JPS62275699A - Dry-type analyzing element for determination of amylase activity - Google Patents

Abstract

PURPOSE:To determine amylase activity preventing the interference of hemoglobin, etc., by using the titled analyzing element composed of a detection layer, a glucosidase-containing reagent layer, a light-shielding layer, a liquid-developing layer containing oligosaccharide substrate and a base supporting the above layers. CONSTITUTION:A detection layer and a glucosidase-containing reagent layer are applied to the surface of a light-transmitting supporting base made of a polymer such as polystyrene, cellulose ester, etc. The laminate is further laminated with a light-shielding layer containing a light-reflecting or light-absorbing particle at an amount to cause a specular transmission density of >=1.0 and <=2.3. The light-sheilding layer is coated with a water-permeable layer containing self-developing oligosaccharide substrate capable of dissociating trisaccharide, disaccharide or monosaccharide bonded with p-nitrophenol in the presence of amylase. The reaction reagent layer may contain a hydrophilic polymer such as starch, cellulose, etc., and a buffering agent such as carbonate, borate, etc.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention [Field of the Invention] The present invention relates to a dry multilayer analytical element for measuring enzyme activity, particularly amylase activity. I will do it.

[Background of the Invention] Self-closing groups that dissociate paranitrophenol dyes have been widely developed in recent years. In particular, various oligo Ns (e.g. G5, G7) are introduced with palani-1 to lophenol dyes (abbreviated as PNP), such as PNP-G5 and PNP.
-07 and a glucosidase enzyme, a conventional dye starch substrate in which starch is dyed with a reactive dye, such as blue starch or amylopectin azure, is used to generate a precipitate by utilizing the difference in molecular weight distribution. It is superior in terms of quantitative accuracy and accuracy compared to the "pigmented starch method" (for example, see Methods of Enzymatic Analysis by Bergmeyer, U, Bergmeyer, p. 894). It has the advantage of allowing continuous monitoring and analysis of enzyme reactions (late assay in a narrow sense).

However, when a self-closing substrate such as PNPG5 or PNP-07 and a glucosidase enzyme are contained in the same layer, dissociation of the self-clasping group by the glucosidase enzyme occurs even in the absence of amylase to be detected. P.N.
Coloration due to P is observed, forming a background density.

In addition, PNP has a maximum absorption wavelength around 410 nm (400 nm in the solution method), and it is difficult to absorb hemoglobin (8i! maximum absorption wavelength 414 nm) and bilirubin (8i! maximum absorption wavelength 430 and 450 nm) that may coexist in the sample solution. Since it overlaps with the spectral absorption region, it is subject to interference from these regions. In the reaction rate method commonly used to measure enzyme activity, interference from hemoglobin and bilirubin causes negative errors in the results of measuring amylase activity. Especially due to illness,
Alternatively, hemoglobin interference appears when hemolysis occurs during the preparation of serum from a blood sample.

[Object of the Invention] An object of the present invention is to provide a dry multilayer analytical element for measuring amylase activity in which interference of hemoglobin and bilirubin is effectively prevented in the reaction rate method.

Another object of the present invention is to provide a dry multilayer analytical element for measuring amylase activity that does not generate a background concentration due to PNPs caused by dissociation of an autochromic substrate by a glucosidase enzyme even in the absence of amylase to be detected. It is in.

[Structure of the Invention] The above-mentioned objects have at least one water-permeable layer on -F of the light-transmitting support, and at least one of the water-permeable layers is coated with para-nitrochloride in the presence of amylase. containing an autochromic oligosaccharide substrate capable of dissociating a phenol-bound trisaccharide, trisaccharide or it sugar (optionally a tetrasaccharide);
Another water-permeable layer provided between the water-permeable layer containing the colored oligosaccharide substrate and the support contains glucosidase,
and between the layer containing the oligosaccharide substrate and the layer containing glucosidase, a light-reflecting or light-absorbing T1'1.
! : Achieved by a multilayer dry liquid analysis element characterized by containing layers within a range that does not exceed

In particular, the objects of the present invention have been preferably achieved by the multilayer dry liquid analytical element described above, in which the layer containing the oligosaccharide substrate does not substantially contain glucosidase.

As the glucosidase, α-glucosidase or β-glucosidase can be used as necessary.

The present invention can be applied to various known dry analysis elements. In particular, it can be applied to an element containing a solid carrier through which both a self-closing substrate and a test liquid can pass. The element may be a 5-layer structure consisting of a support, a detection layer, an advance layer, a reaction reagent layer, a porous liquid spreading layer, an adhesive layer, a filtration layer, a water absorption layer, an undercoat layer, and other known layers. Good examples of such analysis elements include U.S. Pat. No. 3,992,158, U.S. Pat.
There is something disclosed in each specification of No. 356.

When a support is used, the configurations that can be practically explored for the flexible analytical element of the present invention are (1) a reagent layer on the support, a pre-spraying layer on top of the reagent layer, and a liquid spreading layer on top of the reagent layer; in this case, , the reagent layer contains glucosidase, and the liquid rM layer contains oligosaccharide substrate.

(2) A support having a detection layer, a reagent layer, an advance layer, and a liquid spreading layer on the j-plane. In this case, the reagent layer contains glucosidase, and the liquid@interlayer contains the oligosaccharide substrate.

(3) Second X drug layer, advance layer, first reagent layer, and liquid layer 17F on the support! Those with layers in this order. in this case,
The second reagent N layer contains glucosidase, and the first reagent layer contains an oligosaccharide substrate.

(4) A device having a detection layer, a second X-reagent layer, an advance layer, a first reagent layer, and a liquid@T'1FII layer on the support in this order. In this case, the second X drug layer contains glucosidase and the first reagent layer contains the oligosaccharide substrate.

In the present invention, the above rrq arrangement (2) is preferred.

In (1) to 4) above, between the detection layer and the reagent layer,
A filtration layer may be further provided between the reagent layer and the liquid@interlayer or between the second reagent layer and the first reagent layer.

In the present invention, the self-chromochromic substrate may be contained in two or more layers (e.g., the reagent layer and the liquid developer R). In this case, the self-chromochromic substrate may be contained in a layer far from the support or It is also possible to have more distribution in any of the nearby layers.

Light transmittance that can be used in the dry analytical element of the present invention
Examples of water-impermeable supports include polyethylene terephthalate, polycarbonates of bisphenol A, polystyrene, cellulose esters (e.g., cellulose triacetate-1, cellulose acetate propionate, etc.)
A transparent support in the form of a film or sheet made of a polymer such as the like and having a thickness ranging from about 50 μm to about 1 μl, preferably from about 80 μl to about 300 μl, can be mentioned.

If necessary, an undercoat layer may be provided on the surface of these supports to strengthen the adhesion between the support and the detection layer or other layer provided on the support. Further, instead of the undercoat layer, the surface of the support may be subjected to physical (eg, glow discharge, corona discharge) or chemical activation treatment to improve the adhesive strength.

In the integrated multilayer analytical element of the present invention, a detection layer may be provided north of the light-transparent/water-impermeable support (possibly via another layer such as a subbing layer). The detection layer provided in the dry analytical element is preferably a layer made of a hydrophilic binder, that is, a layer whose layer forming component is a hydrophilic polymer that swells upon absorbing water.

Hydrophilic polymers that can be used in the detection layer generally have a swelling rate of about 15 to 20.1 T at 30°C upon absorption of water.
Preferably, it is a natural or synthetic hydrophilic polymer in the range of about 25 to 15%. Examples of such hydrophilic polymers include gelatin (e.g., acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (e.g., phthalated gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinyl and Lolidon etc.

The dry thickness of the detection layer is preferably in the range of about 1 μl to about 50 μm, more preferably in the range of about 3 μl to about 30 μl. Furthermore, the detection layer can also contain a surfactant (cationic, amphoteric, or nonionic) or a new material, if necessary.

An adhesive layer for adhering and laminating the spreading layer may be provided between the liquid spreading layer and layers such as the reaction reagent layer, the light guide layer, and the aperture layer. The adhesive layer is preferably made of a hydrophilic polymer that is capable of adhering the spreading layer when wetted with water or swollen with water. Examples of hydrophilic polymers that can be used in the adhesive layer include:
Similar hydrophilic polymers may be used in the detection layer.

Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. The dry film thickness of the adhesive layer generally ranges from about 0.5 μl to about 20 μl, preferably from about IBy to about 10 μl. Note that the adhesive layer may be provided not only on the water absorption layer but also on other desired layers in order to improve the adhesion between the layers. The adhesive layer can be provided by applying an aqueous solution containing a hydrophilic polymer and a surfactant added if necessary to the detection layer, contact layer, etc., using a known method.

The reaction reagent layer of the dry analytical element of the present invention may contain a hydrophilic polymer, a buffer, light-shielding (reflective or absorbing) fine particles, etc., as necessary.

Examples of hydrophilic polymers that can be contained in the reaction reagent layer of the flexible analytical element of the present invention include starch, cellulose, agarose, gelatin and derivatives thereof (-, hydroxymethylated, hydroxypropylated, etc.), acrylamide, etc. Polymers, copolymers of acrylamide and various vinyl monomers, polyvinyl alcohol, copolymers of vinylpyrrolidone and various vinyl monomers, acrylate-1 polymers, and copolymers of acrylate-I and various vinyl monomers. Examples include polymers. Among the above hydrophilic polymers, gelatin, gelatin derivatives, polyvinyl alcohol, vinylpyrrolidone polymers, acrylamide polymers, and cellulose derivatives are preferred.

Examples of buffering agents that can be contained in the reaction reagent layer of the flexible analytical element of the present invention include carbonates, phosphates, and well-known l1l(
'R agents can be mentioned. These buffers are described in "Basic Experimental Methods for Protein Enzymes" (written by Takeshi Horio et al., Nankodo, Japan).
It can be operated and used with reference to known documents such as 1981).

The reaction reagent layer of the dry analytical element of the present invention may further contain light-shielding particles, which will be described later.

The light shielding layer is preferably a water-permeable layer in which light-reflecting Y&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, etc., and also functions as a light reflecting layer or a 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 diameter of about 0.1 μl to about 1.2 μl.
μ degree microcrystalline particles, etc.), barium sulfate microparticles, aluminum microcrystalline particles, etc. Examples include gamma molecule. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferred.

Examples of hydrophilic polymer binders used in the light shielding layer include the hydrophilic polymers used for the detection layer described above, as well as weakly hydrophilic regenerated cellulose and cellulose acetate.
I., etc.3 can be mentioned.

Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. A known hardening agent can be added to gelatin or gelatin derivatives.

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

In the present invention, subicular optical layer formation (DIl
/11) is 1.0 or more and 2.3 or less, that is, the vertical transmittance is 0.5% or more, but does not exceed 10-6, so that the light-reflecting fine particles are substantially uniform in the light shielding layer. distributed.

Preferably, the spreading layer is a spreading layer having a liquid metering effect. Having a liquid metering effect means that the liquid sample that is dotted onto the surface of the sample can be distributed in a single manner (per square meter) in the lateral (horizontal) direction, without causing the portions contained therein to be distributed unevenly in real snow. It has the effect of spreading at a nearly constant rate.

Materials constituting the matrix of one layer include non-woven fabric, t! & fabrics (e.g. plain weave such as broadcloth, boblin, etc.), knitted fabrics (e.g. tricot knit), P paper, glass JU fiber F paper, membrane filter 1 made of plush polymer or tertiary material made of polymer microbeads, etc. An original lattice structure or the like can be used. Among these, it is preferable to use woven fabrics and knitted fabrics.For details, see JP-A-55-1.6.
No. 4356, No. 57-66359 and No. 60-22
All you have to do is look up No. 2769.

The woven fabric or knitted fabric that can be used in the dry analysis element of the present invention can be treated with degreasing treatment such as washing with water to remove at least threads.
It is preferable that oils and fats attached during the production of the woven or knitted fabric be substantially removed.

When the above-mentioned woven or knitted fabric is used as a spreading layer of the analytical element of the present invention, the woven or knitted fabric is further subjected to a physical activation treatment (preferably a glow discharge treatment) as disclosed in JP-A No. 57-66359. treatment or corona discharge treatment, etc.) on at least one side of the fabric, or hydrophilic polymer impregnation treatment disclosed in JP-A-55-164356, JP-A-57-66359, etc., JP-A-60-22
By applying a hydrophilic treatment such as the method disclosed in No. 2770, the R fabric or knitted fabric can be made hydrophilic and the adhesive force with the lower layer (the side closer to the support) can be strengthened.

Expansion of an integral multilayer analytical element made of l&r' or knitted fabric: In order to adhere and laminate the I layer to the aforementioned detection layer, reaction reagent WJ or adhesive layer, etc.
56 and Japanese Patent Application Laid-Open No. 57-66359.

That is, water (or water containing a small amount of surfactant) is suddenly and uniformly supplied to the water-absorbing layer or adhesive layer while it is still wet after being applied, or to the dry layer to swell the layer, and then the fabric or adhesive layer is coated. The knitted fabric is bonded, laminated, and integrated by applying light pressure suddenly and uniformly on the wet or swollen layer.

The development layer is brush volumer or membrane filter.
When it is made of paper or non-woven fabric, it is published in Japanese Patent Publication No. 53-21677, etc.; when it is a three-dimensional lattice tlIl structure made of polymer microbeads, it is published in Japanese Patent Publication No. 55-90859, and when it is made of paper or non-woven fabric, it is published in Japanese Patent Publication No. 57-148250, etc., according to the methods described respectively.

When the hydrophilic polymer binder such as the reagent layer, light shielding layer, or adhesive layer described above is gelatin or a gelatin derivative, the spreading layer is formed while the gelatin (or derivative) is in an undried gel state after application of the layer. A method of bonding, laminating, and integrating materials can be adopted.

If necessary, the above-mentioned light-reflecting fine particles may also be included in the developing layer.

When the liquid spreading layer contains a reactive reagent such as a self-closing base material and, if necessary, a hydrophilic polymer, a buffering agent, light-shielding fine particles, etc., a coating solution containing the above is applied or coated onto the spreading layer. There are methods for spraying and drying.

Further, when the liquid developing layer M is made of woven fabric, knitted fabric, nonwoven fabric, paper, etc., a method can be used in which the developing layer is impregnated in a solution containing the above-mentioned reagents, etc., and then dried. In addition, when the above-described spread layers are laminated to form an integrated multilayer analysis element, the immersed spread 17FIN'j as described above is covered with other layers in a dry or semi-dry state. A method of integration can also be used.

In the case of a layer formed by coating, for example, a layer made of plush polymer, etc., the coating solution for this layer is coated with the underwing solution or FI1. M, may be mixed and applied.

When containing reagents and the like in the developing layer, different methods can be used for each reagent or reagent group.

The present invention will be explained in more detail below with reference to Examples.

[Example 1] An aqueous solution having the following composition (a) was applied onto a polyethylene terephthalate 1 to colorless transparent smooth film with a thickness of 1.80 Jim and coated with gelatin so that the thickness after drying was 7 μm, dry.

(a) Gelatin 300g surfactant
5g (Ozone $4sur4a
ctant IOG) Bolico (styrene-N-methylmorpholinium methylstyrene-divinylbenzene) Polymerization ratio 55.43.2 +5$”7tex solution?l!280 g water
2150 g (adjust pH to 7.0 with dilute NaOH solution) Next, an aqueous solution having the following composition (b) is applied onto the gelatin layer so that the thickness after drying becomes 5 μm and dried.

(b) Gelatin 200g surfactant
5g (Ozone? JSurfa
cLanLIOG) α-glucosidase
3.5 million IU water
Adjust the pH to 7 with 2600 g (Tr Na O)T solution.
．． Next, an aqueous solution having the following composition (c) was applied to the above gelatin layer so that the thickness after drying was 3 μm, and the gelatin layer was dried and knuffed.

(C) Gelatin 30゜surface active (i) 4g (manufactured by Ozone Co., Ltd. 5urfa
ctanL IOG) Titanium oxide (anatase type)
20g water
950 g (7.0 g of P I (7.0
The coating layer containing titanium oxide has a subicular density of about 13.

Next, about 30 g /
After uniformly supplying moisture to the entire surface at a ratio of m 2 to moisten it, a tricot knitted fabric (made of polyester, 40 gauge) is laminated on top of it by applying light pressure, and it is dried.

Next, apply 150 cc of an aqueous solution of the following composition (d) to this cloth.
It was coated almost uniformly at a ratio of 0.0 m2 and dried to produce an integrated multilayer analytical element for measuring amylase.

(d) Paranitrophenyl α-D-mal 1-pentaoside 34g water
1600g potassium phosphate 60g polyvinylpyrrolidone
140g (average molecule Jit 100,000) (adjust pH to 7.3 with dilute NaOH solution) [ratio f2
For comparison, an analytical element for measuring amylase was prepared in the same manner except that a solution having the following composition (Co) was used instead of the solution prepared in (C) above.

(Co) Gelatin 50g Surfactant
4g (Olin %5urfac
Lant IOG) water
950g (diluted Na○■ (pH 70 with solution)
) The subicular density of the above coating layer is approximately 0.1.
It is.

[Measurement rIA1] Control serum with different amylase activity values (commercially available control serum or human saliva amylase added thereto as needed) and IT were used as shown in Table 1 for the analytical elements of the present invention and for comparison. 10 μp of human hemoglobin added at the concentration shown in was applied and the change in reflection density per minute between 3 and 6 minutes was measured at a wavelength of 400 mμ when kept at 37°C. The amylase concentration was calculated from a standard curve prepared in advance using control serum to which no hemoglobin was added. The results are shown in Table 1.

Table 1 As is clear from Table 1, the analytical element of the present invention is less affected by hemoglobin than the comparative analytical element.

[Moisture determination example 2] The present invention prepared in Example 1 and the comparative analytical element prepared in Comparative example 1 were mixed with 7% human amylase solutions with different amylase activity values (human saliva amylase was added as necessary). 10 μl of bilirubin added to III and V at the concentration shown in Table 2 was applied and kept at 37°C between 3 and 6 minutes later. The reflection density change per minute was measured at a wavelength of 400m7z, and the amylase activity was calculated from a calibration curve prepared in advance using a 7% human albumin solution containing amylases of different activities without the addition of bilirubin. The results are shown in Table 2.

Table 2 As is clear from Table 2, the analytical element of the present invention is less affected by bilirubin than the comparative analytical element.

[Ratio lF2@2] For comparison, instead of the solution of composition (b), use the following composition (
An analytical element for measuring amylase was prepared in the same manner as above except that a solution having the following composition (d'') was used instead of the solution having the composition (d).

(bo) Gelatin 200g surfactant
5g (Olin 5ur4ac
tant IOG ＞Water
Adjust the pH to 7.0 with 2600g Wei Na OH solution.
Adjust to 0.

(d') Paranitrophenyl α-D-maltopentaoside 34g α-glucosidase] 500,000 ItJ polyvinylpyrrolidone
140g (average molecule - +i700,000) Water 1600g Potassium phosphate 60g (dilute NaO
RF to pH 7.3 with 14 solution! Jm) [Measurement example 3
] A 7% Ir5A solution having no amylase activity was spotted on the analytical elements of Example 1 and Comparative Example 2 of the present invention, and reflection was performed;
Measurements were made at an external reflection of 4 degrees 171° at a wavelength of 400 rnμ, and the results are shown in Table 3.

Table 3 As is clear from Table 3, it can be seen that the analytical element of the present invention has a very low back ml and hollowness as compared to the analytical element of the comparative example.

Claims (1)

[Claims] It has at least one water-permeable layer on a light-transparent support, and at least one of the water-permeable layers contains a trisaccharide, a disaccharide, or a monosaccharide to which paranitrophenol is bound in the presence of amylase. (optionally, a tetrasaccharide), the water-permeable layer is provided between the water-permeable layer containing the self-developing oligosaccharide substrate and the support. A layer containing glucosidase, and light reflecting or light absorbing particles are placed between the layer containing the oligosaccharide substrate and the layer containing glucosidase, and the specular transmission density is at least 1.0 and does not exceed 2.3. A multilayer dry liquid analysis element characterized by having a layer comprising