GB2118301A - Multilayer element for quantitative analysis of protein - Google Patents

Abstract

An integral multilayer element for quantitative determination of proteins such as albumin and globulin and comprising, in sequence, a water-impermeable transparent support, a reagent layer and a liquid sample spreading layer is characterized in that the reagent layer comprises: an adsorbent for protein such as a fine colloidal silica carrying an indicator capable of showing a photometrically detectable change when combined with a protein, and a non-proteinous binder capable of allowing permeation of the protein, for example a polyacrylamide or acrylamide copolymer binder.

Description

SPECIFICATION Multilayer element sor quantitative analysis of proteins This invention relates to a multilayer element for quantitative analysis of proteins. More particularly, this invention relates to an integral multilayer element for quantitative analysis of proteins employable in a dry-type analytical process.

A variety of methods for quanti2 .tative analysis of proteins were developed and have been improved until now. As the methods for quantitative analysis of proteins in a solution, there are known Kjeldahl method, Phenol reagent method, Lowry improved method, Biuret method, UV method, Dye-combining method and so forth. These methods are employed upon selection in view of the analytical purpose, analytical conditions, sample volume, protein content, coexis .ting substances and so forth.

All of these known analytical methods comprise diluting or dissolving a sample liquid in an aqueous solution containing an appropriate agent in a certain concentration and determining the reaction product colorimetrically or fluorometrically upon completion of the reaction. In the UV method, the constitutional unit of the proteins is directly determined by measuring the absorbance in a UV reagion. Accordingly, the influence of coexisting substances should be carefully co sidered.

The above-mentioned analytical methods are based on the so-called wet chemistry involving determination of proteins upon diluting a sample liquid containing the protein.

Also known and practically em0loyed are test strip methods employing a reagent-retainable paper strip impregnated with a color indicator, which comprise spotting a liquid sample on the strip and determining the produced color change thereon. The paper strip methods are not appropriate for precisely determining an analyte such as protein. However, these are widely employed for semi-quantitative analysis or simple identification, because these are less expensive and easy in the determination procedure.

Recently, a multilayer element for chemical analysis in the form of a film or sheet has been proposed and employed practically in certain fields. The multilayer element comprises a fundamental structure in which a reagent layer and a liquid sample spreadinl layer consisting of a porous medium are provided onto a transparent support.

The procedure for quantitative analysis of an analyte using the multilayer element involves spotting a liquid sample on the liquid sample spreading layer and measuring photometrically the color production or color change produced in the reagent layer. Thus, the analytical method using the multilqayer element is based on a dry analytical process which employs no liquid reagent. Nevertheless, the multilayer element is apprtpriate for quantitative analysis with high accuracy by means of photometric (e.g., colorimetric or fluorometric) measurement.

In the art using the multilayer element, the use of a color indicator capable of producing color upon combined with proteins is also known. As the color indicator for protein, the following is generally employed: Bromocresol Green, Bromophenol Blue, Bromocresol Purple, Amido Black, Orange G, Methyl Orange or Buffalo Black.

All of these indicators are substantially water-soluble and relatively low in the molecular weight. For the reasons, the indicator becomes diffusive and easily moves throughout the multilayer element during the measurement process. In contrast, the protein (analyte) is high in the molecular weight, and accordingly is less diffusive in the element, as compared with the color indicator.In consequence, when a sample liquid is spotted on a liquid sample spreading layer of a conventional multilayer element which comprises, in sequence, a support, a reagent layer containing the above-mentioned color indicator, and a liquid sample spreading layer (liquid sample receiving layer), the color indicator contained in the reagent layer moves quickly toward the protein still remaining in the liquid sample spreading layer because the diffusive movement of the color indicator into the spreading layer takes place more rapidly than the diffusive movement of the protein into the reagent layer. In this respect, Sanford et al.

reported the properties of a multilayer analytical element consisting of a transparent support, a reagent layer containing Bromocresol Green, and a reflective white spreading layer (Clinical Chemistry, 26(7), 1059(1980) and Japanese Patent Provisional Publication (JPPP) No. 57(1982)-50660 (corresponding to U.S. Pat. No.4,333,733)). According to the disclosures contained therein, the protein in the sample liquid remains in the spreading layer and Bromocresol Green contained in the reagent layer moves into the spreading layer so as to form an albumin-indicator complex in combination.

Further known is a multilayer element for quantitative analysis of proteins, which is based on Biuret reaction (JPPP No. 54(1979)-101398 (corresponding to U.S. Pat. No.4,132,528)). The analytical method based on the photometric measurement of Biuret reaction of proteins, as such, has been known and employed in practice until now. The multilayer element disclosed in the Publication consists of a transparent support, a reagent layer and a diffusion layer, and is characterized in that the reagent layer comprises a mixture of an alkali-protective polymer and a base in an amount enough for rendering the element alkaline of not lower than pH 12 under the working conditions and that the reagent layer contains substantially no sodium ion.Its embodiment shows that the reagent layer contains Biuret reagent consisting of cupper sulfate, iithium hydroxide and tartaric acid, and an alkali-protective polymer such as agarose, or acrylamide-vinylpyrrolidone copolymer. In the Publication, it is also disclosed that the color reaction proceeds mainly in the diffusion layer.

Eikenberry et al. reported the analyses of all proteins present in serum using an analytical element prepared according to the above-mentioned art (Clinical Chemistry, 25(6), 1125(1979)). The report describes that the Biuret reagent contained in the reagent layer quickly moved into the spreading layer and reacted with the protein to produce color when a liquid sample was spotted, and then the measurement was carried out through reflective photometry to determine the amount of protein.

U.S. Pat. No.4,230,456 discloses a multilayer analytical element containing a detector reagent supported on a carrier. Thus, this art employs a non-diffusive detector reagent and utilizes the fact that the binding property and fluorescence strength vary dependent upon a combination between the carrier and albumin. In more detail, the art disclosed therein is required to involve four stages; that is, (1) provision of a detector reagent onto a carrier; (2) liberation of the detector reagent upon introduction of albumin; (3) a combining reaction between the albumin and the detector reagent; and (4) quantitative measurement of increase or decrease of signals produced upon the combining reaction between the detector reagent and the albumin. In the Patent, the terminology "detectable species" is given in Claims and Specification.However, detector reagents illustrated in the description of embodiments and working examples are all fluorescence indicators capable of emitting an increased fluorescence strength upon combined with albumin: Moreover, as the carriers there are mentioned only polyvinyl alcohol, gelatin and cationic mordant polymers. No mention is given to detailed description of the carrier, detector reagent and conditions of the detector reagent supported on the carrier. Thus, this art is understood to disclose quantitative analysis of proteins, particular ly albumin, based on increase of fluorescence strength caused by binding of the protein with a fluorescence indicator contained in a polymer matrix capable of keeping the indicator from diffusion.

The multilayer element for quantitative analysis of proteins according to the present invention is formu lated on the following: a color indicator combinable with proteins resulting in color reaction, namely, color formation, color change or disappearance of color, or a fluorescence indicator combinable with proteins resulting inchange in fluorescence strength or change in the wavelength is carried on a carrier and retained within a reagent layer; proteins (analyte) introduced into the spreading layer in the form of a protein-containing liquid sample is diffused or accelerated to diffuse into the reagent layer; the proteins react with the indicator carried on the carrier under influence of attraction force between the carrier and proteins; and then the change in absorbance or fluorescence strength caused by the reaction is measured to give the quantitative deter mination.

The present invention provides an integral multi layer element for quantitative analysis of proteins comprising, in sequence, a water-impermeable transparent support, a reagent layer and a liquid sample spreading layer, which is characterized in that the reagent layer comprises: a protein adsorbent carrying an indicator capable of showing a photometrically detectable change upon combined with a protein, and a non-proteinous binder capable of allowing permeation of the protein.

The invention is further described hereinbelow.

The water-impermeable transparent support of the integral multilayer element for quantitative analysis of proteins (referred to hereinafter as "multilayer analytical element" or "analytical element") according to the invention is a support in the form of a film, sheet, thin plate or the like. This support is so transparent as to transmit electromagnetic radiation having line or band spectrum in the range of about 200 nm to about 900 nm, namely, ultraviolet rays, near-ultraviolet rays, visible rays, or near-infrared rays, to the extent of at least about 40%, preferably at least about 65%, and is substantially kept from permeation of water. Examples of the support material include polymers such as cellulose acetate, cellulose acetate butyrate, cellulose acetate propionate, polyethylene terephthalate, polycarbonate of Bisphenol A, and polystyrene, and glass.

If integral laminated layers comprising the spreading layer and the reagent layers but the support are self-supporting, these layers can serve also as the support, whereby allowing unemployment of the above-mentioned support.

A surface of the support can be provided with a known subbing layer to facilitate or reinforce adhesion between the support and the reagent layer (or, with other functional layer or layers) to prepare the integral laminated structure. Otherwise, a known chemical treatment such as acid treatment or alkali treatment, or a known physicochemical treatment such as corona discharge treatment, glow discharge treatment, ultraviolet ray irradiation or flame treatment can be applied to the surface of the support for the above-mentioned adhesion purpose.

The indicator to be employed in the invention is a color indicator combinable with proteins resulting in color reaction such as color formation, color change or color disappearance, or a fluorescence indicator combinable with proteins resulting in change in fluorescence strength or change in the wavelength.

The indicator prefer. ably is substantially water-solu ble.

Examples of the color indicator include Amido Black 10B (Color Index (Cl) Constitution No. 470), Acid Orange R (Cl Constitution No. 16 100), Buffalo Black (Cl Constitution No. 480), Orange G (Cl Constitution No. 230), Methyl Orange (Cl Constitution No. 025), Bromo Phenol Blue (cs, - Bis(3,5 - dibromo - 4 - hydroxyphenyl) - a - hydroxy - o - toluenesulfonic acid, y - sultone), Bromo Cresol Green (a,a - Bis(3,5 - dibromo - 4 - hydroxy - o - tolyl) - a - hydroxy - o - toluenesulfonic acid, y - sultone), Azosulfathiazole, and 2 - p - toluidinonaphthalene - 6 - sulfonate. Examples of the fluorescence indicator include Eosine Y (Cl Constitution No. 45380), Safranine Orange (Cl Constitution No. 50 240), trinitrobenzene sulfonic acid, 1 - anilinonaphthalene - 8 - sulphonic acid, 5 - (4' - aresenoanilino) - 2 chloro - 7 - methoxyacridine, Fluorescamine (CAS Registry No. 38183-12-9), Thiamine and 1 - (dimethy lamino) - naphthalene - 5 - sulfonyl chloride.

These indicators, as well as the protein adsor bents, are described in "Experimental Methods of Biochemistry VII: The Assay of Proteins" edited by K Sugawara and M. Fukusima (published by Gakkai Publication Center, Japan, 1979), pp. 147-165 and pp 179-188.

The color indicator or fluorescence indicator is generally included in the reagent layer in the amount ranging from about 0.2 to about 5.0 g/m2. Preferred is the range of about 0.4 to about 3.0 g/m2.

In the reagent layer of the analytical element according to the invention, the above-mentioned protein indicator is included under carried on a protein adsorbent. The analytical element of the invention is characteristic in that albumin or other proteins having high molecular weight are permeable into the reagent layer. Thus, the multilayer analytical element of the invention utilizes the diffusion of proteins contained in a sample liquid into the reagent layer and the combining reaction between the proteins and the indicator on the surface ofthe protein absorbent.

The protein absorbent to be included in the analytical element of the invention is one capable of adsorbing and fixing thereonto an indicator which is inherently more diffusable than proteins and capable of causing thereon a color reaction (color formation, color change or disappearance of color) or a change of fluorescence strength or change win wavelength of fluorescence between the indicator and proteins when a protein-containingliquid sample is applied onto the analytical element and the protein is diffused into the reagent layer.

Examples of the protein adsorbent include a variety of known protein adsorbents such as various anion exchange resins, various cation exchange resins, silica gel, calcium phosphate gel, hydroxyapatite gel, alumina gel and activated white clay.

When the protein adsorbent is employed as carrier for the indicator and the environmental conditions such as pH, ion strength, and temperature are adjusted appropriately for the color indicator or fluorescence indicator employed, the desired reaction between the indicator and the introduced proteins is caused on the carrier. The thus caused reaction is photometrically determined to enable the quantitative analysis.

In the invention, DEAE-cellulose type protein adsorbent and a colloidal silica are preferred as the indicator carriers. More preferred is a finely particulated colloidal silica. Such finely particulated colloidal silica is available on the market under the trade names of Aerosil (DEGUSSA, West Germany), Silanox (tradename of Cabbot Corp., USA), Siloid (Fuji Davison Chemical Co., Ltd., Japan), and Snowtex (a polar solvent dispersion, Nissan Chemical Industries, Ltd., Japan). The finely particulated colloidal silica prominently adsorbesthe indicator, hardly disturbs the reaction of indicator, and is well combinable with proteins. Accordingly, Aerosil is particularly preferred.

When an indicator is placed on an adsorbent, the indicator is generally stabilized resulting in deterioration of the sensitivity for producing a color change or the like. For instance, a strong ion exchange resin is highly combinable with proteins, as well as indicators. However, the strong ion exchange resin having adsorbed a color indicator shows a color reaction (blue reaction), and hardly shows a color change in relation to an amount of an introduced protein. For this reason, the pH conditions and ion strength are necessarily adjusted appropriately when the strong ion exchange resin is employed as the carrier. Such adjustments sometimes deteriorate the storage stability of the analytical element and cause formation of fog, whereby deteriorating the reliability of the analytical element.

In contrast, the finely particulated colloidal silica never shows color change upon receiving an indicator and has appropriate protein-adsorbing capacity.

Further, the fine colloidal silica is a highly hydrophilic colloid and well dispersable in an aqueous medium. The fine colloidal silica hardly disturbs the photometric measurement because it becomes nearly transparent upon coated with a binder and dried.

Furthermore, the finely particulated colloidal silica shows so-called thixotropy due to the hydrogen bond-formation property on its activatee surface.

Accordingly, the fine colloidal silica shows low flowability and is easily set when subjected to the coating procedure, whereby enabling formation of a well stabilized coating layer. These properties are very advantageous in the preparation of the multilayer analytical element of the invention.

The binder employable for the preparation of a reagent layer in the multilayer analytical element of the invention is ought to be hydrophilic, of substantially no or little property to combine with proteins, and of property for allowing permeation of proteins.

Naturally the binder neither disturbs the analyticaily detectable reaction nor causes analytical errors.

Also, the binder shows substantially no or little interaction with the indicator.

Appropriate polymers for the binder of the reagent layer of the invention are polyacrylamide and acrylamide copolymers containing the acrylamide monomer units. Examples of the acrylamide copolymers include acrylamide - N - vinylpyrrolidone copolymer, acrylamide - N - (hydroxyphenol)acrylamide copolymer, and acrylamide - 2 - hydroxyethylacrylate copolymer. Acrylamide polymers swell with water to form large voids within the reagent layer so that macromolecules such as proteins can easily permeate into the reagent layer. Acrylamide polymers are further advantageously used because of their small interactit witt proteins and indicators.

Other preferably employable polymers are, for instance, agarose, poly - N - vinylpyrrolidone, carboxymethylcellulose, methylcellulose, maleic acid copolymers, and meleinamide copolymers.

Cationic polymers such as styrene - N - vinylbenzyl - N,N - dimethyl - N - benzylammonium chloride divinylbenzene terpolymer and styrene - N - vinylbenzyl - N,N - dimethyl - N - benzylammonium chloride copolymer may be employed as the binder of the analytical element of the invention. However, these cationic polymers sometime disturb the color reaction between the indicator and protein, and therefore need careful selection in the practical uses.

The reagent layer ranges in thickness from about 7 to about 50 pom (thickness upon dried), and preferably from about 10 to about 30 pom.

In carrying out the quantitative analysis of proteins using the analytical element of the invention, environmental conditions such as pH and ionic strength are preferably adjusted to the optimum conditions so as to obtain well and reliable analytical results. For this purpose, the functional layer of the element such as the spreading layer, the reagent layer, or a light-shielding layer (or, radiation block ing layer) preferably contains one or more of other reagents such as an acid, alkali, salt, buffer reagent, dissociation reagent, surface active agent, and the like. The amounts and kinds of these additional reagents are selected according to the purpose of the analytical procedure, because these selections are highly dependent on the purpose.

The reagent layer preferably contains an acid or a buffer reagent so that the binding reaction between the analyte and the indicator proceeds under acidic conditions in the pH range of from about 2.0 to about 7.0, preferably about 3.0 to about 6.5. In the case of albumin as the analyte, the pH range is adjusted to about 2.0 to about 4.0, preferably about 3.0 to about 3.5.

Examples of the acid and buffer reagent employable for the pH adjustment of the reagent layer include aliphatic hydroxycarboxylic acids (examples: glycoiic acid, lactic acid, a - hydroxybutyric acid, glyceric acid, tartronic acid, malic acid, tartaric acid, and citric acid, etc.), aliphatic dicarboxylic acids (examples: malonic acid, succinic acid, 3,3 dimethylglutaric acid, and cx,cx' - dimethylglutaric acid, etc.), aliphatic monocarboxylic acids (exam ples: acetic acid, propionic acid), and butyric acid, etc.), inorganic acids (examples: sulfuric acid, and phosphoric acid, etc.), buffer reagents described in "Kagakubinran, Kiso - hen (Chemistry Handbook, Fundamentals)" edited by the Chemical Society of Japan (Maruzen Tokyo, 1966), pp.1312-1320 (exam ples: potassium hydrogen citrate-citric acid, and potassium dihydrogenphosphate - potassium hyd rogenphosphate, etc.), and buffer reagents de scribed in "Hydrogen Buffers for Biological Research" reported by Norman E. Goods et al.: Biochemistry, 5(2), 467-477 (1966) (example: 2 - (N morpholino)ethanesulfonic acid, etc.), and other buffer reagents (examples: sodium or potassium hydrogenmalate, and sodium or potassium monohydrogen - 3,3 - dimethylglutarate, etc.).

Among these reagents, citric acid, tartaric acid, malic acid, and 3,3 - dimethylglutaric acid are preferred.

The layers other than the reagent layer may contain these acids or buffer reagents, if necessary.

In the ciinical analysis, specific proteins within a body fluid, namely albumin and globulin, are necessarily analyzed separately. In this analysis, an indicator reactive specifically to each protein is employed.

For instance, Bromocresol Green is advantageously employed for quantitative analysis of albumin, because it changes markedly in color upon combining with albumin. Otherwise, a specific protein only can be introduced into the reagent layer by providing an additional layer such as a light-shielding layer or an adhesive layer containing a protein-permeable hydrophilic binder and a diffusion - controlling binder, because globulin is less diffusible than albumin in the layer. The diffusion controlling binder can be employed in the amount of about 3 to about 20% by weight of the protein - permeable binder. The diffusion controlling binder preferably is a hydrophi lic polymer. A representative example of the diffu sion - controlling binder is gelatin.

The sample fluid spreading layer (referred to herein as "spreading layer") of the analytical ele ment of the invention is arranged at the outermost position of the analytical element. In other words, the spreading layer is provided at the outermost position far from the support via the reagent layer.

The liquid sample is applied or spotted onto the spreading layer. The function of this layer is to supply a liquid sample together with albumin or other water-soluble proteins to the reagent layer at an approximately constant volume per unit area regardless of its applied volume, that is to say, "metering effect". Thus, this layer acts as a spreader for a liquid sample. Because of the spreading action, a volume of the liquid sample supplied to the reagent layer per unit area is automatically adjusted at a certain value regardless of its applied volume.

This means that a liquid sample can be analyzed quantitatively without precise measurement of the volume when applied to the analytical element of the invention. However, it should be understood that the use of the analytical element of the invention never excludes precise measurement of a sample liquid in carrying out the quantitative analysis. The precise measurement of a sample liquid sometimes increases accuracy of the analysis.

Examples of the spreading layer of the invention include non-fibrous, isotropically porous layers as disclosed in Japanese Patent Provisional Publication (JPPP) No.49 (1974)-53888 (Japanese Patent Publication No. 53 (1978)-21677) and U.S. Pat. No.

3,992,158, for instance, membrane filters, blush polymer layers, an isotropically porous layer comprising voids defined by fine spheres or particles connected to each other through binders; an isotropically porous layer having continuous voids and being formed by three - dimensional matrix in which fine spherical beads are bound in point - to - point contact in all directions through binder not sweiling with water, as disclosed in JPPP No. 55 (1970)-90859 (corresponding to U.S. Pat. No. 4,258,001); a fibrous, anisotropically porous spreading layer consisting of water-washed fabrics or hydrophilically processed fabrics, as disclosed in JPPP No. 55 (1970)-164356 (corresponding to U.S.Pat. No.4,292,272); a fibrous, anisotropically porous spreading layer consisting of fabrics having phisically activated surfaces, as disclosed in JPPP No. 57 (1982)-66359; and a fibrous, anisotropic porous spreading layer consisting of paper, paper filter or no-woven fabrics containing synthetic polymer fiber pulps, as disclosed in JPPP No. 57 (1982)-66359. Any of these spreading layers can be provided to the analytical element of the invention in manners disclosed in these patent specifications. Otherwise, a membranefilter or blushed polymer layer containing titanium dioxide, zinc oxide, or barium sulfate in the form of fine powder disclosed in JPPP No.49 (1974)-53888 can be employed in the analytical element of the invention for serving as a spreading layer and also as a light-shielding layer (radiation-blocking alyer, white background layer, or light-reflecting layer), details being given hereinafter. Further, a membrane filter our a blushed polymer layer containing carbon black can be employed as a spreading layer which also serves as a light-shielding layer, as described hereinafter.If a sample liquid is a whole blood, the spreading layer preferably is the aforementioned isotropically porous layer having continuous voids and being formed bythree-dimensional matrix, or the fibrous, anisotropically porous spreading layer.

The multilayer analytical element of the invention can be provided with a protein-permeable, lightshielding layer (radiation-blocking layer, background layer, or light-reflecting layer) between the reagent layer and the spreading layer.

The light-shielding layer is advantageously provided if the analytical element is employed for analysis of a sample liquid containing colored particles such as the whole blood containing red corpuscles. In more detail, colored particles positioned on one side of the light-shielding layer are photometrically shielded by the light-shielding layer from observation through the transparent support.

Accordingly, the colorimetric or fluorometric measurement is not interfered by the presence of colored particles. The light-shielding layer can be composed of a fine powder such as finely particulated titanium dioxide, barium sulfate, zinc oxide, aluminum or carbon black dispersed within a waterand protein-permeable, hydrophilic polymer binder.

The light-shielding layer has the thickness in the range of from 5 to 100 um, preferably 5 to 30 um, and allows a liquid sample and an analyte passing therethrough. The polymer binder for the preparation of the light-shielding layer can be optionally selected from the protein-permeable hydrophilic polymers described hereinbefore in connection with the binder for the reagent layer.

The light-shielding layer can be a porous lightshielding layer consisting of membrane filter (blushed polymer layer) containing light-shielding particles such as finely particulated titatnium dioxide, zinc oxide, barium sulfate and carbon black. The porous light-shielding layer can be provided to the analytical element in the manner disclosed in Japanese Patent Provisional Publication (JPPP) No.

49 (1974)-53888 (Japanese Patent Publication No. 53 (1978)-21677) orJPPP No. 54(1979)-29700 (corresponding to U.S. Pat. No.4,166,093).

The multilayer analytical element of the invention can be provided with an adhesive layer for superposing the spreading layer on the reagent layer, the light-shielding layer, or other optionally-placed layers under increased adhesion to form an integral laminated structure. The adhesive layer is preferably provided if the spreading layer is made of porous sheet, porous film or porous membrane.

The adhesive layer can be produced from one or more of the water- and protein-permeable hydrophilic polymer described hereinbefore in connection with the binder for the reagent layer. The adhesion of the spreading layer to the adhesive layer can be carried out by placing a porous sheet, film or membrane under pressure on an adhesive layer consisting of half-dried hydrophilic polymer or wetted with water or an aqueous solution containing a surface active agent. The adhesive layer has the thickness in the range of from 0.5-1 5pm, preferably 0.5 to 5 my.

The integral multilayer element for quantitative analysis of proteins of the invention can be provided, if desired, with a variety of layers disclosed in Japanese Patent Provisional Publication No. 49 (1974)-53888 (Japanese Patent Publication No. 53 (1978)-21677); U.S. Pats. No. 3,992,158, and No.

4,110,079; Japanese Patent Provisional Publications No. 54 (1979)-101398 (corresponding to U.S. Pat. No.

4,132,518), No.55 (1980)-90859, No. 55 (1980)-164356, and No. 57 (1982)-50660; and Japanese Utility Model Provisional Publication No.

57 (1982)-42951. Moreover, one or more of these layers can be modified by the use of a water- and protein-permeable hydrophilic polymer binder.

The integral multilayer analytical element of the invention comprises, in sequence, a spreading layer, a reagent layer, and a transparent support. Preferred are an arrangement comprising, in sequence, a spreading layer, a light-shielding layer, a reagent layer, and a transparent layer, and an arrangement comprising, in sequence, a spreading layer, an adhesive layer, a light-shielding layer, a reagent layer, and a transparent support.

The multilayer analytical element of the invention can be prepared in the manners disclosed in the aforementioned patent specifications. Examples of the detailed procedures are described in the hereinafter-given working examples.

The analytical element of the invention can be employed in quantitative analysis of proteins contained in a liquid sample in the same manners as disclosed in the aforementioned patent specifications. The analytical element of the invention is preferably received in a slide frame and employed in the form of an analytical slide as disclosed in Japanese Utility Model Provisional Publications No.

54 (1979)-162294, and No. 56 (1981)-142454, Japanese Patent Provisional Publiration No. 54 (1979)-156079 (corresponding to U.S. Pat. No.

4,169,751), and Japanese Patent Application No. 55 (1 980)-1 38100. The analytical element in the form of a slide is preferred in all aspects, nemely, preparation, transpotation, storage, measurement procedure and so forth.

EXAMPLE 1 In 20 ml. of water was dispersed 1.5 g of finely particulated silica gel (Aerosil, tradename), and the dispersion was processed by means of a superstni etulsifier for 30 seconds. The dispersion obtained was slightly turbid but nearly clear. To the dispersion were successively added, in sequence, the following reagents under stirring: 2 ml. of 10% aqueous solution of polyoxyethylene lauryl ether (nonionic surface active agent); 300 mg. of Bromocresol Green in a mixture of 2 ml. of water and 2 ml. of ethanol; 26 g. of 10% aqueous polyacrylamide solution; 0.5 ml. of 10% aqueous citric acid solution; and 20 ml. of water.

The resulting solution was a yellowish, transparent and viscous solution.

The solution was then coated over a transparent polyethylene terephathalate (PET) film (thickness: 185 pom) having a gelatin subbing layer. The coated solution was dried to give a layer having the thickness of 14 pom.

On the so produced layer was coated a lightshielding layer-producing dispersion of 3.4 g. of titanium dioxide fine powder, 0.34 g. of gelatin, 3 g.

of polyacrylamide and 1 g. of citric acid in 33 g. of water. The coated dispersion was then dried to give a light-shielding layer having the thickness of about 5calm.

The so obtained multilayerfilm was wetted with 0.2% aqueous solution of octylphenoxypolyethoxyethanol (nonionic surface active agent). Immediately, a porous cellulose acetate membrane filter having the average pore size of 5 pom and the thickness of 140 pom (Fuji Microfilter FM-500, tradename of Fuji Photo Film Co., Ltd., Japan) was pressed on the wetted light-shielding layer to give a multilayer sheet for quantitative analysis of albumin. This sheet was cut into a square piece (15 mmx 15 mm), which was in turn inserted into a rectangular plastic mount (24 mm x28 mm) having a circular opening of the diameter of 10 mm. Thus, an analytical slide was prepared.

On the spreading layer, namely, the porous ceilulose acetate membrane filter layer, of the analytical slide was spotted 10111. of a physiological saline solution (pH 7.2) containing a known concentration of albumin. The analytical slide was subsequently incubated for 10 minutes at room temperature (about 23"C), and subjected to measurement of reflection optical density at 600 nm (wavelength).

The results are set forth in Table 1. Thus, it was confirmed that the color reaction took place in proportion to the amount of albumin.

Table 1 (Albumin Concentration (g/de) 0 0.5 1.0 2 4 6 8 10 Reflection Optical 0.22 0.25 0.27 0.36 0.47 0.58 0.68 0.77 Density (600 nm) EXAMPLE 2 The reagent layer was provided onto the transparent pet film in the same manner as in Example 1 except that polyacrylamide (binder) was replaced with acrylamide - N - (m - hydroxyphenyl)acrylamide copolymer (copolymerization ratio, 95:5) and that formalin (37% aqueous formaldehyde solution was included in the amount of 1.5% by weight of the copolymer.

The spreading layer was then superposed on the reagent layer in the same manner as in Example 1 except that the membrane filter was replaced with a broadcloth woven from cotton yarn of 100 counts to give an analytical slide.

On the spreading layer of the slide was spotted 10 p1. of the same physiological saline solution as in Example 1. The slide was subsequently incubated for 10 minutes at 370C, and subjected to measurement of reflection optical density at 600 nm (wavelength). The photometric measurement was carried out from the PET film slide.

The relationship between the albumin concentration and the reflection optical density Is graphically illustrated in Figure 1. Thus, satisfactory relationship was confirmed.

EXAMPLE 3 A slide for quantitative analysis of proteins was prepared in the same manner as in Example 2 except that Bromocresol Green was replaced with 200 mg. of Methyl Orange.

A physiological saline solution was added to 2 ml.

of human serum to prepare 4 sample solutions having different protein content. The total protein contents was determined by Biuret method for each sample liquid.

On the spreading layer of the slide was spotted 10 pI. of the sample liquid. The slide was subsequently incubated for 10 minutes at 37"C and subjected to measurement of reflection optical density (wavelength: 550 nm).

The relationship between the total protein content and the reflection optical density is set forth in Table 2. Thus, satisfactory relationship was confirmed.

Table 2 Total Protein Concentration (g/de) 1 4 8 10 Reflection Optical 0.32 0.56 0.83 0.97 Density (550 nm)

Claims (15)

1. An integral multilayer element for quantitative analysis of proteins comprising, in sequence, a water-impermeable transparent support, a reagent layer and a liquid sample spreading layer, which is characterized in that the reagent layer comprises: a protein adsorbent carrying an indicator capable of showing a photometrically detectable change upon combined with a protein, and a non-proteinous binder capable of allowing permeation of the protein.

2. The integral multilayer element as claimed in Claim 1, in which the protein adsorbent is selected from the group consisting of an anionic ion-exchange resin, a cationic ion-exchange resin, silica gel, calcium phosphate gel, hydroxyapatite gel, alumina gel and activated clay.

3. The integral multilayer element as claimed in Claim 2, in which the protein adsorbent is selected from the group consisting of a DEAE-cellulose type protein adsorbent and colloidal silica.

4. The integral multilayer element as claimed in Claim 3, in which the protein adsorbent is a finely particulated colloidal silica.

5. The integral multilayer element as claimed in Claim 1, in which the indicator is selected from the group consisting of a color indicator and a fluorescent indicator.

6. The integral multilayer element as claimed in Claim 1, in which the non-proteinous binder is selected from the group consisting of polyacrylamide and acrylamide copolymers.

7. The integral multilayer element as claimed in Claim 6, in which the non-proteinous binder is selected from the group consisting of polyacrylamide, acrylamide - N - vinylpyrrolidone copolymer, acrylamide - N - (hydroxyphenyl)acrylamide copolymer, and acrylamide - 2 - hydroxyethyl acrylate copolymer.

8. The integral multilayer element as claimed in Claim 1, in which the reagent layer has the dry thickness ranging from about 7 to about 50 pom.

9. The integral multilayer element as claimed in Claim 8, in which the reagent layer has the dry thickness ranging from about 10 to about 30 pom.

10. The integral multilayer element as claimed in Claim 1, in which a radiation-blocking layer or a light reflecting layer is provided between the reagent layer and the liquid sample spreading layer.

11. The integral multilayer element as claimed in Claim Sin which said indicator is substantially water soluble.

12. The integral multilayer element as claimed in Claim 8 or 9, in which said reagent layer contains said color indicator in the amount ranging from about 0.2 g/m2 to about 5.0 g/m2.

13. The integral multilayer element as claimed in Claim 10, in which said radiation blocking layer or light reflecting layer contains a non-proteinous hydrophilic binder capable of allowing permeation of the protein.

14. The integral multilayer element as claimed in Claim 13, in which said radiation blocking layer further contains a diffusion-controlling binder.

15. The integral multilayer element as claimed in Claim 14, in which said diffusion-controlling binder is gelatin.