JPH0413661B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to analytical chemistry, and more particularly to analytical elements for analyzing proteins in fluid samples. In recent years, in the field of analytical chemistry, dry multilayer analytical elements have been developed that have significantly improved quantitative performance and operability compared to methods in which analytical reactions are carried out in a solution system. For example, as described in US Pat. No. 3,992,158, a porous spreading layer containing all the reagents necessary for analysis is laminated on a reagent layer of a hydrophilic colloid material. However, it is intended to be used in the field of clinical chemistry, and it is disclosed that the PH conditions are relatively mild, neutral, weakly alkaline, and weakly acidic. However, in the field of analytical chemistry, various useful reactions are known that allow analytical reactions to be carried out under severe conditions, such as under strong alkalinity. For example, in JP-A-54-101398, high PH
Elements for liquid analysis under conditions are proposed. The above patent states that a stable layer is formed by a combination of an alkali substantially free of sodium ions and an alkali-protecting polymer. However, the alkali-protecting polymers used in these elements cannot sufficiently protect alkalis, and when stored for a long period of time, they deteriorate due to moisture in the air, carbon dioxide, etc., resulting in a decrease in analytical sensitivity. have. In addition, the analytical elements disclosed in the above patent include:
It also has the serious disadvantage of low color density, which significantly impairs quantitative performance. An object of the present invention is to eliminate the above-mentioned drawbacks and provide an element capable of analysis under strong alkalinity. As a result of extensive research in line with the above objectives, the present inventors have discovered a liquid-impermeable, light-transparent support and at least one reagent that reacts with components in a fluid sample located on one side of the support. and at least one porous spreading layer located on the opposite side of the reagent layer from the support, wherein at least one of the reagent layer and the porous spreading layer is provided. Either one of the layers contains an alkaline mixture of at least two or more selected from a water-soluble cupric salt, a copper chelating agent, and an alkali metal hydroxide or an alkali metal or alkaline earth metal salt. We were able to achieve the above objectives using a multilayer analytical element for protein detection. In an embodiment of the present invention, it is preferable to use copper sulfate as the water-soluble cupric salt and tartaric acid or a salt thereof as the chelating agent. Hereinafter, the analytical element of the present invention will be explained in more detail. The reagent layer according to the present invention is formed by coating a support with at least one layer of a binder containing at least one reagent that reacts with components in a fluid sample. It is also possible to form a layer having interconnecting voids by coating powdered filter paper carrying the binder on its surface, or by coating fibers that have been beaten to a uniform length, or by coating microscopic polymer beads. The porous spreading layer according to the present invention is a protein macromolecule formed by coating at least one layer of fibrous or polymeric microbeads similar to those used in the reagent layer carrying a binder on the reagent layer. It is a layer with interconnecting voids through which the pores flow. The cupric salt, copper chelating agent, and alkaline mixture according to the present invention may be contained alone or in a mixture in the reagent layer and the porous development layer. The other reagent layer according to the invention, which does not contain an alkaline mixture, is preferably composed of a hydrophilic colloid material. Depending on the characteristics of the reagent used, the reagent can be contained in the reagent layer by a dispersion method such as an oil protect dispersion method or a direct dispersion method, or by dissolution, which are known in the photographic industry. The cupric salt according to the present invention includes water-soluble organic salts such as copper chloride, copper bromide, copper acetate, copper sulfate, copper sulfate, etc.
Inorganic cupric salts may be used, but copper sulfate is preferred since it retains a purity that ensures reliability of quantitative analysis. Further, as the copper chelating agent, ethylenediamine, nitriloacetic acid, citric acid, tartaric acid, and salts thereof can be used, but tartaric acid or a salt thereof is preferable from the viewpoint of protein color development. The alkali metal hydroxides according to the present invention include sodium hydroxide, potassium hydroxide, and lithium hydroxide, and the alkali metal or alkaline earth metal salts include sodium, potassium, lithium, calcium, magnesium, barium, Beryllium carbonates, sulfates, nitrates, phosphates,
Examples include borates, acetates, chlorides, bromides, iodides, fluorides, and the like. In the present invention, containing two or more of the above-mentioned compounds means that in the case of a combination of alkali metal hydroxides, any proportion may be used, but at least 1% or more of one of the above compounds is contained. In the case of a combination of an alkali metal hydroxide and an alkali metal or alkaline earth metal salt, the proportion of the alkali metal hydroxide is 99-40%, preferably 95-50%. The alkaline mixture can be contained in various concentrations relative to the binder, but it is not more than 100 times the weight, preferably not more than 70 times the weight. The alkaline mixture, water-soluble cupric salt, and copper chelating agent according to the present invention can be added alone to at least one of the reagent layer and the developing layer of the analytical element of the present invention, or can be added as a mixture in the same layer. For example, it can be added to an organic solvent or aqueous solution containing a binder, dispersed or dissolved, and then applied as a desired layer. Especially when containing an alkaline mixture in the reagent layer,
Depending on the purpose and effect, it can be placed in the same layer as other reagents or in a separate layer. In addition, JP-A-58-70163 and JP-A-58-58 have the function of accommodating macromolecules such as proteins.
It is also possible to apply the reagent layer with interconnecting voids as described in No. 123458. In addition, as a porous expansion layer, for example,
No. 21677, JP-A-55-164356, JP-A No. 57-125847,
It is possible to use those described in No. 57-197466, No. 58-90167, etc., and those using a spread layer consisting of polymer micro beads or fibers with dense openings on the surface. is preferred. The binder used in the present invention includes polymeric substances soluble in organic solvents, such as polystyrenes, polyacrylic esters, polymethacrylic esters, alkyl celluloses such as methyl cellulose and ethyl cellulose, polyvinyl butyral, polyvinyl carbonate, etc. Or water-soluble polymeric substances, such as water-soluble cellulose derivatives such as carboxymethyl cellulose and hydroxyethyl cellulose, pullulan derivatives such as pullulan and carboxymethyl pullulan, water-soluble vinyl polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide. It will be done.
Furthermore, water-soluble polyamides, etc. can also be used, and the above-mentioned water-soluble monomers can also be used in various ways. For example, vinyl acid amides such as acrylamide and methacrylamide, N-vinylpyrrolidone,
Examples include vinyl heterocycles such as N-vinylimidazole. It is also possible to mix two or more types of the above-mentioned binders depending on the purpose, and other vinyl compounds can also be copolymerized as long as the purpose does not deviate from the purpose. Furthermore, various additives that can be used additionally in the present invention, such as preservatives, buffering agents, and surfactants, can be added as desired. In particular, surfactants can be effectively used to adjust the permeation rate when a fluid sample is applied to the element of the present invention. As usable surfactants, it is possible to use both ionic (anionic or cationic) and nonionic surfactants, but nonionic surfactants are preferably effective. . Examples of nonionic surfactants include 2,5-
Examples include polyalkylene glycol derivatives of alkyl-substituted phenols such as di-t-butylphenoxypolyethylene glycol, p-octylphenoxypolyethylene glycol, p-isononylphenoxypolyethylene glycol, and polyalkylene glycol esters of higher fatty acids. It will be done. These surfactants have the effect of regulating the rate of penetration of the fluid sample into the reagent layer and at the same time suppressing the occurrence of undesirable "chromatographic phenomena." The surfactants can be used in widely selected amounts, but can be used in amounts ranging from 10 weight percent to 0.005 weight percent, preferably from 6 weight percent to 0.05 weight percent, based on the weight of the coating fluid. The liquid-impermeable, light-transparent support (hereinafter referred to as the support according to the present invention) used in the analytical element of the present invention may be of any type as long as it is liquid-impermeable and light-transparent. For example, cellulose acetate, polyethylene terephthalate, polycarbonate,
Alternatively, various polymeric materials such as polystyrene are suitable for this purpose. The thickness of the support in this case is arbitrary, but is preferably about 50 microns to 250 microns. Further, one side surface of the support according to the present invention on the observation side can be arbitrarily processed depending on the purpose. Next, when the reagent layer according to the present invention is provided on the support, it can be directly coated, but in some cases, a light-transparent undercoat layer may be used between the reagent layer and the support. It is effective to increase the adhesion of The analytical element of the present invention can be arranged in various different configurations.
It is possible to take any one. Furthermore, the reagent layer of the present invention and various functional layers, reagent-containing layers, and members, such as the reagent layer described in U.S. Pat. No. 3,992,158,
Reflective layers, subbing layers, radiation blocking layers as described in U.S. Pat. No. 4,042,335, barrier layers as described in U.S. Pat. No. 4,066,403, registration layers as in U.S. Pat. No. 4,144,306, migration prevention layers as described in U.S. Pat. The scintillation layer described in U.S. Pat. No. 4,127,499, the cleaning layer described in JP-A-55-90859, the breakable pot-like member described in U.S. Pat. It is possible to construct an analytical element. The various layers described above can be coated to any desired thickness by using slide hopper coating, extrusion coating, dip coating, etc., which are conventionally known in the photographic industry. After obtaining an analytical result as a detectable change using the analytical element of the present invention, it is measured by reflection spectrophotometry. The amount of the unknown test substance can be determined by applying the measured value thus obtained to a calibration curve prepared in advance. The analytical element of the present invention configured as described above is
The purpose can be achieved by supplying a fluid sample from the developing layer and then observing the analytical reaction in the reagent layer from the transparent support side. The amount of fluid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably about 50μ.
to about 5μ, more preferably about 20μ to about 5μ. It is usually preferred to apply a fluid sample of about 10μ. The analytical element of the present invention is suitable for carrying out the Buillet reaction for detecting proteins under highly alkaline conditions, and has excellent color development, storage stability, and quantitative performance. The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto. Example 1 A multilayer analytical element of the present invention was prepared by sequentially coating layers having the compositions listed in Table 1 on a transparent primed polyethylene terephthalate support having a film thickness of about 180 microns.

[Table] Multilayer analytical elements having the above configuration were prepared, and each was designated as analytical elements (1) to (7) of the present invention. Furthermore, for comparison, a multilayer analytical element was prepared by coating a polyethylene terephthalate film support with a reagent layer containing alkali-protecting polymers such as agarose, copper sulfate, and tartaric acid, and a developing layer consisting of microcrystalline cellulose particles. It was made into a multilayer analytical element. The above multilayer analytical element of the present invention and comparative multilayer analytical element were prepared immediately after manufacturing the above element, and at 25°C and 55%
Prepare a solution that has been stored at RH for 60 days, and add 10μ of each aqueous solution containing bovine serum albumin of 2, 5, 7, and 10g/dl.
After dropping it onto the developing layer and incubating at 37°C for 7 minutes, the reflection density was measured from the support side at 540 nm. The results are shown in Table 2 below.

【table】

[Table] As is clear from Table 2 above, the analytical element of the present invention exhibits better color density compared to known comparative analytical elements, and also exhibits good sensitivity with almost no decrease in sensitivity even after long-term storage. It can be seen that it is a multilayer analytical element. Example 2 A multilayer analytical element of the present invention was prepared by sequentially coating layers having the following composition on a transparent, primed polyethylene terephthalate support having a film thickness of about 180 microns.

【table】

[Table] Multilayer analytical elements having the above configuration were prepared, and each was designated as analytical elements (8) to (10) of the present invention. In the same manner as in Example 1, the above devices immediately after manufacture and those stored at 25°C and 55% RH for 60 days were prepared, and aqueous solutions containing bovine serum albumin of 2, 5, and 10 g/dl were placed on a 10μ developing layer. After dropping and incubating at 37°C for 7 minutes, the reflection density was measured from the support side at 540 nm. The results are shown in Table 4 below.

[Table] As is clear from the results in Table 4 above, the analytical element of the present invention has the same effect as shown in Example 1 even when only a hydrophobic polymer substance (reagent dispersion coating) is used as a binder in the reagent layer. It can be seen that it is a good multilayer analytical element that exhibits good color density like the multilayer analytical element of the present invention, and shows almost no decrease in sensitivity even after long-term storage. Example 3 A multilayer analytical element of the present invention was prepared by sequentially coating layers having the following composition on a transparent, primed polyethylene terephthalate support having a film thickness of about 180 microns.

【table】

[Table] Multilayer analytical elements having the above configuration were prepared, and each was designated as analytical elements (11) to (13) of the present invention. In the same manner as in Example 1, the above devices immediately after manufacture and those stored at 25°C and 55% RH for 60 days were prepared, and aqueous solutions containing bovine serum albumin of 2, 5, and 10 g/dl were placed on a 10μ developing layer. After dropping and incubating at 37°C for 7 minutes, the reflection density was measured from the support side at 540 nm. The results are shown in Table 6 below.

[Table] As is clear from the results in Table 6 above, the analytical element of the present invention exhibits good color density even when a reagent layer having interconnected voids made of polyvinyl alcohol/styrene polymer particles is applied. Furthermore, it can be seen that the multilayer analytical element shows almost no decrease in sensitivity even after long-term storage. Example 4 A multilayer analytical element of the present invention was prepared by sequentially coating layers having the compositions listed in Table 7 on a transparent primed polyethylene terephthalate support having a film thickness of about 180 microns.

【table】

[Table] Multilayer analytical elements having the above configuration were prepared, and each was designated as analytical elements (14) to (16) of the present invention. Furthermore, for comparison, a multilayer analytical element was prepared by coating a polyethylene terephthalate film support with a reagent layer containing alkali-protecting polymers such as agarose, copper sulfate, and tartaric acid, and a developing layer consisting of microcrystalline cellulose particles. It was made into a multilayer analytical element. The above multilayer analytical element of the present invention and comparative multilayer analytical element were prepared immediately after manufacturing the above element, and at 25°C and 55%
Prepare a solution that has been stored at RH for 60 days, and add 10μ of each aqueous solution containing bovine serum albumin of 2, 5, 7, and 10g/dl.
After dropping it onto the developing layer and incubating at 37°C for 7 minutes, the reflection density was measured from the support side at 540 nm. The results are shown in Table 8 below. As is clear from the results in Table 8, the multilayer analytical element of the present invention can withstand long-term storage.

【table】

Claims (1)

Claims: 1. A liquid-impermeable, light-transparent support; at least one reagent layer located on one side of the support and containing at least one reagent that reacts with a component in a fluid sample; In a multilayer analytical element comprising at least one porous development layer located on the opposite side of the reagent layer from the support, at least one of the reagent layer and the porous development layer contains water-soluble cupric cupric. 1. A multilayer analytical element for protein detection, comprising an alkaline mixture of at least two selected from a salt, a copper chelating agent, and an alkali metal hydroxide or an alkali metal or alkaline earth metal salt. 2. The multilayer analytical element for protein detection according to claim 1, wherein the water-soluble cupric salt is copper sulfate, and the copper chelating agent is tartaric acid or a salt thereof.