JP2003270249A - Dry analytical element avoiding influence of globulin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a dry analytical element which avoids the occurrence of a positive error caused by a reaction between an albumin detecting indicator and a globulin contained in a sample. <P>SOLUTION: In this dry analytical element for albumin, a water absorbing layer and a developing layer are formed on a substrate in this order. The analytical element contains a bromcresol purple (BCP) as the albumin detecting indicator. In addition, the pH value of the developing layer is set to the isoelectric point of the globulin or its vicinity. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a dry analytical element for albumin which avoids the influence of globulin present in a sample. The dry analytical element of the present invention is useful in the field of clinical examination.

[0002]

2. Description of the Related Art As one form of a conventional dry analytical element, a water absorbing reagent layer containing a color reaction reagent and a hydrophilic polymer binder is provided on a transparent support, and a porous developing layer is provided as an outermost layer. Many monolithic multi-layer analytical elements have been proposed. The spreading layer of the multilayer analytical element is an aqueous liquid sample (for example, biological fluid such as blood, cerebrospinal fluid, saliva, lymph, or urine; spotted water supplied to the upper surface (the surface remote from the support); drinking water. Alcoholic beverages, river water, industrial wastewater, etc.) are spread laterally without causing the components contained in the aqueous liquid sample to be substantially unevenly distributed, and the water-absorbing hydrophilic property is a constant volume per unit area. It is a layer having a function (metering function) of supplying to a reagent layer or a water absorption layer containing a polymer.

Methods for measuring or quantifying albumin in biological fluids (eg, blood, spinal fluid, saliva, lymph, urine, etc.) include buffered bromcresol green (BCG) solutions or US Pat. A method using a test piece described in US Pat. No. 3,485,587 and US Pat. No. 3,485,587 is known. BCG is a kind of acid-base indicator dye belonging to the sulfonephthalein dye which changes color by binding to protein. BCG changes color in response to the amount of protein (eg, albumin) present in the aqueous liquid.

Acid-base indicators such as BCG are not specific to albumin. Globulins present in human body fluids,
Transferrin and other proteins compete with the acid-base indicator dye for binding, interfering with albumin analysis and causing errors in measurements. This competitive interference is particularly pronounced for low concentrations of albumin. Normal human serum has a total protein concentration of about 6-8 g / dL, of which about 2.5-3.
5 g / dL is globulin and the rest is albumin.
Abnormal human serum has a total protein concentration of about 2-6 or about 8-
It may be 12 g / dL, and globulin may be 10 g / dL or more in hyperglobulinemia and the like.

JP-A-57-50660 (US Pat. No. 4,33
3,733), a reagent layer (reagent zone) in which BCG and a buffer are dispersed in a non-protein binder polymer on a transparent support.
And a porous integrated layer (reaction layer; reaction zone) are adhered and laminated in this order, a dry integrated multi-layer analytical element in which competitive interference is reduced is proposed. In this element, when human serum is spotted on the spreading layer, water is supplied from the spreading layer to the reagent layer to bring both layers into a liquid contact state, BCG diffuses from the reagent layer to the spreading layer, and preferentially with albumin. Since it binds and changes to a color peculiar to the BCG-albumin binding, it is a dry operation in which the albumin content is obtained by measuring the color density after discoloration and by the principle of colorimetric measurement. However, it has been found that even if this element is used, the measurement time is prolonged (about 3 to about 7 minutes) and a measurement error due to competitive interference of interference components such as globulin appears to a non-negligible level.

Further, in JP-A-61-243364 and JP-A-62-137564, a pH buffer-containing non-protein hydrophilic polymer binder layer (pH buffer-containing water absorbing layer) and BCG and the like are formed on a transparent support. A dry-integrated multi-layer analytical element for quantitative determination of albumin has been proposed, which has a structure in which a porous spreading layer (reagent spreading layer) impregnated with an indicator is adhered and laminated to further reduce the competitive interference of globulin. Japanese Patent Laid-Open No. 62-2766
As a modification of the above-mentioned element, element 4 in which a dicarboxylic acid such as glutaric acid, adipic acid, pimelic acid or a derivative thereof is contained as a pH buffering agent in the water absorbing layer containing a pH buffering agent is proposed in No. 4 above. It was found that even in these multi-layered analytical elements, the globulin competitive interference appeared.

Further, in JP-A-62-24150, a non-proteinaceous hydrophilic polymer binder layer containing a pH buffer agent (water absorbing layer containing a pH buffer agent) and an indicator such as BCG are made of a reactive polymer or the like on a transparent support. A dry-integrated multi-layer analytical element for quantifying albumin has been proposed, which has a structure in which a fibrous porous development layer (reagent development layer) which is solidified and contained is adhesively laminated to further reduce the competitive interference of globulin. It has been found that this multi-layer analytical element has a problem that the sensitivity is low because the indicator is immobilized by a reactive polymer or the like and the color change reaction rate is slow.

Further, in Japanese Patent Publication No. 6-68494, at least one hydrophilic polymer binder layer and a porous developing layer containing an indicator are integrally formed in this order on a light-transmitting water-impermeable support. In the multilayer integrated albumin analysis element for analyzing albumin, an indicator that changes color by binding to albumin dispersed in a hydrophilic polymer or a mixture of a hydrophilic polymer and a hydrophobic polymer in the spreading layer, An integrated multi-layer analytical element for albumin analysis has been proposed which is characterized by containing and holding a pH buffer composition. However, even if this integrated multilayer analytical element for albumin analysis is used, the effect of globulin is exerted in the case of a sample of a subject having hyperglobulinemia.

[0009]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art. That is, it is an object of the present invention to provide a dry analytical element in which an indicator for detecting albumin and a globulin in a sample react to prevent a positive error.

[0010]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that bromcresol purple (BCP) is used as an indicator for detecting albumin, and at the same time, the pH of the developing layer is adjusted to that of globulin. It has been found that a dry analytical element that solves the above problems can be provided by setting the isoelectric point, and has completed the present invention.

That is, according to the present invention, a dry analytical element for albumin having a water absorbing layer and a spreading layer on a support in this order from the side of the support contains bromcresol purple (BCP) as an indicator for detecting albumin. The dry analytical element for albumin is provided, wherein the pH of the developing layer is set near the isoelectric point of globulin.

Preferably, the spreading layer is made of a polymer having a high affinity for globulin. Preferably, bromcresol purple (BCP) is contained in the water absorbing layer. Preferably, the dry analytical element for albumin of the present invention is bromcresol purple contained in the water absorbing layer.
(BCP) is processed so as not to move to the development layer.
Preferably, the pH of the water absorbing layer containing bromcresol purple (BCP) is not more than the pKa of BCP.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments and methods for carrying out the present invention will be described in detail below. The dry analytical element of the present invention has a water absorbing layer and a developing layer on a support in this order from the support side, contains bromcresol purple (BCP) as an indicator for detecting albumin, and has a pH of the developing layer of globulin. It is characterized in that it is set near the isoelectric point. The pH of the spreading layer is specifically in the range of about 3.5 to about 7.0, preferably about 3.5 to about 6.0, more preferably about 3.7 to 6.0. P of the development layer
H is particularly preferably in the range of 5.0 to 6.0,
For example, it can be set to around 5.5.

In the present invention, the specificity of binding to albumin can be improved by using bromcresol purple (BCP) as an indicator for detecting albumin. In the present invention, BCP may be contained in the water absorbing layer, the developing layer, or the layer provided separately from these layers. Also, BC
P may be included in one of the above layers,
You may include in two or more layers. According to a preferred aspect of the present invention, BCP is contained in the water absorption layer. When the water absorbing layer contains bromcresol purple (BCP), the pH of the water absorbing layer is preferably pKa of BCP (pKa = about 6.0) or less. The amount of BCP used in the present invention is 1
m ² per about 0.2g to about 5.0 g, preferably about 0.
It is in the range of 4 g to about 3.0 g, more preferably about 0.6 g to 2.0 g.

The dry analytical element of the present invention can be used for the determination of albumin. The dry analytical element of the present invention generally has a constitution having a support and two or more layers coated thereon.

The layer coated on the support is
(1) A water-absorbing layer composed mainly of a hydrophilic polymer that absorbs and swells water; and (2) an aqueous liquid sample, which is spot-supplied on the upper surface of a dry analytical element, is contained in the aqueous liquid sample. It has a function of spreading the above-mentioned components in the lateral direction without being substantially unevenly distributed and supplying it to the water-absorbing layer containing the water-absorbing hydrophilic polymer at a ratio of a substantially constant volume per unit area (metering function). Layers: In addition, an intermediate layer such as an adhesive layer may be provided between the support and the layer provided thereon, and between each layer provided on the support.

The water absorbing layer and the spreading layer may be provided as separate layers, but they may be provided as a single layer by simultaneously providing the same layer with two or more functions. That is, the water absorbing layer and the spreading layer may be provided as the same layer. Hereinafter, constituent elements that may be present in the dry analytical element of the present invention will be described in order.

The support used in the present invention is preferably a light-transmitting water-impermeable support. As the light-transmitting water-impermeable support, a known support used for a conventional integrated multilayer analytical element can be used. Specific examples thereof include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, and cellulose ester (for example,
Cellulose diacetate, cellulose triacetate,
A thickness of about 50 μm to about 1 mm, preferably about 8 μm, which is made of a polymer such as cellulose acetate propionate).
A transparent, planar support can be used that is transparent in the range of 0 μm to about 300 μm, for example electromagnetic radiation in the wavelength range of at least some of the wavelengths in the range of about 200 nm to about 900 nm. A known undercoat layer or an adhesive layer may be provided on the surface of the support to strengthen the adhesion with the water absorbing layer.

The water absorbing layer is a water absorbing layer containing a hydrophilic polymer binder capable of swelling in contact with water and absorbing water as a main component. When the water in the aqueous liquid sample spotted on the spreading layer during the analytical operation reaches the upper surface of this layer,
It has the effect of improving the development of the aqueous liquid sample in the development layer.

The polymer binder used in the water absorbing layer is a non-protein hydrophilic polymer having a property of swelling and absorbing water when brought into contact with water. Examples of the non-protein hydrophilic polymer include polyacrylamide, agarose and JP-A-57-5.
0660, acrylamide-N-described in JP-A-58-77664, etc.
Acrylamide-based copolymers such as vinylpyrrolidone copolymer, methallyl alcohol described in JP-A-62-137564 and acrylamide or a derivative thereof, acrylic acid or a derivative thereof, methacrylic acid or a derivative thereof, or N-vinyl-2-pyrrolidone 2 Methacrylic alcohol-based copolymers such as original or ternary copolymers (methallyl alcohol-based copolymers are crosslinkable and curable. For example, acrylamide-N-vinyl-2-pyrrolidone-methallyl alcohol terpolymer).

Among these non-protein hydrophilic polymers, acrylamide-based copolymers such as acrylamide-N-vinylpyrrolidone copolymer or methallyl such as acrylamide-N-vinyl-2-pyrrolidone-methallyl alcohol terpolymer. Alcohol-containing copolymers are preferred. The coating amount of the polymer binder used in the water absorbing layer is in the range of about 5 g to about 100 g, preferably about 7 g to about 70 g per 1 m ² . If necessary, two or more polymer binders can be mixed and used.

The water absorbing layer may contain various components that do not adversely affect the ability of BCP. An example thereof is a nonionic surfactant. Specific examples of the nonionic surfactant include those similar to the surfactant that can be contained in the reagent layer or the spreading layer described later. By including a nonionic surfactant in the water-absorbing layer, water in the aqueous liquid sample is likely to be absorbed into the water-absorbing layer substantially uniformly during the analytical operation, and the liquid contact with the reagent developing layer is rapid and substantial. Become uniform. Further, the water absorbing layer may contain a buffering agent described later.

The water absorbing layer may contain a cross-linking agent (also called a curing agent or a film hardening agent). As the cross-linking agent, various kinds of inorganic and organic cross-linking agents well known in the field of organic polymer chemistry can be used. Dimethylurea is an example of an organic crosslinking agent for polyvinyl alcohol, and formaldehyde is an example of an organic crosslinking agent for a methallyl alcohol-containing polymer. The content of the cross-linking agent in the water absorbing layer can be selected according to the coating amount of the water absorbing layer to be cross-linked and cured and the degree of curing, but generally the coating amount is about 50 mg / m ² to about 5000.
mg / m ² , preferably about 100 mg / m ² to about 2000
It is in the range of mg / m ² .

According to a preferred embodiment of the present invention, the bromcresol purple (BCP) contained in the water absorbing layer is treated so as not to move to the spreading layer. An example of such a treatment includes crosslinking and curing the absorbing layer using the above-mentioned crosslinking agent.

Two or more water-absorbing layers can be provided if necessary. When two or more water absorbing layers are provided, a layer near the reagent developing layer can contain a high molecular weight pH buffer or a high molecular weight acid. Examples of high molecular weight acids that can be used include known carboxyl group-containing polymers and sulfonic acid group-containing polymers. When two water absorbing layers are provided, not only the non-protein hydrophilic polymer described above but also a wide range of hydrophilic polymers can be used in the water absorbing layer near the support. An example of such a hydrophilic polymer is deionized gelatin. The dry thickness of the water absorbing layer is generally about 1 μm to about 100 μm, preferably about 3 μm to about 50 μm.

The spreading layer may be, for example, Japanese Patent Laid-Open No. 55-1.
64356, a woven fabric spreading layer described in JP-A-57-66359 (for example, plain fabric such as broad and poplin), and a knitted fabric development layer described in JP-A-60-222769 (for example,
Tricot knitting, double tricot knitting, Milanese knitting, etc.), a developing layer made of papermaking paper containing organic polymer fiber pulp described in JP-A-57-148250, JP-B-53-2.
1677, a membrane filter (brush polymer layer) described in U.S. Pat. No. 3,992,158, polymer microbeads, glass microbeads, a continuous microvoid-containing porous layer in which diatomaceous earth is held by a hydrophilic polymer binder, and the like. Non-fiber isotropic porous spreading layer, JP-A-55-90
Non-fiber isotropic porosity comprising a continuous microvoid-containing porous layer (three-dimensional lattice-like granular structure layer) obtained by adhering the polymer microbeads according to Item 859 in a point contact manner with a polymer adhesive that does not swell with water. A spreading layer or the like can be used.

According to a preferred embodiment of the present invention, it is preferable to use a polymer having a high affinity for globulin as the spreading layer. Examples of the polymer having a high affinity for globulin include acrylics (polyacrylamide etc.), celluloses (carboxymethyl cellulose, hydroxymethyl cellulose etc.) and the like.

Preferably, as the polymer having a high affinity for globulin, a hydrophilic vinyl polymer or copolymer can be used, and for example, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, etc. can be used. The coating amount of the hydrophilic polymer is about 0.1 g / m ² to about 30 g / m ² , preferably about 0.1 g / m ² .
It is in the range of 5 g / m ² to about 20 g / m ² .

Further, the water-absorbing layer and the spreading layer together with BCP are used as an aqueous liquid sample (for example, whole blood, plasma,
The pH value of the area where the serum, lymph fluid, spinal fluid, and biological fluid typified by urine are spotted and developed is about 3.5 to about 7.
0, preferably about 3.5 to about 6.0, more preferably about 3.7 to 6.0, an organic acid or an acidic pH buffer containing an organic acid (hereinafter sometimes referred to as a buffer). ) Can be included.

The organic acid is at least one selected from the group consisting of hydroxylcarboxylic acid and dicarboxylic acid.
Certain organic acids are used. Examples of hydroxycarboxylic acids include JP-A-57-50660 and JP-A-61-243.
364, malic acid described in JP-A-62-27664 and the like,
Lactic acid, succinic acid, malonic acid, citric acid, tartaric acid. Examples of dicarboxylic acids are malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, 3,3-dimethylglutaric acid. Of these, malic acid is preferred. In addition to the above-mentioned organic acids, "Chemical Handbook Basic Edition" edited by The Chemical Society of Japan (Tokyo, Maruzen, 1966), 1312-1320, U.S. Pat. No. 3,438,7
Other pH buffer composition described in 37 etc. can also be used.

The content of the buffer in the water absorbing layer or the developing layer is in the range of about 30 meq to about 500 meq, preferably about 50 meq to about 300 meq per m ² . In the case of malic acid, the content is about 0.5 g / m ²
It is in the range of about 35 g, preferably about 0.5 g to about 20 g. The buffering agent is preferably dispersed together with the reagent in the hydrophilic polymer and dispersed and held in the water absorbing layer, the reagent layer and / or the developing layer.

As a method of incorporating a hydrophilic polymer that disperses and holds BCP and a buffer into the reagent layer or the spreading layer,
Aqueous solution containing BCP and polymer, water-organic solvent mixed solvent solution or organic solvent solution (example of organic solvent: aliphatic alcohol such as methanol, ethanol, isopropyl alcohol; dialkyl ketone such as acetone and methyl ethyl ketone; dialkyl ether such as dimethyl ether; A method in which an aliphatic cyclic ether such as tetrahydrofuran or dioxane; acetonitrile; hexane; β-methoxyethanol; ethylene glycol etc.) is applied or sprayed substantially uniformly on the spreading layer by a known method, and BCP. , A buffering agent, a hydrophilic polymer is dipped in a solution for a developing layer, and the resultant is dried or semi-dried to be laminated on a water absorbing layer (hydrophilic polymer binder layer) to be integrated. When BCP, a buffer, or a hydrophilic polymer solution is applied or sprayed on the spreading layer, the solvent that dissolves the three is a solvent that does not dissolve the hydrophilic polymer layer of the water-absorbing layer, but one that can dissolve or disperse the three. It is preferable to appropriately select from the above.

Further, a surfactant is contained together with BCP, a buffer and a hydrophilic polymer so that the BCP and the polymer are uniformly contained and held in the spreading layer, and the BCP at the time of spotting the aqueous liquid sample.
The release rate of CP can be controlled. Further, the development of the aqueous liquid sample can be made uniform. As the surfactant, any of anionic surfactant, cationic surfactant, nonionic surfactant and amphoteric surfactant can be used, but nonionic surfactant is preferred. Specific examples of the nonionic surfactant include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate,
There are p-nonylphenoxypolyglycidol, octyl glucoside and the like. Of these, polyoxyethylene oleyl ether is preferred. The content of the nonionic surfactant in the spreading layer is about 20 mg to about 10 per 1 m ^2.
g, preferably in the range of about 30 mg to about 5.0 g.

The dry analytical element of the present invention can be prepared by methods known to those skilled in the art. The dry analytical element of the present invention is cut into small pieces each having a side of about 15 mm to about 30 mm, a square or the same size, and the like.
1, U.S.A. 56-142454, JP-A-57-6345.
2, actual development Sho 58-32350, special table Sho 58-50114
It is preferable to use it as a chemical analysis slide by enclosing it in the slide frame described in 4 etc. from various viewpoints such as production, packaging, transportation, storage and measurement operation. Depending on the purpose of use, it can be used by storing it in a cassette or magazine in the form of a long tape, or by sticking or storing a small piece on a card having an opening.

Quantitative analysis of albumin in a liquid sample can be carried out by carrying out the operations described in the above-mentioned various patent specifications using the analytical element of the present invention. For example, about 5 μL
To about 30 μL, preferably about 8 μL to about 15 μL, of an aqueous liquid sample such as whole blood, plasma, serum, lymph, urine, etc. is spotted on the spreading layer, and is substantially in the range of about 20 ° C. to about 40 ° C. Incubation at a constant temperature, preferably at a substantially constant temperature near 37 ° C., for about 1 minute to about 10 minutes, preferably about 2 minutes to about 7 minutes, from the light-transmissive support side to the dry analytical element side. It is possible to obtain the content of the measurement target component in the liquid sample according to the principle of the colorimetric measurement method by measuring the detectable change such as the color change and the color development. When albumin is analyzed using the analysis element of the present invention,
The optical density of the reagent development layer is measured by reflection using the light having the absorption maximum wavelength of the albumin-BCP bond or a wavelength in the vicinity thereof,
The albumin content in the liquid sample can be determined by the principle of the colorimetric measurement method using a calibration curve prepared in advance. Quantitative analysis of the components to be measured can be performed with high accuracy by keeping the amount of the aqueous liquid sample spotted, the incubation time and the temperature constant. The measuring operation is disclosed in JP-A-60-12554.
3, JP-A-60-220862, JP-A-61-2943
67, the chemical analyzer described in JP-A-58-161867 and the like enables highly accurate quantitative analysis with extremely easy operation. The present invention will be described more specifically by the following examples, but the following examples are for illustrating the present invention and not for limiting the scope of the present invention.

[0036]

Examples Example 1: 180 μm gelatin subbed
The polyethylene terephthalate colorless transparent smooth film of was coated with an aqueous solution having the following composition so that the thickness after drying was 40 μm, and dried. (Water absorption layer) Polymer AAm-VP-MAOH 40.0 g / m ² Malic acid 2.0 g / m ² Surfactant 1.0 g / m ² Here, the surfactant is polyoxy (2-hydroxy).
Propylene nonyl phenyl ether (Surfactant 10G,
Aurin Co.) was used. The pH of the aqueous solution was set to 4.0. The polymer AAm-VP-MAOH represents a polyacrylamide-N-vinyl-2-pyrrolidone-methallyl alcohol ternary copolymer (monomer molar ratio 57: 38: 5 (manufactured by Fuji Photo Film Co., Ltd.)) (hereinafter the same).

Next, water was supplied to the entire surface of the film at a supply rate of about 30 g / m ² to wet it, and then 50 denier of polyethylene terephthalate spun yarn was knitted with 36 gauge tricot knitted fabric with light pressure. It was laminated and dried.

Next, an ethanol solution (OC solution) having the following composition was applied so that the amount of each component was as shown below, and dried to prepare an integrated multilayer analytical element. (Development layer) Buffer solution Malic acid 1.0 g / m ² Polyvinylpyrrolidone 0.5 g / m ² BCP 1.5 g / m ² Surfactant 0.5 g / m ² Here, the surfactant is polyoxyethylene oleyl ether (BO-7 Nikko Chemicals) was used. After spotting
The pH was adjusted to 4.0. The integrated multi-layer analytical element is cut into 12 mm × 13 mm square chips and placed in a slide frame (described in JP-A-57-63452) to prepare a dry slide for ALB analysis (Example 1) according to the present invention. It was made.

Example 2 A 180 μm polyethylene terephthalate colorless transparent smooth film undercoated with gelatin was treated with an aqueous solution having the following composition:
It was applied and dried so that the thickness after drying was 40 μm. (Water absorption layer) Polymer AAm-VP-MAOH 40.0 g / m ² Malic acid 2.0 g / m ² Surfactant 1.0 g / m ² Here, the surfactant is polyoxy (2-hydroxy).
Propylene nonyl phenyl ether (Surfactant 10G,
Aurin Co.) was used. The pH of the aqueous solution was set to 4.0.

Next, water was supplied to the entire surface of the film at a supply rate of about 30 g / m ² to wet it, and then 50 denier of polyethylene terephthalate spun yarn was knitted with 36 gauge tricot knitted fabric with light pressure. It was laminated and dried.

Next, an ethanol solution (OC solution) having the following composition was applied so that the amount of each component was as shown below and dried to prepare an integrated multi-layer analytical element. (Development layer) Buffer solution Succinic acid 1.0 g / m ² Polyvinylpyrrolidone 0.5 g / m ² BCP 1.5 g / m ² Surfactant 0.5 g / m ² Here, the surfactant is polyoxyethylene oleyl ether (BO-7 Nikko Chemicals) was used. After spotting
The pH was adjusted to 5.5. The same applies as in Example 1 below.

Example 3 A 180 μm polyethylene terephthalate colorless transparent smooth film undercoated with gelatin was treated with an aqueous solution having the following composition:
It was applied and dried so that the thickness after drying was 40 μm. (Water absorption layer) Polymer AAm-VP-MAOH 40.0 g / m ² Malic acid 2.0 g / m ² Surfactant 1.0 g / m ² Here, the surfactant is polyoxy (2-hydroxy).
Propylene nonyl phenyl ether (Surfactant 10G,
Aurin Co.) was used. The pH of the aqueous solution was set to 4.0.

Next, water was supplied to the entire surface of the film at a supply rate of about 30 g / m ^{2 to} moisten the surface of the film, and a 50-denier polyethylene terephthalate spun yarn of 36 gauge knitted tricot knitted fabric was lightly pressed. It was laminated and dried.

Next, an ethanol solution (OC solution) having the following composition was applied so that the amount of each component was as shown below, and dried to prepare an integral type multi-layer analytical element. (Development layer) Buffer solution Malic acid 1.0 g / m ² Polyvinylpyrrolidone 10.0 g / m ² BCP 1.5 g / m ² Surfactant 0.5 g / m ² Here, the surfactant is polyoxyethylene oleyl ether (BO-7 Nikko Chemicals) was used. After spotting
The pH was adjusted to 4.0. The same applies as in Example 1 below.

Example 4 A 180 μm polyethylene terephthalate colorless transparent smooth film undercoated with gelatin was treated with an aqueous solution having the following composition:
It was applied and dried so that the thickness after drying was 40 μm. (Water absorption layer) Polymer AAm-VP-MAOH 40.0 g / m ² Malic acid 5.0 g / m ² BCP 1.5 g / m ² Surfactant 1.0 g / m ² Here, the surfactant is polyoxy (2-hydroxy).
Propylene nonyl phenyl ether (Surfactant 10G,
Aurin Co.) was used. The pH of the aqueous solution was set to 4.0.

Next, water was supplied to the entire surface of the above-mentioned film at a supply amount of about 30 g / m ² to wet it, and then 50 denier of polyethylene terephthalate spun yarn was knitted with 36 gauge tricot knitted fabric with light pressure. It was laminated and dried.

Next, an ethanol solution (OC solution) having the following composition was applied so that the amount of each component was as shown below, and dried to prepare an integral type multi-layer analytical element. (Development layer) Buffer solution Succinic acid 1.0 g / m ² Polyvinylpyrrolidone 7.0 g / m ² Surfactant 0.5 g / m ² Here, polyoxyethylene oleyl ether (BO-7 manufactured by Nikko Chemicals) is used as the surfactant. Using. After spotting
The pH was adjusted to 5.5. The same applies as in Example 1 below.

Example 5 A 180 μm polyethylene terephthalate colorless transparent smooth film undercoated with gelatin was treated with an aqueous solution having the following composition:
It was applied and dried so that the thickness after drying was 40 μm. (Water absorption layer) Polymer AA m-VP-MAOH 40.0 g / m ² Malic acid 5.0 g / m ² BCP 1.5 g / m ² Formalin 2.2 g / m ² Surfactant 1.0 g / m ² Where, the surfactant is Polyoxy (2-hydroxy)
Propylene nonyl phenyl ether (Surfactant 10G,
Aurin Co.) was used. The pH of the aqueous solution was set to 4.0.

Next, water was supplied to the entire surface of the film at a supply rate of about 30 g / m ² to wet it, and then 50 denier polyethylene terephthalate spun yarn of 36 gauge knitted tricot knitted fabric was lightly pressed. It was laminated and dried. Leave at 70 ° C for 5 hours to accelerate the cross-linking reaction with formalin. As a result, BCP does not substantially move to the development layer.

Next, an ethanol solution (OC solution) having the following composition was applied so that the amount of each component was as follows, and dried to prepare an integrated multi-layer analytical element. (Development layer) Buffer solution Succinic acid 1.0 g / m ² Polyvinylpyrrolidone 7.0 g / m ² Surfactant 0.5 g / m ² Here, polyoxyethylene oleyl ether (BO-7 manufactured by Nikko Chemicals) is used as the surfactant. Using. After spotting
The pH was adjusted to 5.5. The same applies as in Example 1 below.

Comparative Example 1 A 180 μm polyethylene terephthalate colorless transparent smooth film undercoated with gelatin was treated with an aqueous solution having the following composition:
It was applied and dried so that the thickness after drying was 40 μm. (Water absorption layer) Polymer AAm-VP-MAOH 40.0 g / m ² Malic acid 2.0 g / m ² Surfactant 1.0 g / m ² Here, the surfactant is polyoxy (2-hydroxy).
Propylene nonyl phenyl ether (Surfactant 10G,
Aurin Co.) was used. The pH of the aqueous solution was 3.1.

Next, water was supplied to the entire surface of the film at a supply rate of about 30 g / m ² to wet it, and then 50 denier of polyethylene terephthalate spun yarn was knitted with 36 gauge tricot knitted fabric with light pressure. It was laminated and dried.

Next, an ethanol solution (OC solution) having the following composition was applied so that the amount of each component was as shown below and dried to prepare an integral type multi-layer analytical element. (Development layer) Buffer solution Malic acid 1.0 g / m ² Polyvinylpyrrolidone 0.5 g / m ² BCG 1.0 g / m ² Surfactant 0.5 g / m ² Here, the surfactant is polyoxyethylene oleyl ether (BO-7 Nikko Chemicals) was used. After spotting
The pH was adjusted to 3.1. The integrated multi-layer analytical element is cut into a 12 mm × 13 mm square chip and placed in a slide frame (described in Japanese Patent Laid-Open No. 57-63452) for comparison.
A dry slide for B analysis (Comparative Example 1) was prepared.

Comparative Example 2 A 180 μm polyethylene terephthalate colorless transparent smooth film undercoated with gelatin was treated with an aqueous solution having the following composition:
It was applied and dried so that the thickness after drying was 40 μm. (Water absorption layer) Polymer AAm-VP-MAOH 40.0 g / m ² Malic acid 2.0 g / m ² Surfactant 1.0 g / m ² Here, the surfactant is polyoxy (2-hydroxy).
Propylene nonyl phenyl ether (Surfactant 10G,
Aurin Co.) was used. The pH of the aqueous solution was 3.1. Next, on the above film, water was supplied to the entire surface at a supply rate of about 30 g / m ² to wet it, and then 50 denier equivalent polyethylene terephthalate spun yarn was woven with 36 gauge tricot knitted fabric with light pressure to be laminated. Dried.

Next, an ethanol solution (OC solution) having the following composition was applied so that the amount of each component was as shown below and dried to prepare an integral type multilayer analytical element. (Development layer) Buffer solution Malic acid 1.0 g / m ² HEC 5.0 g / m ² BCG 1.0 g / m ² Surfactant 0.5 g / m ² Where, the surfactant is polyoxyethylene oleyl ether (BO-7 Nikko Chemicals Co., Ltd.) was used. After spotting
The pH was adjusted to 3.1. HEC is hydroxyethyl cellulose. Hereinafter, the same as in Comparative Example 1 is applied.

Comparative Example 3 A 180 μm polyethylene terephthalate colorless transparent smooth film undercoated with gelatin was treated with an aqueous solution having the following composition:
It was applied and dried so that the thickness after drying was 40 μm. (Water absorption layer) Polymer AAm-VP-MAOH 40.0 g / m ² Malic acid 2.0 g / m ² BCG 1.0 g / m ² Surfactant 1.0 g / m ² Here, the surfactant is polyoxy (2-hydroxy).
Propylene nonyl phenyl ether (Surfactant 10G,
Aurin Co.) was used. The pH of the aqueous solution was 3.1. Next, on the above film, water was supplied to the entire surface at a supply rate of about 30 g / m ² to wet it, and then 50 denier equivalent polyethylene terephthalate spun yarn was woven with 36 gauge tricot knitted fabric with light pressure to be laminated. Dried.

Next, an ethanol solution (OC solution) having the following composition
Each component is applied and dried in the following amounts,
An integrated multi-layer analytical element was made. (Development layer) Buffer solution Malic acid 1.0 g / m ² Polyvinylpyrrolidone 7.0 g / m ² Surfactant 0.5 g / m ² Here, polyoxyethylene oleyl ether (BO-7 Nikko Chemicals) is used as the surfactant. Using. After spotting
The pH was adjusted to 3.1. The same applies to Comparative Example 1 below.

Measurement Example 1: Globulin concentration of 3, 5, 9 g / d
10 μL of the serum added so as to give L is spotted on the slides prepared in Examples 1 to 5 and Comparative Examples 1 to 3 (the content of each slide is shown in FIG. 1). Then 3
While incubating at 7 ° C. for 5 minutes, the reflection density at 625 nm was measured by Fuji Dry Chem (manufactured by Fuji Photo Film Co., Ltd.) approximately every 10 seconds. The reflection density for 5 minutes at that time was obtained. The albumin error per g / gL of globulin was as follows.

[0059]

[0060]

According to the technique of the present invention, the specificity of binding to albumin can be increased by using bromcresol purple (BCP). Further, by setting the pH of the spreading layer near the isoelectric point of globulin, the binding between globulin and bromcresol purple (BCP) can be inhibited, and the albumin value can be accurately measured. In addition, when a polymer having an affinity for globulin is used as the polymer of the spreading layer, it delays the migration of globulin or globulin bound with bromcresol purple (BCP) to a layer below the spreading layer. The effect of globulin can be avoided. Also bromcresol purple
When (BCP) is formulated in the layer below the spreading layer, only albumin that has moved to the layer below the spreading layer preferentially reacts with bromcresol purple (BCP), and the effect of globulin can be avoided.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of analytical elements prepared in Examples 1 to 5 and Comparative Examples 1 to 3.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Osamu Soshimoto             3-11-46 Izumi, Asaka-shi, Saitama Fuji Photographic Group             Within Ilum Co., Ltd. F term (reference) 2G042 AA01 BD20 CA10 CB03 DA08                       FA11                 2G045 AA13 AA16 BA01 CA25 CA26                       CB03 CB07 CB30 DA37 DA38                       FB11 GC11                 2G054 AA07 CA23 CE01 EA05

Claims (5)

[Claims] 1. A dry analytical element for albumin having a water absorbing layer and a spreading layer on a support in this order from the side of the support, containing bromcresol purple (BCP) as an indicator for detecting albumin, and pH of the spreading layer. Is set near the isoelectric point of globulin, a dry analytical element for albumin. 2. The dry analytical element for albumin according to claim 1, wherein the spreading layer is composed of a polymer having a high affinity for globulin. 3. The dry analytical element for albumin according to claim 1, wherein bromcresol purple (BCP) is contained in the water absorbing layer. 4. The dry analytical element for albumin according to claim 4, wherein bromcresol purple (BCP) contained in the water absorbing layer is treated so as not to move to the developing layer. 5. The pH of the water absorbing layer containing bromcresol purple (BCP) is not more than pKa of BCP.
Or the dry analytical element for albumin according to 4.