REAGENT SHEET AND INTEGRAL MULTILAYER ANALYTICAL ELEMENT FOR MEASUREMENT OF GAMMA-GLUTAMYL TRANSFERASE ACTIVITY

Abstract: A reagent sheet for the measurement of GGT (γ-glutamyl transferase) activity comprising a porous sheet containing a cationic surfactant and a self-developable substrate upon contact with γ-glutamyl transferase. An integral multilayer analytical element for the measurement of GGT actitivity, comprising a porous spreading layer containing a cationic surfactant and a self-developable substrate in response to contact with γ-glutamyl transferase, a water-absorbing layer and a support in a laminated form.

Description: The present invention relates to a reagent sheet for use in the measurement of γ-glutamyl transferase activity in a liquid sample, particularly for use in the analysis of aqueous sample solutions, particularly in the analysis of γ-glutamyl transferase activity in body fluids in clinical test. It is very important to measure the activity of γ-glutamyl transferase in human body fluids in clinical test. As enzyme activity relating to a cancer of the liver, bile duct or the pancreas, there is frequently done the measurement of the activity of γ-glutamyl transferase (hereinafter referred to sometimes as GGT) in body fluids in the diagnosis of said cancer. As the method of measurement of GGT activity, there had been reported a GGT activity measuring method using a synthetic substrate by A. Szewczuk & M. Orlowski in 1960 [see, Clin. Chem. Acta, 5; 680 - 688, (1980)]. Since then, various synthetic compounds for the measurement of GGT activity had been synthesized, systems of conjugation reactions with color-forming reactions had been developed, and various methods are widely used at the present time [see, Japanese Patent Provisional Publication Nos. 54(1979)-133191, 53(1978)-111793, 54(1979)-132533, 56(1981)-148298, 56(1981)-148299, 56(1981)-164796, 56(1981)-23897, 56(1981)-92845, 56(1981)-158745, 56(1981)-169597, 56(1981)-30956, 53(1978)-147034, 54(1979)-28195 and 55(1980)-26870]. Most of the aforementioned methods use consecutive reactions in which pH of the reaction system is changed to form a color or there is added a color reagent which has a fear of said reagent itself or an intermediate inhibiting an enzyme reaction. Though there are only a few cases at present, there are synthesized some substrates which allows the enzyme reaction itself to be directly observed, that is, some self-developable substrates having velocity method applicability. As the self-developable substrates for the measurement of GGT activity, there are known γ-glutamyl-p-nitroanilide developed by M. Orlowski [see, Arch. Immunol. Ther. Exp., 13 , 538 (1985)] and γ-glutamyl-3-carboxy-4-nitroanilide disclosed in Japanese Patent Publication No. 54(1979)-7781. The latter is superior in solubility and used as a substrate for IFCC standard method. Although the former is poor in solubility, it has been early developed and its manufacturing cost is low, so that the substrate has been most studied, its clinical knowledge is rich, and it is widely used presently. In an analytical method using γ-glutamyl-p-nitroanilide as self-developable substrate, there is used a surfactant for dissolving said γ-glutamyl -p-nitroanilide which is difficultly soluble. However, it is known that the use of a cationic or anionic surfactant alone inhibits GGT activity. Thus, in conventional analytical methods, there is a serious problem how to dissolve γ-glutamyl-p-nitroanilide without inhibiting GGT activity. As a method for dissolving γ-glutamyl-p-nitroanilide without inhibiting GGT activity, there is disclosed in Japanese Patent Publication No. 57(1982)-24759 a method in which γ-glutamyl-p-nitroanilide is dissolved in an aqueous solution containing both a cationic surfactant and a nonionic surfactant. Further, Japanese Patent Provisional Publication No. 55(1980)-153600 discloses a freeze-dried agent which can be almost instantaneously dissolved in a bufferred test solution at 20 to 25 o C by adding a stoichiometric or excess amount of anionic surfactant to a base equal amount of γ-glutamyl-p-nitroanilide to form a protonized substrate and freeze-drying it. Conventional analytical methods including the above-described methods are those which are a so-called wet analytical method in which an analyte reacts with a reagent in a diluted aqueous solution. An analytical method which can be performed with only a small amount of a sample and can be easily operated with high accuracy, has been highly desired by medical persons such as medical doctors in clinical test. Therefore, GGT activity-measuring methods using a dry analytical method in place of said conventional wet analytical method have been developed in recent years. In the dry analytical methods, reagents required for detection reaction are preserved in a dry state and the reaction of these reagents with a small quantity of a sample solution is detected to make the measurement. Among the dry analytical methods, there have been developed analytical methods using an integral multilayer analytical element which is easy to handle and the improvements of the element are being made. The integral multilayer analytical element for the measurement of GGT activity comprises a porous spreading layer containing a substrate which is self-developable in response to contact with γ-glutamyl-p-nitroanilide, a support layer and optionally other intervening functional layers such as a water-absorbing layer prepared by laminating them for integration. The dry analytical methods using such an integral multilayer analytical element was reported by H.G. Curme, et al. [Clim. Chem., 24 , 1335 - 1350 (1978)] and B. Walter [Analytical Chemistry, (1983) 55 , 493A]. However, when the activity of γ-glutamyl transferase is measured by these dry analytical methods, there are caused other problems in addition to those associated with the conventional wet analytical methods in which γ-glutamyl-p-nitroanilide is poor in solubility and the surfactants inhibits GGT activity. Unlike the wet analytical method, the substrate and other reagents are preserved in dry state in the dry analytical method. Therefore, when the substrate preserved in a dry state is brought into contact with a small amount of water in a liquid sample, the substrate must be readily re-dissolved in order to improve the accuracy of the measurement of GGT activity and to make the measurement over a wide range. In US-A-4 050 898, the incorporation of a surfactant into a dry type analytical element is described. In WO-A-8 400 978, the analysis of GGT in a wet system utilizing a solution of gamma-glutamyl-p-nitroanilide is disclosed. However, no mention is given with respect to a dry type analytical element. EP-A-0 022 240 describes an analytical element designed specifically for bilirubin analysis utilizing diazonium salts. EP-A-0 159 638 describes a multizone analytical element for the determination of an analyt in an aqueous liquid comprising a registration zone and a porous spreading zone to receive in transport the aqueous liquid. The spreading zone contains a non-polymeric quarternary ammonium compound. The present inventors have made studies in the measurement of γ-glutamyl transferase activity by the dry analytical method and have now surprisingly found that the cationic surfactant hardly inhibits GGT activity in the dry analytical method, even when the surfactant is used at a concentration at which the surfactant inhibits GGT activity in the conventional wet analytical method. It is an object of the present invention to provide a reagent sheet for the measurement of GGT activity which is free from the problems associated with the measurement of the activity of γ-glutamyl transferase in the conventional dry analytical method. It is another object of the present invention to provide a reagent sheet for the measurement of GGT activity, which is easy to handle, can be readily operated and makes it possible to easily make the measurement of the activity of γ-glutamyl transferase in a liquid sample even by unskilled persons such as medical doctors and nurses in clinical test. Accordingly, the present invention provides a reagent sheet for the measurement of activity of gamma-glutamyl transferase comprising a porous sheet containing gamma-glutamyl-p-nitroanilide or comprising a porous spreading layer containing gamma-glutamyl-p-nitroanilide, a water absorbing layer and a support layer, characterized in that the porous sheet or porous spreading layer further contains a cationic surfactant of quarternary ammonium salt in an amount ranging from 0.2 g/m² to 5 g/m² and a hydrophilic polymer. In the reagent sheet for the measurement of the activity of γ-glutamyl transferase according to the present invention, the porous reagent sheet or the porous spreading layer (hereinafter referred to as spreading layer) contains γ-glutamyl-p-nitroanilide as a self-developable substrate as well as a cationic surfactant of quarternary ammonium salt in an amount in the range of 0.2 to 5g./m². The cationic surfactant of quarternary ammonium salt has an effect of stably dispersing the self-developable substrate in the reagent sheet or the spreading layer and uniformly containing it in an amorphous state with a high dissolving rate. Further, the cationic surfactant has an effect of improving the spreadability of a sample on the reagent sheet or the spreading layer. The range of 0.2 to 5 g./m² for the cationic surfactant content of the reagent sheet or the spreading layer is a range within which the cationic surfactant functions effectively and does substantially not inhibit GGT activity. When in the conventional wet analytical method the cationic surfactant is used in such an amount that it functions effectively, it inhibits GGT activity to such an extent that the accuracy of the measurement is seriously affected, while in the dry analytical method using the reagent sheet of the invention, said cationic surfactant functions effectively and substantially does not inhibit GGT activity without requiring any other particular means. The above unique structure of the reagent sheet or the spreading layer makes it possible that the reagent sheet for the measurement of the activity of γ-glutamyl transferase according to the invention are allowed to conduct measurement over a wide range and can be easily operated with high accuracy. Figure 1 is a graph showing the correlation between the amount of cetyltrimethylammonium bromide used in the Comparison Example and the value of the relative activity of GGT measured in the solution method. The values of the relative activity shown in Figure 1 are calculated by taking as 100 the value of the relative activity where the amount of cetyltrimethylammonium bromide is 0.25 g. The porous reagent sheet for the measurement of the activity of γ-glutamyl transferase according to the invention has preferably a metering effect (i.e., metering the spotted liquid sample). The term "reagent sheet and spreading layer capable of metering a liquid sample" herein used refers to a layer having a function capable of spreading an applied liquid in such a manner that the spread area of the liquid is approximately in proportion to the amount of the liquid when the liquid is applied thereon. When the layer is spreading layer, it also has a function capable of supplying an uniform quantity of the liquid per area to a water-absorbing layer described hereinafter. Further, the reagent sheet and the spreading layer of the present invention preferably have spaces which allow γ-glutamyl transferase as an analyte to pass therethrough. Therefore, it is preferred to use filter paper, woven fabric, nonwoven fabric, knitted fabric, glass fiber filter, membrane filter or a three-dimensional lattice structure composed of a polymer microbead as a material constituting a matrix of the reagent sheet or the spreading layer of the analytical element of the present invention. Examples of the woven fabrics (woven cloth) which can be used for the reagent sheet or the spreading layer include those disclosed in Japanese Patent Provisional Publications No. 55(1980)-164356 and No. 57(1982)-66359. Among the woven fabrics, plain weave fabrics made of warp and weft are preferred. Among plain woven fabrics, thin cloth, muslin, broadcloth and poplin are preferred. Examples of yarns for woven cloth include those composed of the same materials as those constituting knitted cloths as described in more detail hereinafter. Any of filament yarn and spun yarn (twist yarn) can be used, and the spun yarn is preferred. The yarn diameter of the woven fabric is generally in the range of about 20S to about 150S, preferably about 40S to about 120S in terms of cotton spinning yarn count or in the range of about 35 to about 300D, preferably about 45 to about 130D in terms of silk thread denier. The thickness of the woven fabric is generally in the range of about 100 to about 500 µm, preferably about 120 to 350 µm. The voids of the woven fabric are generally in the range of about 40 to about 90%, preferably about 50 to about 85%. Examples of the knitted fabrics which can be used for the reagent sheet or the spreading layer include many kinds of knitted fabrics, among which warp knitted fabric and weft knitted fabric are preferred. Examples of the warp knitted fabrics include single atlas knitted cloth, tricot knitted cloth, double tricot knitted cloth, milanese knitted cloth and rashar knitted cloth. Examples of the weft knitted fabrics include plain weave knitted cloth, pearl knitted cloth, rib stitch cloth, and double face knitted cloth. Examples of the yarns for knitted fabrics include yarns of natural fibers such as cotton, silk and wool; yarns composed of fine fibers or single fibers of regenerated cellulose (e.g. viscose rayon and cupra), semi-synthetic organic polymer (e.g. cellulose diacetate and cellulose triacetate), synthetic organic polymer (e.g., polyamide such as nylon, acetalated polyvinyl alcohol such as vinylon, polyacrylonitrile, polyethylene terephthalate, polyethylene, polypropylene and polyurethane), and yarns composed of fiber blends of a natural fiber and a regenerated cellulose or a semi-synthetic or synthetic organic polymer fiber. Any of filament yarn and spun yarn can be used, and spun yarn is preferred. The diameter of the yarn for knitted fabric is generally in the range of from about 40 to 150S, preferably about 60 to about 120S in terms of cotton spinning yarn count, or in the range of about 35 to about 130D, preferably about 45 to about 90D in terms of silk thread denier. The number of knitting gauge of the knitted fabric is generally in the range of about 20 to about 50. The thickness of the knitted fabric is generally in the range of about 100 to about 600 µm, preferably about 150 to about 400 µm. The voids of the knitted fabric are generally in the range of about 40 to about 90%, preferably about 50 to about 85%. The warp knitted fabric, tricot knitted cloth, rashar knitted cloth, milanese knitted cloth and double tricot knitted cloth are preferred, because shrinkage in the wale's direction is small, the operation in the lamination stage of knitted goods is easy and the stitches are not easily loosened during cutting. Woven fabric or knitted fabric is preferably a fabric from which fat is substantially removed when the yarn or the fabric is prepared. The fabrics are more preferably processed to be hydrophilic to enhance the adhesion to an underlying layer, when the fabric is used as the spreading layer. Examples of such process to make the fabric hydrophilic include physical activating process (preferably glow discharge process or corona discharge process) disclosed in Japanese Patent Provisional Publication No. 57(1982)-66359 and hydrophilic polymer permeating process disclosed in Japanese Patent Provisional Publication Nos. 55(1980)-164356 and 57(1982)-66359. The spreading layer constituted of woven fabric or knitted fabric can be laminated on a water-absorbing layer or adhesive layer according to the process disclosed in Japanese Patent Provisional Publications No. 55(1980)-164356 and No. 57(1982)-66359. The process is that woven fabric or knitted fabric is laminated under the substantially uniform light pressure on the wet or swelling buffer layer or adhesive layer which has been still wet condition after coating or has been supplied with water (or water containing small amount of detergent) after drying. The reagent sheet or the spreading layer constituted of a brush polymer or a membrane filter can be provided according to the process disclosed in Japanese Patent Publication No. 53(1978)-21677, the reagent sheet or the spreading layer having a three-dimensional lattice structure constituted of polymer microbead can be provided according to the method disclosed in Japanese Patent Provisional Publication No. 55(1980)-90859, and the reagent sheet or the spreading layer constituted of filter paper or nonwoven fabric can be provided according to the process disclosed in Japanese Patent Provisional Publication No. 57(1982)-148250. When a water-absorbing layer or an adhesive layer described hereinafter is made of gelatin or gelatin derivatives, the spreading layer made of woven fabric or knitted fabric is preferably laminated on the wet or swelling gelatin (or its derivatives) of water-absorbing layer or adhesive layer which has been still wet condition after coating. The reagent sheet of the present invention contains a self-developable substrate. Self-developable substrate used herein refers to a substance which is γ-glutamyl-p-nitroanilide. The reagent sheet for the measurement of the activity of γ-glutamyl transferase contains a cationic surfactant, which is a quaternary ammonium salt type cationic surfactant. Examples of such quaternary ammonium salt type cationic surfactants include those having the following formula: wherein n is 1 or 2; R¹ is an alkyl group having 12 or more carbon atoms; R², R³ and R⁴ are the same or different from each other, and each is methyl, ethyl or benzyl group, or R², R³ and R⁴ may form a piperidinium group together with the neighboring nitrogen atom; R⁵ is a single bond or a group of -CONHR⁶- (wherein R⁶ is methylene or ethylene group); and X is halogen ion, sulfate ion or methanesulfonate ion. Examples of the surfactants having the above formula where R⁵ is a single bond, include lauryltrimethylammonium chloride, stearyltrimethylammonium bromide, cetyltrimethylammonium chloride, lauryltrimethylammonium sulfate, cetyldimethylbenzylammonium bromide, lauryldimethylbenzylammonium chloride, myristyldimethylbenzylammonium chloride, cetylpyridinium bromide and laurylpyridinium chloride. Other examples of the surfactants having the above formula where R⁵ is -CONHR⁶- include stearamidomethylpyridinium chloride and those which are commercially available under the trade name of Sapamine MS, Sapamine BCH and Caternack SN. Said cationic surfactant in the range of 0.2 to 5 g./m², preferably about 0.5 to about 3 g./m² is incorporated in the reagent sheet or the spreading layer of the present invention. The reagent sheet and the spreading layer further contain a hydrophilic polymer. If desired, the reagent sheet and the spreading layer of the present invention may contain a nonionic surfactant, reagents such as enzyme activator and buffering agent, fine light-blocking particles, etc. in addition to said self-developable substrate and said cationic surfactant. Any of nonionic surfactants may be used in the present invention without particular limitation. However, nonionic surfactants having HLB value [Hydrophile-Lipophile-Balance; according to the definition of J. Soc. Cosmet. Chem., 1,311 (1949) or "Kagaku" 23,546 (1953)] of 10 or more are preferred. The nonionic surfactant having the HLB value may be selected from ethylene oxide adducts (i.e., condensation products) of polyhydric alcohol ester, polyethylene glycol monoester, polyethylene glycol diester, ethylene oxide adducts (condensation products) of higher alcohol, ethylene oxide adducts (condensation products) of alkylphenol, and alkanol amides of higher fatty acid. A combination of two or more surfactants may be used as the nonionic surfactant. HLB value of the surfactant can be adjusted by using said combination of the surfactants (including surfactants having HLB value of less than 10). Examples of the nonionic surfactant having HLB value of 10 or more and their HLB values are described in the following Table 1. Nonionic Surfactant HLB value POE (20) sorbitan monooleate 15.0 POE (10) sorbitan monooleate 13.5 POE (4) sorbitan tristearate 10.5 POE (4) trioleate 11.0 POE (30) stearate 16.0 POE (40) stearate 16.9 POE (100) stearate 18.8 PEG (400) monostearate 11.6 PEG (400) monolaurate 13.1 PEG (1000) dilaurate 14.1 PEG (1540) distearate 14.8 Condensation product of lauryl alcohol and 6 mol. of EO 11.8 Condensation product of lauryl alcohol and 10 mol. of EO 14.1 Condensation product of lauryl alcohol and 30 mol. of EO 17.4 Condensation product of lauryl alcohol and 20 mol. of EO 15.3 Condensation product of cetyl alcoholand 20 mol. of EO 15.7 Nonionic Surfactant HLB value POE (9 - 10) octyl phenyl ether 13.5 POE (15) octyl phenyl ether 15.1 POE (30) octyl phenyl ether 17.4 POE (40) octyl phenyl ether 18.7 POE (10) nonyl phenyl ether 11.7 POE (12) nonyl phenyl ether 14.1 POE (20) nonyl phenyl ether 16.0 POE (50) nonyl phenyl ether 19.5 Triethanolamine oleate 12.0 In the Tabel 1, POE is polyethylene oxide, PEG is polyethylene glycol, EO is ethylene oxide, and the numbers in parentheses are condensation numbers of ethylene oxide units. Said nonionic surfactant preferably in an amount in the range of 0.1 to 3 g./m², more preferably 0.2 to 2 g./m² is incorporated in the reagent sheet or the spreading layer according to the present invention. Said nonionic surfactant (particularly surfactant having HLB value of 10 or more) has an effect of improving the spreadability of a sample on the reagent sheet or the spreading layer. Examples of the hydrophilic polymers which are incorporated in the reagent sheet or the spreading layer according to the invention include starch, cellulose, agarose, gelatin and their derivatives (e.g., hydroxymethylated and hydroxypropylated derivatives), acrylamide polymer, copolymers of acrylamide with other vinyl monomer, vinylpyrrolidone polymer, copolymers of vinylpyrrolidone with other vinyl monomer, acrylate polymer, and copolymers of an acrylate with other vinyl monomer. Among them, acrylamide polymer and cellulose derivatives are preferred. Particularly preferred examples of the cellulose derivatives are cellulose ethers where part or the whole of hydroxyl groups is etherified with an alkyl group having 1 to 3 carbon atoms or a hydroxyl group-substituted lower alkyl group having 1 to 4 carbon atoms. Examples of such cellulose ethers include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and hydroxybutylmethylcellulose. The hydrophilic polymer in an amount in the range of 0.5 to 15 g./m², preferably 2 to 10 g./m² is incorporated in the reagent sheet or the spreading layer according to the present invention. Said hydrophilic polymer has an effect of preserving small amount of water in the spotted sample to accelerate the catalytic reaction of analyte enzyme. The enzyme activators which can be incorporated in the reagent sheet or the spreading layer according to the present invention are those serving as an acceptor for γ-glutamyl groups to be transferred from the substrate by the action of γ-glutamyl transferase. Examples of such γ-glutamyl group acceptors include various amino acids and oligopeptides. However, when a sufficient amount of such component serving as an acceptor is contained in a sample solution, it is not necessary to incorporate such an acceptor in the reagent sheet or the spreading layer. Such γ-glutamyl group acceptors are described in literatures such as Clinical Chemistry, Vol. 24, No. 6 (1978) 905 - 915 by L.M. Shaw. Preferred examples of the γ-glutamyl group acceptors include D-methionine, L-methionine, L-glutamine, L-leucine, L-alanine, L-lysine, L-cysteine, glycylglycine and glycylglycylglycine, among which glycylglycine is particularly preferred. Examples of light-blocking and light-reflecting particles which can be incorporated in the reagent layer or the spreading layer of the analytical element of the present invention include titanium dioxide fine particles (rutile-type, anatase-type or brookite-type; mean size ranging from approx. 0.1 µm to approx. 1.2 µm), barium sulfate fine particles, aluminum fine particles and fine flakes thereof. Examples of light-blocking particles include carbon black, gas black and carbon microbeads. Titanium dioxide fine particles and barium sulfate fine particles are preferred and anatase-type titanium dioxide fine particle is most preferred. The self-developable substrate, the cationic surfactant, the nonionic surfactant, the hydrophilic polymer, the reagents such as enzyme activator and the buffering agent and the fine light-blocking particle can be incorporated in the reagent sheet or the spreading layer by spraying a coating solution containing said ingredients over the reagent sheet or the spreading layer and drying it. When the reagent sheet or the spraying layer is composed of woven fabric, knitted fabric, filter paper, non-woven fabric or glass fiber filter paper, the reagent sheet or the spreading layer is immersed in a solution containing said ingredients and dried. When the spreading layer is composed of materials to be laminated, the materials are immersed in said solution and are laminated in a dry or semi-dry state on each other for integration. When the spreading layer is to be formed by coating, for instance, the spreading layer is composed of a brush polymer layer or a three-dimensional lattice microbead structure, a mixture of a coating solution for the spreading layer and a coating solution containing said ingredients may be coated. These ingredients can be incorporated in the reagent sheet or the spreading layer by using different methods for every ingredient, or by applying separately every ingredient. However, it is desirable that a solution containing both the self-developable substrate (γ-glutamyl-p-nitroanilide) and the cationic surfactant is prepared, so that the cationic surfactant sufficiently reacts with the substrate. The material of the support of the reagent sheet of the invention is preferably a light-transmissive, liquid-impermeable support. Examples of the liquid-impermeable, light-transmissive supports include substantially water-impermeable transparent supports in the form of a film or sheet made of a polymer such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene and cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate etc.). The thickness of the support generally ranges from 50 µm to 1 mm, preferably from 80 µm to 300 µm. The reagent sheet for the measurement of the activity of γ-glutamyl transferase according to the present invention can be laminated onto the support to integrate them. The support to be laminated onto the reagent sheet is preferably a support which does not transmit light or contains a light-reflecting colorant. Such a light-non-transmitting support or a light reflecting colorant-containing support can be easily prepared by incorporating a light-reflecting colorant in said polymer, by coating the surface of a transparent support composed of said polymer with a light-reflecting colorant, or by sticking a pressure-sensitive adhesive tape containing a light-reflecting colorant on a support. There may be provided an undercoating layer on the support to enhance the adhesion between the support and a water-absorbing layer or the reagent sheet. Instead of providing the undercoating layer, the surface of the support may be activated by a physical or chemical process to enhance the adhesion. There is provided a water-absorbing layer on the support (optionally, intervened by other layers such as undercoating layer). The water-absorbing layer is a layer comprising a hydrophilic binder which is preferably a hydrophilic polymer which absorbs water to swell. The hydrophilic polymer preferably shows swelling ratio ranging from 150 % to 2,000 %, more preferably from 250 % to 1,500 %. at 30 o C. Examples of the hydrophilic polymer satisfying the above-described conditions include gelatin (e.g., acid-processed gelatin, deionized gelatin etc.), gelatin derivatives (e.g., phthalated gelatin, hydroxyacrylate grafted gelatin etc.), agarose, pullulan, pullulan derivatives, dextran, polyacrylamide, polyvinyl alcohol, and polyvinyl pyrrolidone. The dry thickness of the water-absorbing layer is in the range of 1 to 100 µm, preferably 3 to 30 µm. Preferably, the water-absorbing layer should be substantially transparent. If desired, a buffering agent may be incorporated in the water-absorbing layer. Examples of such buffering agents include conventional buffering agents such as carbonates, borates, phosphates and good's buffering agents. These buffering agents can be chosen by referring to literatures such as "Fundamental Experimental Method of Protein·Enzyme" by T. Horio et al. (Nankodo, 1981) written in Japanese. There may be provided a light-blocking layer on the water-absorbing layer. The light-blocking layer is a water-permeable layer in which light-blocking (or light-reflecting) fine particles are dispersed in small amount of a film-forming hydrophilic polymer binder. The light-blocking layer may function as light-reflecting layer or background layer as well as blocker to the color of an aqueous liquid spotted on the spreading layer, such as the red of hemoglobin in a whole blood sample, when a detectable change (a color change or a color development etc.) in the water-absorbing layer is measured from the side of the transparent support reflection photometry. Examples of light-blocking and light-reflecting particle include fine titanium dioxide particles (rutile-type, anatase-type or brookite-type; mean size ranging from 0.1 µm to 1.2 µm), fine barium sulfate particles, fine aluminum particles and their flakes. Examples of light-blocking particle include carbon black, gas black and carbon microbeads. Fine titanium dioxide particles and fine barium sulfate particles are preferred, and anatase type titanium dioxide particles are most preferred. Examples of the film-forming hydrophilic polymer binder include a weakly hydrophilic polymer such as regenerated cellulose and cellulose acetate as well as the hydrophilic polymer employable in the reaction layer. Most preferred are gelatin, gelatin derivatives and polyacrylamide. Gelatin and gelatin derivatives may be used as a mixture with a known hardening agent (cross-linking agent). The light-blocking layer can be provided in such a manner that an aqueous dispersion containing the light-blocking particle and the hydrophilic polymer is coated on the reaction layer and then dried by any of the conventional methods. Instead of the light-blocking layer, the light-blocking particles may be incorporated into the spreading layer. There may be provided an adhesive layer on the water-absorbing layer or optionally added other layer (e.g., light-blocking layer) to enhance the adhesion of the spreading layer. The adhesive layer is preferably constituted by a hydrophilic polymer which can bond the spreading layer to other layer to make all of the layers integrated while the polymer is wetted or swelled with water. Examples of the hydrophilic polymer include the polymers employable in the water-absorbing layer. Most preferred are gelatin, gelatin derivatives and polyacrylamide. The dry thickness of the adhesive layer generally ranges from 0.5 µm to 20 µm, preferably from 1 µm to 10 µm. The adhesive layer may be provided on other layers as well as the water-absorbing layer. The adhesive layer can be prepared in such a manner that a solution of a hydrophilic polymer and optionally added other agent such as a surfactant is coated on the water-absorbing layer or other layer. The reagent sheet of the present invention may optionally contain other functional layers in addition to the above-described essential layers. It is preferred from the viewpoint of manufacture, packaging, transportation, preservation and measuring operation that the reagent sheet of the present invention is cut into pieces of about 15 to 30 mm square or a circle of about 15 to 30 mm in diameter and put in a slide frame to provide an analytical slide as disclosed in Japanese Patent Provisional Publication Nos. 57(1982)-63452 and 54(1979)-156079, Japanese Utility Model Provisional Publication Nos. 56(1981)-142454 and 58(1983)-32350 and Japanese Patent Provisional Publication No. 58(1983)-501144. 5 to 30 µℓ, preferably 8 to 15 µℓ of an aqueous liquid sample is deposited (spotted) on the reagent sheet or the porous spreading layer of the integral multilayer analytical element of the invention, and if necessary, the reagent sheet or the analytical element is incubated at a substantially constant temperature of 20 to 45 o C. A detectable change such as color change or color formation in the sheet or the element is measured (from the side of the light-transmissive support when the analytical element is used) by reflection photometry to thereby analyze a substance to be measured in the liquid sample according to colormetry. The following examples and comparison example are provided to illustrate the present invention without limiting it thereto. Example 1 The surface of a transparent polyethylene terephthalate support of 180 µm in thickness was treated to make it hydrophilic. The hydrophilic surface of the support was coated with the coating solution having the following composition and dried to form a water-absorbing layer of 15 µm in dry film thickness. Coating Solution for Water-absorbing Layer Alkali-treated deionized gelatin 10 g. Octylphenoxy polyethoxyethanol 0.5 g. Water 100 mℓ 1,2-Bis(vinylsulfonylacetamido)ethane 0.15 g. The surface of said water-absorbing layer was coated with the coating solution having the following composition and dried to form an adhesive layer of 3 µm in dry film thickness. Coating Solution for Adhesive Layer Gelatin 12 g. Water 290 g. Nonylphenoxy polyglycidol 1.3 g. The surface of said adhesive layer was wetted with a 0.4 % aqueous solution of nonylphenoxy polyglycidol. A tricot fabric composed of a spun yarn of polyethylene terephthalate (36 gauge, 50 denier, about 230 µm thick) was laminated onto said wetted surface under pressure to form a spreading layer. The following substrate coating solution was applied to the spreading layer in a coating amount of 120 mℓ/m² and dried. Preparation of Substrate Coating Solution A solution of 2.28 g. of γ-glutamyl-p-nitroanilide dissolved in a solution of 1 mℓ of 2 N hydrochloric acid and 1 mℓ of ethanol was added to the following uniform solution, and pH of the resulting solution was adjusted to 8.3 by the addition of 1.3 mℓ of 6 N hydrochloric acid to prepare the substrate coating solution. Uniform Solution Tris(hydroxyethyl)aminomethane 3.03 g. Glycylglycine 0.651 g. Cetyltrimethylammonium bromide 0.500 g. Water 20.0 g. Polyacrylamide (10 wt.% aqueous solution) (viscosity at 25 o C: 2500 cps.) 25 g. The surface of the spreading layer was coated with the following titanium dioxide coating solution in a coating amount of 112 mℓ/m² and dried. Titanium Dioxide Coating Solution Anatase type titanium dioxide 2.5 g. 0.5 % hydroxylethyl cellulose 50 mℓ In this way, a reagent sheet (the cationic surfactant [cetyltrimethylammonium bromide] content of the spreading layer: about 1 g./m²) for the measurement of GGT activity according to the present invention was prepared. Example 2 The procedure of Example 1 was repeated except that the amount of cetyltrimethylammonium bromide used in the uniform solution was 0.250 g. in place of 0.50 g. to prepare an analytical element (cationic surfactant [cetyltrimethylammonium bromide] content of the spreading layer: about 0.5 g./m²) for the measurement of GGT activity according to the present invention. Example 3 The procedure of Example 1 was repeated except that the amount of cetyltrimethylammonium bromide used in the uniform solution was 1.0 g. in place of 0.50 g. to prepare an analytical element (cationic surfactant [cetyltrimethylammonium bromide] content of the spreading layer: about 2 g./m²) for the measurement of GGT activity according to the present invention. Each of the reagent sheets obtained by Examples 1 to 3 was cut into a piece 15 mm square and put in a plastic mount (disclosed in Japanese Patent Provisional Publication No. 57(1982)-63452) to prepare a slide for the analysis of GGT. GGT of bovine kidney, which had a GGT activity of 1900 U/L from the measurement with RA 1000 (a wet-process analyzer) manufactured by Technicon Co. Ltd., was dissolved in commercial available control serum, and the resulting solution was added to each of the above slides. The resulting control solution was used as a specimen. 10 µℓ of the control solution was applied (dropped) on each slide, the hole of the deposited side was closed by a pressure-sensitive adhesive tape (Mylar) to prevent water from being evaporated, each slide was heated on a 37 o C constant heated block, and the reflection density of each slide on the transparent support was measured with a light of 400 nm after 2 and 5 minutes, respectively. The results are shown in Table 2. Analytical Slide OD (5 min. - 2 min.) Example 1 0.223 Example 2 0.230 Example 3 0.222 It is apparent from the above results that there are no much fluctuations in GGT activity over a cetyltrimethylammonium bromide content range of 0.5 to 2 g./m². Comparison Example The procedure of Example 1 was repeated except that polyacrylamide was omitted from the uniform solution and the amount of cetyltrimethylammonium bromide was 0.25 to 2.0 g. to prepare a uniform solution. In a similar manner to that described in Example 1, γ-glutamyl-p-nitroanilide was added to each of the uniform solutions to prepare a substrate solution. 30 µℓ of each substrate solution was put in a cuvette, 10 µℓ of the same control serum as that of Example 3 was added thereto, the cuvette was kept at 37 o C and the transmitted optical density thereof was measured with a light having a central wavelength of 410 nm. The relative activity value of GGT was determined from the resulting optical density. The results are shown in Figure 1. Figure 1 is a graph showing the correlation between the amount of cetyltrimethylammonium bromide used in the Comparison Example and the value of the relative activity of GGT measured in the solution method. The values of the relative activity shown in Figure 1 are calculated by taking as 100 the value of a relative activity where the amount of cetyltrimethylammonium bromide is 0.25 g. It is apparent from Figure 1 that when the cetyltrimethylammonium bromide content is 0.5 g., the ratio of inhibition in the activity of GGT is 30 %; when the content is 0.75 g., the ratio is 10 %; and when the content is 1.0 g. or higher, cetyltrimethylammonium bromide inhibits the activity to such an extent that the measurement is practically impossible. Therefore it is also apparent that even when cetyltrimethylammonium bromide is used in an amount which does almost not inhibit the activity in the dry analytical method of the present invention, such an amount is to cause a serious inhibition to the measurement in the solution method. Example 4 The analytical element was cut into pieces of 15 mm square and each was put into a plastic mount (disclosed in Japanese Patent Provisional Publication No. 57(1982)-63452) to prepare an analytical slide for the analysis of GGT. While heating said slide on a 37 o C constant heated block, 10 µℓ of each of commercially available control serums containing bovine kidney GGT having GGT activity of 10, 150, 290, 600 and 1150 U/L was deposited (dropped) on the slide, and the color density thereof was measured with a light of 410 nm after 2 and 5 minutes, while preventing water from being evaporated. The values of the color density were those calculated from a color density to p-nitroaniline conversion formula which was prepared by using an aqueous p-nitroaniline solution having a known concentration. The measured and calculated results are shown in Table 3. The values of Table 3 are calculated from the following formula: Y = 1.5692 x 10⁻³X + 0.16853 wherein X is color density and Y is the concentration of p-nitroaniline. GGT activity (U/L) Amount of p-nitroaniline formed 10 0.090 mM 150 0.405 mM 290 0.700 mM 600 1.184 mM 1150 1.916 mM The coefficient γ of correlation is 0.99425 from the results of Table 3. It is apparent from Table 3 that the slides having the analytical element of the invention produce p-nitroaniline in well correlation to GGT activity and hence, the analytical element of the present invention is effective in measuring GGT activity. Example 5 The procedure of Example 1 was repeated except that an equal amount of each of cationic surfactants given in Table 4 was used in place of cetyltrimethylammonium bromide used in the uniform solution to prepare an analytical element. Slides for the analysis of GGT were prepared therefrom, and it was found that any of the slides was effective in the measurement of GGT activity as in Example 4. No. Cationic Surfactant 5-1 cetyltrimethylammonium chloride 5-2 cetylpyridinium bromide 5-3 cetylpyridinium chloride 5-4 tetradecyldimethylbenzylammonium chloride 5-5 octadecyltrimethylammonium bromide Example 6 A glass fiber filter of 200 µm in thickness was impregnated with the substrate coating solution of Example 1 in a coating amount of 100 mℓ/m², and cut into a 1 cm² sheet which was then fixed to a flat plastic plate containing a white pigment incorporated therein to obtain a reagent sheet for the measurement of GGT activity. 10 µℓ of serum was deposited on said reagent sheet. After 10 minutes, the reflection density was measured with Macbeth reflection densitometer and there was detected an yellow color density in proportion to the GGT activity of the deposited solution. Example 7 The procedure of Example 1 was repeated except that the following titanium dioxide coating solution was used in place of that used in Example 1 to prepare a reagent sheet (the spreading layer containing both cationic surfactant and nonionic surfactant) for the measurement of GGT activity according to the present invention. Titanium Dioxide Coating Solution Anatase type titanium dioxide 2.5 g. 0.5 % hydroxylethyl cellulose 50 mℓ POE (50) nonyl phenyl ether (HLB value; 19.5) 2.5 g. Example 8 The procedure of Example 7 was repeated except that 50 mℓ of 0.5 % hydroxylmethyl cellulose was used in place of 50 mℓ of 0.5 % hydroxylethyl cellulose used in the titanium dioxide coating solution and each of nonionic surfactants given in Table 5 was used in place of POE(50) nonyl phenyl ether used in the titanium dioxide coating solution to prepare a reagent sheet. No. Nonionic Surfactant (HLB value) Content 8-1 POE (40) octyl phenyl ether (18.7) 2.5 g. 8-2 POE (50) nonyl phenyl ether (19.5) 2.0 g. 8-3 POE (9 - 10) octyl phenyl ether (13.5) 0.5 g. 8-4 POE (9 - 10) octyl phenyl ether (13.5) 3.0 g. 8-5 POE (40) octyl phenyl ether (18.7) 1.0 g. 8-6 POE (20) sorbitan monooleate (15.0) 3.0 g. Each of the analytical elements obtained by Examples 7 and 8 was cut into a piece 15 mm square and put in a plastic mount (disclosed in Japanese Patent Provisional Publication No. 57(1982)-63452) to prepare a slide for the analysis of GGT. 10 µℓ of each of control solution having protein content of 4 % and 12% and GGT activity of 580 U/L was deposited (dropped) on each slide, and the color density thereof was measured after 2 and 5 minutes. The results are shown in Table 6. In Table 6, OD is the difference of values measured after 2 and 5 minutes. Analytical Slide OD (12 %) / OD (4 %) Example 7 95 % Example 8-1 97 % Example 8-2 96 % Example 8-3 92 % Example 8-4 98 % Example 8-5 95 % Example 8-6 97 % It is apparent from the above results that analytical elements using nonionic surfactant in addition to cationic surfactant have the improved spreadability of the spreading layer and that values measured by such elements are almost not interferred by protein content in sample solution.

Claims: A reagent sheet for the measurement of activity of gamma-glutamyl transferase comprising a porous sheet containing gamma-glutamyl-p-nitroanilide or comprising a porous spreading layer containing gamma-glutamyl-p-nitroanilide, a water absorbing layer and a support layer, characterized in that the porous sheet or porous spreading layer further contains a cationic surfactant of quarternary ammonium salt in an amount ranging from 0.2 g/m² to 5 g/m² and a hydrophilic polymer. The reagent sheet as claimed in claim 1, wherein said cationic surfactant is a compound having the formula: wherein n is 1 or 2; R¹ is an alkyl group having 12 or more carbon atoms; R², R³ and R⁴ are the same or different from each other, and each is methyl, ethyl or benzyl group, or R², R³ and R⁴ may form a piperidinium group together with the neighboring nitrogen atom; R⁵ is a single bond or a group of -CONHR⁶- (wherein R⁶ is methylene or ethylene group); and X is halogen ion, sulfate ion or methanesulfonate ion. The reagent sheet of claim 1, wherein the porous sheet or porous spreading layer further contains a nonionic surfactant. The reagent sheet of claim 1, wherein the porous sheet or porous spreading layer further contains a cellulose derivative.

Inventors: KAGEYAMA, SHIGEKI, KATSUYAMA, HARUMI
Applicants: FUJI PHOTO FILM CO., LTD.
Application year: 1986
Main classification: C12Q1
IPC codes: G01N33, C12Q1