JPH0713635B2 - Manufacturing method of integrated multi-layer analytical element for calcium analysis - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing an integrated multilayer analytical element for analyzing calcium in an aqueous liquid sample, and more particularly to a biological fluid such as blood (whole blood, plasma, serum). ), Spinal fluid, lymph fluid, saliva, urine and the like, and an improved method for producing an integrated multi-layer analytical element which is dry-operable and particularly useful for clinical diagnosis for quantitative analysis of calcium in an aqueous liquid sample.

[Conventional technology]

Various types of integrated multi-layer analytical elements have been developed, but when the indicator etc. that reacts with the analyte and causes an optical change are water-soluble, the porous development layer from the reagent layer that should originally exist to the upper layer (Hereinafter, it may be simply referred to as “expanded layer”.) Etc. (this diffusion is referred to as “migration”), which may reduce the analysis accuracy. This problem is fatal to the analytical element when the indicator is well soluble in water, for example, in the case of calcium analysis, the analysis accuracy is significantly reduced due to migration, while the fluctuation range of calcium in blood is not so large. It was a thing.

Therefore, various studies have been carried out in order to improve such a defect. For example, a migration prevention layer has been introduced into the analysis element disclosed in Japanese Patent Laid-Open No. 54-29700. This migration-preventing layer was prepared by mordanting a water-soluble indicator with a polymer mordant to immobilize it to prevent diffusion.

On the other hand, it is also recognized that the aqueous liquid sample spotted on the porous spreading layer spreads too widely in the layer, and it is recognized as a problem in JP-A-60-222770.
It has been shown that a nonionic surfactant having an HLB value of 10 or more is effective as a development control agent. Although this development control agent is contained in the development layer as an aqueous solution, it is also shown that the nonionic surfactant can be contained in an organic solvent solution such as acetone.

[Problems to be solved by the invention]

Although the migration prevention layer described in JP-A-54-29700 described above can prevent the migration of the indicator, the mordant generally interferes with the color development and has not been a fundamental solution. Further, it is not easy to use, and it is costly.

That is, it is an object of the present invention to provide a means for producing an integrated multilayer analytical element which prevents migration of the indicator in the integrated multilayer analytical element for calcium analysis using a water-soluble indicator and does not reduce the analysis accuracy.

Another object of the present invention is to provide a means for easily producing an integrated multilayer analytical element for a calcium analysis using a water-soluble indicator, which does not reduce the analysis accuracy.

Still another object of the present invention is to provide an inexpensive means which does not lower the analysis accuracy of the integrated multilayer analytical element for calcium analysis using a water-soluble indicator.

[Means for solving problems]

As a result of intensive studies to solve these problems, the present inventor found that after providing a porous development layer on the reagent layer (directly or via an adhesive layer or the like), a development control agent was added to the porous development layer. It has been found that the above-mentioned object can be achieved by containing the above-mentioned substance by a method such as coating and dissolving it in an organic solvent in which the indicator is insoluble and then adding it to prevent migration of the indicator.

That is, the present invention provides a development control agent and a reagent layer containing a water-soluble indicator capable of reacting with calcium to cause an optically detectable change on a light-permeable, water-impermeable support. In producing an integrated multi-layer analytical element in which a porous developing layer containing the layer is laminated in this order, after providing the porous developing layer on the reagent layer, the development controlling agent in an organic solvent that does not dissolve the indicator. The present invention relates to a method for producing an integrated multi-layer analytical element for calcium analysis, which comprises dissolving the solution in a porous development layer and then drying the solution.

As the light-transmissive water-impermeable support, a light-transmissive (transparent) water-impermeable support conventionally used in a multilayer analysis element can be used. Specific examples thereof include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose ester (eg, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), and a thickness of about 50 μm to about 1 mm, preferably Is about 80 μm to about 300
Transparent in the μm range, ie wavelengths from about 200 nm to about 900 nm
It is possible to use a film-shaped (sheet-shaped) or flat-shaped support having a smooth surface that transmits electromagnetic radiation in at least a part of the wavelength range within the above range. If necessary, titanium dioxide fine particles, barium sulfate fine particles, carbon black and the like may be dispersed and contained in the support to control the optical performance. If necessary, a publicly known undercoat layer or an adhesive layer may be provided on the surface of the support to strengthen the adhesion between the support and the water absorbing layer or the reagent layer provided on the support.

The reagent layer comprises a reagent composition containing at least one indicator which reacts with calcium in an aqueous liquid sample to produce a detectable color change (preferably in the visible light region) in a hydrophilic polymer as a polymer binder. It is a water-absorbing, water-permeable layer that is substantially uniformly dispersed.

The hydrophilic polymer used in the reagent layer has a swelling rate upon absorption of water at 30 ° C. of about 150% to about 2000%, preferably about 250% to about 1500%. Specific examples of hydrophilic polymers include acid-treated gelatin described in JP-A-59-171864 and JP-A-60-115859, gelatin such as deionized gelatin, phthalated gelatin, gelatin derivatives such as hydroxyacrylate-grafted gelatin, and JP-A- 59-171864, JP-A-60
-115859 and the like, agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, Japanese Patent Application No. 60-171134 (JP-A-62-32).
No. 109) and methallyl alcohol binary or ternary copolymers. These hydrophilic polymers can be used alone or in combination of two or more. In the reagent layer, it is generally preferable to use gelatin or a gelatin derivative, polyacrylamide, polyvinyl alcohol, etc. Among these, gelatin (deionized gelatin) is most preferable. The thickness of the reagent layer when dried is approximately 5 μm
To about 50 μm, preferably about 7 μm to about 30 μm, and the coating amount is about 5 g / m ² to about 50 g / m ² , preferably about 7 g / m ² .
The range is from m ² to about 30 g / m ² .

The indicator in the reagent composition contained in the reagent layer is capable of reacting with calcium to cause an optically detectable change. The wavelength used for detection is not limited to the visible part, and may be ultraviolet light or infrared light as long as the wavelength can be transmitted by the support. The indicator is water-soluble because the method of the present invention was developed to prevent migration when the indicator is water-soluble. The method of the present invention is particularly effective when the solubility is 5 mg / 100 g water (25 ° C.) or more. On the other hand, it is also necessary that the solvent is insoluble in the organic solvent that dissolves the development control agent. Such an indicator is appropriately selected from known ones, and includes those which form a complex by binding with calcium.

The analyte calcium, which exists in the form of ions or fatty acid salts, may be bound to proteins. The liquid sample containing such calcium is not particularly limited, for example, various biological fluids, food and drink,
Examples include alcoholic beverages and pharmaceuticals.

An example of the indicator is o-cresolphthalein complexone (3,3'-bis [[di (carboxymethyl) amino] methyl] -o-cresolphthalein [2411-89-
4], optimum pH value of about 10.5; the numbers in [] below are Chemical
Abstracts Registry Number), Eriochrome Black T (1- (1-hydroxy-2-naphthylazo) -6-nitro-2-hydroxynaphthalene-4-sulfonic acid monosodium salt [1787-61-7]), methyl Thymol blue complexone (3,3'-bis [[di (carboxylmethyl) amino] methyl] thymol sulfonephthalein tetrasodium salt [1945-77-
3]), thymolphthalein complexone (3,3'-
Bis [di (carboxylmethyl) amino] methyl] thymolphthalein [1913-93-5]), Arsenazo III
(2,7-bis [(2-arsonophenyl) azo] -1,8-
Dihydroxynaphthalene-3,6-disulfonic acid [1668-0
0-4]) (ArsenazoIII), chlorophosphonazo III (2,7
-Bis [(4-chloro-2-phosphonophenyl) azo]
-1,8-Dihydroxynaphthalene-3,6-disulfonic acid [1914-99-4]) (Chlorophoshonazo III) etc. "Doutite Reagent General Catalog 12th Edition" (Kumamoto City, Dojindo Laboratories Co., Ltd., 1980) Issuance) etc. Among these indicators, o-cresolphthalein and arsenazo III are preferable because they enable the most accurate quantitative analysis of calcium. If necessary, add 2 parts of the reagent composition.
It is also possible to include them in separate layers (for example, a reagent layer and a water absorption layer).

The reagent layer may further contain a pH buffer, which will be described later, a known basic polymer and the like to adjust the pH during the analysis operation and the like. It is preferable that the reagent layer and / or the water absorption layer described later be substantially transparent, but if necessary, a small amount of titanium dioxide fine particles, barium sulfate fine particles, carbon black, etc. are dispersed in the layer to adjust the optical performance. You can also do it.

The porous spreading layer is disclosed in JP-A-55-164356 and JP-A-57-66.
359, etc., (for example, plain weaves such as broad, poplin, etc.), knitted layers (for example, tricot knitting, double tricot knitting, Milanese knitting, etc.) described in JP-A-60-222769, etc. Development layer consisting of papermaking paper containing organic polymer fiber pulp described in Kaisho 57-148250, membrane filter (brush polymer layer), polymer microbeads, glass microbeads, diatomaceous earth described in Japanese Patent Publication Sho 53-21677, U.S. Pat. Is a non-fiber isotropic porous development layer such as a continuous microvoid-containing porous layer held by a hydrophilic polymer binder, a polymer adhesive in which the polymer microbeads described in JP-A-55-90859 do not swell with water. It is possible to use a non-fibrous isotropic porous development layer composed of a continuous microvoid-containing porous layer (three-dimensional lattice-like granular structure layer) adhered in a point contact manner.

The woven fabric, knitted fabric or paper for use in the porous spreading layer is subjected to physical activation treatment represented by glow discharge treatment or corona discharge treatment described in JP-A-57-66359 on at least one side of the cloth. Or, the washing and degreasing treatment described in JP-A-55-164356, JP-A-57-66359 and the like, hydrophilic treatment such as impregnation of hydrophilic polymer, or a combination of these treatment steps is carried out sequentially to form a cloth material. Hydrophilized,
It is possible to increase the adhesive force with the lower layer (the side closer to the support).

The porous spreading layer contains a spreading control agent. The development control agent controls the aqueous liquid sample so as not to spread too much in the porous development layer, and is a water-soluble polymer and / or
Alternatively, an appropriate nonionic surfactant is selected.

The water-soluble polymer is polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, polyacrylic acid, water-soluble cellulose derivative and the like. The water-soluble cellulose derivative is a cellulose ether in which a part or all of the hydroxyl groups are etherified by a lower alkyl group having 1 to 3 carbon atoms or a hydroxy-substituted lower alkyl group having 1 to 4 carbon atoms. Examples of cellulose ethers include water-soluble methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxybutyl methyl cellulose. Among these, preferred water-soluble polymers are polyvinylpyrrolidone, polyvinyl alcohol, and water-soluble cellulose ethers. Two or more water-soluble polymers can be used in combination. The content of the water-soluble polymer in the porous spreading layer is in the range of about 0.5 g to about 15 g, preferably about 0.7 g to about 10 g per 1 m ^{2 of the} porous spreading layer.

Nonionic surfactants are polyhydric alcohol ester ethylene oxide adducts (condensates), polyethylene glycol monoesters, polyethylene glycol diesters, higher alcohol ethylene oxide adducts (condensates), alkylphenol ethylene oxide adducts (condensates) and higher fatty acid alkanolamides. Etc. Two or more kinds of these nonionic surfactants can be used in combination. When a nonionic surfactant is used in combination with a water-soluble cellulose derivative, an HLB value of 10 or more is preferable.

Specific examples of the nonionic surfactant are given below. It goes without saying that the nonionic surfactant used in the present invention is not limited to these compounds.

POE (20) sorbitan monooleate POE (10) sorbitan monooleate POE (4) sorbitan tristearate POE (4) trioleate POE (30) stearate POE (40) stearate POE (100) stearate PEG (400) ) Monostearate PEG (400) Monolaurate PEG (1000) Dilaurate PEG (1540) Distearate Lauryl alcohol EO 6 mol Condensate Lauryl alcohol EO 10 mol Condensate Lauryl alcohol EO 30 mol Condensate Oleyl alcohol EO 20 mol Condensate Cetyl alcohol EO 20 mol Condensate POE (10) Octyl phenyl ether POE (15) Octyl phenyl ether POE (30) Octyl phenyl ether POE (12) Nonyl phenyl ether POE (20) Nonyl phenyl ether Triethanolamine oleate (Note) POE: Polyethylene oxide PEG : Polyethylene glycol EO: ethylene oxide () is the number of condensation of ethylene oxide units. The content of nonionic surfactant in the porous expanded layer is
It is in the range of about 0.1 g to about 3 g / m ² , preferably about 0.2 g to about 2 g.

In addition to these layers, the multilayer analysis element can have layers. A water absorbing layer can be provided between the support and the reagent layer. The water-absorbing layer is a layer mainly composed of a hydrophilic polymer that absorbs and swells water, and is a layer that can absorb water of an aqueous liquid sample that has reached or penetrated the interface of the water-absorbing layer. Has a function of promoting permeation of blood plasma, which is an aqueous liquid component, into the reagent layer. The hydrophilic polymer used for the water absorbing layer may be selected from those used for the reagent layer described above. Generally gelatin or gelatin derivatives,
It is preferable to use polyacrylamide and polyvinyl alcohol, and of these, gelatin (deionized gelatin) is most preferable.

The dry thickness of the water absorbing layer is in the range of about 3 μm to about 100 μm, preferably about 5 μm to about 30 μm, and the coating amount is about 3 g / m 2.
² to about 100 g / m ² , preferably in the range of about 5 g / m ² to about 30 g / m ² . The water-absorbing layer may contain a pH buffer, which will be described later, a known basic polymer and the like to adjust the pH during use (at the time of carrying out an analytical operation).

A reflective layer can also be provided between the spreading layer and the reagent layer. The reflective layer is a hydrophilic polymer binder similar to that used for the water-absorbing layer or a cross-linked hydrophilic polymer binder in which titanium dioxide, light-reflecting fine particles such as barium sulfate are dispersed and contained, and light incident from the reagent layer side Is a layer that reflects light and thereby shields the color of an aqueous liquid sample such as blood in the spreading layer to provide a stable background. Generally, gelatin or a gelatin derivative, polyacrylamide, polyvinyl alcohol and the like are preferably used for this reflective layer, and among these, gelatin (deionized gelatin) is most preferable. The hydrophilic polymer used in the reflective layer can be appropriately crosslinked and cured with a crosslinking agent (curing agent) to give a reflective layer.
Examples of the cross-linking agent include known vinyl sulfone-based cross-linking agents such as 1,2-bis (vinylsulfonylacetamido) ethane and bis (vinylsulfonylmethyl) ether for gelatin, aldehydes, aldehydes for methallyl alcohol copolymer, and two There are glycidyl group-containing epoxy compounds and the like. The dry thickness of the reflective layer is about 5 μm to about
50 μm, preferably in the range of about 7 μm to about 30 μm, and the coating amount is about 5 g / m ² to about 50 g / m ² , preferably about 7 g / m ² to about
It is in the range of 30 g / m ² . The reflective layer may contain the above-mentioned pH buffer, a known basic polymer, etc. to adjust the pH during the analysis operation. The amount of fine particles of titanium dioxide, fine particles of barium sulfate, etc. in the reflective layer can be limited to a range that does not impair the non-porous property, and can also have a protein permeation blocking function.

On the reflective layer, a known adhesive layer made of a hydrophilic polymer similar to that used for a water absorbing layer typified by gelatin can be provided for the purpose of firmly adhering and integrating the spreading layer. The dry thickness of the adhesive layer is in the range of about 0.5 μm to about 5 μm.

On the other hand, for reagents other than those mentioned above, the pH of the site where the reaction accompanied by an optically detectable change is carried out is the optimum of the reaction.
There is a buffer to maintain the pH range. It goes without saying that this optimum pH differs depending on each reaction system. For example, when calcium is analyzed using o-cresolphthalein as an indicator, the pH range for coloring is in the range of pH 9 to 10.5, so a buffering agent in this range is selected. To do.

As a buffering agent that can be used, "Chemical Handbook Basic Edition" edited by The Chemical Society of Japan, Tokyo, Maruzen Co., Ltd., 1966, 1312-1320
Page, RMC Dawson et al. Eds. “Data for Biochemical Res
earch "Second Edition (Oxford at the Clarendon Press, 1969
Issued pp. 476-508, "Biochemistry" 5 , pp. 467 et seq. (1966), "Analytical Biochemistry" 104 , 300-31
There is a pH buffer system described on page 0 (1980).

Specific examples of pH buffers in the range of pH 8.0 to 11.0, particularly pH 9.0 to 10.5, include buffers containing tris (hydroxymethyl) aminomethane (Tris); buffers containing phosphates; buffers containing borate. A buffer containing carbonate; a buffer containing glycine; N, N-bis (2-hydroxyethyl) glycine (Bici
ne); N-2-hydroxyethylpiperazine-N'-2-
Hydroxypropane-3-sulfonic acid (HEPPS) Na salt or K salt; N-2-hydroxyethylpiperazine-N '
-3-Sulfonic acid (EPPS) Na salt or K salt; 3- (cyclohexylamino) -1-propanesulfonic acid (CAP
S); N- [Tris (hydroxymethyl) methyl] -3-
Aminopropanesulfonic acid (TAPS) Na salt or K salt; N
2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) Na salt or K salt and the like; and an acid, alkali or salt optionally combined with any of these. Specific examples of preferable buffering agents include Tris-sodium borate; Bicine; HEPPS; HEPPS sodium salt; EPPS; E
PPS sodium salt; CAPS; CAPS sodium salt; TAPS; TAPS sodium salt and the like. When gelatin or a gelatin derivative is used as the hydrophilic polymer binder of the reagent layer, the water-absorbing layer or the reflective layer, the vinyl sulfone structure-containing cross-linking agent can appropriately strengthen the cross-linking of these layers, and the coating stabilizes these layers. A pH buffer containing boric acid or sodium borate is preferable from the viewpoint of being provided and capable of performing highly accurate quantitative analysis. The pH buffer can be contained in the reagent layer, the water absorption layer, the reflection layer and the like.

In addition to the spreading layer, the reagent layer, the water absorbing layer, the reflecting layer, the adhesive layer and the like may contain a surfactant. An example thereof is a nonionic surfactant. Specific examples of the nonionic surfactant include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylenesorbitan monolaurate, p-nonylphenoxypolyglycidol, octylglucoside, and the like. There is.
By including a nonionic surfactant in the developing layer, the developing action (metering action) of the aqueous liquid sample becomes better. By containing a nonionic surfactant in the reagent layer or the water absorption layer, water in the aqueous liquid sample is easily absorbed into the reagent layer or the water absorption layer substantially uniformly during the analytical operation, and the liquid contact with the developing layer is made. Quickly and substantially uniform.

The multilayer analytical element of the present invention can be prepared by known methods described in the above-mentioned patent specifications.

The method of the present invention is characterized in that the above-mentioned expansion control agent is dissolved in an organic solvent that does not dissolve the above-mentioned indicator and added to the porous development layer. The type of organic solvent may be one that satisfies such conditions, but in general, the boiling point is 100
A polar solvent of ℃ or less is preferable, for example, aliphatic alcohol (eg, methanol, ethanol, propanol, butanol, isopropyl alcohol), dialkyl ketone (eg, acetone), dialkyl ether (eg, dimethyl ether), aliphatic cyclic ether (eg, Tetrahydrofuran, dioxane) and the like are suitable. Among these, resin group alcohols are preferable, and in consideration of the working environment and the like, alcohols such as ethanol, propanol, butanol, and isopropanol which are less toxic to the human body are particularly desirable. For example, o-cresolphthalein can be used as an indicator for the measurement of calcium, and the above-mentioned alcohol is preferable as the organic solvent for this compound. This compound alone is poorly soluble in water and well soluble in alcohol, but it is well soluble in water under alkaline conditions around pH 10.5, which is the optimum pH for color development, but not in alcohol at all.

The concentration of the solution is preferably as high as possible within the range where it can be easily contained and can be uniformly permeated into the porous spreading layer, and this depends on the type of the spreading control agent and the like, but is, for example, about 0.2. % To about 10%, preferably about 0.3% to about 7% is suitable. The solution may be prepared by a conventional method. Water can be mixed with an organic solvent to the extent that the water-soluble indicator does not substantially migrate to the spreading layer.

When both the water-soluble polymer and the surfactant are contained, a mixed solution of the both may be prepared, or separate solutions may be prepared and contained separately.

The development control agent is contained in the porous development layer after the development layer is laminated. In the case where the spreading layer is a structure such as a microfilter, knit or woven fabric, it may be contained first and then laminated, but in that case, there is a problem that it is difficult to control the content. Therefore, it is advantageous in terms of analysis accuracy and manufacturing cost to include the development control agent in the development layer after providing the development layer on the reagent layer (directly or via an adhesive layer or the like). The content may be applied or sprayed uniformly on the porous spreading layer by a known method.

Drying may be performed by air drying, reduced pressure drying, or the like.

The multilayer analytical element obtained by the method of the present invention is cut into a small piece such as a square having a side of about 15 mm to about 30 mm or a circle of about the same size, and is disclosed in Japanese Examined Patent Publication No. 57-28331, Japanese Utility Model Publication No. 56-142454, and Japanese Unexamined Patent Publication No.
It is preferable to use it as a chemical analysis slide by putting it in a slide frame described in -63452, Jitsukai Sho 58-32350, Tokukaisho Sho 58-501144 and the like from the viewpoints of various aspects 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.

The analysis of the test component in the liquid sample using this multilayer analysis element can be carried out by the operations described in the above-mentioned patent specifications and the like. That is, from about 5μ to about 30μ, preferably 8μ
To 15μ of whole blood, plasma, serum and other aqueous liquid sample droplets are spotted on the spreading layer, and the temperature is about 20 ° C in the range of 1 to 10 minutes.
To a substantially constant temperature in the range of about 40 ° C., preferably
Incubate at a substantially constant temperature near 37 ° C,
Reflection measurement of the optical density of the reagent layer or water absorption layer using visible light (or near-ultraviolet light) from the light-transmissive support side, and making a test in a liquid sample according to the principle of colorimetry using the calibration curve prepared in advance. The component content can be determined. By making the amount of the aqueous liquid sample to be spotted, the incubation time and the temperature constant, the quantitative analysis of the test component can be performed with high accuracy.
This measurement operation can be performed with high precision by a chemical analyzer described in JP-A-56-77746, JP-A-58-21566, JP-A-58-161867 and the like with extremely easy operation.

[Work]

Since the method of the present invention has conventionally used an aqueous solution for inclusion in the porous development layer, an indicator or the like contained in the reagent layer partially migrates to the development layer due to this inclusion, which contributes to a decrease in analysis accuracy. It is based on the knowledge that

In the method of the present invention, by using an organic solvent as the solvent of the development control agent to prevent the migration of the indicator when the development control agent is contained, further migration of the indicator during the color development reaction or the migration of the color developing material to the development layer by the development control agent. The accuracy of analysis is improved by lowering it.

〔Example〕

Example 1 180 μm thick colorless transparent polyethylene terephthalate (P
On the ET) film (support), coating layers were sequentially coated with an aqueous solution so as to have a coating amount of the following composition, dried, provided and laminated.

Water absorption layer Deionized gelatin 4.8 g / m ² Nonylphenoxypolyethoxyethanol (containing 10 oxyethylene units on average) 0.11 g / m ² 1,2-bis (vinylsulfonylacetamide) ethane 0.5
The pH was adjusted to 6.3 with g / m ² NaOH, then applied in an aqueous system and dried.

Reagent layer deionized gelatin 23.9 g / m ² polyoxyethylene nonylphenyl ether (average 10 oxyethylene unit ^{content) 0.41g / m 2 CAPS 3.81g /} m 2 o- cresol phthalein complexone 0.15 g / m ² 8- human Roxyquinoline-5-sulfonic acid 0.54 g / m ² aqueous solution was adjusted to pH 10.6 with NaOH.

Adhesive layer Deionized gelatin 1.46 g / m ² Polyoxyethylene nonyl phenyl ether (containing 10 oxyethylene units on average) 0.10 g / m ² Titanium dioxide fine particles 0.85 g / m ² Water is then supplied almost evenly on the surface of the adhesive layer. And wet it, and the thickness of PET spun yarn equivalent to 100S is about 250μ.
The tricot knitted fabric of m was laminated uniformly and lightly with pressure to form a spreading layer. Then, the following polymer ethanol solution was applied onto the spreading layer so as to have the following coating amount and dried to prepare an integral type multilayer analytical element for calcium ion determination.

Polymer ethanol solution coating composition Polyvinylpyrrolidone (average molecular weight 360,000) 1.01 g / m ² polyoxyethylene nonylphenyl ether (containing 40 oxyethylene units on average) 2.11 g / m ² Dissolved in ethanol and applied.

Comparative Example 1 An integrated multilayer analytical element for quantitative analysis of calcium ions was prepared in the same manner as in Example 1. However, the following points are different.

Reagent layer o-cresolphthalein complexone 0.46 g / m ² 8-humanroxyquinoline-5-sulfonic acid 1.65 g / m ² Others are the same as in Example 1 Polymer aqueous solution coating composition Methylcellulose (2% aqueous solution at 20 ° C. viscosity 100cps) 5.75g / m ² nonylphenoxy polyethoxy ethanol (average 40 oxyethylene unit content) 7.44 g / m ² titanium dioxide fine particles 15.5 g / m ² dissolved in water and dispersed in the coating.

Example 1 and Comparative Example 1 A commercially available control serum (MONITOL IX, manufactured by Dade) was used to evaluate the performance of the integrated multilayer analytical element for quantitative determination of calcium. The results are shown in Table 1. As shown here, Example 1 made by the manufacturing method of the present invention.
As for the analysis element, the variation in the measurement result is extremely small. Therefore, it is clear that the manufacturing method of the present invention produces an analytical element with high analytical accuracy.

Claims (10)

[Claims] 1. A reagent layer containing a water-soluble indicator capable of reacting with calcium to cause an optically detectable change and a development control agent on a light-permeable, water-impermeable support. In producing an integrated multi-layer analytical element in which a porous spreading layer is laminated in this order, the spreading control agent is added to an organic solvent that does not dissolve the indicator after the porous spreading layer is provided on the reagent layer. Is dissolved, the solution is contained in a porous spreading layer, and then the porous spreading layer is dried, which is a method for producing an integrated multilayer analytical element for calcium analysis. 2. The integrated multi-layer analytical element according to claim 1, wherein the step of incorporating a solution of a development control agent into the porous development layer is carried out by applying the solution from above the porous development layer. Manufacturing method of 3. The integrated multilayer analytical element according to claim 1, wherein the step of incorporating a solution of a development control agent into the porous development layer is carried out by spraying the solution on the porous development layer. Manufacturing method of 4. The method for producing an integrated multilayer analytical element according to claim 1, wherein the solubility of the indicator at 25 ° C. is 5 mg / 100 g water or more. 5. The method for producing an integrated multilayer analytical element according to claim 1, wherein the development control agent is one or both of a water-soluble polymer and a nonionic surfactant. 6. The water-soluble polymer is polyvinylpyrrolidone,
Polyvinyl alcohol, polyacrylic acid, methyl cellulose, or ethyl cellulose, the nonionic surfactant is a polyhydric alcohol ester ethylene oxide adduct, polyethylene glycol mono- or di-fatty acid ester, higher alcohol ethylene oxide adduct, alkylphenol ethylene oxide adduct, Or a higher fatty acid alkanolamide, the method for producing an integrated multilayer analytical element according to claim 5. 7. The method for producing an integrated multilayer analytical element according to claim 1, wherein the organic solvent is a polar organic solvent having a boiling point of 100 ° C. or lower. 8. The method for producing an integrated multilayer analytical element according to claim 8, wherein the polar organic solvent is an aliphatic alcohol, a dialkyl ketone, a dialkyl ether or an aliphatic cyclic ether. 9. The polar organic solvent is methanol, ethanol,
The method for producing an integrated multilayer analytical element according to claim 7, which is propanol, butanol or isopropanol. 10. The method for producing an integrated multilayer analytical element according to claim 1, wherein the water-soluble indicator is o-cresolphthalein complexone, arsenazo III or chlorophosphonazo III.