JPS63233373A - Element and method for analyzing complete blood - Google Patents
Element and method for analyzing complete bloodInfo
- Publication number
- JPS63233373A JPS63233373A JP6751187A JP6751187A JPS63233373A JP S63233373 A JPS63233373 A JP S63233373A JP 6751187 A JP6751187 A JP 6751187A JP 6751187 A JP6751187 A JP 6751187A JP S63233373 A JPS63233373 A JP S63233373A
- Authority
- JP
- Japan
- Prior art keywords
- layer
- dye
- whole blood
- composition
- analytical element
- 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.)
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- 239000000203 mixture Substances 0.000 claims abstract description 32
- 238000001914 filtration Methods 0.000 claims abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims description 30
- 210000000601 blood cell Anatomy 0.000 claims description 25
- 230000007480 spreading Effects 0.000 claims description 22
- 239000012491 analyte Substances 0.000 claims description 16
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- 238000001514 detection method Methods 0.000 claims description 6
- 238000003556 assay Methods 0.000 claims 1
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Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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、肝臓障害、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, liver 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
−E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)は、正確な分析結果を得るなめ
にサンプルから予め除去するか、分析要素中で何らかの
手段で分離しなければならない。Dry chemical analysis is superior to 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) must be removed from the sample in advance or separated by some means in the analytical element in order to obtain accurate analysis results. Must be.
全血中の血球成分から血清または血漿を分離する操作の
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
に時間と学問がかかり、また血漿または血清がろ過層を
通過する時に分析物質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 for separation of 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 very time-consuming and scientifically tedious; When plasma or serum passes through the filtration layer, a portion of the analyte is lost in the filtration layer,
This is because the analysis may become 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ji層、帽
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, in order, a recording layer, a radiation blocking layer, and a reagent layer 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1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 high extinction coefficient per minute. 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 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、水浸透性層のうち少なくとも1つが全血球
ろ過層であり、該分析物質と相互牛用した時に該相互作
用組成物が6000m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。[Structure of the Invention] The dry multilayer analytical element of the present invention for detecting a specific component in whole blood includes a composition that interacts with the component to be analyzed, and at least one of the water-permeable layers contains a composition that interacts with the component to be analyzed. a whole blood cell filtration layer, characterized in that the interactive composition, when interacted with the analyte, is capable of producing a dye that can be detected spectrophotometrically at wavelengths of 6000 m or more.
好ましい分析要素は、分析要素が有する水浸透性層の一
つが液体展開層であり、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全血球
ろ過層を有するが、前記液体展開層が全血球ろ過層でも
ある多層分析要素である。試薬層に前記相互作用組成物
の全部を含むこともできる。In a preferred analytical element, one of the water-permeable layers of the analytical element is a liquid spreading layer, and the other is a reagent layer containing at least a portion of the interacting composition capable of producing the dye, and It is a multilayer analytical element that has a whole blood cell filtration layer between the liquid development layer and the reagent layer, and the liquid development layer is also a whole blood cell filtration layer. The reagent layer can also contain all of the interactive composition.
本発明の分析方法の特徴は、
(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
より検出され得る染料を生成せしめ(B)該染料を60
0nmまたはそれ以上の波長において検出する
工程を含んで成る全血中の分析物質の定量または検出方
法である。The characteristics of the analysis method of the present invention are as follows: (A) bringing the whole blood sample into contact with the analysis element;
(B) produce a dye that can be detected spectrophotometrically at wavelengths of 600 nm or greater;
A method for quantifying or detecting an analyte in whole blood, comprising a step of detecting at a wavelength of 0 nm or more.
[本発明の効果]
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0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 samples.According to the present invention, there is no need to pre-separate the cellular components of blood from plasma or serum, and there is no need to separate the cellular components of blood 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まな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。検体血液のヘマト
クリット値の変動に対しても比較的影響を受けにくい。The dye produced by interaction with the analyte is 600
Since it is detected spectrophotometrically at wavelengths up to 100 nm or longer, 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 relatively unaffected by fluctuations in the hematocrit value of sample blood.
本発明は全血中の種々の分析物質の定量に有用である0
本発明は、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−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 blood.
The present invention provides, for example, glucose, cholesterol, uric acid,
It is useful for quantifying metabolites such as glycerol, triglycerides, uric acid, and 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(例
えば、1〜20μl)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 provides a layer that has two functions: a liquid spreading layer that can accommodate or absorb a whole blood sample (e.g., 1-20 μl), and that does not require wiping off excess blood; and by the use of a dry multilayer analytical element that essentially has a color-forming layer in which a dye is formed in response to the presence of the analyte. 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. .
液体展開層は、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 spreading layer is constructed from 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60−222769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 U.S. Pat. No. 4,292,272 and the knitted fabrics described in JP-A-60-222,769.
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Go −222770号、特願昭61−12287
5号。In the developing layer, in order to adjust the developing area, developing speed, etc., there are
No. 5.
81−122878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。It may contain a hydrophilic polymer or a surfactant as described in Nos. 81-122878 and 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、この相互作用組成物
は、分析成分を含む食代のサンプルが展開層に接触した
時に1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)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
が生成されることである。このような染料は600nm
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 contains at least one interactive composition, and this interactive composition is capable of producing one analyte when a food sample containing an analytical component comes into contact with the spreading layer. or with the reaction or decomposition products of the analyte, or with each other. or indirectly (via other reactions) to produce a dye that can be detected spectrophotometrically at wavelengths of 600 nm or higher. Such dyes are 600nm
The dye may be produced by interaction of the dye-forming substance with the desired specific components of the blood. and may also be produced 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 indicating the presence or concentration of.
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0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 are those containing substances with oxidase activity, such as glucose oxidase, cholesterol oxidase, pyruvate oxidase, etc., in the reagent layer or liquid of an analytical element for the analysis of components that are substrates for enzymes. Can be included in the deployment layer.
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0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1〜リアリ
一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. Compositions that produce dyes by oxidation of leuco dyes are useful in the analytical elements of the invention. Examples of leuco dyes include 1- to aryl-limidazole leuco dyes as described in U.S. Pat. Can be done.
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0例えばヒドロキシル基をもつ種々の化
合物、例えば0−アミンフェノール類、4−アルコキシ
ナフトール類、4−アミノ−5−ピラゾロン票、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(Tbe Tl+eory or the PboL
oHrapl+1cProcess) +ミーグ(He
es)及びジエームズ(James)著、第3版(19
66年)中、特に第17章に記載されている。The dye-forming composition may also contain, as a compound capable of forming a dye, a compound which forms a dye when oxidized, either intramolecularly or by coupling with its reduced form, e.g. Various compounds including 0-amine phenols, 4-alkoxynaphthols, 4-amino-5-pyrazolones, bresols, pyrogallol, guaiacol, oxytools,
Catechol, phloroglucinol, p-dihydroxydiphenyl gallic acid, pyrocatechin 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 (Tbe Tl + theory or the PboL
oHrapl+1cProcess) +Mig(He
es) and James, 3rd edition (19
(1966), especially in Chapter 17.
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、ρ−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(にosar )著、ライト・センシティブ・システ
ムズ(Light 5ensitive 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, ρ- 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 described in the literature, for example in the Meig and James article cited above and in Light 5-sensitive Systems by Osar.
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4,251,829号、同4,260゜67
9号及び同4,396,714号、特公昭5B−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1−ルイジン化合物の例としては次のものが
挙げられる:
N−エチル−N−2−スルホエチル1−トルイジン、N
−エチル−N−2−カルポキシエチル−−−トルイジン
、N−2−カルボキシエチル−輸−トルイジン、N−ス
ルホメチル−p−)ルイジン、
N−メチル−N−(2,3−ジヒドロキシプロピル)−
嬶−トルイジン
など。U.S. Pat. No. 4,251,829, U.S. Pat.
No. 9 and No. 4,396,714, Special Publication No. 5B-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 1-luidine compounds include: N-ethyl-N-2-sulfoethyl 1-toluidine, N
-Ethyl-N-2-carpoxyethyl--toluidine, N-2-carboxyethyl-transport-toluidine, N-sulfomethyl-p-)luidine, N-methyl-N-(2,3-dihydroxypropyl)-
- Toluidine etc.
池の有用な発色剤としては、ジしドロインドール類、テ
トラヒドロキシキノリン類、1.7−ジヒド。Useful color formers include dihydroindoles, tetrahydroxyquinolines, and 1,7-dihydre.
ロキシナフタレン、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、トメチル−N−ス
ルホプロピルアニリン、その他害業界で公知の化合物を
挙げることができる。Mention may be made of loxinaphthalene, substituted aniline compounds such as 8-anilino-1-naphthalenesulfonic acid, tomethyl-N-sulfopropylaniline, and other compounds known in the art.
染料生成組成物は5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 5-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.
あるいはオリゴ糖に染料分子を形成し得る置換基の結合
した化合物も利用できる。Alternatively, a compound in which a substituent that can form a dye molecule is bonded to an oligosaccharide can also be used.
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1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. One of these can be easily determined by a person 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 spreading 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
間に吸水層を設けてもよい0本発明で好ましいのは上記
(1)のN構成である。上記(1)ないしく3)におい
て液体展開層または光反射層が全血球ろ退店でない場合
は、試薬層と液体展開層の間に血球ろ退店を設ける。上
記(3)および(4)において液体展開層または光反射
層が全血球ろ退店でない場合は、液体展開層と試薬層ま
たは光反射層との間に、上記(5)において液体展開層
または光反射層が全血球ろ退店でない場合は、試薬層と
光反射層の間に、血球ろ退店を設ける。In the above (1) or (3), a water absorption layer may be provided between the support and the reagent layer. In the present invention, the N configuration in the above (1) is preferred. In (1) to 3) above, if the liquid spreading layer or the light reflecting layer does not have a whole blood cell filter, a blood cell filter is provided between the reagent layer and the liquid spreading layer. If the liquid spreading layer or light reflecting layer in (3) and (4) above does not filter whole blood cells, the liquid spreading layer or light reflecting layer in (5) above is between the liquid spreading layer and the reagent layer or light reflecting layer. If the light-reflecting layer is not a whole blood cell filter, a blood cell filter is provided between the reagent layer and the light-reflecting layer.
本発明の分析要素において、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することがより有利
であるが、この層として例えば特開昭58−70163
号、特開昭61−4959号、特願昭60−25640
8号、同60−279859号、同60−279880
号、同60−279881号等に記載されたような多孔
性層は好適である。In the analytical element of the present invention, it is more advantageous to have a layer that substantially filters and removes whole blood cells separately from the liquid spreading layer.
No. 61-4959, patent application No. 60-25640
No. 8, No. 60-279859, No. 60-279880
A porous layer such as that described in No. 60-279881 is suitable.
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。また液
体展開層や血球ろ退店を、同時に光反射層として機能さ
せてもよい、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
層または背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、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、
また液体展開層、試薬層、血球ろ退店等に酸化チタン等
の粒子を含有させて、これらの層を光反射層として機能
させてもよい。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. In addition, the liquid development layer and the blood cell filtration layer may function as a light reflection layer at the same time. When performing reflection photometry from the support side that has a polarity, the color of the test liquid applied to the developing layer, especially when the sample is whole blood, the red of hemoglobin, the yellow of bilirubin, etc. is shielded, and the light reflection layer is also used. Or act as a background 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. Preferred curable polymers, such as gelatin, may include a hardening agent.
Further, particles such as titanium oxide may be contained in the liquid spreading layer, the reagent layer, the blood cell filter, etc., so that these layers function as light reflecting layers.
本発明の要素の1種またはそれ以上の層は、1mまたは
それ以上の池の種々の任意の成分、例えば界面活性化剤
、発色剤溶剤、!!衝剤、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崖で存在
できる0代表的な要素成分は1例えば米国特許4,25
8,011号、同3,992゜158号、同4,042
,335号、同4,144,308号、同4゜132.
528号、同4,050,898号、同4,275,1
52号、゛ 特開昭82−299号等に記載されてい
る。One or more layers of the elements of the invention may contain a variety of optional ingredients such as surfactants, color formers, solvents,! ! It may contain a buffering agent, a binder, a hardening agent, and the like. These components can be present in cliffs known to those skilled in the art. Typical elemental components are 1 e.g. U.S. Pat.
No. 8,011, No. 3,992゜158, No. 4,042
, No. 335, No. 4,144,308, No. 4, 132.
No. 528, No. 4,050,898, No. 4,275,1
No. 52, ``Japanese Unexamined Patent Publication No. 82-299, 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(加熱)を要素に対して行なう
ことができる。After applying the sample whole blood, incubation (heating) 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]
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)(PET)フィルム(支持体)の上に、
乾燥後の膜厚が7μ厘になるようにゼラチン水溶液を塗
布し、乾燥して吸水層を形成した。[Example 1] On a gelatin-subbed 180 μl colorless transparent polyethylene terephthalate (PET) film (support),
An aqueous gelatin solution was applied so that the film thickness after drying was 7 μm, and dried to form a water absorption layer.
次に吸水層表面を約25℃の水でほぼ一様にぬらした後
、富士写真フィルム(株)製ミクロフイ、ルタFM30
0 (最小孔径3.0μl、厚さ140μl、空隙率約
80%のセルロースアセテートメンブレンフィルタ)を
ラミネートし、乾燥させて、吸水層とFM300フィル
タを一体化させた。Next, after wetting the surface of the water absorbing layer almost uniformly with water at about 25°C,
0 (a cellulose acetate membrane filter with a minimum pore size of 3.0 μl, a thickness of 140 μl, and a porosity of about 80%) was laminated and dried to integrate the water absorption layer and the FM300 filter.
次にこのFM300フィルタの上から下記組成物1およ
び組成物2を表示の割合で順次塗布し、乾燥して第1非
繊維多孔質層とした。Next, Composition 1 and Composition 2 below were sequentially applied on top of this FM300 filter at the indicated ratios and dried to form a first non-fibrous porous layer.
!!LtLL:
ゼラチン 0.64 g7x2
ポリオキシエチレン
ノニルフェニルエーテル(n=40) 2.527m”
トリスヒドロキシ
メチルアミノメタン 0.46 gem”す
:yvi2水素1 力!J ’/ ム0.46 gem
”L−アスコルビン酸 2.5 g7a2
塩化マグネシウム(無水) 0.3 g/lペ
ルオキシダーゼ 6,400 U/、w2フ
ラビンアデニンジヌクレオチド 2867m2チアミン
ピロリン酸 118 x11/瀧2α−ケ
トグルタル酸 500 mg7x2オキ
ザロ酢酸
デカルボキシラーゼ 12,6001J/x2ピ
ルビン酸オキシダーゼ 35,000 U/J12
(pH=7.5の水溶液)
(組羞」11)
ロイコ色素(下記構造) 1.8 g/β2
ポリオキシエチレンノニル
フェニルエーテル(n=40) 0.6 ge
m2(エタノール溶液)
色素:
2−(3,5−dimethoxy−4−hydrox
yphenyl)−4−pbenethyl−5−(4
−dimethylaminopl+e++yl)i+
1idazole
次に富士写真フィルム(株)製ミクロフィルタFM30
0の表面に、100メツシユの網点(面積率約20%)
を通してスクリーン印刷法でデンプンのりを固形成分3
g/I12の割合で付着させたものとラミネートし、乾
燥して第2非繊維多孔層とした。! ! LtLL: Gelatin 0.64 g7x2
Polyoxyethylene nonylphenyl ether (n=40) 2.527m"
Trishydroxymethylaminomethane 0.46 gem"S:yvi2hydrogen1 force!J'/mu0.46 gem
"L-ascorbic acid 2.5 g7a2
Magnesium chloride (anhydrous) 0.3 g/l peroxidase 6,400 U/, w2 flavin adenine dinucleotide 2867 m2 thiamine pyrophosphate 118 x11/Taki2 α-ketoglutarate 500 mg7x2 oxaloacetate decarboxylase 12,6001 J/x2 pyruvate oxidase 35 ,000 U/J12
(Aqueous solution with pH = 7.5) (Kumihiro” 11) Leuco dye (structure below) 1.8 g/β2
Polyoxyethylene nonylphenyl ether (n=40) 0.6 ge
m2 (ethanol solution) Dye: 2-(3,5-dimethoxy-4-hydrox
yphenyl)-4-pbenethyl-5-(4
-dimethylaminopl+e++yl)i+
1idazole Next, micro filter FM30 manufactured by Fuji Photo Film Co., Ltd.
100 mesh dots on the surface of 0 (area ratio approximately 20%)
Through the screen printing method, starch paste is added to the solid component 3.
It was laminated with the material deposited at a ratio of 12 g/I and dried to form a second non-fibrous porous layer.
次に10O8相当のPET紡績糸からなる厚さ約250
μ肩のトリコット編物生地を、第2非繊維多孔層上に上
記網点接着法により接着し、一体化させ、GOT定量定
量体型多層分析要素とした。Next, a thickness of approximately 250 mm made of PET spun yarn equivalent to 10O8
The μ shoulder tricot knitted fabric was adhered and integrated onto the second non-fibrous porous layer by the dot bonding method described above to form a GOT quantitative multilayer analysis element.
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22単位/l 400単位
71 800単位/4 1600単位/lの4種類の血
液を作成した。Next, porcine GOT (Sigma, USA) was added to fresh whole blood collected from humans (heparinized blood, hematocrit value 44%), and the GOT activity was 22 units/l 400 units 71 800 units/4 1600 units/l Four types of blood were created.
面層分析要素を1.5X1.5cm角に切断し、プラス
チックマウントに挿入し、これに上記4種類の血液を点
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2.5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(Clinica
l C1+emistry、 Vol、24゜1335
(1978)の原理による)を用いてGOT活性値を
算出した。結果を第1表に示す。The surface layer analysis element was cut into a 1.5 x 1.5 cm square, inserted into a plastic mount, and the four types of blood mentioned above were spotted on it. After the 0 point, the element was reacted at 37°C to measure the absorption at 640 nm from the PET support side. After 2.5 minutes and 4 minutes, reflectance measurement was carried out.
Value converted to 0Dt (transmission optical density) (Clinica
l C1+emistry, Vol, 24°1335
(1978)) to calculate the GOT activity value. The results are shown in Table 1.
第1表
[参考rIA]
組成物1からペルオキシダーゼ、オキザロ酢酸デカルボ
キシラーゼおよびピルビン酸オキシダーゼを除いた他は
、実施例1と全く同様にして、G O’r”に応答しな
い参照用要素を作製した。これに前記の血液N091を
点着し、37℃に4分放置後、波長640 n mでの
反射光学濃度をPF、T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。また参照用要素
に血清を点着して同様測定した。その結果は第2表の通
りである。Table 1 [Reference rIA] A reference element that does not respond to G O'r'' was prepared in the same manner as in Example 1, except that peroxidase, oxaloacetate decarboxylase, and pyruvate oxidase were removed from Composition 1. The blood N091 mentioned above was spotted on this, and after being left at 37°C for 4 minutes, the reflection optical density at a wavelength of 640 nm was measured from the PF and T support sides.The analytical element of Example 1 was also measured in the same way. .Serum was also spotted on the reference element and the same measurements were carried out.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
差はなく、また実施例の分析要素は背景に対し充分高い
光学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 a sufficiently high optical density relative to the background.
く出願人 富士写真フィルム株式会社〉手続補正書
1、事件の表示 昭和62年特願第67511号2)
発明の名称
全血分析要素及び全血の分析方法
3、補正をする者
事件との関係 特許出願人
住所 神奈川県南足柄市中沼210番地富士写真フィ
ルム株式会社 東京本社
電話 03(406) 2537
4、補正の対象
明細書の「発明の詳細な説明」の欄
5、補正の内容
明細書第21ページ第8行の「L−アスコルビン酸」を
「L−アスパラギン酸」と補正する。Applicant: Fuji Photo Film Co., Ltd. Procedural Amendment 1, Indication of Case: 1988 Patent Application No. 67511 2)
Name of the invention Whole blood analysis element and whole blood analysis method 3. Relationship with the case of the person making the amendment Patent applicant address 210 Nakanuma, Minamiashigara City, Kanagawa Prefecture Fuji Photo Film Co., Ltd. Tokyo Head Office Telephone: 03 (406) 2537 4. Amendment In column 5 of "Detailed Description of the Invention" of the subject specification, "L-ascorbic acid" in line 8 on page 21 of the specification of the contents of the amendment is amended to "L-aspartic acid".
以上that's all
Claims (6)
定成分に対し相互作用する組成物を含み、全血中の前記
成分を検出するための多層分析要素であつて、前記層の
うち少なくとも1つが全血球ろ過層であり、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 having at least two water-permeable layers, containing a composition that interacts with a specific component in whole blood, and for detecting the component in whole blood, wherein the layer comprises at least two water-permeable layers. a multilayer assay, wherein at least one of the layers is a whole blood cell filtration layer, and the interacting composition, when interacting with the analyte, is capable of producing a dye that can be detected spectrophotometrically at a wavelength of 600 nm or greater. element.
開層である多層分析要素。(2) The multilayer analytical element according to claim (1), wherein the outermost layer is a liquid spreading layer.
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
層と光反射層とを有する多層分析要素。(3) A multilayer analytical element according to claim (1), comprising a layer containing at least a portion of the interactive composition capable of producing the dye and a light-reflecting layer.
る水浸透性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
層との間に全血球ろ過層を有するか、前記液体展開層が
全血球ろ過層でもある多層分析要素。(4) In claim (1), one of the water-permeable layers included in the analytical element is a liquid spreading layer, and the other one is a portion of the interactive composition capable of producing at least the dye. a reagent layer comprising: a whole blood cell filtration layer between the liquid spreading layer and the reagent layer, or the liquid spreading layer is also a whole blood cell filtration 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全血球ろ
過層を有する多層液体分析要素に全血検体を接触させて
、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 containing a composition that interacts with the specific component in whole blood; and when the interacting composition interacts with the component, the interaction composition
a multilayer liquid analytical element capable of producing a dye spectrophotometrically detectable at wavelengths of m or more and having a layer comprising at least a portion of said interacting composition capable of producing said dye and a whole blood cell filtration layer; A detection method comprising the steps of: contacting a whole blood specimen to produce 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.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP6751187A JPS63233373A (en) | 1987-03-20 | 1987-03-20 | Element and method for analyzing complete 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 |
---|---|---|---|
JP6751187A JPS63233373A (en) | 1987-03-20 | 1987-03-20 | Element and method for analyzing complete blood |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS63233373A true JPS63233373A (en) | 1988-09-29 |
Family
ID=13347078
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP6751187A Pending JPS63233373A (en) | 1987-03-20 | 1987-03-20 | Element and method for analyzing complete blood |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS63233373A (en) |
-
1987
- 1987-03-20 JP JP6751187A patent/JPS63233373A/en active Pending
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