JPS63233373A - Element and method for analyzing complete blood - Google Patents

Abstract

PURPOSE:To enable the rapid quantification of a substance to be analyzed in a complete blood by a method wherein at least one of water-permeable layers is formed of a complete blood filtering layer and a composition of mutual action is produced by a dye detected at a wavelength of 600mn or above by a spectrophotometric method and on mutual action with the substance. CONSTITUTION:A liquid developing layer formed of textile or the like being used a complete blood sample is made to contact an analyzing element, and thereby a dye which can be detected by a spectrophotometric method is produced at a wavelength of 600nm or above. The dye is detected at the wavelength of 600nm or above. Since the dye produced by the mutual action with a substance to be analyzed is detected at the wavelength of 600nm or above by the spectrophotometric method, no disturbance by hemoglobin or bilirubin takes place. Accordingly, the substance to be analyzed in a complete blood can be quantified rapidly.

Description

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.

[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.

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.

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.

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.

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.

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).

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.

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.

However, if the pore structure is too rough, the element becomes mechanically unstable (destruction).

[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.

[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.

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.

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.

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.

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.

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.

[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. .

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.

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.

It can also be constructed using non-fibrous isotropic porous materials (eg, plush polymers) as described in US Pat. No. 3,992,158.

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.

In the developing layer, in order to adjust the developing area, developing speed, etc., there are
No. 5.

It may contain a hydrophilic polymer or a surfactant as described in Nos. 81-122878 and 61-143754.

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.

The interaction composition described above depends on the analytical reaction system chosen. Useful interactive compositions include, for example, substances with dehydrogenase activity.

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.

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.

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.

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.

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.

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.

Useful color formers include dihydroindoles, tetrahydroxyquinolines, and 1,7-dihydre.

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.

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.

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.

Alternatively, a compound in which a substituent that can form a dye molecule is bonded to an oligosaccharide can also be used.

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.

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) A device having a detection layer, a reagent layer, and a liquid development layer in this order on a support.

(3) A support having a reagent layer, a light reflection layer, and a liquid development layer in this order.

(4) A support having a detection layer, a reagent layer, a light reflection layer, and a liquid development layer in this order.

(5) A support having a detection layer, a light reflection layer, a reagent layer, and a liquid development layer in this order.

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.

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.

The analytical element of the invention may have a light reflective layer.

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.

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.

After applying the sample whole blood, incubation (heating) can be carried out on the element in order to obtain test results quickly or accurately.

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.

Examples will be described below to further specifically explain the present invention.

[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.

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.

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: 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.

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.

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.

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.

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.

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.

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".

that's all

Claims (6)

[Claims] (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. (2) The multilayer analytical element according to claim (1), wherein the outermost layer is a liquid spreading layer. (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. (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. (5) The multilayer analytical element according to claim (4), wherein the reagent layer contains all of the interaction composition. (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.