収して試薬のｐＨが低下し
てしまうばかりか、バルビタール自体不安定で、特に硫
酸亜鉛共存下、室温以上で保存すると、経日的にｐＨの
低下を招き測定値が高値になってしまう。一方、ｐＨ安
定性を改善した方法として例えば両性電解質を使用する
方法が提案されている（特開昭６２−１５３７５９
号）。しかし、この方法も高温下、長期間保存した場合
に於ける安定化効果は未だ十分では無く、従来の標準法
と相関性が良好でしかも経日安定性により優れたＺＴＴ
用試薬組成物の開発が長年渇望されてきた。In the above method, preparation of reagents and standard solutions,
Reaction conditions and measurement methods are strictly specified. In particular, the reaction according to this measuring method is greatly influenced by pH,
Between 8 and 8, the lower the pH, the higher the turbidity, so strict pH control is required when adjusting the test solution. However, if the ionic strength of the test solution is increased for that reason, the turbidity in the vicinity of the normal value becomes high, while in the abnormal region, the turbidity is decreased and the diagnostic efficiency is decreased. Therefore, in the standard method, 2.6 mM barbital buffer is used to maintain the pH.
At this buffer concentration, the pH-maintaining ability is extremely low. When the cap is opened, carbon dioxide gas in the air is absorbed and the pH of the reagent decreases, and barbital itself is unstable. If it is stored in, the pH value will decrease with time and the measured value will become high. On the other hand, as a method for improving pH stability, for example, a method using an ampholyte has been proposed (Japanese Patent Laid-Open No. 62-153759).
issue). However, this method still does not have sufficient stabilizing effect when stored at high temperature for a long period of time, and it has good correlation with the conventional standard method and has excellent stability over time.
There has been a long-felt need for the development of a reagent composition for use.
【手続補正４】[Procedure amendment 4]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００６[Correction target item name] 0006
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【０００６】[0006]
【発明の構成】本発明は、硫酸亜鉛と、緩衝剤としての
ビス（２−ヒドロキシエチル）イミノトリス（ヒドロキ
シメチル）メタンと、目的のｐＨに調整するための酸性
化剤とを含んで成ることを特徴とするＺＴＴ用試薬組成
物の発明である。即ち、本発明者らは標準法と良く相関
しながらも、ｐＨによる影響を受けず、経日安定性に優
れたＺＴＴ用試薬組成物を得るべく鋭意研究の結果、緩
衝剤としてビス（２−ヒドロキシエチル）イミノトリス
（ヒドロキシメチル）メタン（以下、ビストリスと略記
する。）を用いると、ｐＨの変動が少なく、しかも経日
安定性に優れたＺＴＴ用試薬組成物が得られることを見
い出し、本発明を完成した。The present invention comprises zinc sulfate, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane as a buffer, and an acidifying agent for adjusting the pH to a desired level. It is an invention of a characteristic ZTT reagent composition. That is, the present inventors have earnestly studied in order to obtain a reagent composition for ZTT which is not affected by pH and is excellent in stability over time even though it correlates well with the standard method. The present invention found that a hydroxyethyl) iminotris (hydroxymethyl) methane (hereinafter abbreviated as bistris) is used to obtain a ZTT reagent composition having little pH fluctuation and excellent stability over time. Was completed.
【手続補正５】[Procedure Amendment 5]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００９[Correction target item name] 0009
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【０００９】[0009]
【実施例】 実施例１ ［試薬の調製］ 試薬１（ビストリス−硼酸緩衝液） ビストリス ２．０９２ｇ 硼酸 ５８７ｍｇ 硫酸亜鉛７水塩 ２８ｍｇ 精製水１リットルに上記試薬類を溶解し、ｐＨを７．５
０に調整した。 試薬２（ビストリス−塩酸緩衝液） ビストリス ２．０９２ｇ １Ｎ塩酸 ０．９ｍｌ 硫酸亜鉛７水塩 ２４ｍｇ 精製水１リットルに上記試薬類を溶解し、ｐＨを７．５
０に調整した。 試薬３（ビストリス−ＥＰＰＳ緩衝液） ビストリス ２．０９２ｇ ＥＰＰＳ ７８２ｍｇ 硫酸亜鉛７水塩 ２８ｍｇ 精製水１リットルに上記試薬類を溶解し、ｐＨを７．６
０に調整した。 ［測定方法］日立７０５０形自動分析装置により、以下
の方法で測定した。ヒト血清１０μｌを反応セルにと
り、調製した試薬１、試薬２又は試薬３を５００μｌ加
えて３７℃で１０分間加温した後、主波長６００ｎｍ、
側波長７００ｎｍに於ける吸光度を測定した。１６クン
ケル単位のヒト血清を標準として用い、検体の吸光度を
クンケル単位に換算した。該測定は、各試薬毎に同じ検
体について各１０回測定を行い、得られたクンケル単位
の最大値及び最小値、測定値の幅、平均値、標準偏差及
び変動係数を求めた。結果を表１に示す。Example 1 [Preparation of reagent] Reagent 1 (bistris-borate buffer solution) Bistris 2.092 g Boric acid 587 mg Zinc sulfate heptahydrate 28 mg Purified water was dissolved in 1 liter of the above reagents to a pH of 7.5.
Adjusted to 0. Reagent 2 (bis-Tris-hydrochloric acid buffer solution) Bis-Tris 2.092 g 1N hydrochloric acid 0.9 ml Zinc sulfate heptahydrate 24 mg Dissolve the above reagents in 1 liter of purified water and adjust the pH to 7.5.
Adjusted to 0. Reagent 3 (Bistris-EPPS buffer) Bistris 2.092 g EPPS 782 mg Zinc sulfate heptahydrate 28 mg Dissolve the above reagents in 1 liter of purified water, and adjust the pH to 7.6.
Adjusted to 0. [Measurement method] The measurement was carried out by the following method using a Hitachi 7050 type automatic analyzer. After taking 10 μl of human serum in a reaction cell and adding 500 μl of the prepared reagent 1, reagent 2 or reagent 3 and heating at 37 ° C. for 10 minutes, a main wavelength of 600 nm,
The absorbance was measured at a side wavelength of 700 nm. The absorbance of the sample was converted into Kunkel units using 16 Kunkel units of human serum as a standard. In the measurement, the same sample was measured 10 times for each reagent, and the maximum value and the minimum value of the obtained Kunkel unit, the width of the measured value, the average value, the standard deviation, and the coefficient of variation were obtained. The results are shown in Table 1.
【手続補正６】[Procedure correction 6]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】００１２[Correction target item name] 0012
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【００１２】実施例２ 表２に記載のビストリス緩衝剤（試薬１〜８）を用い、
ヒト血清２０〜３０検体について実施例１と同様にして
クンケル値の測定を行った。得られた測定結果［Ｙ］
と、標準法（試薬Ａを用いた方法）［Ｘ］との相関関係
を表わす回帰式及び相関係数を表２に示す。標準法
［Ｘ］は、１６クンケル単位の血清を標準にたててお
り、検討法［Ｙ］は測定吸光度を標準法［Ｘ］で得られ
た標準血清の吸光度を用いてクンケル単位に換算した。Example 2 Using the Bis-Tris buffer (reagents 1 to 8) shown in Table 2,
The Kunkel value was measured in the same manner as in Example 1 for 20 to 30 samples of human serum. Obtained measurement result [Y]
Table 2 shows the regression equation and the correlation coefficient indicating the correlation between the standard method and the standard method (method using reagent A) [X]. The standard method [X] uses 16 Kunkel units of serum as a standard, and the examination method [Y] converts the measured absorbance into Kunkel units using the absorbance of the standard serum obtained by the standard method [X]. .
【手続補正７】[Procedure Amendment 7]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】００１５[Name of item to be corrected] 0015
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【００１５】比較例２ ［試液の調製］ 試薬Ｂ（高濃度バルビタール緩衝液：１５ｍＭ） バルビタール １．１７ｇ バルビタールナトリウム １．１１ｇ 硫酸亜鉛７水塩 ２４ｍｇ 煮沸して（ＣＯ２を除去した精製水１リットルに上記試
薬類を溶解し、ｐＨを７．６０に調整した。 試薬Ｃ Ｎ，Ｎ−ビス（２−ヒドロキシエチル）−２−アミノエタンスルホン酸（Ｂ ＥＳ） ２１３ｍｇ トリス（ヒドロキシメチル）アミノメタン １３３ｍｇ 硫酸亜鉛７水塩 ２４ｍｇ 精製水１リットルに上記試薬類を溶解し、ｐＨを７．６
０に調整した。 ［測定方法］試薬Ｂ、又は試薬Ｃを用い、ヒト血清１０
検体について、実施例１と同様の方法でクンケル値の測
定を行った。得られた測定結果［Ｙ］と、標準法（試薬
Ａを用いた方法）［Ｘ］との相関関係を表わす回帰式及
び相関係数を表２に併せて示す。また、同じく比較例と
して表２に記載の各緩衝液（試薬Ｄ〜Ｏ）を用いて同様
に測定を行った結果も表２に併せて示す。更に、標準法
（試薬Ａを用いた方法）と試薬Ｂを用いた結果との相関
図を図４に（１０検体）、標準法と試薬Ｃを用いた結果
との相関図を図５に（１０検体）夫々示す。Comparative Example 2 [Preparation of Reagent] Reagent B (high-concentration barbital buffer: 15 mM) Barbital 1.17 g Barbital sodium 1.11 g Zinc sulfate 7-hydrate 24 mg Boiled (1 liter of purified water from which CO 2 was removed) The above reagents were dissolved in and the pH was adjusted to 7.60. Reagent C N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (B ES) 213 mg Tris (hydroxymethyl) aminomethane 133 mg Zinc sulfate heptahydrate 24 mg Dissolve the above reagents in 1 liter of purified water and adjust the pH to 7.6.
Adjusted to 0. [Measurement Method] Using the reagent B or the reagent C, the human serum 10
The Kunkel value of the sample was measured in the same manner as in Example 1. Table 2 also shows the regression equation and the correlation coefficient showing the correlation between the obtained measurement result [Y] and the standard method (method using reagent A) [X]. In addition, similarly, as a comparative example, the results obtained by the same measurement using each of the buffer solutions (reagents D to O) described in Table 2 are also shown in Table 2. Furthermore, a correlation diagram between the standard method (method using reagent A) and the result using reagent B is shown in FIG. 4 (10 samples), and a correlation diagram between the standard method and the result using reagent C is shown in FIG. 10 samples are shown respectively.
【手続補正８】[Procedure Amendment 8]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】００１６[Correction target item name] 0016
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【００１６】表２から明らかな如く、本発明に係る試薬
組成物を用いた場合は、何れも標準法処方試薬を用いた
場合と、よい相関を示した。一方、他の緩衝剤を用いた
場合、回帰式から明らかなように、相関性が悪く、ＺＴ
Ｔ測定用に好ましくないことが判る。また、図１、図
２、図３からも、本発明に係る試薬を用いた場合は、測
定値が標準法処方試薬を用いた場合と高い相関関係にあ
ることが判るが、一方、図４から明らかな如く、標準法
処方試薬に更にバルビタールの濃度を上げると、濁度が
低くなり、標準法処方試薬を用いた場合よりも測定値が
低値となる傾向が見られる。また、図５から明らかな如
く、緩衝剤にＢＥＳを用いると、濁度が高くなり、測定
値が高値となる傾向がある。これらのことから、バルビ
タール，ＢＥＳ共に、ＺＴＴの測定には好ましくないこ
とが判る。As is clear from Table 2, when the reagent composition according to the present invention was used, each showed a good correlation with the case where the standard method prescription reagent was used. On the other hand, when other buffering agents were used, the correlation was poor and ZT
It turns out that it is not preferable for T measurement. Further, it can be seen from FIGS. 1, 2 and 3 that the measured values have a high correlation with the case where the standard method prescription reagent is used when the reagent according to the present invention is used. As is clear from the above, when the concentration of barbital is further increased in the standard formulation reagent, the turbidity becomes lower, and the measured value tends to be lower than when the standard formulation reagent is used. Further, as is clear from FIG. 5, when BES is used as the buffer, the turbidity tends to be high and the measured value tends to be high. From these, it is understood that both barbital and BES are not preferable for the measurement of ZTT.
Claims (6)
ロキシエチル）イミノトリス（ヒドロキシメチル）メタ
ンと、目的のpＨに調整するための酸性化剤とを含んで
成ることを特徴とする硫酸亜鉛混濁試験用試薬組成物。1. Sulfuric acid comprising zinc sulfate, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane as a buffer, and an acidifying agent for adjusting to a desired pH. Reagent composition for zinc turbidity test.
１に記載の組成物。2. The composition according to claim 1, wherein the zinc sulfate is zinc sulfate heptahydrate.
酸、塩酸、硫酸又は硝酸である、請求項１に記載の組成
物。3. The composition according to claim 1, wherein the acidifying agent for adjusting the pH to a desired value is boric acid, hydrochloric acid, sulfuric acid or nitric acid.
酸である、請求項１に記載の組成物。4. The composition according to claim 1, wherein the acidifying agent for adjusting the pH to a desired value is boric acid.
酸、琥珀酸又は乳酸である、請求項１に記載の組成物。5. The composition according to claim 1, wherein the acidifying agent for adjusting to a desired pH is acetic acid, succinic acid or lactic acid.
-ヒドロキシエチルピペラジン-N'-2-ヒドロキシプロパ
ン-3-スルホン酸（ＨＥＰＰＳＯ）又はN-2-ヒドロキシ
エチルピペラジン-N'-3-プロパンスルホン酸（ＥＰＰ
Ｓ）である、請求項１に記載の組成物。6. The acidifying agent for adjusting the pH to a desired value is N-2.
-Hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid (HEPPSO) or N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid (EPP
The composition of claim 1, which is S).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13998393A JP3237310B2 (en) | 1993-05-19 | 1993-05-19 | Reagent composition for zinc sulfate turbidity test |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13998393A JP3237310B2 (en) | 1993-05-19 | 1993-05-19 | Reagent composition for zinc sulfate turbidity test |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06331624A true JPH06331624A (en) | 1994-12-02 |
| JP3237310B2 JP3237310B2 (en) | 2001-12-10 |
Family
ID=15258214
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP13998393A Expired - Lifetime JP3237310B2 (en) | 1993-05-19 | 1993-05-19 | Reagent composition for zinc sulfate turbidity test |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3237310B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU661540B2 (en) * | 1992-01-27 | 1995-07-27 | Mitsubishi Denki Kabushiki Kaisha | Air conditioner |
-
1993
- 1993-05-19 JP JP13998393A patent/JP3237310B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU661540B2 (en) * | 1992-01-27 | 1995-07-27 | Mitsubishi Denki Kabushiki Kaisha | Air conditioner |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3237310B2 (en) | 2001-12-10 |
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