JPH0260264B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] 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. BACKGROUND OF THE INVENTION Many methods have been developed in the past for analyzing specific components in biological 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 the specification of 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. In this case, it has been disclosed that the specificity of the dye for albumin can be increased by adjusting the pH and the transfer rate of bromocresol green. Furthermore, JP-A-58-179359 discloses a method of incorporating a protein adsorbent carrying an optically detectable indicator into a non-protein binder layer that is permeable to proteins to form a reagent layer. ing. 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. [Object of the Invention] The object of the present invention is to provide a multilayer analytical element for albumin measurement that can measure albumin in a biological fluid sample without being affected by other interfering proteins such as globulin, and is stable in terms of manufacture. The goal is to provide the following. [Structure of the Invention] The above object of the present invention is to provide a liquid impermeable material, and
A compound that causes a detectable color change when combined with albumin under specified pH conditions in a multilayer analytical element consisting of a non-protein hydrophilic polymer layer and a porous development layer sequentially laminated on a light-transparent support. of,
This is achieved by using a multilayer analytical element that is contained in the porous spreading layer without substantially moving within and/or between the layers. [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, bromophenol blue, amide black 10B, acid orange R, buffer arrow black, orange G, azosulfathiazole,
Examples include 2-p-toluidylnaphthalene-6-sulfonate. Examples of fluorescent indicators include eosin Y, safranin orange, trinitrobenzenesulfonic acid, 1-anilinonaphthalene-8-sulfonic acid,
5-(4'-Arsenoanilino)-2-chloro-7-
Examples include methoxyacridine, fluoresamine, thiamine, 1-(dimethylamino)naphthalene-5-sulfonyl chloride, and the like. Among these, particularly preferably used color indicators include, for example, 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 that does not substantially move within the layer and/or between the layers. As a method for incorporating the compound according to the present invention into a porous spreading layer, for example, Japanese Patent Application Laid-Open No. 57-197466
(U.S. Pat. No. 4,427,632), in the case of a porous spreading layer consisting of discrete fibers and a polymer binder having reactive groups, the fibers are pre-impregnated with the compound according to the present invention, or
Alternatively, a method in which the compound according to the present invention is finely dispersed is effective. 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, regardless of whether it is impregnated or finely dispersed. Also, Special Publication No. 53-21677 (U.S. Patent No. 3992158)
In the case of a non-fibrous isotropic porous layer (for example, a brushed polymer layer) disclosed in No. 1, it is preferable to incorporate the compound according to the present invention by fine dispersion. Furthermore, in the case of a woven fabric that has been subjected to a hydrophilic treatment as disclosed in JP-A-55-164356, the woven fabric can be impregnated and then immobilized with, for example, the above-mentioned copolymer. However, among these porous spread layers, the spread layer disclosed in JP-A-57-197466 is easy to manufacture and is useful because both impregnation and fine dispersion can be selected. The compound according to the invention is present in the porous spreading layer about
0.1 g/m ² to about 5.0 g/m ² , preferably about 0.5 g/m 2
The content ranges from about 3.0 g/m ² to about 3.0 g/m ² . Further, the thickness of the porous spreading layer is preferably in the range of about 100 microns to about 350 microns. For this purpose, acids, alkalis, salts,
A buffer, a dissociating agent, a surfactant, etc. can be included as appropriate. Particularly in albumin analysis, the binding reaction between the compound of the present invention and albumin to be used is greatly influenced by pH, so the selection must be made carefully. PH of binding reaction with albumin in 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
The pH ranges from about 3.0 to about 6.5. An acid or a buffer for controlling pH is contained in the non-protein hydrophilic polymer layer, but can be contained in a layer other than the hydrophilic polymer layer 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, carboxymethyl cellulose, etc.
Examples include sodium salts, hydrophilic cellulose derivatives such as hydroxyethylcellulose and 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 thickness of the non-proteinaceous hydrophilic polymer layer is generally about 5 microns to about 50 microns, preferably about 10 microns to about 30 microns.
in the micron range. When quantifying albumin as described above, pH, ionic strength, etc. are important factors, so setting these factors to optimal conditions is important in obtaining good analytical results. Examples of acids or buffers used to control the pH in the porous spreading layer include aliphatic hydroxycarboxylic acids (e.g., 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-dimethylglutaric acid, α,α′-dimethylglutaric acid), aliphatic carboxylic acids (e.g. acetic acid,
(propionic acid, butyric acid), buffers made from combinations of salts of these acids and the above acids, and the like. Preferred acids or buffers with acids and their own salts include malic acid, lactic acid, succinic acid,
Examples include malonic acid, citric acid, and tartaric acid. The liquid-impermeable and light-transparent support that can be applied to the multilayer analytical element of the present invention includes wavelengths from about 200 nm to about
Electromagnetic radiation (ultraviolet, near ultraviolet, infrared,
A support in the form of a film, sheet, thin plate, or other form that transmits at least about 40%, preferably at least about 65% of (or near infrared rays) and is substantially impermeable to liquids, such as water; for,
Typical examples include polymers such as cellulose acetate, cellulose acetate butyrate, cellulose acetate propionate, polyethylene terephthalate, polystyrene, polycarbonate, and glass. According to the present invention, the non-protein hydrophilic polymer layer 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 a slide hopper coating method, a dip coating method, and a curtain coating method. Furthermore, as disclosed in JP-A No. 55-164350, in the case of a hydrophilized fabric, the porous spreading layer may be made of, for example, the non-proteinaceous hydrophilic polymer layer of the present invention or a similar material; 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 sheet-like material used as a spreading layer; A film or membranous 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. Examples of surfactants that can be used include alkyl phenoxypolyethoxyethanol [e.g., Triton Enoxyglycerin [e.g. Surf Actant 10G
(Olean Corporation)] is cited as a typical example. The multilayer analytical element of the present invention can have various functional layers and layer configurations as desired. For example, it is possible to arbitrarily select the layers and layer configurations described in JP-A-51-40191 (JP-A-58-18628), US Pat. No. 4,110,079, JP-A-58-131,565, and the like. The multilayer analysis element manufactured in this way can be made into various shapes depending on the analysis method. It can take a variety of configurations, including, for example, a stretchable tape of the desired width, a sheet, or a slide mounted on a plastic mount. The multilayer analytical element of the present invention contains the compound of the present invention in a porous spreading layer in a form that does not substantially migrate, so that albumin in a biological fluid sample is not affected by other interfering proteins. It can be measured without The multilayer analytical element of the present invention will be explained in detail below using experimental examples and examples, but the present invention is not limited thereto. Experimental Example In the multilayer analytical element for albumin analysis of the present invention, it was confirmed that the compound according to the present invention does not migrate within and/or between layers. Porous spreading layer of the present invention (1) Triton X-100 Rhom & Hass Co. 3.0 in 84 ml xylene
g. Dissolve 45 g of styrene-glycidyl methacrylate copolymer (9:1), mix with 0.585 g of bromocresol green, add glass beads, stir with a sand stirrer for 5 hours to disperse, and then mix with gauze. After filtration, the dispersion was separated from the glass beads. 10.5 g of powder filter paper C (Toyo Roshi Co., Ltd., 300 mesh or more) was mixed with 30 ml of this dispersion, and after ultrasonic dispersion, it was placed on a subbed polyethylene terephthalate support with a thickness of 180 microns.
Application is performed using a doctor blade with a gap of 375 microns, and the film thickness is 180 microns (dry).
A porous spread layer was formed and dried. Porous development layer of the present invention (2) (Preparation of bromocresol green impregnated fiber) Add 0.5 g of bromocresol green to a mixed solvent of 4.25 ml of acetone and 40 ml of xylene and dissolve with stirring, then add 4 ml of pure water and stir. Transfer the bromocresol green to the aqueous phase. Add 17.4 g of powdered filter paper C (Toyo Roshi Co., Ltd., 300 mesh or more) to this, mix, stir, and let stand. After confirming that the bromocresol green coloring has disappeared from the supernatant, filter and dry. (Creation of porous development layer) Add Triton X-100 to 84 ml of xylene.
(Rohm & Haas) 28.9 g of the bromocresol green impregnated fiber was added to a solution containing 3.0 g of styrene-glycidyl methacrylate copolymer (9:1) and 4.5 g of styrene-glycidyl methacrylate copolymer (9:1), and dispersed by ultrasonic irradiation while stirring. It was made into a liquid. 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, 32.28 g/m ² of polyacrylamide and 1.08 g of bromocresol green were prepared on a 180 micron subbed polyethylene terephthalate support according to the element manufacturing described in JP-A-57-50660.
A reagent layer was prepared containing 0.32 g/m ² of Surf Actant 10G (Olin-Mathieson). However, to prevent polyacrylamide from dissolving in water, formaldehyde 37% is added to polyacrylamide.
% aqueous solution was added at 1.0%. Each of the three types of films was cut to a certain area, immersed in 3 ml of distilled water, and the absorbance at 435 nm was measured at each time using a Hitachi spectrophotometer model 220-A with n=3. The results are shown in Table-1.

[Table] From Table 1 above, it can be seen that in the comparative reagent layer described in JP-A-57-50660, bromocresol green, which is a coloring indicator, quickly migrates from the layer.
The porous development layers (1) and (2) used in the present invention do not transfer dyes, which are color indicators, at all. Furthermore, since no difference was observed in the absorbance results even when the film was shaken during dipping, it is considered that bromocresol green did not migrate between or within layers. Example 1 As a multilayer analytical element for albumin 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 citric acid to 35 g of 10% polyacrylamide aqueous solution.
2.5g, Emulgen 120 (manufactured by Kao Atlas Co., Ltd.) 50%
After adding 5 ml of aqueous solution and stirring and dissolving the solution, add 30% aqueous sodium hydroxide solution to adjust the pH to 3.7.
It was made up to 50 ml with distilled water, 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 spread over the non-proteinaceous polymer layer prepared above.
Each was applied to a film thickness of 180 microns (when dry) using a 375 micron doctor blade and allowed to dry.
These are used in the multilayer analysis element () and () of the present invention.
And so. (Preparation of Comparative Multilayer Analytical Element) A comparative multilayer analytical element was prepared according to the description in JP-A No. 57-50660. (Reagent layer) Polyacrylamide 32.28 g/m ² Bromocresol Green 1.08 g/m ² Malic acid 5.4 g/m ² Surf Actant 10G (Olean)
Reagent layer consisting of 0.32g/ ^m2 . (Development layer) A development layer consisting of microcrystalline cellulose 53.80g/m ² and polyvinylpyrrolidone 1.35g/m ² . These multilayer analysis elements were cut to 1.5 cm x 1.5 cm and mounted on a 2.4 cm x 2.8 cm rectangular plastic mount with a 1 cm hole in the center to form an analysis slide. The analytical element of the present invention and the comparative analytical element prepared as described above were prepared, and human serum albumin was dissolved in physiological saline at a concentration of 2.0, 3.5, 5.0, 6.5, 7.5 g/dl as a specimen. Add 55% human globulin to the albumin level standard solution above.
Prepare a standard solution containing
Reflection density was measured at 545 nm. The results are shown in Table-2.

【table】

[Table] In the table, only albumin indicates the reflection density when albumin standard solution is dropped, globulin-containing indicates the reflection density when albumin + globulin 5% standard solution is added, and deviation indicates the percentage difference between the two. As is clear from Table 2, 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] 1. 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, albumin was bound under predetermined pH conditions. A multilayer analytical element for measuring albumin, characterized in that a compound that sometimes causes a detectable color change is contained in the porous developing layer in a form that does not substantially migrate within the layer and/or between the layers.