JP2614124B2 - Integrated multilayer analytical element - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a dry integrated multi-layer analytical element for quantitative analysis of biological components, for example, blood components by biochemical analysis or immunoassay. is there.

[Prior art and its disadvantages] Various metabolic components present in body fluids, especially blood, such as glucose, bilirubin, BUN (urea nitrogen), uric acid, cholesterol, LDH (lactate dehydrogenase), creatine kinase, and ALT (alanine) Aminotransferase), AST
Quantitative analysis of (aspartate aminotransferase) is important in clinical medicine and is indispensable for diagnosis and confirmation of disease, follow-up of treatment course, and judgment of prognosis. In a clinical chemistry test using a body fluid such as blood as a sample, it is desirable that a highly accurate test can be performed using a small amount of a liquid sample. Conventionally,
Although wet analysis using a solution-type analysis reagent is widely used, it tends to lack rapidity. Dry analysis, ie, a test piece containing a biochemical or immunoassay reagent in a substantially dry state, a non-integrated multilayer analytical element, or an integrated multilayer analytical element (these are sometimes collectively referred to as dry analytical elements) And other clinical analysis methods have been proposed and put to practical use. The dry analysis is superior to the wet analysis in terms of, for example, the simplicity of the analysis operation, the speed of the analysis, the low overall cost, and the like.

The dry-type integrated multi-layer analytical element was developed as an analytical means that can quickly perform high-precision tests on a small amount of liquid sample. The integrated multi-layer analysis element is disclosed in JP-A-49-53888.
(JP-B-53-21677), JP-A-55-164356, JP-A-60-22
It is known as 2769 mag. One example of the integrated multilayer analysis element is a structure in which a reagent layer, a light reflection layer, and a spreading layer are laminated and integrated in this order on a transparent support. The transparent support is
For example, a thin transparent organic polymer sheet. The reagent layer coated on the transparent support contains a reagent composition that reacts with the analyte (analyte) contained in the liquid sample and develops or changes color to an optical density corresponding to the amount. It is. The light reflecting layer mainly has a role of preventing the influence of the color of the liquid sample spotted on the developing layer (eg, red blood cells contained in whole blood) when measuring the color optical density of the reagent layer. In the developing layer, the spotted liquid sample is uniformly spread over an area approximately proportional to the volume of the liquid and supplied to the reagent layer. To perform quantitative analysis using such a multilayer analysis element, a liquid sample, for example, about 5 μL to about 20 μL of whole blood is spotted on the surface of the developing layer. The blood spread in the developing layer passes through the light reflecting layer and reaches the reagent layer, where it reacts with the reagent, and develops a color or discoloration. After the liquid sample has been spotted, the color analysis or discoloration reaction after the multi-layer analysis element has been kept at a constant temperature (eg, 37 ° C, 6 minutes) for a suitable period of time to allow the color development reaction or color change reaction to proceed sufficiently (the end point method ) Or the rate of change in the optical density of color development or color change that occurs with the progress of the color development or color change reaction (velocity method)
Irradiates the reagent layer with light from the transparent support side, measures the amount of reflected light in a specific wavelength range to determine the reflected optical density or its change rate, and calculates the reflected optical density or the rate of change based on a previously determined calibration curve. The content of the test component is determined by converting the content of

Recently, an integrated multi-layer analysis element has been devised, and it has become possible to use undiluted whole blood as a blood sample. For example, Japanese Unexamined Patent Publication No. Sho 57-66359 discloses a fabric whose surface is physically activated as a spreading layer capable of uniformly spreading a whole blood sample. A method for removing blood cell components from whole blood by a filtration layer provided on an analysis element is described. Also, in order to change the polarity of the red blood cell surface in a whole blood sample and thereby coagulate the red blood cells in order to remove blood cell components efficiently by filtration,
A method for containing an erythrocyte retention substrate consisting of a strongly polar group linked by a linking group serving as a spacer is disclosed in
961. However, this method requires the passage of a whole blood sample over a relatively long distance through a filter medium carrying a retention substrate to filter blood cells.
Moreover, many of the exemplified retention substrates have a tendency to cause hemolysis.

An integrated multilayer analytical element capable of separating blood cell components more efficiently and accurately is disclosed in JP-A-62-138756, JP-A-62-138757,
It was proposed in JP-A-62-138758. This multi-layer analysis element
On the support, a fibrous microporous layer, a non-fibrous microporous layer, a second fibrous or non-fibrous microporous layer are integrally laminated in this order from a side far from the support, The three microporous layers are substantially formed by an adhesive partially arranged so as to form minute penetration portions between adjacent interfaces so that uniform passage of liquid is not substantially impeded. Is a multi-layer analytical element that is adhered to and integrated with
The reagent composition that causes color development or discoloration is contained in any of the three microporous layers, and the non-fiber microporous layer has an average effective pore size of 0.8 μm to 30 μm. However, when analyzing whole blood using this multi-layer analysis element, the separation and removal of blood cell components is still insufficient, and the permeation of plasma into the reaction layer depends on the hematocrit value of the blood (percentage of blood cells in the blood). It was found that the erythrocyte hemolysis occurs depending on the condition of the whole blood sample and the analysis results differ even if the blood contains the same test component in the plasma depending on the condition of the whole blood sample.

In the case of clinical chemistry analysis, whether wet analysis or dry analysis has been used,
In order to make the detection reaction reproducible and stable, a pH buffer has been added to the sample to substantially eliminate the change of the pH value and to keep the pH value constant. When performing dry analysis using an integrated multi-layer analytical element, the pH buffer is contained in the developing layer or the reaction layer containing the reagent composition that reacts with the test component, and is dissolved in the spotted whole blood sample. Thus, the pH around the detection reaction is stabilized. There is no particular limitation on the pH buffer used when analysis is performed using plasma or serum from which red blood cells have been removed as in the past, but in the case of whole blood samples, depending on the type of pH buffer, red blood cells may not be used. Hemolysis was found to occur and the importance of the choice of pH buffer was recognized.

When a whole blood sample is used, an attempt to improve the fluctuation of the analysis result due to the hematocrit value of blood is disclosed in Japanese Patent Application No. 1-30408.
(JP-A-3-16651). This analytical element has a fibrous microporous layer, a non-fibrous microporous layer, and a second fibrous layer which are integrally laminated on a support in this order from a side far from the support. The microporous layer is substantially adhered and adhered by an adhesive partially arranged so as to form a micro penetration portion so that uniform passage of liquid is not substantially hindered between adjacent surfaces. This is a multi-layer analysis element that has been integrated. However, when performing whole blood analysis using this multi-layer analysis element, the hematocrit of blood
It was found that the quantitative analysis value of the test component in the blood plasma decreased from 55% to 60% (blood with the same test component content but blood with a hematocrit value of 60%) decreased. Further, since the second microporous layer is fibrous, the color optical density of the detection layer is lower (lower sensitivity) than in the case of non-fibrous layer.
Was also found.

According to the present invention, the pH value of the liquid component corresponding to plasma or serum (of undiluted whole blood) is about 6.5 to about even when the blood sample to be spotted is diluted with another liquid, for example, physiological saline. It is based on the new finding that red blood cells do not lyse when a diluted blood sample passes through the microporous layer of a multi-layer analytical element if it has a hydrogen ion concentration value in the range of 9.5. It is.

[Problems to be Solved by the Invention] An object of the present invention is to rapidly diffuse a test component in plasma to a reagent layer and to convert a test component to be measured in a whole blood sample into blood. An object of the present invention is to provide an integrated multi-layer analytical element capable of performing quantitative analysis with high sensitivity and high accuracy regardless of the hematocrit value. In addition, to perform quantitative analysis that separates and removes red blood cells in whole blood from plasma without causing hemolysis in an integrated multi-layer analytical element to avoid optical interference and chemical interference during measurement of color optical density by red blood cells. It is also to provide an integrated multi-layer analysis element that can be used.

Other objects of the present invention are described in Japanese Patent Application Laid-Open Nos. 62-138756 and
-30408 (Japanese Unexamined Patent Publication (Kokai) No. 3-16651). Partially arranged so that a minute penetration portion is formed between three adjacent microporous layers so as not to prevent uniform passage of liquid. An object of the present invention is to further improve an integrated multi-layer analytical element which is adhered and adhered substantially in close contact with an adhesive (this adhesion is called partial adhesion or porous adhesion).

[Means for Solving the Problems] The above problems have been solved by any of the following two embodiments.

In two embodiments, each of the integrated multilayer analytical elements comprises at least one reagent layer containing a coloring reagent composition, a non-fibrous microporous layer, and an uppermost fibrous microporous layer on a support. Are laminated in this order, or at least one reagent layer containing a coloring reagent composition, a fibrous or non-fibrous microporous layer, a non-fibrous microporous layer on a support. An integrated multi-layer analytical element in which a first layer and a fibrous microporous layer as the uppermost layer are laminated in this order, wherein the two or three microporous layers are adjacent to each other and the interface thereof is partially adhered. Integrated multi-layer analysis element. The reagent layer is at least one substantially non-porous reagent layer containing a hydrophilic polymer binder and a color reagent composition or at least one fibrous or non-fibrous microporous layer containing a color reagent composition. Of any one of the following reagent layers.

A first aspect is that at least the fibrous microporous layer serving as the uppermost layer contains a pH buffer which shows a hydrogen ion concentration value in the range of 6.5 to 9.5 when dissolved in water. This is an integrated multi-layer analysis element characterized by the features (first invention).

A second embodiment is that at least the tertiary amine and / or quaternary ammonium salt and hydrogen ions having a pH of 6.5 to 9.5 when dissolved in water are added to at least the uppermost fibrous microporous layer. An integrated multi-layer analytical element characterized by containing a pH buffer showing a concentration value (second invention).

[Detailed Description of Means for Solving the Problems] The feature of the integrated multilayer analytical element of the present invention is that at least two microporous layers adjacent to each other (the two microporous layers are made of fibrous or A non-fibrous material may be used, but a preferable arrangement is such that a minute penetration portion is formed between the adjacent interfaces of the non-fibrous material and the fibrous material from the support side to such an extent that uniform passage of liquid is not substantially impeded. A composite microporous layer which is substantially adhered and adhered by an adhesive partially disposed so as to be bonded to the uppermost layer (the outermost layer farthest from the support) In the microporous layer, a solution was added to the whole blood sample that had been spotted and the whole blood (or separated plasma) had a pH value of about 6.5 to
A pH buffer having a pH value of about 9.5 ”(first invention);
It contains an amine and / or a quaternary ammonium salt (second invention).

The integrated multilayer analytical element of the present invention has at least two adjacent microporous layers, for example, on a support,
In order, (1) a coloring reagent layer, a non-fibrous microporous layer, a fibrous microporous layer forming the uppermost layer, (2) a water absorbing layer, a coloring reagent layer, a nonfibrous microporous layer, and a top layer Fibrous microporous layer, (3) detection layer, color reagent layer, non-fiber microporous layer, fibrous microporous layer forming the uppermost layer, (4) color reagent layer, lower fibrous microporous layer Functional layer, non-fibrous microporous layer, fibrous microporous layer as top layer, (5) water-absorbing layer, color reagent layer, lower fibrous microporous layer, non-fibrous microporous layer, top layer (6) a detecting layer, a reagent layer, a lower fibrous microporous layer, a non-fibrous microporous layer, and an uppermost fibrous microporous layer. I have.

The support is not essential when the laminated color reagent layer, the non-fibrous microporous layer, and the uppermost fibrous microporous layer are self-supporting, or the support is composed of a plurality of these. The layer may be peelable from the layer, but an embodiment in which a plurality of these layers are adhesively laminated on a support is preferable. In an embodiment having a support, the support is preferably light-transmissive (transparent) and water-impermeable. Preferred materials for the water-impermeable light-transmitting support are polyethylene terephthalate, polystyrene and polycarbonate of bisphenol A. Cellulose esters such as cellulose triacetate may be used. In order to firmly adhere the hydrophilic layer, the support is usually provided with an undercoat layer or subjected to a hydrophilic treatment on the surface. The detection layer is generally a layer in which a dye generated in the presence of a test component diffuses and is optically detected through a light-transmitting support, and can be composed mainly of a hydrophilic polymer. A mordant (eg, a cationic polymer for an anionic dye) may be included. The water-absorbing layer generally refers to a layer in which a dye generated in the presence of a test component does not substantially diffuse into the inside of the layer, and can be composed mainly of a hydrophilic polymer that easily swells. The reagent composition includes a substance capable of forming a substance that can be optically detected, for example, a dye, in the presence of the test component. A composition containing a leuco dye that produces a dye by oxidation (eg, triarylimidazole leuco dye described in U.S. Pat. No. 4,089,747, diarylimidazole leuco dye described in JP-A-59-193352, etc.); diazonium salt, oxidized Compositions containing a specific chromogenic compound and a coupler compound that sometimes produces a dye upon coupling (eg, a combination of 4-aminoantipyrines (chromogenic compounds) and phenols or naphthols (coupler compounds)); reduction A composition or the like comprising a compound capable of forming a dye in the presence of a type coenzyme and an electron transfer agent can be used. In the case of an analytical element for measuring enzyme activity, for example, p
A self-developing substrate capable of releasing a colored substance such as -nitrophenol or p-nitrophenyl phosphate can be included in the reagent layer or the non-fibrous microporous layer as a coloring reagent.

In order to include a reagent composition that produces a dye and develops a color or a reagent composition that changes color in the presence of the test component in at least one of the microporous layers, an appropriate solution or dispersion of the reagent composition is impregnated in advance. Alternatively, a method in which the applied microporous spreading layer is bonded to another water-permeable layer, for example, a detection layer or a water-absorbing layer by the method described in JP-A-55-164356 is also useful.

After adhering the microporous layer onto another water-permeable layer (for example, an undercoat layer, an adhesive layer, or a water-absorbing layer) by the method described in JP-A-55-164356, a solution or dispersion of the reagent composition is prepared. Can be applied to the microporous layer. Known methods can be used for impregnation or application to the microporous layer. The coating is appropriately selected from known coating methods such as dip coating, extrusion coating, doctor coating, curtain coating and the like. The reagent composition may be entirely contained in one microporous layer, or may be separately contained in a plurality of microporous layers for each component. All reagent compositions may be contained in multiple microporous layers. Part or all of the reagent composition may be contained in a substantially uniform layer (reagent layer) provided with a hydrophilic polymer as a binder and provided closer to the support than the microporous layer. Examples of hydrophilic polymers include gelatin, gelatin derivatives (eg, phthalated gelatin), cellulose derivatives (eg, hydroxypropylcellulose), agarose, polyacrylamide, polymethacrylamide, acrylamide or copolymers of methacrylamide and various vinyl monomers. Can be used. After applying a uniform layer containing a reagent composition having a hydrophilic polymer as a binder, the fibrous microporous layer containing no reagent composition is adhered by the method described in JP-A-55-164356. The composition can be substantially contained in the microporous layer. The reagent composition may contain an enzyme activator, a cross-linking agent, a surfactant, and the like, if necessary. PH that can be contained in the reagent layer
The buffer does not have to be the same as the pH buffer contained in the top layer; rather, the pH value or components should be selected so that they are adjusted to a pH value suitable for chemical or biochemical reactions in the reagent layer. Can be.

JP-B-53-21677, U.S. Patent No. 14 as a non-fiber microporous layer
Preferred is a microporous membrane (membrane filter, brush polymer layer) comprising cellulose esters (eg, cellulose acetate, cellulose acetate butyrate, cellulose nitrate) described in 21341 and the like. Microporous membranes such as polyamides such as 6-nylon and 6,6-nylon, polyethylene, polypropylene and polysulfone described in JP-A-62-27006 can also be used. Other Special Publication No. 53-
21677, Japanese Patent Application Laid-Open No. 55-90859, and the like can also use a microporous layer having continuous voids in which polymer fine particles, glass fine particles, diatomaceous earth, and the like are bonded by a hydrophilic or non-water-absorbing polymer. The effective pore size of the non-fiber microporous layer is from about 0.8 μm to about 3
A range of 0 μm is preferred. The effective pore size of the non-fiber microporous layer is indicated by the pore size measured by the critical bubble pressure method (bubble point method) according to ASTMF316-70. When the non-fibrous microporous layer is a membrane filter made of a so-called brush polymer made by a layer separation method, the liquid passage in the thickness direction is narrowest on the free surface side (glossy surface) during membrane production. Generally, the hole diameter when the cross section of the liquid passage path is approximated to a circle is the smallest near the free surface. The maximum value of the minimum pore size in the thickness direction of the membrane in the unit passing path determines the filtration performance for the particles. Usually, it is measured by the limiting bubble pressure method. When this type of membrane is used as the non-fibrous microporous layer of the analytical element of the present invention, it is preferable to direct the glossy surface of the membrane filter toward the support.

As a material constituting the fibrous microporous layer, filter paper, nonwoven fabric, woven fabric (eg, plain woven fabric), knitted fabric (eg, tricot knitted fabric), glass fiber filter paper, and the like can be used. Of these, woven and knitted fabrics are preferred. The fabric can be subjected to a physicochemical activation treatment such as a glow discharge treatment described in JP-A-57-66359.

In order to adhere and fix the non-fibrous microporous layer on the fibrous microporous layer without substantially obstructing the uniform passage of liquid, the adhesive is partially disposed and the adhesive is not present A minute penetration portion is formed in the portion. The method described in JP-A-62-138756 is useful as the method.

The top layer of the fibrous microporous layer functions not only as a filter for blood cells in whole blood samples but also as a spreading layer for developing whole blood samples.
It is preferable that the layer has an action. The liquid metering action is the action of spreading the liquid sample spotted and supplied on the surface of the layer at a substantially constant rate per unit area in the plane direction without substantially distributing the components contained therein. It is.
The fibrous microporous layer, which is the uppermost layer, is provided in Japanese Patent Application Laid-Open Nos.
397, JP-A-63-112999 and JP-A-62-182652 can be impregnated.

When dissolved in whole blood, when the whole blood (or separated plasma) is dissolved in water as a pH buffer having a pH value of about 6.5 to about 9.5, the water is dissolved. pH value approx.
Any of a variety of known pH buffer compositions that can maintain a substantially constant pH value within the range of 6.5 to about 9.5 can be used. "Basic Chemistry Handbook" (Maruzen, 1984), pp. 474-484, and "Basic Experimental Methods for Proteins and Enzymes", edited by Takeichi Horio (Horitakekazu), et al. 1981), "Biochemistry" 5 (2), 467-477 (1966), and "Analytical Biochemistry" 104 , 300-310 (1980).

Among these known pH buffers, most of the pH buffers mainly composed of inorganic compounds, that is, composed of about 80% or more by weight of inorganic compounds, are the fibrous materials forming the uppermost layer of the integrated multilayer analytical element. It is preferred that it is contained in the microporous layer to substantially prevent erythrocyte hemolysis. Examples of the pH buffer include a pH buffer composition mainly containing an inorganic salt such as carbonate, borate, metaborate, phosphate and pyrophosphate. In the pH buffer composition, the composition comprises about 80% or more by weight of an inorganic compound, and at least about 5 mol% of the constituent anionic species is used.
Particularly preferred are pH buffer compositions that are B ₄ O ₇ ²⁻ , or at least about 20 mol% are HPO ₄ ²⁻ , or contain both in the proportions mentioned. Examples of the counter ion of the anionic species usually include, but are not limited to, Na ⁺ , K ⁺ , and NH ₄ ⁺ . As a specific example of a preferred pH buffer composition, N
There are _{H 3 BO 3 -NaB 4 O 7} ; aH 2 PO 4 -Na 2 PO 4.

Among the polymers having a tertiary amine group used in the second invention, for example, a polymer having a tertiary amine nitrogen in the main chain of the polymer (eg, polyethyleneimine);
Polymers with amine groups (eg, monomer CH ₂ = CH- (C
_{H 2) -NH-N- (CH} 3) 2 homopolymers, the monomers CH ₂
ホ モ CH—CO— (CH ₂ ) —NH—N— (CH ₃ ) ₂ homopolymer, monomer CH ₂ CHCH—CO—NH— (CH ₂ ) ₃ —N— (C
H ₃ ) ₂ homopolymers; copolymers of the above monomers with methyl methacrylate or CH ₂ CHCH—CO—NH ₂ ). In the case of a polymer having a quaternary ammonium salt, it is general that the polymer is quaternized in the state of a monomer and then polymerized. However, the polymer is not limited to the polymer containing a quaternary ammonium group thus formed. Absent. N, N, N ′,
A quaternary ammonium salt oligomer obtained by condensing N'-tetramethylhexamethylenediamine with 1.3-dichloropropane can also be used. Surfactants such as phosphatidylcholine (lecithin) obtained from biological systems can also be used. Tertiary amines or quaternary ammonium salts are also useful for low molecular weight compounds, but in the case of high molecular weight compounds, the uppermost layer of the fibrous microporous layer is adjacent to the non-fibrous porous layer adjacent thereto and further below. Whole blood (or separated plasma) because tertiary amine or quaternary ammonium salt hardly migrates to the reagent layer, water absorption layer, water absorption layer and detection layer of
This is preferable because the influence of the test component contained in the detection reaction on the detection reaction and the like is small.

In the integrated multilayer analytical element of the present invention, a pH buffer (first invention) or a tertiary amine of the above-mentioned pH buffer and a high molecular compound or a high molecular compound is added to the uppermost fibrous microporous layer. Quaternary ammonium salt (second invention). The pH buffer may be added to the second layer from the top layer or the lower layer in addition to the top layer.

The integrated multi-layer analysis element of the present invention comprises glucose in whole blood,
As well as quantification of low molecular components such as urea, uric acid and creatinine, high molecular components such as total protein, albumin and various enzymes,
It is particularly useful for quantifying components bound to proteins such as bilirubin and hydrophobic components such as cholesterol and triglycerides. By including at least one of an antigen and an antibody in the microporous layer, the microporous layer can be used for quantification of the antigen or the antibody by an immunological method.

[Effects of the Invention] In an integrated multilayer analytical element containing a combination of the tertiary amine of the polymer compound and / or the quaternary ammonium salt of the polymer compound and a pH buffer having a pH value of 6.5 to 9.5, whole blood ( Or the separated plasma) has the effect of little effect on the detection reaction, etc., of the test component contained in the sample, and the effect that the measured value has substantially no effect (error) due to the difference in hematocrit value. It is. Furthermore, in the integrated multi-layer analysis element of the present invention, even if whole blood is used as a sample,
As in the case of the serum sample, the effect (error) due to hemolysis on the measured values of the various test components is extremely small, and there is also an effect that there is substantially no effect. In addition, the hematocrit value of whole blood samples ranges from about 25% to about 60%, and the effect (error) of the difference in hematocrit value on the measured values of various test components is very similar to the case of plasma and serum samples. Less,
It also has the effect of being substantially absent.

Reference Example 1 (First Invention) Integrated Multilayer Cholesterol Quantitative Element 1-1 Preparation of Leuco Dye Dispersion A leuco dye solution having the following composition A was prepared.

A: 2- (4-hydroxy-3,5-dimethoxyphenyl)-
4- [4- (dimethylamino) phenyl] -5-phenethylimidazole acetate 5.7 g 2- (4-hydroxy-3,5-dimethoxyphenyl)-
4- [4- (dimethylamino) phenyl] -5-phenethylimidazole hydrochloride 0.8 g N, N-diethyllauramide 104 g An aqueous gelatin solution having the following composition B was prepared.

B: Alkaline-treated gelatin 300 g Water 1900 g Bis [(vinylsulfonylmethylcarbonyl) -amino] methane 3.0 g Preparation of emulsion An aqueous gelatin solution B was added to each of the leuco dye solutions A while stirring at about 5700 rpm using a homogenizer mixer. about
The emulsion was dispersed by stirring for 30 minutes to prepare an emulsion.

1-2 Coating of color-forming reagent layer The emulsion prepared in step 1-1 was coated on a 180 μm-thick transparent polyethylene terephthalate (PET) sheet (support) subbed with gelatin at a rate of 150 g / m ^2. After drying, a non-porous color-forming reagent layer was formed.

1-3 Laminating the first non-fibrous microporous layer and impregnating the reagent composition The surface of the coloring reagent layer formed in the above step is uniformly wetted with water at about 25 ° C (about 30 g / m ^2). ), The glossy surface of the cellulose acetate membrane filter having an effective pore diameter of 5 μm, a thickness of 140 μm, and a porosity of about 80% and the surface of the non-porous reagent layer were laminated, adhered, and dried. Then from the top of the membrane filter, the following cholesterol analysis reagent composition prepared separately, membrane filter 1 m ² per component the following coverage (Distribution Parameter) to become manner by coating impregnated dried to first non A fibrous porous layer (functioning as a microporous reaction layer) was obtained.

Component coating amount of cholesterol analysis reagent composition (per 1 m ² ) Methylcellulose 3.0 g Titanium dioxide fine particles (average particle diameter 0.3 μm) 24 g Lipoprotein lipase 2000 IU Cholesterol esterase 2500 IU Cholesterol oxidase 2500 IU Peroxidase 2500 IU Potassium dihydrogen phosphate 7.3 g Potassium ferrocyanide 700 mg (IU: International Unit of Enzyme) 1-4 Impregnation treatment of the fibrous microporous layer (development layer) as the uppermost layer Tricot knitted fabric made by knitting PET spun yarn equivalent to 50 denier by 36 gauge (thickness: about 250 μm) Was immersed in an aqueous solution of a boric acid-tetraborate pH buffer composition having the following composition to fill the voids, taken out and dried to impregnate the solid components.

Composition of boric acid-tetraboric acid pH buffer impregnating solution Polyethylene glycol (average molecular weight: 50,000) 2.0 g Boric acid 2.0 g Sodium tetraborate 1.0 g Purified water 95.0 g 1-5 development layer and second non-fibrous microporous layer Temperature of the tricot knitted fabric impregnated in step 1-4
A hot-melt adhesive, which was preheated to 80 ° C and heated and melted to a temperature of 130 ° C, was transferred from a gravure roller by gravure printing to adhere in a dot form. The pattern of the gravure roller is that the dots are circular with a diameter of 0.3 mm,
The distance between the centers of the dots was 0.6 mm, and the dot area ratio was about 20%. The amount of the attached hot melt adhesive was about 3 g / m ² .

Immediately after the adhesive has been transferred, the non-glossy surface of a cellulose acetate membrane filter with an effective pore size of 3 μm, a thickness of 140 μm, and a porosity of about 80% is passed through the laminating roller on the surface of the hot fabric immediately after the transfer of the adhesive. Laminated (adhesive integrated).

1-6 Completion of Integrated Multilayer Analytical Element The above-mentioned laminate (the fibrous microporous layer and the second non-fibrous microporous layer functioning as a developing layer) is subjected to the same step-like bonding method as in step 1-3. Was adhered to and integrated with the surface of the first non-fibrous microporous layer integrated by the above. That is, after the heated hot-melt type adhesive is transferred from a gravure roller by a gravure printing method to the surface of a membrane filter, which is the second non-fibrous microporous layer of the laminate, and is attached in a dot form. Immediately, the surface of the first non-fibrous microporous layer (on the support and the reagent layer) was faced, and both were passed through a laminating roller to be laminated (adhesively integrated).

The multi-layer analytical element for total cholesterol determination thus completed comprises a fibrous microporous layer (which also functions as a spreading layer) as the uppermost layer, a second non-fibrous microporous layer, and a first non-fibrous microporous layer. (Functioning as a microporous reaction layer), a color-forming reagent layer, and a transparent support in this order. The uppermost fibrous microporous layer (development layer) and the second non-fibrous microporous layer work together as a blood cell filtration layer. The first non-fibrous microporous layer acts as a microporous reaction layer that produces ferric ions in the presence of cholesterol. The color-forming reagent layer forms a dye by ferric ions generated in the first non-fibrous microporous layer in the presence of cholesterol, and the dye is optically detected through the transparent support.

1-7 Preparation of analytical slide The obtained multilayer analytical element for total cholesterol determination was 15 mm
It was cut into a square chip of 15 mm in size and placed in a slide described in JP-A-58-32250 to complete a slide for biochemical analysis for quantitative determination of total cholesterol.

Comparative Example 1 Multilayer analysis slide for total cholesterol determination according to the prior art In addition to using the Tris buffer system instead of the boric acid-tetraborate pH buffer system used for the impregnation treatment of the second fibrous microporous layer of Reference example 1 Prepared an analysis slide in the same manner as in Reference Example 1.

Composition of Tris buffer impregnating solution Polyethylene glycol (average molecular weight: 50,000) 2.0 g Tris (hydroxymethyl) aminomethane 2.0 g 2N-hydrochloric acid 1.0 g Purified water 95.0 g Performance evaluation test Total cholesterol of Reference Example 1 and Comparative Example 1 Hemolysis of erythrocytes when using quantitation slides
When 20 μL of hematocrit 40% whole blood was spotted and observed from the transparent support side when the whole blood was developed, no coloration due to hemolysis was observed in the integrated multilayer analytical element of the present invention. Coloring due to hemolysis was clearly seen in the integrated multilayer analytical element according to the prior art.

Example 1 (Second Invention) Integrated Multilayer Analysis Element for Quantifying Total Cholesterol 1-1 Preparation of Dispersion of Leuco Dye A leuco dye solution having the following composition A was prepared.

A: 2- (4-hydroxy-3,5-dimethoxyphenyl)-
4- [4- (dimethylamino) phenyl] -5-phenethylimidazole acetate 5.7 g 2- (4-hydroxy-3,5-dimethoxyphenyl)-
4- [4- (dimethylamino) phenyl] -5-phenethylimidazole hydrochloride 0.8 g N, N-diethyllauramide 104 g An aqueous gelatin solution having the following composition B was prepared.

B: Alkaline-treated gelatin 300 g Water 1900 g Bis [(vinylsulfonylmethylcarbonyl) -amino] methane 3.0 g Preparation of emulsion A small amount of leuco dye solution A was prepared while stirring aqueous gelatin solution B with a homogenizer mixer at about 5700 rpm. The emulsion was dispersed by stirring for about 30 minutes to prepare an emulsion.

1-2 Coating of the reagent layer of the source The above emulsion was coated with gelatin under a thickness of 180μ.
m of a transparent polyethylene terephthalate (PET) sheet (support) at a rate of 150 g / m ² and dried to form a color reagent layer.

1-3 Lamination and treatment of the lower fibrous microporous layer [Example 1-1] The surface of the color reagent layer was uniformly wetted with water at about 25 ° C (at a rate of about 30 g / m ² ), A tricot knitted fabric (thickness: about 250 μm) in which a PET spun yarn equivalent to 50 denier was knitted with 36 gauge was superposed and dried, and the fabric was bonded and integrated with the color reagent layer. Next, the following cholesterol analysis reagent composition was placed on the knitted tricot fabric using a membrane filter.
Coating, impregnation, impregnation, and drying were performed so that the following component coating amount per 1 m ² was obtained, and a lower fibrous porous layer (functioning as a microporous reaction layer) was obtained.

Lamination and treatment of lower non-fibrous microporous layer [Example 1-2] Instead of tricot knitted fabric, effective pore diameter 5.0 µm, thickness 1
The cellulose acetate membrane filter with a porosity of about 82% having a porosity of about 82% is adhered to the color reagent layer with the glossy surface facing the color reagent layer, and then the following cholesterol analysis reagent composition is put on the membrane filter from above.
The composition was coated, impregnated, and dried at the following component coverage per 1 m ² to obtain a lower non-fibrous porous layer (functioning as a microporous reaction layer).

Component coating amount of cholesterol analysis reagent composition (per 1 m ² ) Methylcellulose 3.0 g Titanium dioxide fine particles (average particle diameter 0.3 μm) 24 g Lipoprotein lipase 2000 IU Cholesterol esterase 2500 IU Cholesterol oxidase 2500 IU Peroxidase 2500 IU Potassium dihydrogen phosphate 7.3 g Potassium ferrocyanide 700 mg 1-4 Impregnation of the fibrous microporous layer (development layer) as the uppermost layer A tricot knitted fabric (thickness of about 250 μm) obtained by knitting a PET spun yarn equivalent to 50 denier with a gauge of 36 gauge, and an amine pH of the following composition After immersing in a buffer solution and filling the voids with the solution,
Removed and dried to impregnate the solid components.

[Example 1-1; 1-2] Composition of amine-based pH buffer impregnating solution Polyethylene glycol (average molecular weight: 50,000) 2.0 g Boric acid 2.0 g Sodium tetraborate 1.0 g Tertiary amine-containing polymer aqueous solution ^* 250 mg Purified water 94.75 g ^* CH ₂ = CHCONH ₂ and _{CH 2 = CHCO (CH 2)} 2 N (CH 3) 1 and _2: 1
(Molar ratio) 20% aqueous solution of copolymer (viscosity: 400 cps) 1-5 Laminated layer and specific fibrous microporous layer Laminated tricot knit fabric impregnated in step 1-4
A hot-melt adhesive, which was preheated to 80 ° C and heated and melted to a temperature of 130 ° C, was transferred from a gravure roller by gravure printing to adhere in a dot form. The pattern of the gravure roller is that the dots are circular with a diameter of 0.3 mm,
The distance between the centers of the dots was 0.6 mm, and the dot area ratio was about 20%. The amount of the attached hot melt adhesive was about 3 g / m ² .

Immediately after the adhesive is transferred, the non-glossy surface of a cellulose acetate membrane filter with an effective pore size of 3 μm, a thickness of 140 μm, and a porosity of about 80% is passed through the laminating roller on the surface of the hot fabric immediately after the adhesive is transferred. Laminated (adhesive integrated).

1-6 Completion of Integrated Multilayer Analytical Element The above-mentioned laminate (the uppermost layer of the fibrous microporous layer and the non-fibrous microporous layer) was bonded to the lower microporous layer by the same dot-like bonding method as described above. (Fibrous or non-fibrous) surfaces, respectively. That is, after the heated hot-melt type adhesive is adhered to the membrane filter surface of the previous laminate in a dot form by transfer from a gravure roller using a gravure printing method, immediately the lower microporous layer (fiber (Which consists of a fibrous or non-fibrous microporous layer and is above the support and the chromogenic reagent layer) and passed between laminating rollers to laminate (adhesively integrate).

The multi-layer analytical element for total cholesterol determination thus completed is composed of the uppermost microporous fibrous layer (which also functions as a spreading layer), the non-fibrous microporous layer, the lower microporous layer, the color reagent layer, and the transparent support. Laminated in the order of the body. The uppermost microporous fibrous and non-fibrous microporous layers cooperate to act as a blood cell filtration layer. The lower microporous layer acts as a microporous reaction layer that produces ferric ions in the presence of cholesterol. The chromogenic reagent layer forms a dye with ferric ions generated in the lower microporous layer in the presence of cholesterol, and the dye is optically detected through the transparent support.

1-7 Preparation of analytical slide The obtained multilayer analytical element for total cholesterol determination was 15 mm
It was cut into a square chip of 15 mm in size and placed in a slide described in JP-A-58-32250 to complete a slide for biochemical analysis for quantitative determination of total cholesterol.

Comparative Example 2 Tertiary amine and boric acid based polymer compound used for impregnation of the uppermost fibrous microporous layer of Examples 1-1 and 1-2
A biochemical analysis slide for total cholesterol determination was prepared in the same procedure as in Examples 1-1 and 1-2 except that the developing layer was impregnated with the following impregnating solution instead of the impregnating solution containing a pH buffer.

Composition of pH buffer impregnating solution Polyethylene glycol (average molecular weight: 50,000) 2.0 g Sodium tetraborate 2.0 g Purified water 96.0 g Performance evaluation test 1 The color development of each analysis slide by total cholesterol in whole blood was evaluated as follows.

Human plasma containing 145 mg / dL total cholesterol and 25% hematocrit at the same total cholesterol content; 40%,
55% and 60% human whole blood samples were prepared, and 20 μL of each sample was placed on each analysis slide of Examples 1-1, 1-2 and Comparative Example 2. After spotting onto the microporous layer and incubating at 37 ° C. for 3 minutes and 6 minutes, the color optical density of the analytical element was measured with visible light having a center wavelength of 640 nm from the transparent support side with reflection side light. The results shown in the table were obtained.

As is clear from Table 1, Examples 1-1 and 1-2
Approximately the same color (optical density) regardless of the hematocrit from hematocrit 0% (plasma) to 60%
However, in Comparative Example 2, the color development was remarkably reduced as the hemal crit value was increased.

Performance Evaluation Test 2 The cholesterol concentration dependency of the total cholesterol content in whole blood and the color development concentration of the integrated multilayer analytical element was evaluated as follows.

As a whole blood sample, total cholesterol concentration test value 63 mg / d
L to 310mg / dL range, hematocrit 29% to 44%
Eight human whole blood samples in the range described above were used. As a sample having a total cholesterol concentration of 0, physiological saline containing human albumin at 7.0 g / dL was used. 20 μL each of whole blood sample and physiological saline
Was spotted on the fibrous microporous layer as the uppermost layer of the multi-layered analysis element of the analysis slide of Example 2, incubated at 37 ° C. for 6 minutes, and reflected photometrically from the transparent support side with visible light having a center wavelength of 640 nm. The color optical density of the analytical element was measured by the following method. The results are shown in Table 2 and FIG.

From the data in Table 2 and the graph in FIG. 1, it can be seen that, in the multi-layered analytical element of the present invention, since the coloring concentration has a good correlation with the total cholesterol concentration in the sample over a wide range of hematocrit value of the whole blood sample, It is clear that the total cholesterol concentration can be quantified with high accuracy without depending on the hematocrit value for the whole blood sample having the hematocrit value in the same range as in the case of the plasma and serum samples.

[Brief description of the drawings]

FIG. 1 shows the total cholesterol content in whole blood and the reflection optical density value of the color development of the element in the integrated multilayer analytic element for quantifying total cholesterol for whole blood samples of Examples 1-1 and 1-2 of the present invention. It is a graph which shows that it does not depend on a hematocrit value. The horizontal axis of the graph is the total cholesterol concentration in whole blood, and the vertical axis is the color optical density value of the integrated multi-layer analysis element by reflection photometry.

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Masaji Ogawa 3-11-4 Izumi, Asaka-shi, Saitama Fuji Photo Film Co., Ltd. Examiner Hiroyuki Kameda (56) References JP-A-63-50756 (JP, A) JP-A-62-138758 (JP, A) JP-A-62-165152 (JP, A) JP-A-63-119694 (JP, A)

Claims (4)

(57) [Claims] An at least one reagent layer containing a coloring reagent composition, a non-fibrous microporous layer, and a fibrous microporous layer as an uppermost layer are laminated in this order on a support, The two microporous layers are adjacent to each other, the interface thereof is partially adhered, and at least the tertiary amine of the polymer compound and / or the second layer of the polymer compound are added to at least the uppermost fibrous microporous layer. Quaternary ammonium salt, and when dissolved in water, the water has a pH value
An integrated multilayer analytical element comprising a pH buffer having a hydrogen ion concentration in the range of 6.5 to 9.5. 2. A fibrous or non-fibrous microporous layer is provided between the reagent layer and the non-fibrous microporous layer by partial adhesion at an interface with the adjacent non-fibrous microporous layer. The integrated multilayer analytical element according to claim 1, wherein the analytical element is stacked. 3. The integrated multilayer analytical element according to claim 1, wherein the reagent layer is at least one substantially non-porous reagent layer containing a hydrophilic polymer binder and a coloring reagent composition. 4. The pH buffer contains at least 80% by weight of an inorganic compound, and at least 5% of the constituent anionic species.
Mole% of B ₄ O ₇ ²⁻ and / or at least 20 mole% of HPO ₄ ²⁻
The integrated multilayer analytical element according to any one of claims 1 to 3, wherein