JPS6224150A - Multilayer analysis element for albumin measurement - Google Patents

Abstract

PURPOSE:To make it possible to take the measurement of albumin in a biologi cal liquid sample without being affected by other interferring protein such as globulin, by employing a compound that could cause a detectable color change when coupled to albumin under a specified pH condition. CONSTITUTION:A compound that could cause a detectable color change when coupled to albumin under a specified pH condition is contained in such a manner as to be immovable in substance in a porous development layer within the layer and/or between layers. The compound that could causes a detectable color change when coupled to albumin includes a color indicator which causes a coloring reaction in color formation, decoloring, color vanishment or the like and a fluorescent indicator that presents a substantial change in the intensi ty of fluorescene, wavelength and the like being coupled to albumin. The color indicator is, for example, methylorange and the fluorescent indicator, for exam ple, eosine Y. Preferably used among them is the color indicator such as methylorange. This enables the measurement of albumin in a biological liquid sample without being affected by other interferring protein.

Description

[Detailed Description of the Invention] [Industrial Field of Application J] The present invention relates to a multilayer analytical element for quantifying a specific component in a biological fluid sample, and more particularly to a multilayer analytical element for quantifying albumin in a biological fluid sample. The present invention relates to a multilayer analysis element for

[Background of the Invention 1 In the past, many methods have been developed for analyzing specific components in biological and scientific fluid samples. Particularly in the field of clinical chemistry, various analytical instruments have been developed and have been introduced into clinical laboratories of many hospitals. Among these, the multilayer analytical element disclosed in Japanese Patent Publication No. 53-21677 has attracted attention because of its ease of operation and high quantitative performance.

However, it is known that in quantifying proteins, especially albumin, the specificity of binding of the protein-binding dye used for albumin is low, so errors are likely to occur due to interference from other coexisting proteins, such as globulin.

For this reason, in JP-A-57-50660, the above-mentioned dye is contained in the reagent layer, so that the protein in the sample solution remains in the developing layer, and the dye in the reagent layer, for example, bromocresol green, migrates to the developing layer. Here, a complex is formed with protein-bromocresol green. At this time, p
It has been disclosed that adjusting the migration rate of H and bromocresol green increases the specificity of the dye for albumin.

Also, vPl'jq No. 58-179359 Akim IF
t' C. Optical +: A method has been disclosed in which a protein adsorbent carrying a detectable indicator is contained in a layer of non-proteinaceous material that is permeable to proteins to form a reagent layer.

However, it cannot be said that the former method sufficiently eliminates interference with proteins other than albumin, and it is clear that the latter method also poses various difficulties in terms of production.

[Objective of the invention 1 The object of the present invention is to detect albumin in a biological fluid sample.
It is an object of the present invention to provide a multilayer analytical element for measuring albumin that allows measurement without being influenced by other interfering proteins such as globulin and is stable in terms of manufacture.

[Structure of the Invention j The above object of the present invention is to provide a multilayer analytical element comprising a non-protein hydrophilic polymer layer and a porous spreading layer sequentially laminated on a liquid-impermeable and light-transmitting support. A multilayer analytical element in which a compound that causes a detectable color change when combined with albumin under predetermined pH conditions is contained in the porous development layer in a form that does not substantially migrate within the layer and/or between the layers. This is achieved by using

[Specific Structure of the Invention] In the present invention, a compound that causes a detectable color change by binding to albumin (hereinafter simply referred to as a compound according to the present invention) is a compound that undergoes a color reaction such as color development, color change, or decolorization. Examples include color-changing indicators and fluorescent indicators that show large changes in fluorescence intensity, wavelength, etc. when combined with albumin.

Specific examples of color indicators include methyl orange, bromocresol green, bromocresol purple, bromo 7 ether blue, amide black 10B1 acid orange R1 buffer arrow black, orange G1 azosulfathiazole, 2-p-) Louis Luna 7 Talen 6
- Sulfonates, etc.

Examples of fluorescent indicators include eosin Y, 7ranine orange, trinitrobenzenesulfonic acid, 1-7nylinona7talene-8-sulfonic acid, and 5-(4'-fur7noanily7)-2-chloro-7-medoxy. Examples include acridine, fluoresamine, thiamine, 1-(dimethylamino)naphthalene-5-sulfonyl chloride, and the like.

Among these, those that are particularly preferably used are:
Examples include color indicators such as methyl orange, bromocresol green, bromocresol purple, and bromophenol blue.

The compound according to the present invention is contained in the porous spreading layer in a form in which there is substantially no intralayer and/or interlayer movement.

As a method for incorporating the compound according to the present invention into a porous spreading layer, for example, a polymer having discrete fibers and a reactive group disclosed in JP-A-57-197466 (U.S. Pat. No. 4,427,632) may be used. In the case of a porous spreading layer made of pineapple, it is effective to impregnate the fibers with the compound according to the present invention in advance, or to finely disperse the compound according to the present invention.

At this time, the compound according to the present invention is immobilized by a polymer having a reactive group, such as a styrene-glycidyl methacrylate copolymer, whether by impregnation or differentiation.

Also, Japanese Patent Publication No. 53-21677 (U.S. Patent No. 3,992,
In the case of a non-fibrous isotropic porous layer (for example, a single layer of Brassier Borima) disclosed in No. 158), it is preferable to incorporate the compound according to the present invention by fine dispersion.

Furthermore, in the case of a fabric that has been subjected to a hydrophilic treatment as disclosed in JP-A-55-164356, the fabric can be impregnated and then immobilized with, for example, the above-mentioned copolymer.

However, among these porous expansion layers, JP-A-57
The R interlayer disclosed in No. 197466 is easy to manufacture;
Moreover, it is useful because both impregnation and fine dispersion can be selected.

The compound according to the invention has a porous spreading layer of about 0.14
/a+' to about 5.0 g/m2, preferably about 0.5 g
/m2 to about 3.0g/m2.

Further, the thickness J of the porous spreading layer is preferably in the range of about 100 microns to about 350 microns.

For this purpose, acids, alkalis, salts, buffers, dissociating agents, surfactants, etc. can be appropriately contained in the spreading layer, non-proteinaceous hydrophilic polymer layer, and other functional layers.

First, in the analysis of fulpmin, since the binding reaction between the compound of the present invention and fulpmin to be used is greatly influenced by pH, the selection must be made carefully.

The pH of the binding reaction with fulpmin in the porous spreading layer varies depending on the compound according to the invention used, but generally ranges from about 2.0 to about 7.0, preferably at a pH of about 3.0.
to about 6.5. The acid or buffer for controlling pH is contained in one layer of the non-proteinaceous hydrophilic polymer, but can be contained in layers other than the one layer of the hydrophilic polymer, if necessary.

For example, when the compound according to the present invention is bromocresol green, the pH is preferably 4.0, and when the compound is bromocresol purple, the pH is preferably 5.2.

In the multilayer analytical element of the present invention, a non-protein hydrophilic polymer layer and a porous spreading layer containing the compound according to the present invention are laminated on a support.

Examples of the hydrophilic polymer used to form the non-proteinaceous hydrophilic polymer layer include synthetic homopolymers such as polyacrylamide, polyvinylpyrrolidone, and polyvinyl alcohol, and copolymers thereof, carboxymethylcellulose sodium salt, and hydroxyethylcellulose. , hydrophilic cellulose derivatives such as methylcellulose, agarose, maleic acid copolymers, polyacrylic acid, polymethacrylic acid, and salts thereof.

Among these, acrylamide homopolymers and copolymers are preferred.

In the multilayer analytical element of the present invention, the film thickness of the non-proteinaceous hydrophilic polymer debris generally ranges from about 5 microns to about 50 microns, preferably from about 10 microns to about 30 microns.

In the above-mentioned quantitative determination of fulpmin, pH.

Since ion strength and other factors are important factors, it is important to set these factors to optimal conditions in order to obtain good analytical results.

Acids or buffers used to control the pH in the porous aWI layer include, for example, aliphatic hydroxycarboxylic acids such as glycolic acid, lactic acid, α-hydroxybutyric acid, glyceric acid, tartronic acid, malic acid, tartaric acid, citric acid), aliphatic dicarboxylic acids (e.g. malonic acid, succinic acid, 3,3-methylglutaric acid, a% a'
-dimethylglutaric acid #1), aliphatic carboxylic acids (eg, acetic acid, propionic acid, butyric acid), and buffers based on combinations of salts thereof and the above-mentioned acids.

Preferred acids or buffers using acids and their own salts include malic acid, lactic acid, succinic acid, malonic acid, citric acid, tartaric acid, and the like.

The liquid-impermeable and light-transparent support that can be applied to the multilayer analytical element of the present invention has a wavelength of about 200 nm to about 900 nm.
at least about 40%, preferably at least about 65% of electromagnetic radiation #i (ultraviolet, near-ultraviolet, abrasive, or near-infrared) of a wavelength (wavelength or wavelengths) within the range of
A support in the form of a film, sheet, thin plate, or other form that is permeable and substantially impermeable to liquids such as water, specifically, cellulose acetate, cellulose acetate butyrate, cellulose acetate propionate. Typical examples include polymers such as , polyethylene terephthalate, polystyrene, and polycarbonate, and glass.

According to the present invention, the single layer of the non-proteinaceous hydrophilic polymer and the porous spreading layer are sequentially laminated on the support to obtain the multilayer analytical element of the present invention. At this time, in order to improve the adhesion between the support surface and the non-proteinaceous hydrophilic polymer layer, a subbing layer is provided, or the support surface is subjected to chemical treatment (e.g. acid treatment, alkali treatment) or physicochemical treatment. Treatments (for example, corona discharge treatment, glow discharge treatment, ultraviolet irradiation treatment, flame treatment) can be performed.

In manufacturing the multilayer analytical element of the present invention, various known methods can be used, such as slide coating, tube coating, dip coating, and curtain coating. Furthermore, in the case of a textile in which the porous spreading layer is hydrophilized as disclosed in JP-A No. 55-164350, for example, the non-proteinaceous wood-loving layer of the present invention or a similar material may be used. When the adhesive layer laminated on the polymer layer is in a semi-dry state, or after the hydrophilic polymer is wetted with water or water containing a surfactant, a porous layer is used as a spreading layer. A sheet, film, or membrane material can be adhered onto the layer using appropriate pressure.

Furthermore, in manufacturing the multilayer analytical element of the present invention, various surfactants can be added to each layer. Surfactants that can be used include, for example, alkylphenoxybolyethoxylates [e.g. Triton X-10
0 (o-A & Haas)], a', lyethylene oxide alkyl ester [e.g. Emalden-120 (Kao Atlas Co.)], alkyl 7-ethyl/xyglycerin [e.g. Surfactant 10G (Olin Co.) 1 is typical. This is an example.

The multilayer analytical element of the present invention can be used in various ways as desired. fi
It is possible to take various functional layers and layer configurations. For example, JP 51-40191 (JP 58-18628), U.S. Patent No. 4,110,079, JP 58-1315
It is possible to arbitrarily select the layers and layer configurations described in No. 65 and the like.

The multilayer analytical elements produced in this way can be made into a variety of shapes depending on the analytical method, including e.g. a stretchable tape of the desired width, a sheet or a slide mounted on a plastic mount. It can be shaped.

Since the multilayer analytical element of the present invention contains the compound of the present invention in the porous interlayer in a form that does not substantially migrate, the multilayer analytical element of the present invention can absorb fulbumin in biological fluid samples without being affected by other interfering proteins. It can be measured without receiving any radiation.

Below are 6 experimental examples and V examples of the multilayer analytical element of the present invention.
However, the present invention is not limited thereby.

Experimental Example It was confirmed that the compound according to the present invention does not migrate within and/or between layers in the multilayer analytical element for fulpmin analysis of the present invention.

Porous spreading layer of the present invention (1) Xylene 84! Triton
-100 Rhos & Hass
Co,)3. Og, 45 g of styrene-glycitrus methacrylate copolymer (9:1) were dissolved, and 0.585 g of bromocresol green was further mixed, glass beads 1 were added, and the mixture was stirred with a sand stirrer for 5 hours to disperse. The residue was filtered through a filter, and the dispersion was filtered off from the glass beads. Powder filter paper C (
After mixing 10.5 g of Toyo Roshi Co., Ltd. (300 mesh or more) and performing ultrasonic dispersion, it was applied onto a subbed polyethylene terephthalate support with a thickness of 180 microns using a doctor blade with a gap of 375 microns. A porous spreading layer having a thickness of 180 microns (when dried) was formed, and this was dried.

Porous development of the present invention/1 (2) (Preparation of bromocresol green impregnated fiber) Add 0.5 g of bromocresol green to a mixed solvent of acetone 4.25a+f and 1 xylene 40s+ffi, stir and dissolve, then add 4 ml of pure water. Stir to transfer bromocresol green to the aqueous phase. Add 17.4 g of powder filter paper C (Toyo Roshi Co., Ltd., 300 mesh or more) to this,
After mixing and stirring, let stand. After confirming that the supernatant liquid is no longer colored bromocresol green, it is filtered and dried.

(Creation of porous development layer) 84ml of xylene and Triton
-100 (ROHM and Haas) 3. o, and styrene-glycitrus methacrylate copolymer (9:1)
28.9 g of the bromocresol green impregnated edge was added to a solution in which 4.5 g of bromocresol green was dissolved, and ultrasonic waves were irradiated while stirring to obtain a dispersion. This dispersion was applied onto a transparent subbed polyethylene terephthalate support with a thickness of 180 microns using a doctor blade having a gap of 375 microns to form a porous spread layer with a film thickness of 180 microns (when dry). This was dried.

(Preparation of Comparative Reagent Layer) For further comparison, polyacrylamide 32.28 mm was coated on a 180 micron subbed polyethylene tere-7 tallate support according to the element preparation described in JP-A-57-50660.
g/m2, bromocresol green 1.08g.

Surf Actane) 10G (Olin-Macheson) 0.
A reagent layer consisting of 32 g/ω2 was created. However, in order to prevent the polyacrylamide from dissolving in water, 1.0% of a 37% formaldehyde aqueous solution was added to the polyacrylamide.

Each of the three films was cut to a certain area, soaked in distilled water 3T#1, and the absorbance at 435 nm was measured using a Hitachi spectrophotometer model 220-A at n=3 every time.

The results are shown in Table-1.

Margin below From Table 1 above, it can be seen that in the comparative reagent layer described in JP-A No. 57-50660, bromocresol green, which is a coloring indicator, quickly migrates from the layer, but the porous development used in the present invention Layers (1) and (2) showed no migration of the color indicator dye-1, and no difference was observed in the absorbance results even when the film was shaken during seeding. It is considered that bromocresol green does not undergo interlayer or intralayer migration.

Example 1 As a multilayer analytical element for fulpmin analysis of the present invention, a coating solution having the following composition was coated on a 180 micron undercoated transparent polyethylene terephthalate support.

(Non-protein hydrophilic polymer layer) Add 2.5 g of citric acid to 35 g of a 10% polyacrylamide aqueous solution.
Add 5 g and 5 ml of a 50% aqueous solution of Emalden 120 (manufactured by Kako Atlas Co., Ltd.), stir and dissolve the solution and hydroxide) +7
After adjusting the pH to 3.7 by adding a 30% aqueous solution of
It was brought to 50@1° with distilled water and coated onto a 180 micron thick clear subbed polyethylene terephthalate support using a 500 micron gap doctor blade and dried.

(Porous spreading layer) The coating liquids for spreading layers (1) and (2) shown in the experimental example are applied to the non-proteinaceous polymer layer prepared above in a gap 375.
Each coating was applied to a film thickness of 180 microns (when dry) using a micron doctor blade and dried.

These were designated as multilayer analytical elements (1) and (II) of the present invention.

(Preparation of Comparative Multilayer Analytical Element) A comparative multilayer analytical element was prepared according to the description in JP-A-57-50660.

(Reagent layer) Polyacrylamide 32.28g/11
12 bromocresol green 1.08g/m
'Malic acid 5.4 g/So2 Surfactant 10G (Olean) 0.32 g/
Reagent layer consisting of Theory 2 (NH3 layer) Microcrystalline cellulose 53.80g/m2
Polyvinylpyrrolidone, 1.35g/m
These multilayer analytical elements were cut into 1.5 cm x 1.5 cm and 2.4 cm
Attached to a 2.8cm rectangular plastic mount,
It was used as an analysis slide.

Analytical element (I) of the present invention prepared as described above and (
II) and a comparative analysis element, and 2.0.3.5.5.0.6.5.7.5 g of human serum albumin as a specimen.
/ di, and a standard solution containing 5% human globulin in addition to the above albumin level standard solution, and place 10 μl of the standard solution on the porous spreading layer of each analytical element. After inking at 37'C for 7 minutes, the reflection density was measured at 545 nm from the support side. The results are shown in Table-2.

As is clear from Table 2 below, it was found that the multilayer analytical element of the present invention significantly reduced the influence of interfering proteins compared to the comparative analytical element.

Claims (1)

[Claims] In a multilayer analytical element consisting of a non-protein hydrophilic polymer layer and a porous development layer sequentially laminated on a liquid-impermeable and light-transparent support, when bonded to albumin under predetermined pH conditions, Compounds that cause a detectable color change are
A multilayer analytical element for albumin measurement, characterized in that the porous spreading layer contains the porous spreading layer in a form that does not substantially move within the layer and/or between the layers.