JPH0552196B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a dry analytical element for quantifying hydrolytic enzymes in biological fluids that are the subject of clinical chemical analysis. [Prior art] Hydrolytic enzymes, such as amylase, lipase,
For quantitative analysis of phosphatase, glucosidase, and galactosidase, hydrolysis products are further analyzed using chemical reactions or enzyme-chemical coupling reactions.
A number of methods are known to derive colored products and measure their optical density. (e.g. HU Bergmeyer
(Methods of Enzymatic Analysis by HU Bergmeyer) However, detection is more efficient if the hydrolysis products themselves are colored. For example, for the purpose of analyzing alkaline phosphatase, which is one of the hydrolytic enzymes, a method using p-nitrophenol phosphate, which produces p-nitrophenol as a result of an enzymatic reaction, as a substrate is used. Optimal pH of enzyme reaction system
At around pH 9.5, most of p-nitrophenol becomes dissociated and exhibits a yellow color with maximum absorption at 400 nm. However, p-nitrophenol has a maximum absorption wavelength of
The wavelength of hemoglobin (maximum absorption
414 nm) and bilirubin (maximum absorption wavelength 430 and
Because it overlaps with the spectral absorption region of 450 nm), it is subject to interference. In particular, if there is a change in the absorption spectrum due to deterioration of hemoglobin due to a disease, or if hemolysis occurs during the process of preparing serum from a blood sample, hemoglobin interference may appear, causing errors in quantitative analysis. ing. [Object of the Invention] An object of the present invention is to provide a dry analytical element for enzyme activity measurement that eliminates optical interference of biological pigments such as hemoglobin due to hemolysis and has high analysis sensitivity. In particular, in a dry analytical element that contains a self-developing substrate that dissociates a dye in a water-retaining carrier and measures the activity of an enzyme that is active against the substrate, biological pigments such as hemoglobin due to hemolysis, etc. The objective is to eliminate optical interference and achieve high analytical sensitivity. [Summary of the Invention] The above object is a dry analytical element for measuring the activity of an enzyme that contains a self-developing substrate that dissociates a dye in a water-retaining carrier and has an activity against the substrate, A dry method containing a self-developing substrate and a cationic polymer each having a hydroxydiarylazo dye having an absorption maximum at 500 nm or more in the molecule with an ether bond or an ester bond in at least one of the water-retentive carriers. (The substrate and the polymer may be contained in the same carrier or in different carriers in fluid communication with each other). Further, the object of the present invention is to provide a dry analytical element for measuring the activity of an enzyme that contains a self-developing substrate that dissociates a dye in a water-retaining carrier and has an activity against the substrate. has at least a water-impermeable support and a porous liquid spreading layer having a liquid metering action, the self-developing substrate is contained in the liquid spreading layer, and the cationic polymer is contained in the liquid spreading layer, and This was more preferably achieved by a dry analytical element comprising a cationic polymer in another water-permeable layer in liquid contact with the liquid spreading layer. Representative examples of enzymes suitable for analysis according to the present invention are shown in Table 1, corresponding to the self-developing substrates used for each enzyme. The enzymes may be used alone or in combination (eg amylase and glucosidase).

【table】

[Table] Preferred self-developing substrates having a hydroxydiarylazo dye having an absorption maximum at 500 nm or more in a dissociated state in the molecule through an ether bond or an ester bond are represented by the following general formula. A-N=N-B-N In the formula, A represents a phenyl group, a naphthyl group, or a thienyl group, and the sum of the Hammett constants of the substituents is
It is 0.5 or more. B represents a p-phenylene group or a p-naphthylene group. These aromatic groups may be substituted. X is

【formula】

[Formula] (R represents an alkyl group having 9 to 17 carbon atoms) or a conjugated sugar residue. Preferred conjugated sugars are, for example, maltose, maltotriose, maltotetraose, maltohexaose, D-galactopyranose, and glucuronide. Specific examples of the self-developing substrate used in the present invention are shown below. (1) 4-(2'-Methylsulfonyl-4'-nitrophenylazo)-2-chlorophenyl phosphate = sodium (2) 4-(2'-methylsulfonyl-4'-nitrophenyl azo)-2-chlorophenyl phosphate enylazo) phenyl phosphate = sodium (3) 4-(4'-nitrophenylazo) phenyl phosphate = sodium (4) 4-(2'-methylsulfonyl-4'-nitrophenylazo )-5-(3′-Aminosulfonylphenylsulfonylamino)naphthylphosphate = Sodium(5) 3-[5′-Undecanoyloxy-8′-(2″-
Methylsulfonyl-4″-nitrophenylazo)naphthylaminosulfonyl]sodium benzenesulfonate (6) 4-(2′-methylsulfonyl-4′-nitrophenylazo)phenylgalactopyra Structural formula (1) of specific example of noside 520nm (2) 520nm (3) 500nm (4) 644nm (5) 644nm (6) 520nm Cationic polymers that can be used in the present invention include polymers containing secondary and tertiary amino groups,
Polymers with a nitrogen-containing heterocyclic moiety, polymers containing these quaternary cation groups, etc. with a molecular weight of 5000~
200000, especially those between 10000 and 50000. For example, US Patent No. 2548564, US Patent No. 2484430, US Patent No. 3148061
Vinylpyridinium cationic polymers disclosed in U.S. Pat.
Polymers crosslinkable with gelatin etc. disclosed in British Patent No. 1277453, etc.;
No., JP-A No. 54-115228, No. 54-145529, No. 54
Water-based sol-type cationic polymers disclosed in US Pat. No. 3,898,088 and JP-A-55-33172; US Pat. No. 137333/1983) A reactive mordant capable of forming a covalent bond with the dye disclosed in the specification, etc.; Furthermore, U.S. Pat.
No. 3642482, No. 3488706, No. 3557066, No. 3271147, No. 3271148, JP 50-71332
No. 53-30328, No. 52-155528, No. 53-125
Examples include cationic polymers disclosed in No. 53-1024. Other examples include cationic polymers described in US Pat. No. 2,675,316 and US Pat. No. 2,882,156. Among these, those that are difficult to migrate from the hydrophilic colloid layer to other layers are preferred, such as those that crosslink with hydrophilic colloids such as gelatin, water-insoluble cationic polymers, and aqueous sols (or latex dispersions). It can be preferably used. Particularly preferred cationic polymers are shown below. (1) A group that has a quaternary ammonium group and can be covalently bonded to gelatin (e.g., aldehyde group, chloroalkanoyl group, chloroalkyl group, vinylsulfonyl group, pyridiniumpropionyl group, vinylcarbonyl group, alkylsulfonoxy group, etc.) For example, a polymer with (2) A reaction product of a copolymer consisting of a repeating unit of a monomer represented by the following general formula () and a repeating unit of another ethylenically unsaturated monomer, and a crosslinking agent (for example, bisalkanesulfonate, bisarenesulfonate). General formula ()

[Example 1]

A membrane filter (FM300, manufactured by Fuji Photo Film Co., Ltd.) made of cellulose acetate was impregnated with the following aqueous solution and dried. Compound Example 5 1g Sodium Lauric Sulfate/Phosphate PH8.0 Buffer
100ml Sodium cholate 0.5g Polycor (styrene-N-methylmorpholinium-α-methylstyrene-divinylbenzene (55:43:2) latex polymer content 50% 20ml Next, cut this impregnated filter into 10 mm squares and double-sided them. Secure it to a piece of plastic with adhesive tape,
We created a dry analytical element for detecting lipase. When 10 μl of serum with different enzyme activity was spotted on the sample, it eventually became colored blue, and the lipase activity was quantified visually by comparison with a standard colored piece or by using a calibration curve using a reflection densitometer with a measurement wavelength of 640 nm.
A calibration curve was created as follows. 10 μl of serum with known enzyme activity of 10 types is applied and incubated at 37°C for 5 minutes. For each serum, 2
The reflection density was measured after 5 minutes and after 5 minutes, and a calibration curve was created as the relationship between the difference ΔOD and the enzyme activity value. [Example 2] An aqueous solution having the following composition (a) was applied onto a colorless transparent smooth film of polyethylene terephthalate with a thickness of 180 μm and coated with gelatin so that the thickness after drying was 10 μm, and then dried. (a) Gelatin 300g Water 2150g Surfactant 5g (Olin Surfactant 10G) Polyco (styrene-N-methylmorpholinium methylstyrene-divinylbenzene) Polymerization ratio 55:43:2 15% latex solution 280g (with dilute NaOH solution) (Adjust the pH to 7.0) Next, water is uniformly supplied to the gelatin layer at a rate of about 30 g/m ² over the entire surface to moisten it, and then a tricot knitted fabric (polyester 40 gauge) is lightly spread on top of it. Laminate under pressure and let dry. Next, apply 200 c.c. of an aqueous solution of the following composition (b) to this cloth.
An integrated multilayer analytical element for lipase measurement was prepared by applying it almost uniformly at a ratio of m ² and drying it. (b) Specific compound example 5 6g Sodium cholate 1g Water 170g Sodium laurate sulfate/phosphate PH8.0 buffer (0.5M) 50ml For comparison, the following composition (a') was used instead of the solution of the above composition (a). ), and in the same manner as above, an analytical element for measuring lipase activity was prepared. (a') Gelatin 340g Water 2150g Surfactant 5g (Surfactant 10G manufactured by Olin) (Adjust to pH 7.0 with dilute NaOH solution) In Example 2 of the present invention and comparative analysis elements,
10 μl of control serum with different lipase activity values as shown in Table 1 (commercially available control serum or one to which lipase has been added as necessary)
The change in reflection density per minute was measured at a wavelength of 640 nm from 3 minutes to 6 minutes after the sample was applied and kept at 37°C. The results are shown in Table 2. Table 2 Lipase activity Invention Comparison 150 0.020 0.013 450 0.044 0.027 920 0.077 0.047 1200 0.093 0.056 [Example 3] The following composition (a ) of Apply the aqueous solution to a dry thickness of 10 μm and dry. (a) Deionized gelatin 80g Water 1285g Surfactant 9g (nonylphenoxy polyglycidol) Polyco (styrene-N-methylmorpholinium-α-methylstyrene-divinylbenzene) Polymerization ratio 55:43:2 15% latex 500 g of solution 2 g of 1,2-bis(vinylsulfonylacetamido)ethane (Adjust the pH to 7.0 with dilute NaOH solution) Next, apply an aqueous solution of the following composition (b) on the gelatin layer so that the thickness after drying is It was applied to a thickness of 5 μm and dried (adhesive layer). (b) Gelatin 80g Water 1869g Surfactant 8.6g (nonylphenoxy polyglycidol) (Adjust pH to 7.0 with dilute NaOH solution) Next, apply on top of the gelatin layer at a rate of 30g/m ²
After applying a 0.4% nonylphenoxy polyglycidol aqueous solution uniformly and moistening it, a tricot knitted fabric (made of polyester, 36 gauge) was laminated thereon by applying light pressure, and then dried. Next, apply 150 c.c. of the dispersion of the following composition (d) to this cloth.
An integrated multilayer analytical element for measuring alkaline phosphatase was prepared by applying it almost uniformly at a ratio of m ² and drying it. (d) Compound Specific Example 2 Dicyclohexylamine salt 30 mmol Polyvinylpyrrolidone 5 g (average molecular weight 100,000) Ethanol 20 g Acetone 25 g Control serum with different alkaline phosphatase activity values as shown in Table 3 as analytical elements of Example 3 of the present invention Apply 10μl of (commercially available control serum or one to which enzymes have been added as necessary) and measure the change in reflection density (ΔOD) at a wavelength of 540nm between 2 and 5 minutes after keeping it at 37°C. did. Table 3 Alkaline phos ΔOD Fatase activity 300IU/l 0.120 605 0.240 1200 0.375

Claims (1)

[Scope of Claims] 1. A dry analytical element for measuring the activity of an enzyme that contains a self-developing substrate that dissociates a dye in a water-retaining carrier, and that has an activity against the substrate, wherein the dissociated state A dry analytical element containing a self-developing substrate having a hydroxydiarylazo dye having an absorption maximum at 500 nm or more in the molecule with an ether bond or an ester bond, and a cationic polymer in at least one of the carriers each having water-retentive properties. (The substrate and the polymer may be contained in the same carrier or in different carriers in contact with each other). 2 At least a water-impermeable support and a porous liquid spreading layer having a liquid metering function, the self-developing substrate is included in the liquid spreading layer, and the cationic polymer is in liquid contact with the liquid spreading layer. The dry analytical element according to claim 1, which is comprised in another water-permeable layer.