JPH01262470A - Dry process whole blood analysing element - Google Patents

Abstract

PURPOSE:To enhance the accuracy of a quantitative analysis by providing at least two water permeable layers including a reagent layer and porous development layer and incorporating sodium chloride or potassium chloride of a specific range into the porous development layer. CONSTITUTION:At least the water permeable reagent layer and porous development layer are provided to the title element and the sodium chloride or potassium chloride is incorporated in a 5-60g/m<2> range into the porous development layer formed of a fibrous or nonfibrous material. The disturbance by the red blood cells in whole blood is averted in the analyzing element and the diffusion of the components to be inspected in the blood plasma to the reagent layer is rapidly effected. The analysis is, therefore, executed with high accuracy regardless of the hematocrit value of the blood.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a dry chemical analysis element used for quantitatively analyzing a specific substance in blood.

[Prior art and its drawbacks] Various metabolic components present in body fluids, such as glucose,
and rilubin, uric acid, cholesterol, lactate dehydrogenase,
Quantitative analysis of creatine kinase, GOT, GPT, etc.
It is important in clinical medicine and is essential for understanding diseases, tracking the progress of treatment, and determining prognosis. In clinical chemistry tests using samples such as blood, it is desirable to be able to perform highly accurate tests with a small amount of liquid sample. Conventionally, wet methods using solution reagents have been widely used, but they lack speed. .

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 offers advantages over wet chemical analysis (ie, methods that use reagents in solution), such as ease of use, economic savings, and speed of analysis. Dry multilayer analysis elements were developed as an analysis tool that can quickly perform highly accurate tests on minute amounts of liquid samples. For example, the type 92 multilayer analysis element is
No. 677, JP-A-55-164356, JP-A-60-
It is known as No. 222789.

An example of a dry multilayer analytical element is composed of a transparent support, a reagent layer, a reflective layer, and a developing layer. Transparent support (
The reagent layer coated on top of PA (for example, a thin plastic film treated with an undercoat) contains a reagent that reacts with the test component contained in the liquid sample and develops a color with an optical density depending on the amount of the component. .

The reflective layer has the role of preventing the light incident on the reagent layer from reaching the spreading rM layer and preventing it from being affected by the liquid sample spotted on the developing layer during optical measurement of the reagent layer. , spread the spotted liquid sample uniformly over an area approximately proportional to the amount of liquid. For quantitative analysis using such a dry analytical element, a constant N drop of liquid sample f1, for example, whole blood, is dropped onto the surface of the developing layer. The blood developed in the development layer passes through the reflection layer and reaches the reagent layer, where it reacts with the reagent and develops a color.After reaching the 0 point, the chemical analysis slide is kept at a constant temperature for an appropriate period of time to ensure a sufficient color reaction. After this, the reagent layer is irradiated with illumination light from the transparent support side, the amount of reflected light is measured in a specific wavelength range to determine the reflection density, and quantitative analysis is performed based on a predetermined calibration curve.

Conventionally, in both wet and dry chemical analysis, analysis has often been performed using serum or plasma from which red blood cells have been removed as a sample. However, since the process of separating red blood cells from other blood components requires a lot of labor and equipment costs, it is desirable to be able to analyze undiluted whole blood.

In order to perform dry chemical analysis using whole blood as a sample, blood cells (erythrocytes and white blood cells) and other high molecular weight components of the whole blood must be separated by some means in the analytical element, as described in Japanese Patent Publication No. 53-21677. discloses providing a filtration layer to separate high molecular weight components of blood cells and whole blood in an analytical element.

However, as described in JP-A-60-111960, it takes a very long time to remove blood cell components from serum or plasma using a filtration layer provided in a dry analytical element. There was a risk that some of the analyte would be lost in the filtration layer, making one analysis inaccurate.

In the dry analytical element described in Japanese Patent Publication No. 61-61347, red blood cells in whole blood are separated and removed from plasma in a fibrous spreading layer provided on the upper surface of the analytical element, and a reagent for the test component in the plasma is removed. Diffusion into the layer occurs rapidly, making it suitable for analysis of specific components in whole blood samples. However, when performing whole blood analysis using this multilayer analytical element, the analysis results may vary considerably depending on the hematocrit value (volume percentage of blood cells in the blood), even if the plasma component content is the same. I experienced a difference.

Particularly when the hematocrit value of blood is high and the concentration of the component to be detected (analyte) is high, the measured concentration often shows a negative error relative to the true concentration in plasma.

[Technical Problems to be Solved] The present invention avoids interference caused by red blood cells in whole blood in the analysis element, and also allows rapid diffusion of test components in plasma into the reagent layer, so that whole blood sample A technical problem is to provide a dry analysis element that can analyze specific components in blood with high accuracy regardless of the hematocrit value of blood.

The technical challenge is to prevent negative errors in concentration measurements, which occur particularly when the concentration of the detected component (analyte) in blood is high.

[Means for Solving Technical Problems] The above object of the present invention has at least two water-permeable layers, the water-permeable layer includes a reagent layer and a porous spreading layer, and the reagent layer includes a porous spreading layer. at least one of the water-permeable layers includes a reagent composition capable of producing an optically detectable substance in the presence of the analyte component. This problem was solved by a dry analytical element used for quantitative analysis of specific components in blood from which red blood cells have not been removed, and which contains at least 5th" of sodium chloride or potassium chloride in the porous developing layer.

The above-mentioned problem of the present invention is particularly aimed at the porous spreading layer of the analytical element.
This was preferably achieved when containing 0 to 60 g/m2 of sodium chloride or potassium chloride.

The dry analytical element does not need to have a support, but it is preferable to have the water-permeable layer on a water-impermeable transparent support, or to have the water-permeable layer on a water-permeable support. May have.

[Details of Specific Structure of the Invention J The dry analytical element of the present invention has at least a reagent layer and a porous spreading layer, each of which is permeable to water.

It may have a water permeable layer other than these two layers, or it may have another water permeable layer between the reagent layer and the porous development layer.

The porous spreading layer which receives the liquid sample to be analyzed is preferably a layer with liquid metering action.

Liquid metering action is an action that spreads a liquid sample dotted onto the surface at an approximately constant rate per unit area in the direction of the surface without substantially unevenly distributing the components contained therein. It is. The porous spreading layer may be either fibrous or non-fibrous.

A reagent composition is one that is capable of producing an optically detectable substance, such as a dye, within 1 m in the presence of the analyte.

Preferably, the reagent composition is at least partially contained in the reagent layer.

Sodium chloride or potassium chloride used in the present invention belongs to alkali metal hydrochlorides, but not all alkali metal hydrochlorides are suitable for the present invention. 8 It is appropriate that the porous developing layer of the dry analytical element used in the present invention contains 5 to 60 g/v *". 20 to 60 g/m". is preferable.

The dry analysis element used in the present invention can have various configurations. For example, U.S. Pat.
No. 60-279860 and No. 60-279861 can be referred to. When using a light-transmitting support,
Practical configurations of the dry analytical element used in the present invention are, for example: (1) y: drug layer on the support, liquid spread on it! '5F1
(2) A support that has a detection layer, a reagent layer, and a liquid development layer in this order; (3) A support that has a reagent layer, a light reflection layer, and a liquid development layer in this order. , (4) Support L has a detection layer, a reagent layer, a light reflection layer, and a liquid developing layer in this order. (5) A detection layer, a light reflection layer, a reagent layer, and a liquid on the support! E
(6) a second reagent layer, a light reflection layer, a first reagent layer on the support;
It has development layers in this order. A water absorption layer may be provided between the support and the second reagent layer.

(7) It may have a second reagent layer, a first reagent layer, and a developing layer in this order on the support, or it may have a water-absorbing layer between the support and the second reagent layer.

is useful (the support may have an undercoat layer)
The detection layer is a layer in which a dye or the like generated in the presence of a test component diffuses and can be optically detected through a light-transmitting support, and can be composed of a hydrophilic polymer.

For example, a cationic polymer may be included for an anionic dye.

Preferred in the present invention is the layer configuration (3) above.

In (1), (2) or (7) above, the reagent layer and the expansion 1
A protein blocking layer may be provided between the fFI layer or the detection layer, and in (3) to 6) above, the light reflection layer, the detection layer, and the reagent layer seem to be ff! A protein blocking layer may be further provided between the opening layer, between the reagent layer and the detection layer, or between the reagent layer and the opening 1fMM4.

In (1), (3) or <7), a water absorption layer may be provided between the support and the reagent layer, and the water absorption layer generally includes:
A layer in which a dye produced in the presence of a test component does not substantially diffuse, and can be composed of a hydrophilic polymer that easily swells.

In (6) or 7) above, a protein blocking layer is provided between the first reagent layer and the developing layer, between the second reagent layer and the first reagent layer, or between the light reflective layer and the first or second reagent layer. may be provided.

A preferred material for the water-impermeable transparent support is polyethylene terephthalate. In order to firmly adhere the tf1 aqueous layer, which may be a cellulose ester such as cellulose triacetate, an undercoat layer is usually provided, or
Perform hydrophilic treatment.

Reagent compositions include compositions capable of producing optically detectable substances, such as dyes, in the presence of the analyte; compositions producing dyes by oxidation of leuco dyes (for example, Patent No. 4,089,747, Japanese Patent Publication No. 5119:1
352, etc.), diazonium salts, and compositions containing compounds that produce dyes by coupling with other compounds when oxidized (e.g., 4-aminoantipyrines and phenols). or naphthols), reduction! t'! Those consisting of compounds capable of producing a dye in the presence of a coenzyme and an electron transfer agent can be used. In addition, in the case of an analytical element for measuring enzyme activity, a self-developing substrate capable of releasing a colored substance such as p-21 to oral phenol can be included in the reagent layer or rfIt open layer. .

The reagent composition may be contained entirely in one water-permeable layer, such as a reagent layer, or may be contained separately in separate layers. A composition may be included in a reagent layer, and a composition (indicator) that can react with an intermediate to produce a dye or the like may be included in a second reagent layer between the reagent layer and the support to produce an intermediate. The composition may be included in the hemocyte filtration layer or light reflective layer and in the indicator reagent layer. a gas permeable layer between the first reagent layer and the second reagent layer;
An obstruction removal layer (as in U.S. Pat. No. 4,068,403, Lee, No. 58-19062) may be provided;
These layers may be water impermeable if desired.

The reagent composition may be comprised, in part or in whole, in a substantially uniform layer with a hydrophilic polymer as a binder, such as gelatin and derivatives thereof (e.g. phthalated gelatin); Cellulose derivatives (for example, hydroxyethylcellulose), agarose, acrylamide polymer a, methacrylamide polymers, copolymers of acrylamide or methacrylamide with various vinyl monomers, etc. can be used.

A part or all of the reagent composition may be contained in the porous layer. As the reagent layer, a fibrous porous layer such as filter paper or non-silk cloth may be used, but a non-woven porous layer may also be used. can. Non-discussion; ``As a porous layer,
Layers of plush polymers consisting of cellulose esters such as cellulose acetate, cellulose acetate/butyrate, cellulose nitrate are preferred, as described in US Pat.
Japanese Patent Publication No. 62-27006 and Special Publication No. 63-1045
A microporous membrane made of polysulfone as described in No. 2 is also suitable. Others: Special Publication No. 53-21677, Japanese Patent Publication No. 5
A porous layer containing continuous voids in which small polymer particles, glass particles, diatom-F, etc. described in Japanese Patent Application No. 5-90859 are bonded with an H1 aqueous or non-water-absorbing polymer can also be used.

In order to have a porous layer contain a reagent composition that produces color development in the presence of a test component, a porous spreading layer pre-impregnated or coated with a suitable solution or dispersion of the reagent composition is coated with another water-permeable layer. JP-A No. 55164356 on a part of the layer, for example, the reagent layer.
A method of adhesion as shown in No. 2 is also useful. After adhering the porous layer to other water-permeable layers (e.g., undercoat layer, adhesive layer, water-absorbing layer) by the method described in the above-mentioned Tokoku M1 1984-184:156, the reagent composition is Solution or Molecule11. The liquid may be applied to the porous layer.

Known methods can be used to impregnate or apply the reagent composition or a portion thereof to the porous layer. For example, dip coating, doctor coating, hopper coating, curtain coating, etc. are selected and used as appropriate.

After coating a uniform layer containing a reagent composition using a hydrophilic polymer as a binder, a porous layer containing no reagent composition was coated.
By adhering using a method such as that disclosed in No. 5-164356, the reagent composition can be substantially contained in the porous layer.

The reagent may contain an activator, a hardening agent, a surfactant, etc., if necessary.

V that can be contained in the reagent layer of the analytical element of the present invention
Examples of lvr agents include carbonates, borates, phosphorus
Examples include the yi street agent of Gutsudo ((:ood) described in 2 Ning and 1liocbc import 1sLry magazine No. 5, No. 2, pages 467 to 477 (1966). ``Basic Experimental Methods for Proteins and Enzymes'' (Takeshi Horio et al., Hokoedo, 1981)
The selection can be made by referring to Bunju, such as the above-mentioned Biocbemistry, Vol. 5).

The amount of each component or reagent in the aforementioned reagent compositions can vary widely depending on the analyte being measured. These amounts can be readily determined by those skilled in the art.

The reagent composition may contain an enzyme, and the reagent composition may contain an enzyme.
Those described on pages 18 to 20 of the specification of No. 79859 can be used. The reagent composition may include a component that generates fluorescence or decreases fluorescence in the presence of the component to be detected.

lJ! Materials constituting the fiber porous development layer include 1 paper, nonwoven fabric, and a! A fabric (for example, plain weave fabric), a knitted fabric (for example, tricot knit), glass fiber paper, etc. can be used. Among these, woven fabrics, knitted fabrics, etc. are preferable.
Il materials, etc. may be subjected to glow discharge treatment as described in JP-A-57-66359.For the spreading layer, in order to adjust the spreading 1m surface area, spreading speed, etc., JP-A-60-22277 may be applied.
No. 0, Patent Application No. 61-122875, 61-12287
It may contain a hydrophilic polymer or a surfactant as described in No. 6, No. 61-143754.

When using a non-fibrous porous layer as a spreading layer,
6-nylon, 6-nylon, 6-nylon, 6-nylon, 6-nylon, etc. .6-A microporous membrane of polyamide, polyethylene, polypropylene, etc. with a nylon cap may be used.
A microporous membrane made of polysulfone as described in JP-A No. 62-27006 may also be used;
A porous layer having continuous voids in which small polymer particles, glass particles-1, etc. are bonded with a hydrophilic or non-water-absorbing polymer as described in Japanese Patent Application No. 77, No. 11, 1983-90859, etc. can also be used.

An adhesive layer for adhering and laminating the porous layer may be provided on the support, undercoat layer, water absorption layer, detection layer, etc. The adhesive layer adheres the porous layer when swollen with water. It is preferable to use hydrophilic polymers such as gelatin, gelatin derivatives, polyacrylamide, starch and the like.

Adhesives can be used to secure the semiporous layer over other porous or non-porous layers in such a way that uniform transmission of the liquid and the component to be detected is substantially unhindered. There is a need. To do this, the adhesive is placed partially so that minute d-connections are formed in the areas where the adhesive is not present. As a method for this purpose, the method described in JP-A-62-138756 is useful.

The light reflection/shielding layer detects the red color of hemoglobin in red blood cells in whole blood when measuring detectable changes (color change, color development, etc.) that occur in the detection layer, reagent layer, etc. from the light-transmitting support side. It also functions as a light reflective layer or background layer. A non-porous layer or a porous layer containing light-reflecting particles such as titanium oxide or barium sulfate dispersed using a hydrophilic polymer as a binder can be used. As the binder, gelatin, gelatin derivatives, polyacrylamide, etc. are preferable.As the light reflecting/shielding layer, Japanese Patent Application No. 1986-9
Also useful are microcapsules containing light-reflecting particles as described in No. 046.

In order to analyze blood containing red blood cells using the analytical element of the present invention, after applying a sample liquid of approximately constant ji (for example, 3 μl to 30 μl) onto the dry analytical element, place it in an appropriate thermostat or for colorimetric measurement. One analytical method involves optical measurement after incubation (warming) at a certain temperature for a certain period of time in a machine, or at certain time intervals during that time. Either a method of detecting an optical change near the target end point or a method of measuring the reaction rate from the rate of color development during the reaction may be used.

For spectral reflection density measurement, known suitable devices are used, such as U.S. Pat. No. 4,488,810, U.S. Pat. : No. 16, No. 82-198736, No. 62-245141 to No. 245143, Japanese Patent Application No. 61-25582, No. 61-25583, No. 61-109402, No. 61
-144258, 61-185471, 61-
No. 185472, No. 61-207044 to 2070
The equipment described on the cap up to No. 49, "Fuji Drychem 1000.", [Fuji Drychem 200.
0J, 'Fuji Drygem 5000 J Analyzer (all manufactured by Fuji Photo Film Co., Ltd.), 'lEkLachem 400J, rεkta
chem 700J,' E ktachem
A DT-604 analyzer (all manufactured by Eastman Kodak Company) or the like can be used.

The analytical element of the present invention can also be used for stereotyping of antigens or antibodies by immunological methods by containing at least one of an antigen and an antibody in the porous layer.

Effect of the Invention 1 The analytical element of the present invention uses whole blood as a sample, and the blood hematocrit value ranges over a wide range from 25% to 70%, and analysis values for various test components that are relatively independent of the hematocrit value can be obtained. Obtainable. In particular, when using whole blood samples with hematocrit values in a wide range of 25% to 7026, the measured value should be such that the difference from that of whole blood with hematocrit values of 40% to 50% can be practically ignored. Obtainable.

The present invention is applicable not only to the determination of low-molecular components such as glucose, urea, uric acid, and creatinine in whole blood, but also to the determination of high-molecular components such as total protein, albumin, various enzymes, and components bound to proteins such as bilirubin. 8 Applicable analytes The present invention is suitable for quantifying analytes whose concentrations inside and outside blood cells are equal, but whose concentration in plasma is about 1/3 to 3 times that in blood cells. It is particularly useful when sugars such as glucose are contained in high concentrations.

[Example 1] Box 1 (1) Support, second reagent layer The following composition was made into an aqueous solution on a gelatin-subbed, colorless and transparent polyethylene terephthalate (PET) smooth film with a thickness of 180 μm, and the following composition was applied as shown below. Apply it so that it becomes
Dry (second trial layer).

(Second reagent R4 coating liquid) Gelatin 6.0 g/m"
1.7-SHIHIF O-'r SI+ 791/ N0.2
2 g/524-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one 0.40
gem' (2) First Reagent Layer: On the second reagent layer, apply the following composition as an aqueous solution and dry it to the following coating amount (first reagent layer).

(First reagent layer coating solution) Gelatin 6.9 Bi/Rin 2
Peroxidase 23800 tlNIT
/m2 glucose oxidase 10100 UN
IT/+m'styrene/p! (1-methyl-1-piperazino)methyl) styrene/7-divinylbenzene copolymer 2.4 g/brass 2 Polyoxyethylene (40H diethylene units) nonylphenol 0.60 g/brass 2 (3
〉Light reflection/shielding layer The following composition was applied as a water solution to the following amount of N on the first leakage layer and dried to form a light reflection/shielding layer.

(Light reflection/shielding layer coating liquid) Titanium dioxide 8.211/m2
Gelatin 0.81 s/bell 2
Polyoxyethylene nonylphenol (401 xylene ethylene units)
0.23 g/m2 (4> Adhesive Layer An aqueous solution of the following composition was applied onto the optical path MHI to the following T'1M and dried to form an adhesive layer.

<Contact? Layer coating liquid) Gelatin 1.5 Harm
"Polyoxyethylene (40 ethylene units) nonylphenol 0.22 g/I-2 calcium acetate 1.0.52 g/2
(5) Spreading layer Next, the following composition was applied to the entire surface of the adhesive layer at a rate of about 30 g/m2 at 15°C and moistened, and then a tricot fabric made of polyester thread was attached to it, and passed through a laminating roll. Dry.

Water 97.8 gN-(pyrrolidinochloromethyl)pyrrolidinium-β-naphthalenesulfone #2.0g Polyoxyethylene (4
04~di-engineered tyrene unit) nonylphenol
0.2g? L [14 ml of sodium chloride on the cloth]
26 applied as an aqueous solution to a coating amount of 42 g/m',
After drying, an analytical element for glucose determination [11] was completed.

For comparison, a sample was also prepared in which the above-mentioned application to cloth was omitted and used as analysis element [2].

Intoxication1 GlucoseGlucose concentration in fresh whole blood collected from humans is 540.
Add the required amount of glucose so that the ratio is xi/J1, leave it for 30 minutes at room temperature, and then centrifuge a portion and mix the blood cells and plasma appropriately until the hematocrit value is 15.25.40.
．． 55% whole blood was prepared.

Their exact glucose amount was measured using glucose electron method. Analytical elements [1] and [2] prepared above
Four kinds of whole blood (IQ) t1 each were placed on the spreading layer, and after incubation at 37°C for 6 minutes, reflection photometry was performed from the support side using visible light with a center wavelength of 540 nm.
The reflected optical density of the analytical element was measured. Glucose concentrations in four types of sample dishes were measured using calibration curves prepared in advance for each analytical element using standard blood obtained by adding various amounts of glucose to whole blood with a hematocrit value of 40%. The results are shown in Table 1.

Table 1 As is clear from Table 1, the analysis element according to the present invention [1
] has much less influence on the hematocrit value than the comparative analysis element [2], and even for whole blood with a hematocrit of less than 40% or more than 50%, the hematocrit value of 1
Gives glucose concentration measurements that are about the same or very similar to 0°6 whole blood.

[Real a13'12] 4-Amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one of Example 1 (0,40 g/s+")
4-amino-2-methyl-3-phenyl-1 instead of
An analytical element was prepared in the same manner as in Example 1, except that -(2,4,6-)dichlorophenyl-3-pyrazolin-5-one (0.40 g/gu 2) was used.

Claims (7)

[Claims] (1) It has at least two water-permeable layers, the water-permeable layer includes a reagent layer and a porous spreading layer, and the reagent layer is provided on the side opposite to the liquid-receiving surface of the porous spreading layer. and at least one of the water-permeable layers includes a reagent composition capable of producing an optically detectable substance in the presence of the analyte component.
A dry multilayer analytical element used for quantitative analysis of specific components in blood including red blood cells, characterized in that the porous development layer contains at least 5 g/m^2 of sodium chloride or potassium chloride. (2) Claims containing 20 to 60 g/m^2 of sodium chloride or potassium chloride in the porous spreading layer (
1) Analysis element. (3) The analytical element according to claim (1), which is used for analyzing blood containing 25% by volume or more of red blood cells. (4) The analytical element according to claim (1), which is used for quantifying non-hydrophobic components in blood. (5) Claims (1) for use in quantifying sugars in blood
) analytical elements. (6) The analytical element according to claim (1), which is used for determining glucose in blood. (7) The analytical element according to claim (1), comprising at least two water-permeable layers on a water-impermeable transparent support.