JPH02276599A - Production of dry analytical element - Google Patents

Abstract

PURPOSE:To inexpensively obtain the subject element having improved storage stability and analytical precision by adding and mixing a p-NPP salt solution of good solvent through a circular flow opening into turbulent flow of poor solvent having prescribed linear velocity, applying a dispersion obtained by dispersing the deposited solid p-NPP salt into a poor solvent and drying. CONSTITUTION:An amine salt or amino alcohol salt of p-nitrophenyl phosphate (p-NPP) of as tris(hydroxymethyl)aminomethane, etc., is dissolved in water, methanol, etc., which is a good solvent to afford a solution (A). Then a poor solvent such as acetone is fed to a capillary using a 0.5-5.0kg/cm<2> pressure- resistant gear pump, etc., to form turbulence (B) having 2-30m/min linear velocity of flow. Then the ingredient A having weight 10 times that of B ingredient is poured from a flow opening nozzle having opening corresponding to 50mum to 5.0mm diameter into the ingredient B and mixed with the ingredient B and the above-mentioned p-NPP salt is deposited to provide a solid phase. Then a dispersion obtained by dispersing the solid phase into a poor solvent is applied to a liquid permeating layer which is porous developing layer of woven fabric cloth, etc., and the applied liquid permeating layer is dried to produce the dry analytical element for determining alkaline phosphatase.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is useful for quantifying the activity of alkaline phosphatase (ALP) in biological fluids such as blood (whole blood, plasma, and serum) and lymph fluid. This invention relates to a method for manufacturing a dry analytical element.

[Prior Art] A salt of P-nitrophenyl phosphate (p-NP) is
Known as a substrate for alkaline phosphatase. It was already well known around 1965 that disodium salts have poor stability in dry conditions. Furthermore, it has been reported in Japanese Patent Publication No. 45-3487 that certain amine salts, such as tris(hydroxymethyl)aminomethane salt and dicyclohexylammonium salt, are relatively stable reagents.
Known for number 2.

In addition to the above, amines listed as specific examples in the publication include methylamine, ethylamine, dipropylamine,
These are tributylamine, cyclohexylamine, dicyclohexylamine, tricyclohegycylamine, monoethanolamine, benzylamine, dimethylamine, tritylamine, furfurylamine, aniline, jetanolamine, and morpholine. It is also described that these p-NP amine salts can be easily dissolved in water.

However, these water-soluble salts of p-NP were
21677 (U.S. Patent No. 3992158) and Japanese Patent Application Laid-open No. 1987-
When applied as an aqueous solution and impregnated with the dry integrated multilayer analytical element described in No. 164356 (US Pat. No. 4,292,272), the stability is poor. This is JP-A-60-23
This is also pointed out in the 9799 specification.

It is desired to realize a dry analytical element for quantifying alkaline phosphatase with good storage stability, and for this purpose,
No. 0-239799 proposes a method in which the substrate is not dissolved in a solution, but made into fine particles, dispersed in a coating iα, and impregnated into an analytical element. However, with this method of mechanically dispersing the substrate in a dry state or in a poor solvent, it is difficult to obtain results with good reproducibility.In other words, it is difficult to obtain high analytical precision for the analytical elements obtained by this method. .

Furthermore, in JP-A No. 62-265995, an amine salt or an amino alcohol salt of p-nitrophenyl phosphate (p-NPP) is made into a solution in a good solvent (e.g., water, methanol) and mixed with a poor solvent (e.g., acetone). to precipitate,
Alkaline phosphatase determination by separating liquid and solid phases (salt of p-NPP), dissolving or dispersing the separated solid phase, and using it as a coating or dipping composition for a liquid permeable layer such as a liquid spreading layer. A method of manufacturing a dry analytical element for use is shown. According to this, well-known methods can be used to separate the precipitated solid phase from the liquid phase, including gravity sedimentation, centrifugal sedimentation, inertial collision, and filtration. , or filtration is simple,
Pressure filtration or vacuum filtration can also be used for filtration.

However, the step of separating the precipitated solid phase from the liquid phase is labor-intensive in all methods, and it is difficult to maintain a constant yield at all times, resulting in poor reproducibility and high costs.

A simple method for manufacturing work shoes is to use the substrate p-
Although the salt of NPP is used in a good solvent solution as a coating or dipping composition for a liquid permeable layer such as a liquid spreading layer, the storage stability of the dry multilayer analytical element manufactured by this method is extremely low. This is a well-known fact.

Therefore, a manufacturing method with low operating costs, constant yield, and good storage stability of analytical elements was desired.

As a result of study, the present inventor found that when the amine salt or amino alcohol salt of p-nitrophenyl phosphate is used as a solution of its good solvent, and when the good solvent solution and the poor solvent are mixed, the linear velocity of the flow is 2 m The solution of the good solvent is added to the turbulent flow of the poor solvent in the range of 50 μm to 5.0 mm in diameter from 50 μm to 5.0 mm in diameter.
By applying through a circular cross-section nozzle in the range of
The inventors have arrived at the present invention by discovering that the stability of the produced dry analytical element for quantifying alkaline phosphatase during storage is significantly improved.

[Objective of the invention] The object of the present invention is to have good storage stability, high analytical accuracy,
An object of the present invention is to provide a method for producing a dry analytical element for quantifying alkaline phosphatase, which is inexpensive in production cost.

[Structure of the Invention Section 3 The object of the present invention is to dissolve the amine salt or amino alcohol salt of p-nitrophenyl phosphate in a good solvent (for example, water, methanol) to form a solution, and convert the good solvent solution into a poor solvent. (e.g., acetone) to precipitate the salt as a solid phase, disperse the precipitated same phase in a poor solvent to form a dispersion, and use the dispersion to impregnate the liquid-permeable layer with the salt. In the method for producing a dry analytical element for quantifying alkaline phosphatase having a liquid-permeable layer, which includes a step of coating the liquid-permeable layer and drying it, the step of mixing the good solvent solution with the poor solvent has a flow linear velocity of 2a/2. Alkaline phosphatase characterized in that the solution of the good solvent is added to the turbulent flow of the poor solvent at a speed ranging from 50 m/min to 30 m/min through an outflow opening with an opening cross-sectional area corresponding to a circular aperture having a diameter ranging from 50 μm to 5.0 m/min. This was achieved using a method for producing quantitative dry analytical elements.

[Detailed Description of the Structure of the Invention] The dry analytical element manufactured by the above method has good storage stability, high analytical accuracy, and relatively low production cost.

Alkaline phosphatase produced by the method of the present invention (
In dry analytical elements for ALP) quantification, the amine salt or amino alcohol salt of ρ-nitrophenyl phosphate is the substrate for ALP, so it is hydrolyzed by the catalytic action of ALP to produce phosphate and p-nitrophenol. do. Since the produced ρ-nitrophenol has an absorption peak at a wavelength of 405 nm in an alkaline region, it can be measured colorimetrically using light at a wavelength near this absorption peak.

Examples of the amine salt or amino alcohol salt of ρ-NPP include tris(hydroxymethyl)aminomethane, 2-amino-2-hydroxymethyl-1,3-propanediol, and Dicyclohexylamine, methylamine, ethylamine, dipropylamine, tributylamine, cyclohexylamine, dicyclohexylamine, tricyclohexylamine, monoethanolamine, benzylamine,
dimethylamine, tritylamine, furfurylamine,
There are salts such as aniline, jetanolamine, and morpholine. These salts are water soluble. Salts that are also soluble in alcohol, particularly methanol, are 2-amino-2-methylpropane-1,3-diol salt and 2-amino-2-ethylpropane-1,3-diol salt. Either of these salts is dissolved in an appropriately concentrated solution in a good solvent.

Since there are only a few types of good solvents for the above salts, a wide range of poor solvents can be used. Examples of good solvents include water, methanol, and ethanol. Examples of poor solvents include organic solvents such as acetone, methyl ethyl ketone, cyclohexane, benzene, and toluene.

The poor solvent is added to the salt solution in an amount sufficient to precipitate the salt.

The amount of poor solvent is about 11 IO or less, preferably in the range of about 1/100 to about 1/10, of the amount of good solvent by weight.

A pressure-resistant gear pump and cylinder that can apply a pressure of about 5 kg/cm2 to about 5.0 kg/cta2 are used to create a turbulent flow of poor solvent with a linear velocity of about 2 m/min to about 30 m/min. A method of pumping a poor solvent into a thin tube using a pump such as a type pump, rotary pump, or plunger pump to obtain the desired flow rate. Send it in,
To obtain the desired flow rate, place the poor solvent in a suitable container, place a disk-shaped or blade-shaped stirring device in the poor solvent,
There is a method of rotating this stirring device at a rotation speed of about 2,000 m to about 50,000 rpm to obtain the desired flow rate.

An opening with a diameter of 50 μ- to approximately 5.0 m+1 (effective cross-sectional area of the opening approximately 200 μm) into which a good solvent solution of p-NPP salt is added
Various types of outflow openings (nozzles) from 0μII+2 to about 20a+m2 can be used. Examples of nozzles include polymers (polytetrafluoroethylene,
There are nozzles or tubes made of hardened silicone rubber, etc., and stainless steel nozzles or tubes. The nozzle may have one or more outlet openings. The cross-sectional shape of the outflow opening of the nozzle is generally circular or elliptical, but it may also be polygonal, such as a square or hexagon with rounded corners.

In a poor solvent, the precipitated minute crystals tend to aggregate with each other and form a tart-like precipitate, so a surfactant (
- It is preferable to include a nonionic surfactant (preferably a nonionic surfactant) or to add a small amount of a good or weak solvent to a poor solvent. Alternatively, it is also preferable to use a dispersion device that can separate aggregated crystals.

In the method of the present invention, the liquid permeability 1 (e.g., porous developing layer) of the dry analytical element is prepared by a known method. ρ-NP is coated or cast on the layer, dried and coated with ρ-NP.
Contain P salt. In addition, after coating or casting the p-NPP salt dispersion on the liquid-permeable layer, it is also possible to dry it by blowing air with a dew point of 15° C. or lower and a dry bulb temperature of 35° C. or higher, as a preferred embodiment of the present invention. It is.

Preferably, the liquid spreading layer is a porous spreading layer consisting of a woven fabric, a knitted fabric or a cellulosic non-woven fabric, but
It may also be a porous spreading layer made of a plush polymer or a particulate structure.

The manufacturing method of the present invention can be applied to various known dry analytical elements. In particular, it can be applied to an analytical element that includes a solid carrier that is permeable to both a self-developing substrate and a test liquid. The analytical element is formed on a light-transparent (or transparent)/liquid-impermeable support, in addition to a porous liquid spreading layer, an undercoat layer, a water absorption layer, an adhesive layer, a reactive reagent layer, a light shielding layer, a filtration layer,
Such an analytical element, which may have a multilayer structure with other layers and other known layers, is disclosed in US Pat. No. 3,992,158;
U.S. Patent No. 4042335 and Japanese Unexamined Patent Publication No. 164356/1983
(US Pat. No. 4,272,292) There are seven types described in each specification.

When a support is used, the practically possible configurations of the dry analytical element produced according to the present invention are (1) one having a liquid spreading layer on the support.

(2) One having a reagent layer and a liquid spreading layer in this order on a support. In this case, the p-NPP salt may be contained in either the reagent layer or the liquid development layer, but it is preferably contained in the iα form development layer. The reagent layer may consist of two or more reagent layers.

(3) A support having a reagent layer, a light shielding layer, and a liquid spreading layer in this order. In this case, the p-NPP salt may be contained in any of the reagent layer, the light shielding layer, and the liquid development layer, but it is preferably contained in the α-form development layer.

(4) A support having a detection layer, a light shielding layer, and a liquid development layer in this order. Although the p-NPP salt may be contained in either the light shielding layer or the liquid spreading layer, it is preferably contained in the liquid spreading layer.

(5) A support having a detection layer, a light shielding layer, a reagent layer, and a liquid developing layer in this order. Although the p-NPP salt may be contained in any of the reagent layer, light shielding layer, and liquid developing layer, it is preferably contained in the liquid developing layer.

(6) A support having a detection layer, a second reagent layer, a light shielding layer, a first reagent layer, and a liquid spreading layer in this order. ρ-NPP
Although the salt may be contained in any of the reagent layers, the light shielding layer, and the liquid spreading layer, it is preferably contained in the liquid spreading layer.

In (1) to 6) above, between the detection layer and the reagent layer or the liquid spreading layer, between the reagent layer and the liquid spreading layer, between the light shielding layer and the detection layer, the reagent layer or the liquid spreading layer, or between the two. A filtration layer may be provided between the two reagent layers.

A water-absorbing layer may be provided between the support and the detection layer, reagent layer, or liquid spreading layer. Also, between the support and the detection layer, reagent layer or α-form development layer, between the reagent layer and the liquid development layer, between the light shielding layer and the detection layer, between the reagent layer or the liquid development layer, or between the two reagent layers. A pH buffer layer, a dura layer, etc. may be provided between them. The above (1) and (2) are preferable for the production method of the present invention.
Or the layer structure of (3).

In the dry analytical element manufactured by the method of the present invention, 9
- The NPP salt may be contained in two or more layers (e.g. reagent layer and liquid interlayer, or second reagent layer and first reagent layer, first reagent layer and liquid interlayer), in which case , p-NP
It is also possible to contain more P salt in either the layer far from the support or the layer closer to the support than the other.

Examples of light-transparent/water-impermeable supports that can be used in the dry analytical element produced by the method of the present invention include polyethylene terephthalate, polycarbonate to bisfunol, polystyrene, cellulose ester (e.g., cellulose triacetate) , cellulose acetate propionate, etc.) and has a thickness of about 50 μm to about 1000 μm, preferably about 80 μm to about 300 μm.
There are transparent supports in the form of films or sheets in the range of . If necessary, an undercoat layer may be provided on the surface of these supports to strengthen the adhesion between the supports and layers such as a water absorption layer and a reagent layer provided on the supports. In addition, instead of the undercoat layer, the surface of the support may be subjected to physical activation treatment (e.g. glow discharge, corona discharge) or chemical activation treatment (e.g. alkali etching) to improve adhesive strength. can.

In the dry multilayer analytical element produced by the method of the present invention, a water-absorbing layer may be provided on the light-transparent/water-impermeable support (in some cases, via another layer such as an undercoat layer). Be able to do it.

The water-absorbing layer provided in the dry analytical element is preferably a layer made of a hydrophilic binder and containing as a layer-forming component a hydrophilic polymer that swells by absorbing water.

The hydrophilic polymer that can be used in the water absorption layer is generally a natural or synthetic hydrophilic polymer having a swelling ratio of about 1.5 to 20, preferably about 2.5 to 15 at 30° C. upon absorption of water. be. Examples of such hydrophilic polymers include gelatin (e.g., alkali-treated gelatin, acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (e.g., phthalated gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, Examples include polyvinyl alcohol and polyvinylpyrrolidone.

The thickness of the water absorption layer when dry is approximately 1 μm to approx. 00 μm, preferably in the range of about 3 μm to about 30 μm. Furthermore, the water-absorbing layer may contain a surfactant (cationic, amphoteric, or non-ionic) or a buffering agent if necessary. can contain light-shielding (reflective or absorptive) fine particles as necessary.

An adhesive layer for adhering and laminating a porous spreading layer may be provided on layers such as a water absorption layer, a reagent layer, and in some cases a light shielding layer and a filtration layer. Examples of hydrophilic polymers that can be used in the adhesive layer include, preferably, hydrophilic polymers that are capable of adhering the porous interlayer when the porous interlayer is swollen with water. There are similar hydrophilic polymers used in the water absorption layer. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred.The dry film thickness of the adhesive layer is generally about 0.5 μ- to about 20 μm, preferably about 1 μm.
It ranges from μm to about lOμI. Note that the adhesive layer may be provided on a desired layer in order to improve the adhesion force between the eyebrows other than the glabellar layer. The adhesive layer may be provided by coating an aqueous solution containing a hydrophilic polymer and a surfactant (cationic, amphoteric, or nonionic) added as necessary on the water absorption layer, etc. using a known method. I can do it.

Examples of pH buffers that can be contained in the reagent layer of the dry analytical element produced by the method of the present invention include carbonates,
There are known buffering agents such as borates, phosphates and Good's p)flit buffers. What are these buffers? They can be selected and used with reference to known literature such as "Basic Experimental Methods for Proteins and Enzymes" (Takeshi Horio et al., Nankodo, 1981).

The light shielding layer is preferably a water-permeable layer in which light-reflecting fine particles are dispersed using a hydrophilic polymer having film-forming ability as a binder. The light-reflecting fine particles measure the color of the aqueous liquid dotted onto the developing layer when detectable changes (color change, color development, etc.) occurring in the detection layer are measured by reflection photometry from the light-transmitting support side. In particular, when the sample is whole blood, it blocks the red color of hemoglobin and also functions as a light reflective layer or background layer.

Examples of light-reflecting fine particles include pigment fine particles, such as titanium dioxide fine particles (rutile type, anatase type, or brutzite type, with a particle size of about 0.1 μm to about 1.2 μm).
microcrystalline particles (microcrystalline particles, etc.), barium sulfate particles, aluminum particles, etc. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferred. Particularly preferred are rutile titanium dioxide fine particles.

Examples of hydrophilic polymer binders having film-forming ability include weakly hydrophilic regenerated cellulose, cellulose acetate, and the like, in addition to the hydrophilic polymers used in the water absorption layer or detection layer described above. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. Note that a known hardening agent can be added to gelatin and gelatin derivatives.

The light-shielding layer can be provided by applying an aqueous dispersion of light-reflecting fine particles and a hydrophilic polymer onto the detection layer, reagent layer, etc. by a known method and drying it.

In the dry analytical element, the above-mentioned light-reflecting fine particles can also be included in the developing layer if necessary.

The liquid spreading layer is preferably a spreading layer having an iα body metering effect. Liquid metering action refers to a liquid sample that is dotted onto the surface of a spreading layer and is distributed in an approximately constant amount per unit area in the lateral (horizontal) direction without substantially unevenly distributing the components contained therein. It is an action that spreads at a rate of .

Materials constituting the matrix of the spreading layer include filter paper,
Nonwoven fabrics, woven fabrics (e.g., plain woven fabrics such as broadcloth and boblin), knitted fabrics (e.g., tricot 1T fabric, double tricot knitted fabric, Milanese silk fabric), glass fiber filter paper, plush polymer (membrane filter),
Alternatively, a three-dimensional lattice structure made of polymer microbeads or the like can be used.

Among these, it is preferable to use fibrous layers typified by woven fabrics and knitted fabrics. For details of these, please refer to JP-A-55-164356 and JP-A-57-6635.
9 and Japanese Unexamined Patent Publication No. 60-222769.

The woven fabric or knitted fabric that can be used for the dry analysis element produced by the method of the present invention is confirmed to have substantially removed oils and fats attached during the production of yarn, woven fabric, or knitted fabric by degreasing treatment such as washing with water. preferable.

The dry multilayer analysis element produced by the method of the present invention is cut into small pieces such as squares or circles of approximately the same size, with a diameter of about 15 nm to about 3011 Ial, and
Japanese Patent Application Publication No. 54-156079, Utility Application Publication No. 58-125424
, Utility Model Publication No. 58-32350 and Special Publication No. 58-50114
4. It is preferable from all viewpoints of manufacturing, packaging, transportation, storage, measurement operations, etc. to use it as an analysis slide by placing it in the slide frame disclosed in each publication.

The dry analytical element produced by the method of the present invention has an aqueous liquid sample (blood (whole blood, plasma, serum), lymph fluid, etc.) of about 5 μL to about 30 μL, preferably about 8 μL to about 15 μL.
Dot-feed to the developing layer, and heat from about 20℃ to about 4℃ as needed.
Detectable changes in the dry analytical element from one side (from the optically transparent support side in the case of integrated multilayer analytical elements), such as color change, development, etc., during incubation at a substantially constant temperature in the range of 5°C. The activity value of alkaline phosphatase, which is the component to be measured (analyte) in the liquid sample, is analyzed using the principle of colorimetry by reflecting photometry.

[Example 1] Recrystallization and dispersion of p-nitrophenylphosphate bis(tris) salt 15 g of p-nitrophenylphosphate bis(tris(hydroxymethyl)amine salt) was completely dissolved in TSG distilled water. Dissolved. Next, this aqueous solution was mixed with nonylphenoxypolyethoxyethanol (oxyethylene unit average: 40
A dispersion of a salt of p-nitrophenyl phosphate was prepared by adding 5 g of p-nitrophenyl phosphate to 750 g of acetone containing 5 g of p-nitrophenyl phosphate in the following manner.

Nozzle: Oval polytetrafluoroethylene tube with pore diameter of 300 μm x 500 μm (LCP-115 manufactured by Nippon Chromato Industries Co., Ltd.) Stirrer:
18m diameter with 4 fixed trapezoidal blades of height 7mm+
A stirrer with m discs housed in a cylindrical housing (AtJTOHOMOMIXER manufactured by Tokushu Kika Kogyo Co., Ltd.)
MODEL M5PECA) Condition: The tip of the tube (nozzle) was fixed on the bladeless side of the disk, 3 mm from the disk, and facing the disk at right angles. Aqueous solution of ρ-nitrophenyl phosphate salt (good solvent solution) was added from the tip of the nozzle at a flow rate of 3 mL/min using a cylindrical pump while rotating the disc at about 10,000 rpm, and dispersed. ■Preparation of analytical element (Part 1) Transparent polyethylene terephthalate support, thickness 180μ
The surface of (m) was subjected to hydrophilic treatment, and a buffer layer coating aqueous solution was applied on the treated surface to the following coating amount and dried to form a buffer layer.

Buffer layer coverage: Alkali-treated deionized gelatin 8.48/m2
Nonylphenoxy boriglycidol 850m3/m2
Poly[N.[3-(dimethylamino)propyl]-acrylamide] (average molecular weight approximately 100,000) 8.9g/m2
N-(2-hydroxyethyl)ethylenediamine-N,
N',N'-triacetic acid 140mg/m
2Zn(CH3COO) ・2H2040mg/m2M
gSO4φ7H2090g/m2 5N-NaOH aqueous solution 2.1
z/m2 A hardened gelatin layer coating aqueous solution was applied on the buffer layer to the following coating amount and dried to form a hardened ceratin layer.

Coverage of hardened gelatin layer Deionized gelatin 2.78/m2
Nonylphenoxy boriglycidol 30mg/m21
．． 2-His(vinylsulfonylacetamido)ethane 1
60 mg/m2 An adhesive layer coating aqueous solution was applied onto the hardened gelatin in the following coating amount and dried to form an adhesive layer.

Deionized gelatin 2.7z/m
2-nonylphenoxy boriglycidol 30mg/m
2. A 0.4% nonylphenoxy polyglycidol aqueous solution was applied almost uniformly to the surface of the adhesive layer, and then a tricot knitted fabric made of 36 gauge 500 polyethylene terephthalate spun yarn was adhered while applying light pressure. A spread layer was formed.

■Production of analysis element (Part 2) For the above-mentioned development layer, use the water-acetone dispersion of p-nitrophenylphosphate bis(tris) salt recrystallized in step (■), and apply a coating liquid with the following composition. Prepared. This coating solution was applied in a coating amount of 120 mL/m2, and dried by passing through a wind (air flow) drying zone with a dew point of θ°C and a dry bulb temperature of 40°C.

p-nitrophenylphosphate bis(
A water-acetone dispersion of Tris) salt 790 g Polyvinylpyrrolidone (average molecular weight ff110,000) 150 g Ethanol 600 g An integrated multilayer analytical element for measuring ALP activity of the present invention was prepared by the above method.

[Comparative Example 1] An integrated multilayer analytical element for measuring ALP activity was prepared in the same manner as in Example 1, except that step (1) in Example 1 was changed to step (2) below.

(2) Recrystallization of p-nitrophenylphosphate bis(tris) salt 20.7 g of p-nitrophenylphosphate bis[tris(hydroxymethyl)amine] salt was completely dissolved in 25 g of distilled water. 750 g of acetone was added to this aqueous solution while stirring well. After about 5 minutes, the precipitate was suction filtered and washed several times with ethanol. Further, it was washed with acetone and dried by suction. The obtained solid substance was heated to 750
g of acetone and stirred with a glass spoon to disperse.

[Comparative Example 2] ALP activity measurement was carried out in the same manner as in Example 1, except that the composition of the coating liquid applied to the developing layer in the step (■) was changed to the aqueous solution (good solvent solution) in the following (■). A multilayer analytical element was prepared.

'■ Composition of coating water-soluble Tα ρ-nitrophenylphosphate bis(tris) salt 5
g Polyvinylpyrrolidone (average molecular weight: 15)
0g water
1880g [Performance evaluation test] [Test 1] In order to estimate the stability during storage of the multilayer analytical element for measuring ALP activity produced in Example 1 and Comparative Example 1.2,
The reflective optical density values of the elements at a wavelength of 400 nn+ were measured using an integrating sphere spectrophotometer immediately after fabrication and after storage at 45° C. for 7 days after vacuum drying. The results obtained are shown in Table 1.

Table 1 [Test 2] To estimate the stability during storage of the multilayer analytical elements for measuring ALP activity produced in Example 1 and Comparative Example 1.2,
A level of 10 μL was applied to these development layers using a Dry Chem Caliprator manufactured by Fuji Photo Film Co., Ltd., and the mixture was heated at 37°C.
Wavelength 400na for 2 and 3 minutes after incubation start
The reflected optical density value of the element at + was measured using an integrating sphere spectrophotometer. The reflected optical density value was converted to the transmitted optical density. Transmission optical density value created in advance from the difference between transmission optical density values 2 minutes and 3 minutes after the start of the incubation period -
The ALP activity unit was determined from the ALP activity unit calibration curve, and the color development performance was evaluated by comparing the obtained activity unit values. The results obtained are shown in Table 2.

Table 2 From the data in Tables 1 and 2, it can be seen that the multilayer analytical element of Example 1 manufactured by the simplified method of the present invention is superior to the comparison manufactured by the non-simplified method of the prior art. It was found that it had a good storage stability comparable to that of the multilayer analytical element of Example 1.

p-nitrophenylphosphate bis(tris)
The multilayer analytical element of Comparative Example 2, which was manufactured by applying a salt-containing aqueous solution (good solvent solution) to the developing layer and drying it, had better storage stability than the multilayer analytical element manufactured according to the method of the present invention or the prior art. It turned out to be extremely bad.

[Test 3] The particle size of p-nitrophenylphosphate bis(tris) salt dispersed in the coating solution applied to the spreading layer of Example 1 and Comparative Example 1 was observed using an optical microscope. The observation results are shown in Table 3.

Table 3 [Test 4] The coating material to be applied to the spreading layer of Example 1 and Comparative Example 1 was poured into a 50 mL measuring cylinder up to the 50 mL mark, and left to stand for 1 hour. The sedimentation status of fine particles of phenylphosphate bis(tris) salt was observed. The results obtained are shown in Table 4.

Table 4 From the observation results shown in Tables 3 and 4, the fine particle dispersion of p-nitrophenylphosphate bis(tris) salt prepared by the method of the present invention is extremely stable and sedimentation of fine particles is extremely difficult to occur. It is clear that

Claims (1)

[Claims] (1) Dissolve the amine salt or amino alcohol salt of p-nitrophenyl phosphate in its good solvent to form a solution, mix the good solvent solution with a poor solvent to precipitate the salt as a solid phase, and precipitate. An alkaline solution having a liquid permeable layer comprising the steps of dispersing a solid phase in a poor solvent to form a dispersion liquid, applying the dispersion liquid to the liquid permeable layer and drying the liquid permeable layer in order to impregnate the liquid permeable layer with the salt. In the method for manufacturing a dry analytical element for quantification of phosphatase, the step of mixing the good solvent solution with a poor solvent involves introducing the good solvent solution into a turbulent flow of the poor solvent with a linear velocity in the range of 2 m/min to 30 m/min. A method for producing a dry analytical element for quantifying alkaline phosphatase, characterized in that the addition is carried out through an outflow opening with an opening cross-sectional area corresponding to a circular opening with a diameter in the range of 50 μm to 5.0 mm.