JPS63163164A - Multi-layer analysis element - Google Patents

Abstract

PURPOSE:To remove effect of hemolysis, by having a reagent layer containing a catalase activity blocking agent within a range of addition. CONSTITUTION:A multi-layer analysis element is provided with a reagent layer containing a reactive composition that generates a detectable substance with the action of hydrogen peroxide on a non-water permeable and light transmitting support and a porous development layer on the reagent layer. In a multi-layer analysis element thus arranged, a catalase activity blocking agent is contained in the reagent layer at the density of 1X10<-2> mol./m<2> 1X10<-4> mol./m<2>. This method, employing the blocking agent in the reagent layer, makes the mixture of the agent uniform distribution thereof in a larger area as well. This eliminates effects of hemolysis while avoiding a drop in the sensitivity and deterioration in the simultaneous reproducibility.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a film-like multilayer analytical element for quantifying an analyte (analyte s bottom fraction) in a liquid T-sample.

[Conventional technology]

Sheet-like or film-like analytical elements can be easily and quickly added to a liquid sample by incorporating the analytical reagents and some of the necessary operations into the element in dry form, and then spotting the liquid sample onto the element. It is possible to analyze analytes of Among these analytical elements, those that are single-layered and use paper, membrane filters, etc. as the material for the reagent-supporting layer (Japanese Patent Publication No. 56-419B, U.S. Patent No. 5607095)
number etc.).

In addition, as a non-integral multilayer analysis element that assumes simple lamination or peeling, a glass filter or a membrane filter can be used (Japanese Patent Application Laid-open No. 11595/1989), or two
A method in which a mesh is placed on paper (Japanese Patent Application Laid-Open No. 54-151096, US Pat. No. 55264BO) is known.

These single-layer or single-layered multilayer analytical elements are used for semi-quantitative purposes, but it is difficult to obtain sufficient reliability for quantitative purposes.

Placing multiple layers on a liquid-impermeable, ex-permeable support;
A multilayer analytical element in which each layer is laminated in a state where the layers are bonded without any gaps, which is indicated by the term fluid contact between the layers, is disclosed in Japanese Patent Publication No. 53-21677, Japanese Patent Application Laid-Open No. 55-164559,
No. 55-90859, No. 57-197466, No. 5
It is disclosed in Publications No. 7-101761 and No. 57-j01760. In these, a porous development layer and a functional layer are arranged in multilayer form on one side of a transparent support and are integrated in a non-separable manner. The porous spreading layer is located at the outermost layer, i.e., the top layer, and is a layer to which a liquid sample is applied by spreading the applied liquid sample, that is, the liquid spreads approximately in proportion to the volume of the sample. In other words, it is a layer that acts to spread out a liquid sample that has been spotted and supplied so that approximately one foot of sample is distributed per unit area, and is also called a spreading layer or a metering layer. The liquid sample spread by the layer is immediately distributed uniformly (so that a nearly constant amount of sample is distributed per unit area) to the functional layer located below.
Supplied.

The functional layer typically includes a chemical layer, a reaction layer, a detection layer, a former shielding layer, a former reflection layer, a filtration layer, a translucent layer, a barrier layer, a trap layer, a water absorption layer, and a pretreatment layer. , migration prevention layers, and many other types, they have the reagents and functions necessary for analysis. Both the deployment layer and the functional layer may have a single layer or multiple functions. For example, is the development layer part of the analytical reagent? : It may contain a layer and serve as a reagent layer, or it may contain all analytical reagents and serve as a development layer and a reaction layer.

As a material for composing each layer, the development layer is
A single layer of plush polymer described in JP-A-5-21677, a woven or knitted fabric as described in JP-A-55-164559, and a fiber spread layer made of loose fibers and a reactive polymer material as described in JP-A-57-197466. , and JP-A-5
5 = No. 90859, No. 57-101760, No. 57-
The granular structures shown in each publication No. 101760 are common.

Further, the functional layer may be made of hydrophilic polymer materials such as gelatin, polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, agarose, cellulose derivatives, and the like.

A combination of compositions (color reagents) capable of producing hydrogen peroxide using oxidase and producing a colored substance that can be detected by the action of hydrogen peroxide in the multilayer analytical element described above. There are methods for quantitative determination. These include, for example, a reagent composition known as Trinder's reagent used for glucose detection, and others similar thereto. For example, Ann, Chin, Birche
n.

Literature described in Volume 6, pages 24 to 27 (1969), U.S. Patent No. 3,992,158, Japanese Patent Publication No. 1983-
Reference is made to the descriptions in JP-A No. 45599 and the like.

It is known that analysis using this reagent composition is affected by hemolysis. It is known that these are mainly caused by the consumption of hydrogen peroxide by catalase and heme proteins in red blood cells in whole blood. As a method to eliminate such effects, US Pat. No. 3,335,069
No. 5,549,006, No. 5,862,885, and other specifications, a catalase inhibitor is included in a reagent containing oxidase to remove the influence of a substance having catalase activity when a liquid sample and oxidase react, and Reactions with reagents including detectable color reagents are disclosed.

Furthermore, JP-A-59-109192 discloses that a catalase inhibitor is contained in one layer or the developing layer of a multilayer analytical element close to the developing layer. According to this publication, it is disclosed that the catalase inhibitor solution can be contained in the developing layer by impregnating the developing layer material with a catalase inhibitor solution, drying it, or spraying it. These can eliminate the effects of hemolysis as in the US patent.

[Problem that the invention seeks to solve]

However, catalase inhibitors are known to also inhibit peroxidase, which is the catalyst for converting hydrogen peroxide into a color-forming substance that can be detected by a color-forming reagent. As a result, sensitivity is significantly reduced.

This results in deterioration of analysis precision, that is, simultaneous reproducibility.

An object of the present invention is to provide a multilayer analytical element containing a reactive composition reagent system that provides a detectable substance through the action of hydrogen peroxide produced by oxidase, in which the influence of hemolysis is eliminated. It is in.

Another object of the present invention is to provide an integrated multilayer analytical element containing a catalase inhibitor that avoids a decrease in reflection density, a decrease in sensitivity, and a deterioration in simultaneous reproducibility.

[Means for solving problems]

To summarize the present invention, the present invention relates to a multilayer analytical element, which comprises a liquid-impermeable and light-transparent support that undergoes a reaction to produce a detectable substance under the action of hydrogen peroxide. A multilayer analytical element comprising a reagent layer containing a chemical composition and a substance that converts hydrogen peroxide into a detectable colored substance, and a porous development layer above the reagent layer, wherein the reagent layer contains a catalase activity inhibitor. , 1xio″″1 mol/m! ~I
It is characterized by being contained at a concentration of X 10-4 mol/m2.

As a result of repeated experiments, the present inventors have arrived at the present invention by discovering that the above drawbacks can be overcome by including a catalase activity inhibitor in the above reagent layer in the above addition amount range.

The catalase activity inhibitor used in the multilayer analytical element of the present invention may be any known inhibitor of catalase activity, such as cyanide compounds, azide compounds, fluorine compounds, formic acid and its salts, acetic acid and its salts, and aminotriazole. Examples include ascorbic acid, divalent copper ion, semicarbazide, pyrazole, diethyldithiocarbamate, nitrite, N-ethylmaleimide, and the like. In particular, azide compounds, fluorine compounds, aminotriazoles, acetates, and the like are preferred because of their strong inhibitory effects.

The addition of these catalase activity inhibitors was 1×
It may be 10""1 to I x 10"-4 mol/tn", preferably 5 x 10-s to 3 x 10-4 mol/tn".
m”.

The multilayer analytical element of the present invention comprises, on a water-impermeable and light-transparent support, a reagent layer containing a reactive composition that produces a detectable substance under the action of hydrogen peroxide, and a porous support. It has a spreading layer as an essential constituent layer, but if necessary, a functional layer having other functions (for example, a light reflection layer, a filtration layer, a barrier layer, a migration prevention layer, etc.) or a structural auxiliary layer (for example, an adhesive layer, etc.) ).

The catalase activity inhibitor is contained in the reagent layer. By including the inhibitor in the reagent layer, it can be uniformly contained and distributed evenly over a large area without depending on its position.H2O generated under the involvement of oxidase can be detected. For conversion into possible color-forming substances, substances exhibiting peroxidase-like or peroxide activity are required. Generally, peroxidase is used, but the present invention also uses synthetic peroxidase, hemin, methemoglobin, oxyhemoglobin, hemoglobin, hemochromogen, alkaline hematin, hemin derivatives, iron sulfocyanate, iron tannate, iron ferrocyanide, chromate, etc. useful for implementation.

The above-mentioned substance exhibiting peroxidase-like activity or peroxide activity is contained in the reagent layer.

As a porous spreading layer, a fiber spreading layer consisting of loose fibers and a reactive polymer is disclosed in JP-A No. 57-197466, and a non-fibrous porous layer is disclosed in JP-A-53-21677. JP-A-57-101760 and JP-A-57-101760, a so-called plush polymer layer
7-101761 Particle bonded body in which particles disclosed in Equity Publication are bonded directly or through a low molecular compound, JP-A-5
Examples thereof include a granular structure comprising particles and fine adhesive particles disclosed in Japanese Patent Publication No. 5-90859, and a hydrophilized fabric described in Japanese Patent Application Laid-open No. 55-164356.

Naturally, it can contain enzymes and buffers necessary for analyte analysis.

Any suitable coloring composition that causes a detectable change in the presence of hydrogen peroxide can be used. Typical examples that can be used include those containing the following substances along with a coloring agent if necessary.

Monoamines such as aniline and its derivatives, diamines such as O-phenylenediamine, benzidine, etc.
Phenols, Examples 1-1' Phenol, thymol, cresol, naphthol, etc., polyphenols, such as catechol, guaiacol, pyrogallol, etc., aromatic carboxylic acids, such as salicylic acid, gallic acid, etc., leuco dyes, such as leucomalachite green, US patent No. 40897
Triarylimidazoles and triarylmethanes [B, E, pub (B,
E, Babb) and S, Dani x le (D
, S., Danish) et al. May 2, 1984
[including those described in U.S. Pat. acids, tyrosine, etc., and others known to those skilled in the art of clinical chemistry.

Particularly useful color-forming compositions are the leuco dyes mentioned above and especially the triarylimidazole-containing compositions described in the US Pat.

Other preferred color-forming compositions are those based on color-forming color formers that can react with oxidizable color developer compounds to produce a color change. Useful color-forming color formers include U.S. Pat. No. 4,251,629.
No. 4260679 and No. 4596714, Publication No. 1982-22200, European Patent Application No. 68556, British Patent No. 210786
It includes substituted anilines (ie, toluidines), including those described in Specification No. 3 and Japanese Patent Publication No. 58-898. Representative oxidizable color developer compounds include benzidine and its congeners, p-7 22-diamine, p-aminophenol, aminoantipyrine,
For example, 4-aminoantipyrine and the like are included. A preferred color developing compound is 4-aminoantipyrine. Oxidase enzymes that can be incorporated into the present invention include glucose oxidase, uricase, cholesterol oxidase, sarcosine oxidase, pyruvate oxidase, glycerol oxidase, glycerophosphate oxidase, choline oxidase. , lactate oxidase, and the like.

Furthermore, when the analyte is a hydrophobic compound such as cholesterol that is difficult to diffuse into the hydrophilic polymer matrix represented by the reagent layer,
Among the oxidase enzymes, 16-cholesterol oxidase is preferably contained in the spreading layer or a layer adjacent to the spreading layer, and this is an example to which the present invention is preferably applied.

These oxidase enzymes are selected according to the analyte and are used in combination with other enzymes. Additionally, when used in combination with a buffer at a preferred pH range and concentration,
Of course this is desirable.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these examples.

Example 1 A reagent layer having the following composition was entirely coated and dried on a transparent undercoated polyethylene terephthalate support having a thickness of 180 μm.

Sera f 7 15.09 f
7m” Sodium azide See Table 1 4-aminoantipyrine hydrochloride (L823
97m"1.7-')hi)"(1-'Fshi+7.(t,
'y (L612 t/rn" Peroxidase 15500U/yn2 Dimedone α1579/m" Potassium phosphate buffer (pH 6.7-6.9) A 2
27 f 7m” Alkanol XC (product name) 2soI
slF/m"1.2-bis(vinylsulfonyl)ethane 10011F/m" reagent layer with a dry film thickness of 16 μm.

On the above reagent layer, the following coating solution was coated with <v4 to form a developing layer.

Triton X-10014,0097m2Cholesterol oxidase 3000 U/m"Cholesterol esterase 5000 U/m"
Bovine serum albumin 500t
7m” spread layer with a dry film thickness of 300μm.

Furthermore, as comparative analysis elements (1) and (2), sodium azide was added to the composition of the reagent layer in an amount shown in Table 1 below.

The analytical element of the present invention (Il, (21), the comparative analytical element (+1, (2)) has a total cholesterol amount of 180.50
Using 0.450η/dt serum, apply 10μt,
After incubation at 37°C for 7 minutes, 546
Reflection density was measured using a nm filter. Table 2 shows the measurement results of the simultaneous reproducibility of reflection density. Furthermore, red blood cells are separated from human whole blood and subjected to ultrasonication to cause hemolysis.
Remove the red blood cell membrane to make a hemoglobin solution, and the amount of hemoglobin is 0.50.100.200.500.500η7
The addition was made so that dt. However, at this time, I made the total Vscf roll IIH1801Q/dtVcfl. Similarly, after spotting and incubation, 546n
Reflection density was measured at m. The results are shown in Table 3.

Table 1 Table 2 (n=10 each) Table 5 From the results shown above, the analytical element of the present invention has sufficient sensitivity and good simultaneous reproducibility. Furthermore, it is clear that for hemolysis of red blood cells, there is no change in reflection density at any level compared to no hemolysis.

On the other hand, in the comparative analytical element (1), the reflection density decreased significantly at high cholesterol levels, and it is clear that the simultaneous reproducibility was worse than that of the analytical element of the present invention. Furthermore, although comparative analysis element (2) has good sensitivity and simultaneous reproducibility, it is clear that it is affected by hemolysis.

Example 2 A reagent layer having the following composition was applied onto a transparent polyethylene terephthalate support having a thickness of 180 μm and which had been undercoated.

Gelatin 109/m"Peroxidase 7000U/m"'
7! J Curse (derived from e mother) 25
007m” Diptyl phthalate
6.7 f/m” borate buffer (pHa5)
7.597m” Sodium azide
Dry film thickness of 1×10-3 mol/m”
5 μm reagent layer.

Furthermore, a developing layer having the following composition was coated on the above reagent layer.

Triton X-1001α2 f/ryt”
p-Toluenesulfonate 115r
/m” with a dry film thickness of 300 μm.

The above uric acid analysis element was designated as the analysis element (3) of the present invention.

The analytical element (3) of the present invention has a uric acid concentration of 4.5 ■/dz.
.

9.5■/d! ,,'I55 Misaki/6t human serum, 1
After spotting 0 At and incubating at 57°C for 7 minutes, the reflection density was measured using a 546 nm filter. Furthermore, catalase (manufactured by Sigma) was added to human serum with a uric acid concentration of 9.5897 dt at O8, U/d, 1000S.
, U/+d, 2500S, U/-55000S, U/d
, 10,000S, and U/d were added to the serum. All results are shown in Tables 4 and 5.

As shown in Tables 4 and 5, it is clear that the analytical element of the present invention has good sensitivity in uric acid analysis and is not affected by blood catalase.

Example 5 The same analytical element as shown in Example 1 was prepared except that the azide in the reagent layer of the analytical element of the present invention was replaced with the catalase activity inhibitor shown in Table 6 below. .

These cholesterol analysis elements can be used as the analysis element + of the present invention.
41. (It was set at 51.

Table 6 Analytical element of the present invention described above +41. (51, Example-1
Similarly, the total cholesterol amount is 180°300.4
Using 50"P/dtO serum, 10 AL was spotted and incubated at 37°C for 7 minutes, followed by 546"
Reflection density was measured using a nm filter. The results are shown in Table 7.

Table 7 also shows hemoglobin ti (, o, 50.100.
200.500.500"l/r3LlC'lk using serum with a total cholesterol amount of 180q7'az, after spotting 10μ in the same way, after incubation at 57°C for 7 minutes. Reflection density was measured at 546 nm.The results are shown in Table 8.

From the results shown in Table 8, it is clear that even when the catalase activity inhibitor is replaced with sodium fluoride or aminotriazole, the analytical element of the present invention sufficiently eliminates the influence of hemolysis.

〔Effect of the invention〕

As described above, the analytical element of the present invention has the remarkable effect of eliminating the influence of hemolysis and avoiding a decrease in sensitivity and, in turn, a deterioration in reproducibility.

Claims (1)

[Scope of Claims] 1. A reactive composition that produces a detectable substance under the action of hydrogen peroxide and an exhibit that allows hydrogen peroxide to be detected, on a liquid-impermeable and light-transparent support. In a multilayer analytical element having a reagent layer containing a substance to be converted into a color substance and a porous development layer above the reagent layer, a catalase activity inhibitor is added to the reagent layer at a concentration of 1×10^-^2 mol/m^. 2~1×10
A multilayer analytical element characterized in that the content is ^-^4 mol/m^2. 2. The substance that converts hydrogen peroxide into a detectable colored substance is peroxidase, synthetic peroxidase, hemin, methemoglobin, oxyhemoglobin, hemoglobin, hemochromogen, alkaline hematin, hemin derivative, iron sulfocyanate, iron tannate, The multilayer analytical element according to claim 1, which is at least one substance selected from the group consisting of iron ferrocyanide and chromate. 3. The catalase activity inhibitor contains cyanide compounds, azide compounds, fluorine compounds, formic acid and its salts, acetic acid and its salts, aminotriazoles, ascorbic acid, divalent copper ions, semicarbazide, pyrazole, diethyldithiocarbamate, The multilayer analytical element according to claim 1, which is at least one substance selected from the group consisting of nitrates and N-ethylmaleimide. 4. The substance that converts hydrogen peroxide into a detectable colored substance is peroxidase, and the catalase activity inhibitor is at least one selected from the group consisting of azide compounds, fluorine compounds, aminotriazoles, and acetates.
The multilayer analytical element according to claim 1, which is a seed substance.