JPS63250566A - Method and element for analyzing total blood - Google Patents
Method and element for analyzing total bloodInfo
- Publication number
- JPS63250566A JPS63250566A JP8609587A JP8609587A JPS63250566A JP S63250566 A JPS63250566 A JP S63250566A JP 8609587 A JP8609587 A JP 8609587A JP 8609587 A JP8609587 A JP 8609587A JP S63250566 A JPS63250566 A JP S63250566A
- Authority
- JP
- Japan
- Prior art keywords
- layer
- interaction
- dye
- composition
- whole blood
- 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.)
- Pending
Links
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- 239000008280 blood Substances 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 33
- 239000007788 liquid Substances 0.000 claims abstract description 27
- 230000003993 interaction Effects 0.000 claims abstract description 13
- 239000000203 mixture Substances 0.000 claims description 32
- 239000012491 analyte Substances 0.000 claims description 15
- 230000007480 spreading Effects 0.000 claims description 13
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- 238000001514 detection method Methods 0.000 claims description 11
- 230000002452 interceptive effect Effects 0.000 claims description 9
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- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract 4
- 238000000354 decomposition reaction Methods 0.000 abstract 1
- 238000002798 spectrophotometry method Methods 0.000 abstract 1
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- 239000010410 layer Substances 0.000 description 93
- 210000000601 blood cell Anatomy 0.000 description 15
- -1 0-aminophenols Chemical class 0.000 description 10
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
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Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は全血中の特定成分の分析方法およびその目的に
有用な多層分析要素に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for analyzing specific components in whole blood and a multilayer analytical element useful for that purpose.
［従来の技術］
予防または治療のための健康管理を行なうために、医師
は患者の血液中の種々の分析物質を定量しなければなら
ないことが多い０例えば、血液中のグルコース又はコレ
ステロールのレベルは種々の疾患、例えば糖尿病、低血
糖症、肝ｍ障害、甲状腺障害及びアテローム性動脈硬化
症の有効な治療にしばしば重要である。[Prior Art] In order to perform preventive or therapeutic health care, physicians often have to quantify various analytes in a patient's blood. For example, the level of glucose or cholesterol in the blood is It is often important for the effective treatment of various diseases, such as diabetes, hypoglycemia, hepatic disorders, thyroid disorders and atherosclerosis.
従来、このような成分は、全赤血球を除去後に血清また
は血漿中において測定されていた。しかし赤血球を血液
の他の成分から分離するのに必要な操作に付随する労力
及び装置のコストを避けるために、未希釈の全血中の分
析物質を測定できることが望ましい、また、未希釈の全
血を分析に用いれば、より簡易で迅速なサンプルの入手
及び処理が可能になる。これは、分析操作ができる限り
簡易でなければならない家庭での健康管理に特に有用で
ある。Traditionally, such components have been measured in serum or plasma after removal of whole red blood cells. However, to avoid the labor and equipment costs associated with the manipulations required to separate red blood cells from other components of blood, it is desirable to be able to measure analytes in undiluted whole blood; Using blood for analysis allows easier and faster sample acquisition and processing. This is particularly useful in home health care where analytical operations must be as simple as possible.
乾式化学分析、すなわち実質的に乾燥状態の分析要素、
例えば、試験片や多層分析要素中に、分析試薬系を導入
した臨床分析法が知られている。Dry chemical analysis, i.e. the analytical elements in a substantially dry state;
For example, clinical analysis methods are known in which an analytical reagent system is introduced into a test piece or multilayer analytical element.
乾式化学分析は湿式法による化学分析（即ち、溶液中に
試薬を用いる方法）より、例えば使用上の簡易性、経済
上の節約及び分析の迅速さなどの点で優れている。しか
し、乾式化学分析を用いて全血の分析を行うには、血球
（赤血球及び白血球）及び全血の他の高分子量成分は、
正確な分析結果を得るためにサンプルから予め除去する
か、分析要素中で血球を何らかの手段で分離しなければ
ならない。Dry chemical analysis has advantages over wet chemical analysis (ie, using reagents in solution) in terms of, for example, ease of use, economic savings, and speed of analysis. However, in order to analyze whole blood using dry chemical analysis, blood cells (erythrocytes and white blood cells) and other high molecular weight components of whole blood are
In order to obtain accurate analytical results, the blood cells must be removed from the sample or separated by some means in the analytical element.
全血中の血球成分から血清または血漿を分離する必要性
はぜひとも回避しなければならない、ろ過層を用いて血
清または血漿から血球成分を除去する操作は非常に時間
と手間がかかり、また血漿または血清がろ過層を通過す
る時に分析物πの一部分がろ過層中で失われて、分析が
不正確になる可能性があるからである。The need to separate serum or plasma from the corpuscular components of whole blood must be avoided at all costs; the operation of removing corpuscles from serum or plasma using a filtration layer is extremely time-consuming and labor-intensive; This is because when the serum passes through the filtration layer, a portion of the analyte π may be lost in the filtration layer, making the analysis inaccurate.
従来の乾式分析要素のあるものは、血清または血漿を要
素に浸透させた後浸透できない血球成分を拭きとること
により血球成分を除去することを必要とした。全血の大
きな割合を占める血球成分は捕捉するが、血清または血
漿は特定の成分の存在下で検出可能な変化を生ずる試薬
を含む試薬層まで通過させることができる多孔質層を有
する分析要素を用いることにより、全血の分析は可能で
ある。米国特許第４，０４２，３３５号には、多層分析
要素を用いる全血分析について記載されている。ここに
記載された分析要素は、支持体の上に順に記録層、輻射
線阻害層及び試薬層を有している。試薬層は多孔質展開
層としての作用もすることができ、輻射線阻害層は全血
球を除去して記録層から除く透過層として作用すること
ができ、その結果ヘモグロビンによる妨害を回避できる
（上記米国特許の第１図参照）。Some conventional dry analytical elements require the removal of blood cell components by impregnating the element with serum or plasma and then wiping off the impermeable blood cell components. The analytical element has a porous layer that captures the blood cell components that make up a large proportion of whole blood, but allows serum or plasma to pass through to a reagent layer containing reagents that produce detectable changes in the presence of specific components. By using this method, whole blood analysis is possible. US Pat. No. 4,042,335 describes whole blood analysis using a multilayer analytical element. The analytical element described herein has a recording layer, a radiation blocking layer and a reagent layer in sequence on a support. The reagent layer can also act as a porous spreading layer, and the radiation blocking layer can act as a permeable layer to remove whole blood cells from the recording layer, thus avoiding interference by hemoglobin (see above). (See Figure 1 of the US patent).
このような要素は全血中の分析物質を測定するのに使用
できるが、いくつかの問題を有した。第に、全血球透過
層を通って記録層まで急速に拡散でき且つ十分に高い吸
光係数を有する検出可能な物質を用いる必要がある。し
かし、この要求を同時に満足する検出可能な物質（染料
、発色剤等）は数少ない。Although such elements can be used to measure analytes in whole blood, they have had several problems. First, it is necessary to use a detectable substance that can diffuse rapidly through the whole blood cell permeable layer to the recording layer and has a sufficiently high extinction coefficient. However, there are only a few detectable substances (dyes, color formers, etc.) that simultaneously satisfy this requirement.
また分析要素中で血球成分を分離する場合、血液サンプ
ルが接触する面内の気孔率及び気孔の大きさが、分析要
素を目詰りさせずにサンプルを完全に吸収するのに十分
なものでなければならない。In addition, when separating blood cell components in an analytical element, the porosity and pore size of the surface in contact with the blood sample must be sufficient to completely absorb the sample without clogging the analytical element. Must be.
しかし、気孔の構造はあまり粗いと、要素が機械的に不
安定（破壊）になる。However, if the pore structure is too rough, the element becomes mechanically unstable (destruction).
［発明の目的〕
本発明の目的は、全血中の分析物質を迅速にかつ高い精
度で定量するための乾式分析要素および分析方法を提供
することにある。[Object of the Invention] An object of the present invention is to provide a dry analytical element and an analytical method for rapidly and highly accurately quantifying an analyte in whole blood.
［発明の構成］
本発明の、全血中の特定成分を検出するための乾式多層
分析要素および方法は、分析すべき前記成分に対し相互
作用する組成物を含み、該分析物質と相互作用した時に
該相互作用組成物が６００ｎｍまたはそれ以上の波長で
分光光度法によって検出され得る染料を生成できること
を特徴とする。[Configuration of the Invention] The dry multilayer analytical element and method for detecting a specific component in whole blood of the present invention includes a composition that interacts with the component to be analyzed, and a composition that interacts with the analyte. Sometimes the interactive composition is characterized in that it is capable of producing a dye that can be detected spectrophotometrically at wavelengths of 600 nm or greater.
本発明はさらに、
（Ａ）全血のサンプルと前記分析要素とを接触させて、
６００ｎｍまたはそれ以上の波長において分光光度法に
よって検出され得る染料を生成せしめ、
（Ｂ）該染料を６００ｎｍまたはそれ以上の波長におい
て検出する
工程を含んでなることを特徴とする、全血中の分析物質
の定量または検出方法である。The present invention further provides: (A) contacting the whole blood sample with the analytical element;
an assay in whole blood, comprising the step of: producing a dye that can be detected spectrophotometrically at a wavelength of 600 nm or greater; and (B) detecting the dye at a wavelength of 600 nm or greater. It is a method for quantifying or detecting substances.
［本発明の効果コ
本発明の分析方法及び要素は、公知の全血分析に関連す
る前述の問題を克服するものである。即ち、この方法は
簡易、迅速かつ正確であり、しかも未希釈の全血サンプ
ルを分析できる６本発明によれば、血漿または血清から
血液の細胞成分を予め分離する必要がない、また過剰な
血液（即ち、血球成分）を拭きとったり、血液サンプル
を希釈したりする必要がない。Advantages of the Invention The analytical methods and components of the present invention overcome the aforementioned problems associated with known whole blood analyses. That is, this method is simple, rapid, and accurate, and can analyze undiluted whole blood samples6.According to the present invention, there is no need to pre-separate blood cellular components from plasma or serum, and there is no need to separate blood cell components from plasma or serum. There is no need to wipe (ie, blood cell components) or dilute the blood sample.
分析物質との相互作用によって生成される染料は６００
ｎｍまたはそれ以上の波長において分光光度法によって
検出されるので、ヘモグロビンやビリルビンによる妨害
が生じない０本発明の分析方法は極めて迅速である。即
ち、通常数分またはそれ以下で分析が可能で、ある場合
には２分以下での分析も可能である。サンプルごとのへ
マドクリット値の変動に対しても影響を受けにくい。The dye produced by interaction with the analyte is 600
Since it is detected spectrophotometrically at wavelengths of nm or higher, the analytical method of the present invention is extremely rapid, without interference from hemoglobin or bilirubin. That is, analysis can usually be performed in several minutes or less, and in some cases, analysis can be performed in less than two minutes. It is also less susceptible to variations in hemadcrit values from sample to sample.
本発明は全血中の種々の分析物質の定量に有用である１
本発明は１例えばグルコース、コレステロール、尿酸、
グリセロール、トリグリセリド、尿酸、ビリルビンなど
の代謝物質の定量に有用であるのみならず、脱水素酵素
、クレアチンキナーゼ、トランスアミナーゼ（例えばア
ラニンアミノトランスフェラーゼ、アスパラギンアミノ
トランスフェラーゼ）、加水分解酵素（例えばアミラー
ゼ、酸性ホスファターゼ、アルカリホスファターゼ等）
、等の酵素活性の測定にも有用である０本発明は又、特
定の抗体または抗原を用いた免疫分析にも使用できる０
本発明の分析方法は希釈、未希釈いずれの全血の分析に
も適用できるが、本発明の分析方法の利点の一つは、未
希釈の全血を分析できることである。未希釈の全血とは
、生理的塩溶液、血清、血漿などで希釈されていない全
血を意味する。The present invention is useful for quantifying various analytes in whole blood1
The present invention provides 1 for example glucose, cholesterol, uric acid,
It is useful for quantifying metabolites such as glycerol, triglycerides, uric acid, bilirubin, as well as dehydrogenases, creatine kinase, transaminases (e.g. alanine aminotransferase, asparagine aminotransferase), hydrolytic enzymes (e.g. amylase, acid phosphatase, alkaline phosphatase, etc.)
The present invention can also be used for immunoassays using specific antibodies or antigens.
Although the analysis method of the present invention can be applied to the analysis of either diluted or undiluted whole blood, one of the advantages of the analysis method of the present invention is that undiluted whole blood can be analyzed. By undiluted whole blood is meant whole blood that has not been diluted with physiological saline, serum, plasma, or the like.
［具体的実施態様の説明］
本発明は２種の機能をもつ層、即ち、全血サンプル（Ｉ
Ｎえば、１〜２０ｕｌ’）を収容または吸収でき、かつ
過剰の血液を拭きとる必要のない液体展開層及び分析物
質の存在に反応して染料が形成される発色層を必須に有
する乾式多層分析要素の使用によって達成される。この
２種の機能を有する層をそれぞれ２つ以上有してもよい
、この要素は例えば、米国特許４，０４２，３３５号に
記載されたような光遮蔽層またはろ過層を有してもよい
。[Description of Specific Embodiments] The present invention has two functional layers: a whole blood sample (I
Dry multilayer analysis that can accommodate or absorb 1 to 20 ul' of blood, and that requires a liquid development layer that does not require wiping off excess blood and a coloring layer that forms a dye in response to the presence of the analyte. This is accomplished through the use of elements. Each of these two functional layers may have more than one layer; the element may have a light shielding layer or a filtration layer, for example as described in U.S. Pat. No. 4,042,335. .
液体ｉ間層は、全血を吸収できる適当な気孔率及び平均
気孔寸法を有する任意の適当な繊維もしくは非繊維材料
、またはそれらの混合物から構成される。液体展開層は
それが液体接触している隣接する水浸透性層に面した面
において単位面積当たり均一の濃度の全血を提供するも
のが好ましい。The liquid interlayer is comprised of any suitable fibrous or non-fibrous material, or mixtures thereof, having suitable porosity and average pore size capable of absorbing whole blood. Preferably, the liquid spreading layer provides a uniform concentration of whole blood per unit area on the side facing the adjacent water permeable layer with which it is in liquid contact.
有用な液体展開層は、米国特許第４，２９２，２７２号
に記載された織物や特開昭８０−２２２７６９号に記載
された編物のような繊維材料により構成できる。Useful liquid spreading layers can be constructed from fibrous materials such as the woven fabrics described in US Pat. No. 4,292,272 and the knitted fabrics described in JP-A-80-222,769.
また米国特許３，９９２，１５８号に記載されている非
繊維等方性多孔質（例えばプラッシュポリマー）を用い
て構成することもできる。It can also be constructed using non-fibrous isotropic porous materials (eg, plush polymers) as described in US Pat. No. 3,992,158.
しかし本発明の液体展開層は同時に、実質的に全部、ま
たは少なくとも一部の血球をろ過して除去する作用を有
することが好ましく、前記の織物や編み物から成るもの
が有用である。However, it is preferable that the liquid spreading layer of the present invention also has the function of filtering and removing substantially all or at least some of the blood cells, and those made of the above-mentioned woven or knitted fabrics are useful.
展開層には、展開面積、展開速度等を調節するため、特
開昭６０−２２２７７０号、特願昭６１−１２２８７５
号、６１−１２２８７６号、６１−１４３７５４号に記
載したような親水性高分子あるいは界面活性剤を含有し
てもよい。In the developing layer, in order to adjust the developing area, developing speed, etc., there are
It may contain a hydrophilic polymer or a surfactant as described in No. 61-122876 and No. 61-143754.
本発明の分析要素の液体展開層または試薬層には少なく
とも１種の相互作用組成物を含む、相互作用組成物は、
分析成分を含む全血のサンプルが展開層に接触した時に
、分析物質または分析物質の反応生成物もしくは分解生
成物と、または互いに、相互作用する１種またはそれ以
上の活性成分を含んでなる０本発明の分析要素または方
法の特徴は、このような相互作用によって直接にまたは
間接に（他の反応を経て）６００ｎｓまたはそれ以上の
波長において分光光度法によって検出され得る染料が生
成されることである。このような染料は６００ ｎｍま
たはそれ以上の波長において有意な光学濃度が観察でき
るように充分高い吸光度を有するものでなければならな
い、染料は、血液中の所要の特定成分と染料生成物質と
の相互作用によって生成してもよいし、また非拡散性染
料からの拡散性染料の遊離によって生成してもよい、「
相互作用」なる用語は、化学活性、酵素−基質複合体の
形成におけるような触媒活性、抗原−抗体反応における
ような免疫原活性、並びに染料の濃度が直接的また間接
的に特定の分析物質の存在または濃度を示すような検出
可能な染料を遊離、形成または生成できる他の全ての型
の化学的または物理的相互作用を指す。The liquid spreading layer or reagent layer of the analytical element of the present invention includes at least one interactive composition, the interactive composition comprising:
comprising one or more active ingredients that interact with the analyte or reaction or degradation products of the analyte, or with each other when a sample of whole blood containing the analyte contacts the spreading layer. A feature of the analytical elements or methods of the invention is that such interactions directly or indirectly (via other reactions) produce dyes that can be detected spectrophotometrically at wavelengths of 600 ns or longer. be. Such dyes must have sufficiently high absorbance that a significant optical density can be observed at wavelengths of 600 nm or greater; may be produced by action or by liberation of a diffusible dye from a non-diffusible dye,
The term ``interaction'' refers to chemical activity, catalytic activity, such as in the formation of enzyme-substrate complexes, immunogenic activity, such as in antigen-antibody reactions, as well as the ability of dye concentration to directly or indirectly interact with a particular analyte. Refers to any other type of chemical or physical interaction that can liberate, form or produce a detectable dye indicative of the presence or concentration.
上記の相互作用組成物は、選択される分析反応系によっ
て決まる。有用な相互作用組成物は例えば脱水素酵素活
性を有する物質を含むものである。The interaction composition described above depends on the analytical reaction system chosen. Useful interactive compositions include, for example, substances with dehydrogenase activity.
酵素、例えばグリセロールデヒドロゲナーゼ、コレステ
ロールデヒドロゲナーゼ等のようなデヒドロゲナーゼ活
性物質をこのような酵素に対する基質である成分の分析
のための分析要素の試薬層あるいは液体展開層に含ませ
ることができる。Enzymes, such as dehydrogenase active substances such as glycerol dehydrogenase, cholesterol dehydrogenase, etc., can be included in the reagent layer or liquid spreading layer of an analytical element for the analysis of components that are substrates for such enzymes.
相互作用組成物の他の例は、酸化酵素活性を有する物質
を含むものである。例えばグルコースオキシダーゼ、コ
レステロールオキシダーゼ、ピルビン酸オキシダーゼ等
を酵素に対する基質である成分の分析のための分析要素
の試薬層あるいは液体展開層に含ませることができる。Other examples of interactive compositions include substances that have oxidase activity. For example, glucose oxidase, cholesterol oxidase, pyruvate oxidase, etc. can be included in the reagent layer or liquid developing layer of an analytical element for analyzing components that are substrates for enzymes.
相互作用組成物は、染料生成性組成物を含むことが好ま
しい１本発明の分析要素において、ロイコ色素の酸化に
よって染料を生成する組成物は有用である。ロイコ色素
の例としては、米国特許４．０８９，７４７号、特開昭
５９−１９３３５２号等に記載されたようなトリアリー
ルイミダゾールロイコ色素、公知のトリアリールメタン
ロイコ色素その他を挙げることができる。The interactive composition preferably comprises a dye-forming composition In one analytical element of the invention, compositions that produce dyes by oxidation of leuco dyes are useful. Examples of leuco dyes include triarylimidazole leuco dyes such as those described in U.S. Pat.
染料生成組成物はまた、染料を生成することのできる化
合物として、酸化されたときに分子内で、またはその還
元体とのカップリングによって染料を生成する化合物を
含んでもよい１例えばヒドロキシル基をもつ種々の化合
物、例えば０−アミノフェノール類、４−アルコキシナ
フトール類、４−アミノ−５−ピラゾロン頚、ブレゾー
ル類、ピロガロール、グアヤコール、オキシツール、カ
テコール、フロログルシノール、ｐ−ジヒドロキシジフ
ェニル没食子酸、ピロカテキン及びサリチル酸が挙げら
れる。この型の化合物は公知であり、文献に、例えばザ
・セオリー・オブ・ザ・フォトグラフィック°プロセス
（Ｔｈｅ Ｔｈｅｏｒｙ ｏｆ ｔｈｅ Ｐｈｏｔｏｇ
ｒａｐｈｉｃＰｒ。The dye-forming composition may also contain, as a compound capable of forming a dye, a compound which forms a dye when oxidized intramolecularly or by coupling with its reduced form, e.g. one having a hydroxyl group. Various compounds, such as 0-aminophenols, 4-alkoxynaphthols, 4-amino-5-pyrazolones, bresols, pyrogallol, guaiacol, oxytool, catechol, phloroglucinol, p-dihydroxydiphenyl gallic acid, pyro Catechin and salicylic acid are mentioned. Compounds of this type are known and can be found in the literature, for example in The Theory of the Photographic Process.
raphicPr.
ｃｅｓｓ） 、ミーグ（Ｍｅｅｓ）及びジエームズ（Ｊ
ａｍｅｓ）著。cess), Mees and James (J.
ames).
第３版（１９６６年）中、特に第１７１に記載されてい
る。It is particularly described in No. 171 of the 3rd edition (1966).
更に別の例として、染料は被酸化性化合物と発色剤との
縮合生成物を含む染料生成性組成物によって形成されて
もよい、被酸化性化合物の例としてはベンジジン及びそ
の同族体、ｐ−フェニレンジアミノ類、ｐ−アミノフェ
ノール類、アミノアンチピリン、例えば４−アミノアン
チピリンなどを挙げることができる。多数の自己カップ
リング化合物を含む広範囲のこのような発色剤は文献に
、例えば前記のミーグ及びジエームズの文献並びにコザ
ー（Ｋｏｓａｒ ）著、ライト・センシティブ・システ
ムズ（Ｌｉｇｈｔ ５ｅｎｓｉＬｉｖｅ Ｓｙｓｔｅｍ
ｓ） 、１９６５年、２１５〜２４９ページに記載され
ている。As yet another example, the dye may be formed by a dye-forming composition comprising the condensation product of an oxidizable compound and a color former; examples of oxidizable compounds include benzidine and its congeners, p- Examples include phenylene diamino, p-aminophenol, aminoantipyrine, such as 4-aminoantipyrine. A wide variety of such color formers, including a large number of self-coupling compounds, are found in the literature, for example in the Meig and James article cited above and Kosar, Light Sensitive Systems.
s), 1965, pp. 215-249.
系内で過酸化水素が発生する分析に有用な発色剤として
、米国特許４，２５１，６２９号、同４，２６０゜６７
９号及び同４，３９６，７１４号、特公昭５８−２２２
００号、ヨーロッパ特許出願６８，３５６号、英国特許
出願２゜１０７．８６３号、特開昭５８−８９８号等に
記載されているようなトルイジン類を用いることができ
る。有用なトルイジン化合物の例としては次のものが挙
げられる：
トエチルート２−スルホエチル１−トルイジン、トエチ
ルーＮ−２−カルボキシエチル１−トルイジン、Ｎ−２
−カルボキシエチル一一一トルイジン、Ｎ−スルホメチ
ル−ｐ−トルイジン、
Ｎ−メチル−Ｎ−（２，３−ジヒドロキシプロピル）−
ｍ＝トルイジンなど。U.S. Pat. No. 4,251,629, U.S. Pat.
No. 9 and No. 4,396,714, Special Publication No. 58-222
Toluidines such as those described in European Patent Application No. 00, European Patent Application No. 68,356, British Patent Application No. 2.107.863, and Japanese Patent Application Laid-Open No. 58-898 can be used. Examples of useful toluidine compounds include: toethyl-2-sulfoethyl-1-toluidine, toethyl-N-2-carboxyethyl-1-toluidine, N-2.
-Carboxyethyl-1-1-toluidine, N-sulfomethyl-p-toluidine, N-methyl-N-(2,3-dihydroxypropyl)-
m = toluidine, etc.
他の有用な発色剤としては、ジしドロインドール類、デ
ｊ・ラヒドロキシノリン類、置換アニリン化合物、例え
ば８−アニリノ−１−ナフタレンスルホン酸、Ｎ−メチ
ル−Ｎ−スルホプロピルアニリン。Other useful color formers include didroindoles, dihydroxynolines, substituted aniline compounds such as 8-anilino-1-naphthalenesulfonic acid, N-methyl-N-sulfopropylaniline.
７−シヒドロキシナフタレン、その細円業界で公知の１
ヒ合物を挙げることができる。7-hydroxynaphthalene, its thin circle known in the industry as 1
Hypoallergenic compounds can be mentioned.
染料生成組成物は、還元型補酵素と電子伝達剤の存在下
で染料を生成することのできる化合物から成るものでも
よい。The dye-forming composition may consist of a compound capable of forming a dye in the presence of a reduced coenzyme and an electron transfer agent.
染料は拡散性で、試薬層から試薬を特に含まない検出層
へ移動できるものであってもよいが、非拡散性のもので
もよい、非拡散性染料は例えば分子中にいわゆるバラス
ト基を有するものである。The dye may be diffusible and can move from the reagent layer to the detection layer that does not specifically contain the reagent, but it may also be non-diffusible. Non-diffusible dyes include, for example, those that have a so-called ballast group in the molecule. It is.
゛前述の相互作用組成物中の各成分又は試薬の量は測定
される分析物質に応じて広範囲に変化させることができ
る。これらの量は当業者ならば容易に決定できる。The amount of each component or reagent in the aforementioned interaction compositions can vary over a wide range depending on the analyte being measured. These amounts can be readily determined by those skilled in the art.
光透過性支持体を用いる場合、本発明の乾式分析要素の
実用的に採りうる構成は
（１）支持体上に試薬層、その上に・液体展開層を有す
るもの。When a light-transmitting support is used, the practical configuration of the dry analytical element of the present invention is (1) having a reagent layer on the support and a liquid developing layer thereon.
（２）支持体上に検出層、試薬層、液体展開層をこの順
に有するもの。(2) A device having a detection layer, a reagent layer, and a liquid development layer in this order on a support.
（３）支持体上に試薬層、光反射層、液体展開層をこの
順に有するもの。(3) A support having a reagent layer, a light reflection layer, and a liquid development layer in this order.
（４）支持体上に検出層、試薬層、光反射層、液体展開
層をこの順に有するもの。(4) A support having a detection layer, a reagent layer, a light reflection layer, and a liquid development layer in this order.
（５）支持体上に検出層、光反射層、試薬層、液体展開
層をこの順に有するもの。(5) A support having a detection layer, a light reflection layer, a reagent layer, and a liquid development layer in this order.
上記（１）または（３）において、支持体と試薬層との
閘に吸水層を設けてもよい６本発明で好ましいのは上記
（１）の層構成である。上記（１）ないしく３〉におい
て試薬層と検出層または液体展開層の贋に血球ろ過層を
設けてもよい、上記（３）ないしく５）において光反射
層と検出層、試薬層もしくは液体展開層との間、試薬層
と検出層との問、または試薬層と液体展開層の間に、さ
らに血球ろ過層を設けてもよい。In (1) or (3) above, a water-absorbing layer may be provided between the support and the reagent layer.6 The layer structure in (1) above is preferred in the present invention. In the above (1) to 3>, a blood cell filtration layer may be provided on the reagent layer and the detection layer or the liquid spreading layer, and in the above (3) to 5), the light reflection layer and the detection layer, the reagent layer or the liquid A blood cell filtration layer may be further provided between the developing layer, between the reagent layer and the detection layer, or between the reagent layer and the liquid developing layer.
本発明の分析要素は光反射層を有してもよい。The analytical element of the invention may have a light reflective layer.
例えば、試薬層と検出層との間、または試薬層と液体展
開層との間に、光反射層を設けることができる。光反射
層は、検出層、試薬層等に生じた検出可能な変化（色変
化、発色等）を光透過性を有する支持体側から反射測光
する際に、展開層に点着供給された被検液の色、特に試
料が全血である場合のヘモグロビンの赤色、ビリルビン
の黄色等を遮蔽するとともに光反射層または青景層とし
て機能する。光反射層は、親水性ポリマーをバインダー
として、酸化チタン、硫酸バリウム等の光反射性微粒子
が分散された水浸透性の層であることが好ましい、バイ
ンダーとしてはゼラチン、ゼラチン誘導体、ポリアクリ
ルアミド等が好ましい。For example, a light reflecting layer can be provided between the reagent layer and the detection layer or between the reagent layer and the liquid developing layer. The light-reflecting layer is used to measure the detectable changes (color change, color development, etc.) that occur in the detection layer, reagent layer, etc. from the light-transmitting support side. It blocks the color of the liquid, especially the red color of hemoglobin when the sample is whole blood, the yellow color of bilirubin, etc., and also functions as a light reflection layer or a blue-scene layer. The light-reflecting layer is preferably a water-permeable layer in which light-reflecting fine particles such as titanium oxide and barium sulfate are dispersed using a hydrophilic polymer as a binder. The binder may include gelatin, gelatin derivatives, polyacrylamide, etc. preferable.
ゼラチンのような硬化可能なポリマーには硬膜剤を加え
てもよい１分析要素には、必要に応じ展開層、試薬層、
検出層等に酸化チタン等の粒子を含有させてもよい。A hardening agent may be added to hardenable polymers such as gelatin.1 Analytical elements include a spreading layer, reagent layer,
Particles such as titanium oxide may be contained in the detection layer or the like.
本発明の分析要素は、液体展開層とは別に全血球を実質
的にろ過して除去する層を有してもよい。The analytical element of the present invention may have a layer that substantially filters and removes whole blood cells, separate from the liquid spreading layer.
例えば＃１ｒＭ昭５８−７（１１６３号、特開昭６１−
４９５９号、特願昭６０−２５８４０８号、同６０−２
７９８５９号、同６〇−２７９８６０号、同８０−２７
９８６１号等に記載されたような多孔性層は好適である
。For example, #1rM Sho 58-7 (No. 1163, JP-A-Sho 61-
No. 4959, Patent Application No. 60-258408, No. 60-2
No. 79859, No. 60-279860, No. 80-27
Porous layers such as those described in US Pat. No. 9,861 are suitable.
本発明の要素の１種またはそれ以上の層は、ＩＮまたは
それ以上の他の種々の任意の成分１例えば界面活性化剤
、発色剤溶剤、ＭＷＲ剤、結合剤、硬化剤などを含むこ
とができる。これらの成分は当業者に知られた量で存在
できる０代表的な要素成分は、例えば米国特許４，２５
８，０１１号、同３，９９２゜１５８号、同４，０４２
，３３５号、同４，１４４，３０６号、同４゜１３２．
５２８号、同４，０５０，８９８号、同４，２７５，１
５２号等に記載されている。One or more layers of the elements of the invention may include IN or a variety of other optional components such as surfactants, color former solvents, MWR agents, binders, hardeners, etc. can. These components can be present in amounts known to those skilled in the art. Representative component components are described, for example, in U.S. Pat.
No. 8,011, No. 3,992゜158, No. 4,042
, No. 335, No. 4,144,306, No. 4゜132.
No. 528, No. 4,050,898, No. 4,275,1
It is described in No. 52, etc.
試料全血を適用後、試験結果を早く又は正確に得るため
に、インキュベーション（加ｆ！％）を要素に対して行
なうことができる。After applying the sample whole blood, incubation (f!%) can be carried out on the element in order to obtain test results quickly or accurately.
分析成分が存在するならば、サンプル中の分析成分の濃
度に基づく速度で分析物質が相互作用組成物と相互作用
する。染料を検出するための適当な装置に分析要素を通
すことによって、染料の形成速度を測定するか、分析成
分濃度に対応して形成された染料の量を測定する。染料
は当業界において公知の適当な分光光度測定装置、例え
ば米国特許４，５８４，２７５号に記載された装置を用
いて検出できる。If the analyte is present, the analyte interacts with the interactive composition at a rate based on the concentration of the analyte in the sample. By passing the analytical element through a suitable device for detecting the dye, the rate of dye formation is determined, or the amount of dye formed in response to the concentration of the analyte. Dyes can be detected using any suitable spectrophotometric equipment known in the art, such as that described in US Pat. No. 4,584,275.
本発明をさらに具体的に説明するため以下に実施例を記
載する。Examples will be described below to further specifically explain the present invention.
［実施例１］
（支持体、試薬層）
ゼラチン下塗された１８０μｌの無色透明ポリエチレン
テレフタレート（ＰＥＴ）フィルム（支持体）の上に、
下記組成物１を下記被Ｆｌ！量になるように水溶液とし
て塗布し、乾燥して試薬層を形成した（乾燥後の膜厚７
μｍ）。[Example 1] (Support, reagent layer) On a 180 μl transparent colorless polyethylene terephthalate (PET) film (support) coated with gelatin,
The following composition 1 was applied to the following Fl! It was applied as an aqueous solution to the desired amount and dried to form a reagent layer (film thickness after drying: 7
μm).
租襲ｄ１Ｖ：
脱イオンゼラチン １４．２１７ｍ”ポリオ
キシエチレンノニル
フェニルエーテル（ｎ＝４０） ４１０ ｍｇ７
ｍ２ヘルオキシダーゼ ６，５００ Ｕ／
ｍ’フラビンアデニンジヌクレオチド ２２ Ｈ／ｍ”
チアミンピロリン１ｉ１２９３ ｘｇ７ｗ２ピルビン酸
オキシダーゼ １２，３００ Ｕ／ｌＩ２０イコ色
素（下記１１ａ） ２８０ ｍｇ７ｘ２ビス
〔（ビニルスルホニルメチル
カルボニル）アミノ〕メタン １８０ Ｈａｍ２（
ｐｌｌを６．８に調節する）
色素：
２−（３，５−ｄｉｍｅＬｈｏｘｙ−４−ｈｙｄｒｏｘ
ｙｐｈｅｎｙｌ）−４−ｐｈｅｎｅｔｌ＋ｙｌ−５−（
４−ｄｉａｅｔｌ＋ｙｌａｍｉｎｏｐｈｅｎｙｌ）ｉａ
ｉｃｌａｚｏｌｅ
次に下記組成物２および組成物）３を表示の割合で順次
塗布し、乾燥して光反射層および接着層とした。Concession d1V: Deionized gelatin 14.217m” polyoxyethylene nonylphenyl ether (n=40) 410 mg7
m2 heloxidase 6,500 U/
m'flavin adenine dinucleotide 22 H/m"
Thiamine pyrroline 1i1293 xg7w2 Pyruvate oxidase 12,300 U/lI20 Ico dye (11a below) 280 mg7x2 Bis[(vinylsulfonylmethylcarbonyl)amino]methane 180 Ham2(
Adjust pll to 6.8) Dye: 2-(3,5-dimeLhoxy-4-hydrox
yphenyl)-4-phenetl+yl-5-(
4-diaetl+ylaminophenyl)ia
iclazole Next, Composition 2 and Composition 3 below were sequentially applied at the indicated ratios and dried to form a light reflective layer and an adhesive layer.
（光反射層）
発色試薬層の上に、各成分について下記の被覆Ｉから成
る光反射層を、水分散液の塗布・乾燥により設けた。(Light-reflecting layer) A light-reflecting layer consisting of the following coating I for each component was provided on the coloring reagent layer by coating and drying an aqueous dispersion.
区民１１；
アルカリ処理ゼラチン ３．２ ｇ７ｍ’ル
チル型二酸化チタン微粒子 ８．９ ｇ７ｍ’ポリ
オキシエチレンノニル
フェニルエーテル
（平均オキシエチレン４０単位含有） ０．５ ｇｅ
ｍ”（接着層）
光反射層の上に下記の被覆量の接着層を、水溶液の塗布
・乾燥により設けた。Citizen 11: Alkali-treated gelatin 3.2 g7m'Rutile type titanium dioxide fine particles 8.9 g7m'Polyoxyethylene nonylphenyl ether (contains 40 units of oxyethylene on average) 0.5 ge
m'' (adhesive layer) An adhesive layer having the following coverage was provided on the light reflecting layer by coating and drying an aqueous solution.
粧玖１１：
アルカリ処理ゼラチン ３．１ ｇ／１１２
ポリオキシエチレンノニル
フェニルエーテル
（平均オキシエチレン４０単位含有） ０．１５ ｙ
／々２（展開Ｎ）
次に上記接着層表面を約２５℃の水でほぼ一様にぬらし
た後、１００Ｓ相当のＰＥＴｖＪ！？を糸からなる厚さ
約２５０μｌのトリコット！ｉ物生地を軽く圧着して接
着層上に接着させ、一体化させた。Shape 11: Alkali-treated gelatin 3.1 g/112
Polyoxyethylene nonylphenyl ether (contains 40 units of oxyethylene on average) 0.15 y
/2 (Development N) Next, after wetting the surface of the adhesive layer almost uniformly with water at about 25°C, PETvJ! equivalent to 100S was applied. ? Tricot with a thickness of about 250μl made of thread! The i-product fabric was lightly pressed onto the adhesive layer and integrated.
次に下記の被覆量になるように、組成物４（ポリマー合
有水溶液）を展ｒｆｎＭＩの上から塗布し、乾燥して、
グルタメートオキザロ酢酸トランスアミナーゼ（ＧＯＴ
）定量分析用多層分析フィルムを作製した。Next, composition 4 (polymer-containing aqueous solution) was applied over the expanded rfnMI to the following coverage amount, dried,
Glutamate oxaloacetate transaminase (GOT)
) A multilayer analytical film for quantitative analysis was prepared.
紅炎１生：
ポリオキシエチレンノニル
フェニルエーテル １．２ ｙ／１（平均
オキシエチレン４０単位含有）
トリスヒドロキシ
メチルアミノメタン ０．３２ｉｎ２リン酸
２水素１カリウム ０．３６ ｇ７ｚ’Ｌ−ア
スパラギン酸 １．９２ ｇ／ｚ２α−ケ
トグルタル酸 ０．３２ ｇ／履２り化
マグネシウム（無水＞ １８．７ ｇ／ｉ’オ
キザロ酢酸 ・ζ
デカルボキシラーゼ １２，６００ Ｕ／麿２ヒド
ロキシプロピルメチル
セルロース ３．２ ｇ／饋２（希
Ｎ ａ ＯＬ（でｐ Ｈ＝ ７ 、５に調節）（分析ス
ライド）
前記分析フィルムを１．５Ｘ１．５ｃｍ角に切断し、プ
ラスチックマウントに挿入し、ＧＯＴ定量分析用分析ス
ライドを完成した。Koen 1st grade: Polyoxyethylene nonylphenyl ether 1.2 y/1 (contains 40 units of oxyethylene on average) Trishydroxymethylaminomethane 0.32 in dihydrogen monopotassium phosphate 0.36 g7z'L-aspartic acid 1. 92 g/z2α-ketoglutaric acid 0.32 g/magnesium dihydride (anhydrous > 18.7 g/i'oxaloacetic acid/ζ decarboxylase 12,600 U/m2hydroxypropylmethylcellulose 3.2 g/2 (Adjusted to pH = 7, 5 with dilute NaOL (Analysis slide) The analysis film was cut into 1.5 x 1.5 cm squares and inserted into a plastic mount to complete an analysis slide for GOT quantitative analysis.
（ＧＯＴ活性測定）
次にヒトより採血した新鮮全血（ヘパリン採血、ヘマト
クリット値４４％）に、ブタ由来のＧＯＴ（米国シグマ
社）を加え、ＧＯＴ活性が２５単位／ｌ ４００単位
／ｌ ８００単位／ｌ １５８０単位／ｌの４種類
の血液試料を作成した。(Measurement of GOT activity) Next, pig-derived GOT (Sigma, USA) was added to fresh whole blood collected from humans (heparinized blood, hematocrit value 44%), and the GOT activity was 25 units/l, 400 units/l, and 800 units. Four types of blood samples were prepared at 1580 units/l.
この４種類の血液を上記のＧＯＴ定量分析用分析スライ
ドに点着した０点着後３７℃にて反応させ６４０ｎｍの
吸収をＰＥＴ支持体側より２６５分後と４分後に反射測
光し、０Ｄｔ（透過光学濃度）に換算した値（ＣＩ １
ｎｉｃａｌ ＣＩ＋ｅｍｉｓｔｒｙ、 Ｖｏｌ 、２４
゜１３３５ （１９７８） ノ原理による）を用いてＧ
ｏＴ活性値を算出した。結果を第１表に示す。These four types of blood were spotted on the GOT quantitative analysis slide mentioned above, reacted at 37°C after the 0 point, and the absorption at 640 nm was measured by reflection photometry from the PET support side after 265 minutes and 4 minutes. Optical density) converted value (CI 1
nical CI+emistry, Vol, 24
G using the principle of ゜1335 (1978)
The oT activity value was calculated. The results are shown in Table 1.
第１表
［９考例］
組成物１からペルオキシダーゼおよびピルビン酸オキシ
ダーゼを、組成物４からオキザロ酢酸デカルボキシラー
ゼを除いた他は、実施例１と全く同様にして、ＧＯＴに
応答しない参照用要素を作製した。これに前記の血液Ｎ
ｏ、１およびその血清をそれぞれ点着し、３７℃に４分
放置後波長６４０ｎｍでの反射光学濃度をＰＥＴ支持体
側より測定した。実施例１の分析要素についても同様測
定した。その結果は第２表の通りである。Table 1 [9 Examples] A reference element that does not respond to GOT was prepared in the same manner as in Example 1, except that peroxidase and pyruvate oxidase were removed from Composition 1 and oxaloacetate decarboxylase was removed from Composition 4. Created. In addition to the blood N
0, 1 and their serum were spotted respectively, and after being left at 37° C. for 4 minutes, the reflection optical density at a wavelength of 640 nm was measured from the PET support side. The analytical elements of Example 1 were also measured in the same manner. The results are shown in Table 2.
第２表
第２表から、血球の有無により背景の光学濃度に有意の
差はないこと、また実施例の分析要素は背景に対し充分
高い光字濃度を示すことがわかる。Table 2 From Table 2, it can be seen that there is no significant difference in the optical density of the background depending on the presence or absence of blood cells, and that the analytical elements of Examples exhibit sufficiently high optical density relative to the background.
Claims (6)
定成分に対し相互作用する組成物を含む、全血中の前記
成分を検出するための多層分析要素であって、該分析物
質と相互作用した時に該相互作用組成物が６００ｎｍま
たはそれ以上の波長において分光光度法で検出され得る
染料を生成できることを特徴とする多層分析要素。(1) A multilayer analytical element for detecting a specific component in whole blood, the element having at least two water-permeable layers and comprising a composition that interacts with a specific component in whole blood; A multilayer analytical element, characterized in that the interacting composition, when interacting with a substance, is capable of producing a dye that can be detected spectrophotometrically at a wavelength of 600 nm or more.
成物のうち少なくとも前記染料を生成し得る部分を含む
層と他の層との間に光反射層を有する多層分析要素。(2) A multilayer analytical element according to claim (1), which has a light-reflecting layer between a layer containing at least a portion of the interactive composition capable of producing the dye and another layer.
開層である多層分析要素。(3) The multilayer analytical element according to claim (1), wherein the outermost layer is a liquid spreading layer.
くとも２つの水浸透性層を有し、該層の一つは液体展開
層であり、他の一つは前記相互作用組成物のうち少なく
とも前記染料を生成し得る部分を含む試薬層であり、前
記液体展開層と前記試薬層との間に光反射層を有する多
層分析要素。(4) In claim (1), the analytical element has at least two water-permeable layers, one of which is a liquid spreading layer and the other one of which is a liquid spreading layer. A multilayer analytical element, the reagent layer including at least a portion capable of producing the dye, and having a light reflective layer between the liquid developing layer and the reagent layer.
互作用組成物の全部を含む多層分析要素。(5) The multilayer analytical element according to claim (4), wherein the reagent layer contains all of the interaction composition.
血中の特定成分に対し相互作用する組成物を含む全血中
の特定成分を検出するための多層分析要素であって、前
記成分と相互作用した時に該相互作用組成物が６００ｎ
ｍまたはそれ以上の波長において分光光度法で検出され
得る染料を生成でき、かつ前記相互作用組成物のうち少
なくとも前記染料を生成し得る部分を含む層と他の層と
の間に光反射層を有する多層液体分析要素と全血検体と
を接触させて、６００ｎｍまたはそれ以上の波長におい
て分光光度法で検出され得る染料を生成せしめる工程と （Ｂ）該染料を６００ｎｍまたはそれ以上の波長におい
て検出する工程と から成る検出方法。(6) A method for detecting an analyte in whole blood, the method comprising: (A) a multilayer analytical element for detecting a specific component in whole blood, including a composition that interacts with the specific component in whole blood; and when the interacting composition interacts with the component, the interaction composition
a light-reflecting layer between a layer capable of producing a dye spectrophotometrically detectable at wavelengths of m or more and comprising at least a portion of said interactive composition capable of producing said dye and another layer; (B) contacting the whole blood sample with a multilayer liquid analytical element having the composition to produce a dye that can be spectrophotometrically detected at a wavelength of 600 nm or greater; and (B) detecting the dye at a wavelength of 600 nm or greater. A detection method consisting of a process.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8609587A JPS63250566A (en) | 1987-04-08 | 1987-04-08 | Method and element for analyzing total blood |
| US07/171,326 USH600H (en) | 1987-03-20 | 1988-03-21 | Whole blood analytical element and method of analyzing whole blood using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8609587A JPS63250566A (en) | 1987-04-08 | 1987-04-08 | Method and element for analyzing total blood |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63250566A true JPS63250566A (en) | 1988-10-18 |
Family
ID=13877150
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8609587A Pending JPS63250566A (en) | 1987-03-20 | 1987-04-08 | Method and element for analyzing total blood |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63250566A (en) |
-
1987
- 1987-04-08 JP JP8609587A patent/JPS63250566A/en active Pending
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