JPS58175495A - Analytical element for measuring gamma-gtp - Google Patents

Abstract

PURPOSE:To determine the activity of gamma-GTP, and to prevent the transfer of a reagent and a dyestuff, by adding a substance having an oxidizing action, a coupler having diffusion resistance, and an anilide derivative to at least one layer of an analytical element consisting of a reagent layer and a developing layer. CONSTITUTION:A substance having an oxidizing action, a coupler having diffusion resistance, and an anilide derivative shown by the formula or its salt are added to at least one layer of an analytical element having a reagent layer and a developing layer on a substrate. In the formula, R1 and R2 show hydrogen atom or alkyl group, R1 and R2 may be linked to nitrogen atom to form heterocyclic ring, R3-R6 are hydrogen atom, halogen atom, hydroxy, amino group, alkoxy group, acylamide group, arylsulfonamide group, alkane sulfonamide group or alkyl group, and R3-R6 are not hydrogen atom at the same time.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an analytical element, and particularly relates to a useful analytical element for measuring the activity of γ-glutamyl transpeptidase (hereinafter referred to as γ-GTP).

As is well known, γ-GTP is generally specific to chronic liver diseases, and along with enzyme measurements conventionally used clinically, its diagnostic significance is high and it is important to This test is essential for understanding the disease state.

Conventionally, several methods have been reported for quantifying the amine compound produced using a synthetic substrate for r-GTP.

For example, as a colorimetric method for quantifying four aniline derivatives produced by γ-GTP, there is a method of transforming into diazonium salt, TJ
- A method of forming a Schiff base with -dimethylamino-cinnamic altehyde, or a method of forming a color by performing partial condensation with xylenol has been disclosed. However, since a solution is used in the reaction until an analytical result is obtained, handling is complicated and several dispensing operations are required, resulting in measurement errors.

The inventors of the present invention have conducted extensive studies to improve the above drawbacks. That is, the first object of the present invention is to provide an analytical element for fixing γ-GTP activity.5 The second object of the present invention is to provide an analytical element for determining γ-GTP activity.
The purpose of the present invention is to provide an analytical element that does not cause f-induced problems.

The object of the present invention is to provide at least one layer of reagent Jm on a support.
and an analytical element comprising at least one spreading layer, in which at least one layer contains an oxidizing substance, a diffusion-resistant coupler, and an anilide conductor represented by the following general formula or a salt thereof. By using it, the growth was delayed.

(ゝR2 In the formula, R9 and R1 each represent an r1 hydrogen atom or an alkyl group, and R, and R2 together with a ♀ elementary atom
S may be formed, R3+ R4+ 'W and R6
each represents a water AB atom, ), a rogone atom, a human mixture, an amino group, an alkoxy group, an acylamido group, an arylsulfonamide group, an alkyl group, a rusulfonamide F group, or an alkyl group; The alkyl group preferably has 1 to 6 carbon atoms, particularly preferably 1 to 4 carbon atoms. For example, it can extract methyl, ethyl, and butyl. These alkyl groups may have a substituent. Examples of the substituent include ureido group, tetrahydrofuryl, carboxyl, methanesulfonamide, sulfo, methoxy, ethoxy, methoxyethoxy, methoxytetraethquin, methoxy Tetraetkin is caught. R, R, and R6 are preferably a water $ atom, an alkoxy group, an alkyl sulfonamide group, or an arylsulfonamide 4, and more preferably a hydrogen atom. -R is a hydrogen atom, an alkyl group, or an acyl amide group. Preferred is an alkyl group having 1 to 3 carbon atoms, particularly methyl. In addition, the salts of compounds represented by the general formula include p-toluenesulfonic acid, sulfonic acid, sulfinic acid, p-toluenesulfonic acid,
Sulfamic acid, thiosulfate S-ester, carboxylic acid.

Mention may be made of salts of α-acids or inorganic acids such as phosphoric acid esters, amidophosphoric acid, phosphoric acid, phosphorous esters, organoboron compounds, hydrochloric acid and waxic acid, in particular p-)luenesulfonates, hydrochlorides. and sulfates are preferred.

Typical specific examples of the anilide enzyme conductor according to the present invention are shown below, but the present invention is not limited thereto.

[Exemplary compound j +1-1) 1-(L-γ-glutamyl)-4-N,N-diethylamino-2-methylanilide (1-2) 1-(L-r-glutamyl)-4-(N- Carbamidomethyl-N-tetrahydrofurfuryl)amino-2-methyl7nilide (1-3) 1-(L-γ-glutamyl)-4-(N-ethyl-N-
hydroxymethyl)amino-2-methylanilide (1-4) 1-(L-γ-glutamyl)-4-(N-kahamidomethyl-N-ethyl)amino-2-methylfuranilide (1-5) 1-(L -r-glutamyl)-4-(N-ethyl-N-
Tetrahydrofurfuryl)amino-2-methylanilide (1-6) 1-r-glutamyl)-4-(N,N-dimethyl)amino-2-acetylaminoanilide (1-7) 1-(L-glutamyl) -4-(N-ethyl-N-p-
Methanesulfonamidoethyl)amino-2-methylanilide (1-8) 1-(L-γ-glutamyl)-4-(N-ethyl-N-
methoxyethyl)amino-2-methylanilide (1-9) 1-(L-1-glutamyl)-4-(N-ethyl-N-
(2-(2-methoxyethoxy)ethyl)amino-2
-Methylanilide 1-10) 1-(L-γ-glutamyl)-4-N,N-diethylamino-3-methylanilide (1-111 1-(L-γ-glutamyl)-4-(N-ethyl- N
-42-(2-(2-methoxyethoxy)ethoxy]ethyl)]amino-2-methylanilide (1-12) 1-(L-γ-glutamyl)74-(N,N-diethyl)amino-2- Methanesulfonamide ethylanilide The salt of the anilide derivative according to the present invention is generally water-soluble and can be easily dissolved in water or an aqueous buffer solution.
+Although it is added to the element, it is also possible to add it after dissolving it in an organic solvent if the solubility in water is small.

+ The anilide derivative and its salt in the analytical element of the invention are important in determining the measurement range (dynamic range), and are usually contained in the coating dispersion liquid iz. □mM
A concentration of ~700 mM enables good measurement, but preferably 10.0111M to 500 mM is used.

The diffusion-resistant coupler useful in the present invention may be any one that produces a dye through a coupling reaction with the aniline derivative oxide produced from the anilide derivative of the present invention, but the diffusion-resistant coupler It is a compound having a group (hereinafter referred to as ballast &) having a size and steric configuration that can be used as a ballast. phenol compound, the diffusion-resistant acylacetamide compound described in No. 55-171873, and l! ! 15
It is preferable to use the diffusion-resistant pyrazolone compound described in No. 5-171875.

Typical specific examples of the diffusion-resistant power dollar according to the present invention are shown below, but the invention is not limited thereto.

[Example coupler]

(2-1) l-Hydroxy-N-dodecyl-2-naphthamide.

(2-2”) 1-hydroxy-N-(δ-(2,4-di-t-amylphenoxy)butyl]-2-naphthamide (2-3) 1-hydroxy-4-phenoxycarbonylmethquin-
N-(δ-(2,4-di-t-amylphenoxycobutyl)-2-su7tamide(2-4) l-hydroxy-4-(ethoxycarbonylmethoxy)
-N-(δ-(2,4-di-t-amylphenoxy)butyl]-2-naphthamide (2-'5) 1-hydroxy-N,N-dioctadecyl-2-su2doamide (2-6 ) 1-(2,4,6-drichlorophenyl)-3-C3-
<2.4-di-t-amylphenoxyaidamide)
Petzamide]-5-pyrazolone (2-7) 1-(2,4,6-)lichlorophenyl)-3-(2-
Chloro-5-octadecylsuccinimide anilino)-
5-pyrazolone (2-8) 1-(2,4,6-1-lichlorophenyl)-3-(2
-Chloro-5-[α-(3-t-butyl-4-hydroxyphenoxy)nato2decane-a-do]anilino)-5-
Pyrazolone (2-9) 1-(2,4,6-)dichlorophenyl)-3-(2-
Chloro-5-octadecenylsfuciimide-anilino)
-5-pyrazolone (2-10) 1-(2,4,6-1lichlorophenyl)-3-(2-
Chloro-5-tritecanamidoanilino)-5-pyrazolone (2-11) 2.4-dichloro-3-methyl-6-[α-(2,4-
di-t-amylphenoxy)butyramidotaphenol (2-12) 2-(α,α,β,7y-tetrafluoroglobionamide)-4-J-chloroethoxy-5-[α-(2,4
-di-t-amylphenoxy)butyramidotaphenol (2-13) 2-10c2-3-meth-4-ethylcarbamoylmethoxy-6-[α-(2,4-di-t-amylphenoxy)butyl Amidotaphenol (2-14) 2-chloro-3-methyl-4-methoxycarbonylmethoxy-6-[α-(2,4-di-t-amylphenoxy)butyramidotaphenol (2-15) 2- Chloro-3-methyl-4-carboxymethoxy-
6-[α-2,4-di-t-amylphenoxy]butyramide]phenol (21ti) 2-chloro-3-methyl-4-(p-carbomethoxyphenyl carbamoylmethoxy)-6-[α-2,4 −
di-t-amylphenoxy)butylamidotaphenol (2-17) 2-chloro-3-methyl-4-triethoxycarbonylmethoxy-6-[(2,4-di-t-amylphenoxy)ethylamiderophenol (2-11 2-chloro-3-,1'thyl-4-methoxyethylaminocarbonylmethoxycarbonylmethoxy-6-[α-
(2,4-di-t-alumiphenoxy)butylamidotaphenol (2-19) 2.4-dichloro-3-methyl-6-(3-(4-dodecylbenzenesulfonamido)benzamide)phenol (2- 20) 2,4-dichloro-3-methyl-6-(ω-monohydro-octafluoropentanoylamino)phenol (2-21 2,4-dibrom-3-methyl-5-(!1-octadecanoyl amine) phenol (2-22) 2-(n-octylcarbonylamino)-4-chloro-
5-r(2,4-di-t-amicif8 fragment)ethylamidrophenol (2-23) 2-(ω-monohydro-octafluoropentanoylamino)-4-chloro-5-[α -(2゜4-di-t-amylphenoxy)butylamidotaphenol (2-24) 2-(ω-monohydro-octafluoropentanoylamino)-4-chloro-5-[α-(2゜4 -di-t-amylphenoxy)acetaminocophenol (2-25) 2-n-octafluorobutylcarbonylamino-5(
3-(3-n-pentadecanylphenoxy)propylcarbonylaminocophenol (2-26) 2-M-octafluorobutylcarbonylamino-5-
(2-n-cyclyl-2-hydroxycarbonylethylcarbonylamino)phenol (2-27> 2-(n-hephtanylcarbonylamino)-5-[α-
(2,4-di-t-amylphenoxy]butyramide]
Phenol (2-28) 2-(n-octafluorobutylcarbonylamino)-
5-4α-(2,4-di-t-amylphenoxy)hexanoylamino]phenol (2-29) 2-(n-hexafluoroglobulinylponylamino)
-5-Cα-(2,4-di-t-amylphenoxy)pentylcarbonylaminocophenol (2-30) 2-tetrafluoroethylcarbonylamino-5-[α
-(2,4-di-t-amylphenoxy]propylcarbonylamino]phenol (2-31) 2-[α-(2,4-di-t-amylphenoxy]propylcarbonylamino-5-methylphenol (2-32) ) 2-[α-(2,4-di-t-amylphenoxy)propylcarbonylaminophenol (2-33) 2-tetrafluoroethylcarbonylamino-5-methylphenol (2-34) 2-tetrafluoroethylcarbonyl Amino-5-n-
Pentatecanylphenol (2-35) 2- n-octafluorobutylcarbonylamino-5
-[(2,4-di-t-amylphenoxy)methylcarbonylaminocophenol (2-36) 2-trifluoromethyl-5-n-undecanylcarbonylamino-phenol (2-37) 2-phenyl- Di-fluoromethyl-4-10o-5
-n -+77Decanylcarponylaminophenol (2-38) 2-10low 3-methyl-4-methanesulfonyloxy-6-(m-n-dodecanyloxyphenylcarbonylamino)phenol (2-3F) 2- trifluoromethyl-4-methanesulfonyl 7)
'-6-(n-undecanylcarbonyl-N-methyl-
amino) phenol (2-40) 2-chloro-3-methyl-4-(α-phenyl-〇-carboxymethoxy)-6-(α-(2゜4-di-t-butylphenoxy)butyramide]phenol +2 -41 2-chloro-3-methyl-4-n-octadecanyloxycarbonylmethoxy-6-7enylcarbonylaminophenol (2-42) α-benzoyl-2-chloro-5-[γ-(2゜4 -di-t-amylphenoxy)butyramide]acetanilide (2-43) α-benzoyl-2-chloro-5-Cα-(dodecyloxycarbonyl)ethoxycarbonyl]acetanilide (2-44) α-[4-(1- benzyl-2-phenyl-3゜5-dioquine-1,2,4-triaziridinyl)〕-〕α-bivalyl-2-chloro-5-γ-(2゜4-di-amylphenoxy)butyramide]acetanilide (2- 45) α-(2,4-dioxo-5,5-dimethyloxazolidin-3-yl)-α-vivalyl α-chloro5-[
α-(2,4-di-t-amylphenoxy]butyramide]acetanilide (2-46) α-pivalyl α-[4-(1-methyl-2-phenyl-3,5-dioxo-1,2,4- ) riazolidinyl)
]-]2-chloro5-γ-(2゜4-di-t-a, milphenoxy]butyramide]ateanilide (2-47) l-hydroxy-4-(methoxyethylaminocarbonylmethoxy)-N-( δ-(2,4-di-t-amylphenoxy)butyl]-2-su7tamide (2-48) 1-hydroxy-4-(methoxyethylaminocarbonylmethoxy)-N-(n-hexadecanyl)-2 -su7
Toamide (2-49) 1-hydroxy-4-(hydroxycarbonylethylcarbonylamino-p-2 enoxy) -N-[δ-(2
, 4-di-t-7milkenoxy)butylcou-2-naphthamide The amount of diffusion-resistant turnip 2 in the analytical element of the present invention is coating dispersion MII! It is between 0.1 and 10.0 nM, preferably between 0.5 and 0.8 mM.

In addition, the amount of oxidizing substances used is the same as above.
Km! 1.0-20.0 mM in cloth dispersion 811, 69 especially 5.0-15.0 mM is preferred.

Diffusion-resistant couplers used in the present invention are not limited to those mentioned above, but can be selected from a wide range of
The above can also be used together.

The diffusion-resistant coupler of the present invention is generally oil-soluble, and the method for adding the same is as follows: one or more diffusion-resistant couplers of the present invention are added to a high boiling point solvent and/or Dissolved in a low boiling point solvent, mixed with an aqueous solution containing a hydrophilic colloid such as gelatin containing an anionic surfactant and/or nonionic surfactant, and emulsified and dispersed using a high-speed rotating mixer, colloid mill, ultrasonic dispersion device, etc. (hereinafter referred to as oil protection method).

Examples of depletion boiling point solvents include organic acid amides, carbamates, esters, ketones, urinary esters, and especially di-n-butyl phthalate, tricresyl phosphate,
Triphenyl phosphate, di-isooctyl acetate, di-n-butyl sebacate, tri-n-hexyl phosphate, N.

N-di-ethyl-cagrilamidoptyl, N,N-dimethylformamide, n-pentadecyl phenyl ether, sea-octyl 7-thale), n-nonylphenol, 3
-pentadecyl phenylethyl ether, 2,5-
Examples include sec-7miA/7enyl phthyl ether, monophenyl-di-op lorophenyl phosphate, or fluoroparaffin. Among these,
Dialkyl phthalates, especially those having an alkyl group having 1 to 6 carbon atoms, are preferred.

Examples of low boiling point solvents include methyl acetate, ethyl acetate,
Examples include propyl acetate, butyl acetate, butyl propionate, cyclohexanol, diethylene glycol monoacetate, nitromethane, carbon tetrachloride, chloroform, cyclohexane, tetrahydrofuran, methyl alcohol, acetonitrile, dimethylformamide, dioxane, and methyl ethyl ketone.

Examples of anionic surfactants include alkylbenzene sulfonic acid and alkyl lunaphthalene sulfonic acid.
Examples of nonionic surfactants include sorbitan sesquioleate and sorbitan monolaurate.

The analytical element according to the present invention has a component (
As a reagent that generates a dye by reacting with the target substance (detected substance),
Contains the above-mentioned diffusion-resistant coupler anilide derivative or its salt and a substance with oxidizing action. The anilide derivative produced from the anilide derivative of the present invention is oxidized by the action of this oxidizing substance. As a result, the resulting oxidant undergoes a coupling reaction with the diffusion-resistant coupler of the present invention to produce a dye.

Conventionally known substances can be used as the oxidizing substance according to the present invention, but typical examples include ammonium iodate, potassium iodate, sodium methane, Useful are sodium periodate, persulfates, iron thiocyanate, iron stannate, ferric hepterocyanate, nichrome salts adsorbed on silica (e.g. potassium chromium acetate), and the like.

Diffusion-resistant coupler of the invention, in an analytical element comprising an ayufudo derivative represented by the general formula (!) or a salt thereof and a substance with an oxidizing action, the use of a diffusion-resistant coupler in particular is effective in detecting the generated Possible dye substances are also diffusion resistant, which means that they do not migrate to each layer of the integrated analytical element, nor do they cause undesired diffusion within the same layer, thereby increasing the detection concentration. Significantly increases reliability and accuracy.

Furthermore, when the analytical element of the present invention co-contains a substance with an oxidizing effect, ordinary hydrophilic phenols and naphthols undergo coloration such as yellow due to oxidation during the storage period, resulting in poor accuracy. However,
When the diffusion-resistant coupler of the present invention is used, its storage stability is stable and it is effective in maintaining the reliability of the analytical element for a long period of time.

Furthermore, the coexistence of an oxidizing substance has the effect of reducing the effects of interfering substances in the specimen, particularly reducing substances such as ascorbic acid.

As described above, the analytical element of the present invention is found to be easy to use, accurate in measurement values, and excellent in long-term storage.

The various reagents mentioned above typically use a hydrophilic colloid as a binder, and are used by being dispersed in the colloid. Examples of the hydrophilic colloid include gelatin, acid-treated gelatin, deionized gelatin, gelatin derivatives such as acylated gelatin, polyvinyl alcohol, and the like. It is also possible to mix synthetic polymer latex into the hydrophilic colloid substance. These latexes have a total amount of
It is possible to mix about 10% to 70%.

In addition, it is also possible to use not only a reagent layer consisting of a hydrophilic colloid, but also a material having a putamen structure as described in Japanese Patent Application No. 56-155788, which is preferable for measuring proteins such as #1. It is.

The support in the analytical element of the present invention may be liquid-permeable or absorbent, or liquid-impermeable; the support may be light-transmissive or non-light-transparent. Good too. Paper is a typical example of liquid absorbent materials. A liquid-absorbing support can be made into a so-called test piece or test paper by impregnating it with a reagent.

That is, the reagent according to the present invention can be produced by, for example, dispersing the reagent in water, immersing the support in this dispersion, impregnating it with the chain reagent, and then drying it. It is operationally advantageous to use the analytical element formed in this manner, attached to a liquid-impermeable support described below as desired.

When a liquid-impermeable support is used, a reagent layer is generally provided on the support to form an analytical element. As the liquid-impermeable support, plate-shaped bodies such as metal, glass, and plastics can be used, but plastics are usually used. As for plastics,
Various 'M's such as cellulose triacetate, polyethylene terephthalate, polycarbonate, polystyrene, etc.
The material of the rod body is mentioned. The thickness of the support can be selected arbitrarily, but typically ranges from about microns to 350 microns.

The reagent layer may be a single layer containing all the reagents, or it may be divided into multiple layers and contained therein. The thickness of the reagent layer and its degree of permeability can vary widely and depend on the actual use. Dry film thicknesses of about lOμ to 100ρ have been useful. Further, as a measure of the degree of permeability, expressed in swelling degree, it is advantageous to have a degree of about 150% to 500%, particularly about 200% to 350%.

Various treatments can be applied between the support and the reagent layer to improve the adhesion of the reagent layer. For example, applying a high molecular weight undercoating polymer as an auxiliary layer is one such method.

A reflective layer, an overlayer, and a spreading layer may be provided on the reagent layer of the present invention, if necessary. The spreading layer has the function of spreading the fluid sample and uniformly distributing it in the lateral direction, and supplying a constant volume of the fluid sample per unit surface IrR to the reagent layer. The superlayer functions to filter the fluid sample and remove components that would interfere with the dye-forming reaction. The reflective layer has a function of reflecting light that has passed through the support when performing spectral nine-fold analysis of the produced dye as a balagra wand. If these three layers are provided, K
It is preferable to provide a IIK reflective layer in this order from the support side, followed by a spreading layer. Also, two of the three functions above
It is also possible to use a single layer with one function and another layer with one function. A single layer with three functions can also be used.

Spreading layers include, for example, layers of diatomaceous earth dispersed in a binder such as cellulose acetate, layers of natural or synthetic microcrystalline colloidal products, such as cellulose, and inert thin particles of uniform size. , for example, a layer containing glass beads. ' As a layer that functions as a transmissive film and a reflective layer,
Examples include a layer in which titanium dioxide or barium sulfate is dispersed in a binder such as cellulose acetate or gelatin.

In addition, layers having all of the above three functions include the non-fibrous porous media layer disclosed in U.S. Pat. Included are fibrous porous media layers. As a specific example of a non-fibrous porous media layer, plastic and unshed volima are known, and for example, a mixture of cellulose ester and a good solvent with a low boiling point of the same polymer and a poor solvent with a higher boiling point than the good solvent is appropriately combined. - It is possible to form a porous cellulose film by dissolving it in a solvent and applying it, using a phase separation method. Furthermore, instead of applying a single layer of brushed voluminous material, thin layers of microporous filtration membranes having the above three functions may be laminated. Examples of the microporous membrane include a plush polymer made from cellulose ester, such as the commercially available "Millipore" (trade name: Millipore, manufactured by Milliboa Corborecian).

The porosity of the porous medium layer is preferably about 5% or more, more preferably about % to %. Further, the film thickness can be arbitrarily selected from about I to about 500p, but preferably about 1 to 300p.

On the other hand, % Application No. 56-13203, No. 56-65446
Also advantageously used are the fibrous porous media layers described in the US patent application Ser.

The term "fibrous" herein refers to discrete fibers, filaments, or strands, and is a general term for those having a structure in which the aforementioned discrete fibers are intertwined in a three-dimensional K. Therefore, pores in a fibrous porous medium refer to voids created by three-dimensional entanglement of fibers. Examples of such materials include paper, natural or expanded non-woven fabrics, synthetic paper, synthetic fiber paper.

It is effective to mix an ionic (anionic or cationic) or nonionic surfactant into these non-fibrous or fibrous porous media layers.

The analysis probe of the present invention may be combined with ILK and other layers as desired. For example, 1 is unfavorable for quantitative phase separation reactions.
A dialysis layer for irreversibly removing substances and a migration blocking layer may be provided between the reagent layer and the spreading layer to prevent migration of the water-soluble reagent to the spreading layer. As the dialysis layer, for example, cellophane or the like can be used, and as the migration blocking layer, a high molecular weight polymer having an expanding nature colloid substance and a quaternary ammonium salt, such as poly(vinylbenzyl trialkylammonium chloride), etc. can be used. It is possible to use mixtures. These layers may be combined with the other layers described above in various ways as required.

The oxidizing substance, the diffusion-resistant coupler, and the anilide conductor represented by the general formula or its salt according to the present invention may be contained separately in each layer of the analytical element of the present invention, or may be contained in a single layer. , and may be in contact with each other in the analytical element or may be in non-contact with each other 67.

When detecting γ-GTP using the analytical element of the present invention, the analytical element is immersed in a fluid sample as a specimen, or the fluid sample is dropped onto the analytical element, and after a certain period of time, the dye produced is colored into a standard color. It is possible to measure components in a fluid sample by comparing them with a chart, a standard color scale, or by using a spectrophotometer. It's advantageous.

The fluid sample applied to the analytical element of the present invention may be a biological or non-biological fluid sample as long as it contains r-GTP, such as serum (including blood serum). First, in the case of a test piece, the amount of the rIL body sample used is arbitrary, as long as it is at least the amount that allows the fluid sample to sufficiently impregnate the absorbent carrier containing the reagent. On the other hand, Analysis JI! has a reagent layer on the support! ! ! The plain case is also optional, preferably about 50p1 to 5ρ, and further KtFftL,
＜' is about 〃! to about 5 tJ. Normal lθμm
It is preferred to apply a fluid sample of .

The analytical element according to the present invention has r-G T P or
When measuring a fluid sample containing AP, it has the advantage that a uniform colored spot can be stably provided because the intermediate hexagonal layer is hydrophobic, and there is little variation in measured values.

In addition, it is possible to obtain accurate &li St.
It is also excellent in terms of value.

Hereinafter, the present invention will be explained in more detail (Illustrative examples are shown below)
The present invention is not limited to these in any way.

Glycyl glycy 28.3 mM and exemplified compound (
1-1), (1-7), (1-8'l, (1-9).

(1-10), (1-12) 4.6 mM to 0.
058 M) Dissolve in Squirrel slowing solution (pH 8,0) and use it as a substrate solution.

Substrate 111i1. in test tube. After humidifying Od at 9r℃ for about 5 minutes, serum-1 to serum-with known activity values were added.
Add so and osQ, mix, and heat at exactly 37°C for 1 minute. 0.2N hydrochloric acid solution #'13,0- containing iK metacresol 87~ and metaperiod 1A11 sodium 321q is added to the reaction mixture, and the mixture is allowed to stand at room temperature for 10 minutes, and then its absorbance is measured. During the above procedure, the 2-ink absorbance was measured in exactly the same manner except that water was used instead of serum. The measured values thus obtained are shown in Table IK.

In addition, the r-GTp activity value of the serum used was 12 for serum-1.
, Serum-2 is Otsu, Serum-3 is 67, Serum-4 is 149,
Serum-5 is 281.

Table 1 The measured values of the present invention show a good correlation with the known activity values of serum, indicating that the compound mineral of the present invention is an essential substrate for measuring γ-GTP.

Example-2 Diffusion-resistant coupler (N cuffler (2-47)) 4.1 x 5 mol/F on a transparent poly(ethylene terephthalate) film support with a thin adhesive subbing layer
dm2, red blood salt 3.1 '17 / dml! , and SE') + 7215 'lv/dm' was coated and dried to create a reagent layer according to the present invention, and then a fiber dispersion (%M described in No. 1983-65446) was applied. 5.6 X If mol of glycylglycine, 0.85 Ii of tris(hydroxymethyl)aminomethane and the anilide derivative of the present invention [exemplified compound (1-1),
(1-7).

(1-8), (1-9), (1-10), (1-12)
] was provided to prepare analytical element samples 1 to 6 for measuring γ-GTP activity according to the present invention.

Also, for comparison, L-
Comparative sample-1 was prepared in the same manner as sample-1 except that it contained γ-glutamyl-P-(N,N-dimethylamino)anilide (compound described in JP-A-53-147034), and exemplified coupler (2) was prepared in the same manner as sample-1. Comparative sample-2 was prepared in the same manner as sample-6 except that it contained 1-naphthol-2-sulfonic acid in place of 1-naphthol-2-sulfonic acid, and in place of the example Kabcy (2-47)' in comparative sample-1. Comparative Sample-3 was prepared in the same manner as Comparative Sample-1 except that it contained phenol.

The diffusion-resistant coupler was dispersed as follows.

Exemplary coupler (2-47) was dissolved in ethyl acetate and hissibutyl phthalate, and then an aqueous solution of Alkanol XO (trade name, manufactured by Devon) and an aqueous gelatin solution were added and dispersed.

In addition, the developing layer consists of xylene, water, poly(styrene-coglycidyl-methacrylate) (weight ratio 90/lo),
Octylphenoxypolyethoxyethanol, fiber (C
It was prepared using a dispersion of each exemplified compound of K flow drip, Deyu Kokusaku) manufactured by Krug) and anilide derivatives.

Sera with known activity values used in Example-1 for these samples.
1. Add 10 drops of serum-2, blood f#-3, serum-4, and serum-5, and incubate at r°C for 7 minutes.
The reflection at the center of the colored spot was measured once. The results are shown in Table-2.

Furthermore, the above samples and comparative samples were added to serum-3 (the activity of which was determined by the γ-glutamyl-p-nitroanilide method at 67
The sample was heated at 37° C. for 10 minutes after being injected with a 10 μm drop, and the colored area was measured using a Sakura microdensitometer (manufactured by Konishiroku Photo Industry Co., Ltd.). The results are shown in Table-3.

Table-2 All measurements indicate reflection density.

Table 3 As a result, as shown in Table 2, although the comparative samples were not proportional to the activity value VC, the samples of the present invention were all directly proportional to the activity value, showing 9 color intensity, and had good quantitative properties. You can see what it shows.

Further, as shown in Table 3, the density uniformity of the colored area in the sample of the present invention was excellent, and the dye produced from the diffusion-resistant coupler was also found to exhibit excellent diffusion resistance. On the other hand, the comparative sample had a rather low color density, and the phenomenon of uneven density of the coloring part was significant, and when applied to an analytical element, it was found that the quantitative performance was significantly inferior.

Example-3 Example coupler (2-47) in Example-2 was replaced with example couplers (2-6), (2-7), (2-113, (2-
17), (2-19), (2-B), (2-42), (
Samples 3-7 to 2-44), (2-28) and (2-48) were prepared in the same manner as Sample-1 in Example-2 except
Sample 3-16 was prepared and the same operation as in Example-2 was performed.

As a result, as in Example 2, the activity value and color density were well correlated, and no concentration shift phenomenon that would seriously impair quantitative performance was observed.

Example-4 If! on opaque poly(Niger lenterephthalate) film with a thin adhesive subbing layer. fM 1978-15578
Gelatin/copoly(styrene:glycidyl methacrylate = 95:5 weight ratio [weight 12/&
4) 0.35 E/dm and p−/=Lua! /xypholyethylene oxide 0.015 f/ /
dm and example force dollar (2-
47) 2.29/rr? , red blood salt 6.9fl/♂, chrysylglycine 5.6 X IF3mole/dm
, fIl indicated gold compound-1) 8.7 X IF'
Sample 17 was prepared by providing a reagent layer consisting of mole and /d♂, and then adding a layer of the fiber dispersion liquid used in Example-2.

Samples 18 and 19 were prepared in the same manner as Sample 17 except that Exemplary Compound (1-1) in Sample 17 of Example-4 was changed to Exemplary Compounds (1-12) and (1-7).

As a result of carrying out the same operation as in Example 2 for Samples 17 to 19, it was found that there was a proportional relationship between the colored reflection density and the r-GTP activity value.

Agent Yoshi Kuwahara Beautiful procedural amendment) 18F1158'li July Ill↑, νZF I
:l' 1 & White Kazuo Wakasugi 1, ! 31 I'I Indication 11t'j Sum 57 'R Patent Application No. 51664 υ2
Name of the invention Molecular device for r-GTP measurement Relationship with J¥f'1 Patent applicant Office 1 'I' IN 26, Nishi-Shinjuku, Shinjuku-ku, Tokyo
Number 2 Name (+27) Roku Konishi Photography Co., Ltd. Representative Director Yoshihiko Kawamoto Voluntary statement 6. Column ``1lilJ of disclaimer'' and ``Detailed description of the invention'' of the specification to be amended - 7 , Contents of the amendment (1) The scope of the claims shall be amended as shown in the attached sheet.

(2) Amend the llP detailed explanation of the invention as follows・g3
page! I, line 18, page 4, recommended line 6..., sulfonamide in alcohol →..., alkanesulfonamide, page 4, line mentioned..., alkoxy group and sulfone in alcohol →...
・Alkanesulfone with an alcoholic group! Page 116, line 16, 1-r-darutaru)-4--... → 1-(L-r
-Glutamyl)-4-... 8th page II'
5111] [Line 1120 55-171872 → 57-94863m5
Summer al 1st line j15s-171873-4 57-94654g9 summer get ~ 3rd line 4! 5-17187! ! -+ Fable 1 $7-94 @
Sm iris W page armpit cetamide) veraamide]... → cetamide) penzamide]... l1131lilJ 厘...aldenphenogyushi) →...Amurufenogoushi) Eyes...-α-Pivaliru α-→ -α-Vivaliru 2
- Page 1123 II Line 3 The pigment substance is also... → The pigment substance is also 1 diffusion resistant... Page τ Series 17 Line Special 11 [1154-99580-+ 4111215
124576$28, line 16...Patent 115 B6-13! O3, *! ! @-$
5446 → 轡siopen [No. 57-1115847, 57-57-11) 741G Summer 2nd shot...For example, serum (blood →...For example, Jkg (*
Serum.

GT? Contains... 1E32 page lI 5th line Slow @g (p]1lLQ) ---4x buffer @ (
plILO)...$712 Summer Ig 17-18 lines 1 is blood, serum-2 is mu, serum-3 is nail, serum-4 is 1
49. Serum - 5 or 281 0 → 1 is 121 U/L
, serum-2 is 231Er/Le serum-3 is 671U/
L, serum-4 is 149 I'U/L, serum-5 is 2
@I IU/L 0 Calendar n page HI line g Example-1 → Reference example 12th ffth additional suction ytmt-ig is determined. ... → Measure the absorbance at each absorption maximum wavelength. ... Lines 14 to 15 of Fukui, page 5... Add the measured values obtained from this 51CL to Table 1 →... Add the measured values obtained from this 51C to the above 4-N,N-diethylamino-2-methylaniline, 4-(N-ethyl-N-/-methanesulfonamidoethyl)amino-2-methylaniline, 4-(N-ethyl-N) corresponding to the exemplified compounds, respectively. -methoxyethyl)amino-
2-Methylaniline, 4-(N-ethyl-N-[2-ethyl)ethyl]doamino-2-methylaniline, 4-N,N-diethylamino-3-methylaniline and 4-N,N- Table 1 shows the results of calculating the r-GTP activity value in comparison with a calibration sample prepared in advance according to a conventional method using diethylamino-2-/-methanesulfonamide ethylaniline.

Completion of the page... Uninvented compounds are necessary... → ... Uninvented compounds are useful... gas page gLs line Example-2 → Example-1 2nd line of page U (Patent application @ No. 56-65446,...
No. 57-197466--Page 1134, line 4! IJI Chill) Aminomethane 0.85 # # and... →
ethyl)aminomethane 0.851, tris(hydroxymethyl)aminomethane hydrochloride 0.151. Magnesium chloride 0.05 Jl and page 11L! Added row Ruamino) anilide (#Kaisho... → Ruamino)anilide 12)] Contains... → 12)) a, 7
Contains Xl0-' moles...Page 4, line...Alkanol xO... → Alkanol xC
...Page 35, line 7, the developing layer is medium silane, water, lsu... →Also, the fiber fraction liquid is medium silene, poly... Each exemplified compound was dispersed 1
A collection of pages with -1 distributed! Line 13...Used in Example-1... →...Used in the reference example... wE page 36 j Line 12 Its activity is 67 unit... → Its activity is 67
IU/L...Page *-S Exam? '! -2-1-#-21-+ 1[-1~sample-
6 11Ii left page l [4th line Example-3 → Example-2 5th line left Example-2... → Example-1...1st series, 9th line... Example-2... → Example-1...M38
Page 10 line 3-7 to sample 3-16 were created and Example-2...→
7~Create sample-16/Example-1...Nth page n
Line...Example-2... →...Example-1...
Page 1138 l Line 115 Example-4 → Example-3 #I Page 39 Line 3 How many layers 2 and... → Example-1 and... Used in Example-2 on page 7 line A layer of delicate dispersion... → 0.85 N of tris(hydroxymethyl)aminomethane, 0.5J of m-acid tris(hydroxymethyl)aminomethane and 0.0 of magnesium chloride in 42d of fiber dispersion.
Developing layer containing 54F...m39 page #l9Ffth Example-4... → Example-3...1I39 page j
l 13 fj'th・°・Example-2...→Example-1...Attachment Claims A small amount (one reagent layer and at least one
An analytical element having a spread layer, characterized in that at least one of the layers contains a substance capable of forming #IcrII, a diffusion-resistant coupler, and an anilide derivative represented by the following general formula or a salt thereof. GT? 111
Standard analytical element/general formula

Claims (1)

[Claims] At least one reagent layer and at least one reagent layer on a support.
An analytical element having a spread layer, characterized in that at least one of the layers contains an oxidizing substance, a diffusion-resistant coupler, and an anilide inducing group represented by the following general formula or a salt thereof. - Analytical element for GTP measurement. General formula [wherein R and R2 are respectively water IgJJA,
or represents an alkyl group, R1 and R7 may form an a1g group together with an i1 tree atom, and R3+ R4HRff
and R6 each represent a water atom, a halogen atom, a hydrocarbon group, an amino group, an alkoxy group, an acylamide group, an arylsulfonamide group, an alkylsulfonamide group, or an alkyl group, and 1(3,R4゜R3
and R6 are never hydrogen atoms at the same time. ]