JPH04160365A - Immunological analyzing element and method - Google Patents

Abstract

PURPOSE:To quantify the specific component in a fluid sample accurately and precisely with good sensitivity and reproducibility by simple operation using a small amount of a sample solution by using a specific multilayer analyzing element. CONSTITUTION:A multilayer analyzing element is obtained by providing at least one reagent layer and a porous developing layer on a support in this order and at least one reagent layer and the developing layer contain a marker, the substance capable of being specifically bonded to both of the marker and a specific component, the substance acting on color forming reaction by the action of the marker and a reagent essential to color forming reaction as essential reagents. The substance capable of being specifically bonded to both of the marker and the specific component is contained in the reagent layer containing a hydrophilic binder. The marker is contained in a layer other than the layer containing the marker, the substance capable of being specifically bonded to both of the marker and the specific component and the substance acting on the color forming reaction by the action of the marker. As the reagent essential for the color forming reaction, a diffusion-resistant coupler is used and contained in the reagent layer containing the hydrophilic binder.

Description

[Detailed description of the invention] [Industrial application field]

The present invention is an analytical element for analyzing a specific component in a fluid sample, in particular, a specific component in a biological fluid sample is measured by a homogeneous competitive reaction between a label and a substance that can specifically bind to the specific component. The present invention relates to an analytical element and an analytical method.

[Background of the invention]

Various analytical methods have been developed to detect substances contained in trace amounts in biological fluid samples. One of the analytical methods is one that uses immune reactions as its principle. Various measurement methods have been developed using this principle. That is, in 1958, Berson and Yallow measured insulin in serum using live insulin and anti-insulin antibodies in the serum of diabetic patients, which were standardized in +25 summer. Since its success, immunoassays using radioisotopes have been widely used. And from now on, 'I! radioactive isotopes as li substances
-Various things other than elements have also been developed. for example,
Examples include enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, ring chain reactants, metal and organometallic complexes, organic prosthetic groups, chemiluminescent reactants, and fluorescent molecules. One of the important technical problems regarding the above immunoassay method is the separation of substances that have bound (hereinafter abbreviated as B) and substances that have not bound (hereinafter abbreviated as F).
(abbreviated as B/F separation). Conventionally, various assay methods have been developed to solve the problems of B/F separation in immunoassays. For example, JP-A-51-146295, JP-A-55
-2997 publication, Japanese Patent Application Laid-open No. 58-211662,
JP-A-54-20134, JP-A-55-1048
There are homogeneous immunoassay methods described in Japanese Patent Publication No. 96, Japanese Patent Application Laid-open No. 57-150681, and the like. That is, it is a method of measuring a specific component in a fluid sample by a homogeneous competitive reaction using a label and a substance that specifically binds to the specific component, and the label is a substance that specifically binds to the specific component. When bound, a detectable modulation response is exhibited compared to when unbound, and by using such a label, the step of B/F separation can be omitted. In the field of clinical testing, wet analysis using these homogeneous immunoassay methods is performed. However, since the sample solution and each reagent are mixed and reacted in a relatively large amount of aqueous solution, a high level of skill and experience is required to obtain highly reliable measurement results, and sufficient reproducibility is required. I can't say that. In order to solve these operational problems, and to improve the accuracy and reproducibility of measurement results, measurement devices that automate each operation have been developed, but they are complicated by problems such as the liquid delivery mechanism and the prevention of sample contamination. This requires a special mechanism and is a very expensive device. Furthermore, there are problems in preparing reagents, adjusting equipment, and disposing of reagents and sample solutions after measurement, and also requires a large amount of sample solution, requiring skill in collection and operation. By the way, as an alternative to such wet analysis, a dry analysis method has been devised that allows quick measurement with simple operations. A single-layer analytical element is described that is sequentially impregnated with a coloring reagent and dried. By using this, a specific component in a fluid sample can be quickly analyzed using an inexpensive measuring device, but when measuring from a rough surface such as a filter paper, the influence of a large amount of stray light and noise is unavoidable, resulting in poor measurement accuracy. Reproducibility is not sufficient. Furthermore, Japanese Patent Application Laid-Open No. 18167/1983 describes a multilayer analytical element using a homogeneous immunoassay method. In this analytical element, a reaction layer made of a porous medium contains a substance (for example, an antibody) or a label that specifically binds to a specific component. Generally, low-molecular-weight compounds called haptens do not have immunogenicity per se, so they are used as immunogens by covalently bonding them to carrier proteins such as albumin to obtain antibodies. Therefore, the hapten usually shows a stronger affinity for the label to which the labeling substance is attached, rather than for the hapten itself. Furthermore, in the multilayer analytical element described in JP-A-58-18167, the sample container has a capacity of 24 μl to 200 μl.
2, a large amount of solution is required. In order to measure such a large amount of sample solution by dropping it onto the analytical element, the analytical element must contain a large amount of immune reaction reagent corresponding to the amount, which makes the analytical element expensive. I have no choice but to. Also, for example, when the sample solution is serum or plasma,
This requires a large amount of blood to be collected, which is undesirable in terms of blood collection time, response to blood recipients, etc.

[Disclosure of the invention]

An object of the present invention is to provide a technique that allows a specific component in a fluid sample to be quantified with high sensitivity, accuracy, precision, and reproducibility with a simple operation and with a small amount of sample solution. It is an object of the present invention to target a specific component in a fluid sample through a label in which a labeling substance is bound to either the specific component or an analog of the specific component, thereby making it specific for both the specific component and the label. This is achieved by using a specific multilayer analytical element for determination by homogeneous competitive reaction for substances capable of binding to a substrate, which comprises at least one reagent layer on a support and a porous layer above it. At least one of the reagent layers and/or the developing layer includes a label, a substance that can specifically bind to both the label and a specific component, and a coloring reaction caused by the action of the label. An analytical element containing a substance that acts on a substance and a reagent essential for a color reaction, wherein the substance that can specifically bind to both the label and a specific component is contained in a reagent layer containing a hydrophilic binder. and the label is contained in a layer different from a layer containing a substance that can specifically bind to both the label and the specific component and/or a substance that acts on a color reaction due to the action of the label, and An immunological analysis element is used in which a diffusion-resistant coupler is used as an essential reagent for the color reaction, and this coupler is contained in a reagent layer containing a hydrophilic particle. In particular, in this immunological analytical element, the label may be contained in a layer above the substance that can specifically bind to both the label and the specific component, or the support may have at least two layers. A reagent layer is provided, the reagent layer contains a hydrophilic indium, one reagent layer contains a label, and the other reagent layer contains a label that specifically binds to both the label and a specific component. A substance that acts on a color-forming reaction by the action of a substance and a label that can act on the color-forming reaction, and further contains a reagent essential for the color-forming reaction including a diffusion-resistant coupler in at least one of the reagent layers; The body has a first reagent layer, a second reagent layer, and a porous development layer in order from the support side, the first and second reagent layers containing a hydrophilic binder, and the first reagent layer containing a hydrophilic binder. The reagent layer contains a substance that can specifically bind to both the label and the specific component, and a substance that acts on a color reaction due to the action of the label, the second reagent layer contains the label, and the second reagent layer contains the label. Preferably, the first and/or second reagent layer contains a reagent essential for a color-forming reaction, including a diffusion-resistant coupler. Then, using such an immunological multilayer analysis element, a fluid sample solution of 5μI! The object of the invention is achieved by an immunological analysis method characterized in that an amount in the range of .about.20 μi is applied. The measurement method using homogeneous competitive reaction used in the multilayer analytical element of the present invention is a conventionally known technique, for example, in Japanese Patent Application Laid-open No. 55-2
The measuring method described in Japanese Patent No. 997 can be advantageously used. In the multilayer analytical element of the present invention, a non-porous hydrophilic binder is contained in the layer containing a substance capable of specifically binding to both a label and a specific component. The layer is a reaction layer, and is a layer for carrying out the above-mentioned homogeneous competitive reaction, and by providing it separately from the porous expansion layer, the homogeneous competitive reaction can be carried out efficiently and locally. Therefore, it has become possible to perform a sufficient reaction with a small amount of sample solution and to perform measurement with high accuracy. Also, by incorporating the marker into a layer of a non-porous hydrophilic binder,! ! It becomes possible to arbitrarily delay the reaction between the marker and the substance that can specifically bind to both the specific component and the label, and the specific component can be measured with high sensitivity. In the present invention, any form of solution or colloidal solution can be used as the fluid sample, but fluid samples of biological origin are preferably used, such as blood, plasma, serum, brain, cerebrospinal fluid, saliva, etc.
Examples include amniotic fluid, milk, urine, sweat, meat juice, etc. The specific components in a fluid sample that can be measured by the present invention are:
A substance whose presence or absence or amount in a fluid sample can be detected and which specifically binds to that particular component! (substance group). That is, examples thereof include polypeptides, proteins, complex proteins, polysaccharides, lipids, complex lipids, nucleic acids, hormones, vitamins, drugs, antibiotics, agricultural chemicals, and the like. Preferably, it is a low molecular compound with a molecular weight of 6,000 or less. Specifically, the following substances (substance groups) can be mentioned, but are not limited to these. Hormones and hormone-like substances> Follicle-stimulating hormone (FS) I), lutein-stimulating hormone (L)
H), growth hormone (OH), thyroid stimulating hormone (T
SH), adrenocorticotropic hormone (AcTH), melanin-stimulating hormone (MSH), vasopressin, oxytocin, insulin, glucagon, angiotensin I
and ■, prolactin, secreti, dopamine, serotonin, somatostatin, thyroxine (T4), triiodothyronine (Ts), gastrin, cortisol, aldosterone, catecholamine, estrogen, progesterone, testosterone, placental conadotropin,
Placental lactogen, pituitary hormone releasing factor (TRH)
SFSH-RHSCRHlLH-RH, etc.) etc. (vitamins) Biotin, thiamin, vitamin A, vitamin B2, vitamin B6, vitamin Bl! , vitamin C5 vitamin D1
Vitamin E1 vitamin 1 folic acid, etc. (Various drugs and metabolites) Penzoylecgonine, cocaine, codeine, dextrometrophan, heroin, lysergic acid, morphine, quinidine, quinine, amikacin, gecitamycin, kanamycin, neomycin, tobramycin, actinomycin Mycetin, Caloimycin, Chloramphenicol, Chloromycetin, Chlortetracycline, Erythromycin, Oxytetracycline, Penicillin, Cephalosporin, Polymyxin B, Terramycin, Tetracycline, Streptomycin, Diphenylhydantoin, Ethosuximide, Phenobarbital, Brimidone, Secobarbital , acetaminophen, amitributyrin, carbamazepine, digoxin, shisopyramide, lidocaine, methotrexate, N-acetylprocainamide, phenytoin, procainamide, Brobra 10-
le, theophylline, canapinol, tetrahydrocanapinol, cholinergic suppressants, antihistamines, atropin, butyrophenone, caffeine, chloropromacin, palpyrate, amphetamines, catecholamines, epinephrine, griseofulvin, imipramine, L-
Dopa, meperidine, meprobamate, metatone, narcein, nortributyrin, oxazeban, papaverine, prostaglandin, tegretol, valproic acid, etc. and their metabolites; Pesticides> Halogenated biphenyls, phosphate esters, thiophosphates, and these Metabolites and substances that specifically bind to a specific component or label in a fluid sample that can be applied to the present invention include antibodies, antigens, lectins, protein A, etc. depending on the target to be measured. It is particularly preferred that the binding reaction of the binding substance is an antigen-antibody reaction. The origin of the antibodies used in the present invention is not particularly limited. Antiserum obtained by administering and immunizing mammals with antigens, ascites fluid as is, or sulfuric acid using a conventionally known method. It can be purified and used by sodium precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc. (Refer to Shunsuke Migita, "Immunochemistry J Chuhaku Shoten PP74-88)" Alternatively, monoclonal antibodies can be created by obtaining hybrid cells (hybridomas) from lung cells or myeloma cells (myeloma) of mammals (e.g., mice) infected with the antigen, and then using these to specifically bind to specific components. It is preferable to use these antibodies as substances because they improve specificity.Furthermore, IgG, IgM, and IgA fractions can be used for these antibodies, or these antibodies can be treated with enzymes to produce F.
It may also be used in the form of active antibody fragments such as ab, Fab' or F(ab')z. Furthermore, these antibodies may be used alone or in combination. Used in the present invention! I-identifiers include, for example, prosthetic groups, coenzymes, etc., but preferably flavin adenine dinucleotide, conitinamide adenine dinucleotide and its reduced form, nicotinamide adenine dinucleotide phosphate and its reduced form, and adenosine. There are phosphoric acid and flavin mononucleotides. Particularly preferred is flavin adenine dinucleotide (hereinafter abbreviated as FAD). A labeled body in which a specific component or an analog of a specific component and the above-mentioned labeling substance are bonded is described by D. L. Morris et al.
;Analytical Chemistry, Vo
l 53, pp658-665 (1981), T.
J, Richard at al; C11nica
l Chemistry Vol 27, pp14
99-1504 (1981) and JP-A-55-299
It can be created using the methods described in Japanese Patent Application Laid-Open No. 58-46072, etc. In order to detect the signal caused by the label in the homogeneous immunoassay applied to the present invention, the remaining label that is not bound to the substance (antibody) that can specifically bind to both the label and the specific component is used. A substance that acts on the coloring reaction by the action of the body, that is, an apoenzyme from which prosthetic groups and coenzymes have been removed is required. As the apoenzyme, JP-A-55-2997, JP-A-5
Those described in JP 7-103055 and the like can be used. For example, in the form of holoenzymes, glucose oxidase, glutathione reductase, lipoamide dehydrogenase, glucose-6-phosphate dehydrogenase,
Examples include glutamate dehydrogenase, alcohol dehydrogenase, and lactate dehydrogenase. Preferably it is glucose oxidase. When using apoglucose oxidase, it is desirable to use an anti-glucose oxidase antibody described in JP-A-57-166980 to stabilize the enzyme. Furthermore, it is advantageous to measure the activity of these enzymes colorimetrically using a color reaction. For example, taking glucose oxidase as an example, hydrogen peroxide produced by glucose oxidase can be detected using glucose as a substrate and peroxidase as a catalyst. A method of detection by forming a pigment is widely used. As a coloring reaction reagent, for example, JP-A-64-35369
JP-A-57-94653, JP-A-57-94653, JP-A-57-94653.
An aromatic primary amine compound or a salt thereof described in JP-A No. 94654, JP-A-57-94655, and JP-A-57-94656, a diffusion-resistant coupler, and a color-forming reagent by campling reaction; Furthermore, the compounds shown below can be used. 0-Dianisidine 0-1P-) Aniline derivatives such as luidine 0-phenylenediamine, N,N'-dimethyl-p-phenylenediamine, benzidine, 3.3°. 5.5°-Aromatic diamines such as tetramethylbenzidine Phenol derivatives such as catechol, guaiacol, orcinol, and pyrogallol Aromatic carboxylic acids such as salicylic acid, pyrocatechinic acid, and gallic acid Leucomalachite green, leucophenolphthalene, etc. combination of leuco dye 4-aminoantipyrine and phenol or naphthol derivative 3-methyl-2-benzothiazoline hydrazone and N,N
' - Combination with dimethylaniline Combination of P-anisidine and 8-hydroxyquinoline 2.2°-Azinodi(3-ethyl-6-sulfobenzothiazoline) 2-(4-hydroxy-3-methoxyphenyl)-4,
5-bis(p-dimethoxyaminophenyl)imidazole These coloring reaction reagents are dissolved in water or an organic solvent and can be easily incorporated into the detection layer described below. Further, if necessary, a polymer having a carboxyl group as described in JP-A-58-87461 may be included to stabilize the coloring reaction reagent and the formed dye. The aromatic primary amine compound that reacts with the coupler may be contained in any layer, but it is preferably contained in the developing layer (porous developing layer). The aromatic primary amine compounds used in the present invention include those widely used as color developing agents in various color photographic processes, such as 〇- or p-aminophenol compounds and 〇- Alternatively, p-phenylenediamine compounds may be mentioned. Preferred are 〇- or P-phenylenediamine compounds, with p-phenylenediamine compounds being particularly preferred. Preferred p-phenylenediamine compounds are those represented by the following general formula. In the general formula, A and Bo each represent a hydrogen atom or an alkyl group, A and Bo may form a heterocycle together with a nitrogen atom, D'', E, G and J are a hydrogen atom, a halogen atom, It represents a hydroxy group, an amino group, an alkoxy group, an acylamido group, an arylsulfonamide group, an alkylsulfonamide group, or an alkyl group.The alkyl group represented by A and Bo preferably has 1 to 6 carbon atoms, Particularly those of 1 to 4 i (preferably, these alkyl groups also include those with substituents,
Examples of the substituent include a ureido group, a tetrahydrofurfuryl group, a carboxyl group, a methanesulfonamide group,
Examples include sulfo group, methoxy group, ethoxy group, methoxyethoxy group, methoxyethoxyethoxy group, methoxytetraethoxy group, and the like. D', G and J are hydrogen atoms, alkoxy groups,
An alkylsulfonamide group and an arylsulfonamide group are preferred, and a hydrogen atom is more preferred. E is preferably a hydrogen atom, an alkyl group, or an acylamido group, more preferably an alkyl group having 1 to 3 carbon atoms, and these alkyl groups are the same as the substituents for the alkyl group represented by A and Bo above. may have a substituent. Furthermore, the compound represented by the above general formula is generally used in the form of a salt because it is more stable than in a free state. These salts are
For example, hydrochloric acid, sulfuric acid, P-)luenesulfonic acid, sulfonic acid, sulfinic acid, sulfuric acid ester, sulfamic acid, thiosulfuric acid S-ester, carboxylic acid, phosphoric acid, phosphoric acid ester, amidophosphoric acid, phosphite ester, organic boron Mention may be made of salts of inorganic or organic acids such as compounds, with hydrochlorides, sulfates and p-1 luenesulfonates being particularly preferred. Hereinafter, typical examples of the aromatic primary amine compound used in the present invention will be shown, but the present invention is not limited thereto. The signal caused by the label can be detected by absorbance method (colorimetric method), fluorescence method, or luminescence method.Measurement methods include rate measurement method that measures changes in the signal over time or signal after a certain period of time. can be measured using endpoint assays that measure The absorbance method is preferable, and the absorbance method (colorimetric method) can utilize ultraviolet light, visible light, and near-infrared light. For example, when serum and plasma are used as fluid samples, the absorption of light by the serum and plasma is It is preferable to use green light, red light or near-infrared light to reduce the influence. The multilayer analytical element of the present invention is preferably laminated on a light-transmissive support, and such supports include, for example, cellulose acetate, polyethylene terephthalate, polycarbonate, and polyvinyl compounds (for example, polystyrene). Examples include transparent inorganic compounds such as high molecular weight compounds and glass. The fibrous porous development layer in the present invention is a layer for developing a fluid sample solution and uniformly supplying it to the reaction layer. Examples of its materials include valves, powder filter paper, cotton, linen, silk, etc.
Wool, chitin, chitosan, cellulose ester, viscose rayon, copper ammonia rayon, polyamide (6
- nylon, 6-row nylon, 610-nylon, etc.),
Polyester (polyethylene terephthalate, etc.), polyolefin (polypropylene, vinylon, etc.), glass,
Fibers such as asbestos, for example, vegetable, animal, mineral, synthetic, semi-synthetic, recycled fibers, may be used, and these may be mixed or made into textiles. It is also possible to use Western paper, Japanese paper, filter paper, plush polymer, or woven fabric, nonwoven fabric, synthetic paper, etc. made of the above-mentioned fibers alone or in combination. The spreading layer in the present invention has interconnecting pores (preferably short diameter 1 am-i00tIm) that can freely contact the fluid sample.
It is necessary that there be a porous structure having
The material is not particularly limited as long as it satisfies this condition, but
A preferred example is a structure made of a material containing granules with a size of 1 to 300 μm. Materials for this granular material include diatomaceous earth, titanium dioxide, barium sulfate, zinc oxide, lead oxide, microcrystalline cellulose, silica sand, glass, silica gel, cross-linked dextran, cross-linked polyacrylamide, agarose, cross-linked agarose, various synthetic resins (
polystyrene, etc.), as well as JP-A-57-101760
Examples include self-bonding particles made of a compound having a reactive group as described in JP-A-57-101761 and JP-A-57-101761. Furthermore, several types of these granules can be used in combination. Such a porous spreading layer can be formed by coating and/or forming a film using these fibers and granules. Granular particles that do not have self-bonding properties can be formed into a film by point-adhering the particles to each other using an appropriate adhesive, for example, as disclosed in JP-A-49-53888 and JP-A-55-908.
Methods such as those disclosed in Japanese Patent Application Laid-Open No. 57-67860 can be applied. Organic polymer particles having self-bonding properties are disclosed in JP-A-57-101760 and JP-A-5.
For 0wA fibers or fiber-particle mixtures that can be similarly formed into films by the methods described in JP-A No. 7-101761 and JP-A-58-70163, JP-A-57-1258
A porous layer can be formed by applying a fiber dispersion liquid described in Japanese Patent Application Laid-Open No. 57-197466 and the like. Alternatively, a fiber and/or granular dispersion using a water-soluble binder such as gelatin, polyvinylpyrrolidone, or polyvinyl alcohol may be applied as in the method described in JP-A No. 60-173471. can be formed. The water-soluble binder can be applied in widely selected amounts, but from 0 to 100% based on the weight of the granules and/or fibers. 1 to 25 wt%, preferably 1°0 to 20
wt% used. A number of methods can be used alone or in combination to produce such dispersions.6 For example, one useful method is to add a surfactant to the liquid carrier, to form the granules and /or Dispersion and stabilization of fibers in a dispersion can be promoted. Examples of typical surfactants that can be used include
There are nonionic surfactants and ionic surfactants such as :/X-100 (manufactured by Rohm and Haas; octylphenoxypolyethoxyethanol) and surfactant 10C (manufactured by Olean; nylphenoxypolyglycidol). The above-mentioned surfactants can be used in widely selected amounts, but up to 20% by weight based on the weight of the granules and/or fibers.
wt%~0.005wt%, preferably 15wt%~0
．． 1 wt% can be used. Furthermore, other methods include ultrasonication, physical mixing, and physical stirring of the fibers and particle units and a liquid carrier, and pH adjustment. Combining these with the methods described above is even more useful. Furthermore, the development layer and reaction layer in the present invention may contain proteins that are not involved in the specific reaction to be measured, if necessary, for the purpose of eliminating non-specific reactions in the immune reaction. Typical examples include mammalian normal serum proteins, albumin, gelatin, and their decomposition products. A non-porous hydrophilic hydrophilic substance containing a label according to the present invention, a substance that can specifically bind to both the label and a specific component, an ll1FI material that acts on a color reaction due to the action of the label, and a reagent essential for a color reaction. At least one type of hydrophilic colloid having film-forming properties is used as the material for the binder layer. Examples of hydrophilic compounds include gelatin derivatives such as gelatin and phthalated gelatin, synthetic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinylimidazole, polyacrylamide, and sodium polyacrylate;
Multi-flAM of cellulose derivatives such as hydroxyethylcellulose, carboxymethylcellulose sodium salt
It also includes sodium alginate, etc. Preferably, gelatin derivatives such as gelatin and phthalated gelatin are used. By coating and/or forming a film of these hydrophilic colloids, a label, a substance that can specifically bind to both the label and a specific component, a substance that acts on a color reaction due to the action of the label, and a color reaction A non-porous hydrophilic binder layer containing essential reagents can be formed. The label can be easily incorporated by dissolving it in water or an organic solvent. Further, in the multilayer analytical element of the present invention, a non-porous layer made of a polymeric substance is provided as a detection layer for detecting a signal caused by a label, and a detection layer containing the coloring reagent is provided. It is preferable. Furthermore, the non-porous layer made of a polymeric substance containing a label and the detection layer containing the coloring reagent can be the same layer, and the coloring reagent can be contained in the non-porous layer containing the label. . Furthermore, in order to improve the physical properties of the detection layer, such as swelling degree and heat solubility, the polymer substance of the detection layer according to the present invention may be partially combined with other water-dispersible polymers, that is, polymer latex. Can be replaced. As preferred examples of polymer latexes, those described in, for example, JP-A-57-116258 and JP-A-58-99752 are useful. These polymeric latexes can replace up to 70% of the hydrophilic colloid binder, but preferably about 50% or less. Other additives such as buffers, preservatives, surfactants, mordants, etc. can be added to the detection layer depending on the purpose. Further, the film thickness is 3 to 50 μm, preferably 5 to 30 μm.
It is m. A buffer is included to maintain a pH suitable for specific binding reactions, enzyme reactions, coloring reactions, and the like. Buffers that can be used include Nippon Kagaku Yosen, "Basic Chemical Handbook" (Tokyo, Maruzen 1966) pp1312-1320, N, E.
, Good et al.; Bi. Chemistry Vol 5, P467
(1966L Imamura, Saito; Area of Chemistry, Vo130 (
2), p79 (1976), W, J, Fergus
on etc. Anal, Biochem, Vol 104
, p300 (1980) and the like. Specific examples include citrate, borate, phosphate, carbonate, trisbarbyl,
Examples include glycine and gudo buffer. These buffering agents may be contained in layers other than the detection layer, if necessary. Preservatives are included to stabilize the storage of the substrate coloring reagent, and include antioxidants and the like. Further, in order to maintain the activity of the label contained in the layer, the layer contains an immobilized enzyme, an adsorbent for affinity chromatography, an immobilized antibody, and a preservative used for preserving proteins, enzymes, and the like. The substance is
Japanese Biochemistry Extra Line “Biochemistry Experiment Account 1, Protein Chemistry 1”
(Tokyo Kagaku Doujin @1976) pp66-67, Experiments and Applications [Affinity Orthomatography JPP16-10
4. Examples include those described in JP-A-60-149927. Specific examples include gelatin, gelatin decomposition products, albumin, cyclodextrins, non-reduced @fructose (chefrose, trehalose), polyethylene glycol, amino acids, various ions, sodium azide, and the like. A hardening agent can be added to the polymer material layer made of hydrophilic colloids such as gelatin or gelatin derivatives, and materials commonly used in the photographic industry can be used as the hardening agent. , JameslirThe The
ory of the Photographic
Examples include those described in "Process" 4th, pp. 77-87. Specific examples include aldehydes, active olefins, and active esters. By adding a hardening agent to a non-porous layer made of a polymeric substance containing a label, the elution and diffusion of the label to another reaction layer can be controlled arbitrarily, and the label and specific It is useful to delay reactions with substances that can bind to. Examples of the surfactant include those mentioned above. Other reagents contained in the layer include solubilizers, blocker reagents, and the like. These additives are added in appropriate amounts as necessary. A mordant is a substance that concentrates the detection substance for enzyme activity measurement in the detection layer, increases the ocular luminosity coefficient when the detection substance is a dye, or shifts the wavelength, and exhibits strong interaction with the detection substance. . Cationic polymers, anionic polymers and latexes of these polymers are used. The multilayer analysis element of the present invention can further include auxiliary layers such as a blood cell separation layer useful when the fluid sample is blood (whole blood), an adhesive layer provided as necessary, a protective layer, and a timing layer. The positions of these layers are determined according to their functions.

【Example】

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited by these Examples. 〔Example! The FADI-identified theophylline (FAD-Theo) and apoglucose oxidase (apo-COD) are disclosed in Japanese Patent Application Laid-Open No. 58-46072 and U.S. Patent No. 4.23.
It was prepared according to the method described in No. 8.565. 1-(1) Preparation of monoclonal anti-theophylline antibody 6-chlor-1゜3- in 250jd of absolute ethanol
50g of dimethyluracil and 37g of 6-aminocaproic acid
was added, and further 28 g of triethylamine was added, and the mixture was reacted under heating under reflux for 200 hours. After ethanol was distilled off, the mixture was recrystallized from water to obtain a uracil derivative into which a (5-carboxypentyl)imino group had been introduced. 30g of this compound in 100d of water
To this was added 1'4" g of sodium nitrite dissolved in 50 id of water, and the mixture was reacted for 1 hour at 50°C, and collected after cooling. 32 g of this collected material was dissolved in 400 ml of methanol and oxidized. Platinum was reduced with hydrogen as a catalyst.After identifying the catalyst,
Further, a formamidated uracil derivative was obtained by reacting with methyl formate. Next, 18 g of this was placed in 1 liter of 0-dichlorobenzene and heated under reflux for 4 hours under a nitrogen atmosphere to obtain 9-
(5-carboxypentyl)-1°3-dimethylxanthine was obtained. This compound was then bound to bovine serum albumin and used as an immunogen according to the method described in JP-A-58-46072. Approximately 6 week old Balb/c mice (81) were immunized with the above immunogen. That is, 200 μg of the above immunogen was made into an emulsion using a complete fluorocarbon adjuvant, and the emulsion was injected into the abdominal cavity of a mouse. Two weeks later, 150 μg of the immunogen was made into an emulsion using an incomplete Freund's Ajer band and injected into the peritoneal cavity of the mouse. Furthermore, 0.15M after 2 weeks
Mice were injected intravenously with 100 μg of immunogen dissolved in saline. Three days later, the lungs were removed and lung cells were collected. These lung cells (4 x 102 cells) and azaguanine-resistant mouse myeloma cells X63-Ag 8-6°5.3 (1,
2 x 10' cells) and cell fusion was performed using 50% polyethylene glycol 4000 (manufactured by Merck & Co.). The fused cells were plated in a 96-well plate and incubated with HAT medium (.0,4
The cells were cultured in RPMI-1640 medium containing μM aminopterin, 16 gM thymidine, and 100 μM hyboxanthin. Half of the medium was replaced with fresh IAT medium every 2 weeks, and after 2 weeks, the medium supernatant of each well was assayed by the EIisa method, and positive ones were cloned by the limiting dilution method to obtain hybrid cells. Next, these cells were grown intraperitoneally in mice to obtain mouse ascites containing monoclonal anti-theophylline antibodies. 3.2 ml of this mouse ascites was used in a multilayer analysis element. 112) Preparation of apoglucose oxidase 25 mg of lyophilized apoglucose oxidase was combined with 4 ml of anti-glucose oxidase antibody obtained by immunizing a goat with glucose oxidase and used in a multilayer analytical element. 1-(3) Creation of analytical element for measuring theophylline Coating liquid (1) with the following composition was applied onto a transparent undercoated polyethylene terephthalate film with a thickness of 180 μm.
It was dried to form a detection layer. The film thickness after drying is 16μ.
It was m. Coating solution (1) Deionized gelatin (Gel) 4.0 g Triton anti-Theo) 3.2ml Apo-glucose oxidase containing anti-glucose oxidase antibody (apo-GOD/a nti-C,
OD) 4. 0m1
1.2-bis(vinylsulfonyl)ethane 7 mg Distilled water 21.0 g Next,
A coating liquid (2) having the following composition was applied onto this detection layer and dried to form a non-porous layer made of a gelatin binder containing FAD-labeled theophylline. The film thickness after drying was 16 μm. Coating solution (2) Deionized gelatin 3.0g distilled water
35.0g1.2-bis(vinylsulfonyl)ethane 4mg Alkanol XC (surfactant, trade name; manufactured by DuPont)
0.14g dibutyl phthalate (DBP) 0.26g 7-chloro-3-(
2-hexyldecylsulfonylethyl)-6-methyl-
0.50 g of pyrazolo-(3゜2-c)-s-1-lyazole (coupler After emulsifying and dispersing using HEPES (manufactured by Isotope Kagaku) 0.78gF
AD labeled Theofine'JFin (100tt g/m + aqueous solution) 600, 1/1 was added to prepare a coating solution (2). Next, the following coating liquid (3) was applied as an adhesive layer on this layer and dried. Coating liquid (3) Luhiskol VA-281.0g n-butanol 38.0g Furthermore, coating liquid (4) having the following composition was applied on top of these layers.
It was dried to form a fibrous porous spread layer. The film thickness after drying was 280 μm. Coating liquid [4] Filter paper powder (D Seni, manufactured by Toyo Roshi) 91, Og Styrene-glycidyl methacrylate copolymer (polymerization ratio 9:1) 23.0 g Triton X-1
009.1g xylene 280g dimedone 10.4g aromatic primary amine compound (hydrochloric acid 4-N,N-diethylamino-2(
2゛-methanesulfonamidoethyl)aniline
0.12g Then, the analytical element constructed in this way was cut into pieces of 1°5cm x 1.5cm, with a diameter of 8m in the center.
Plastic mount with M holes (2.5cm x 3
．． (Ocm) to form a multilayer analytical element-1. A schematic diagram of the structure of this multilayer analytical element-1 is shown in FIG. [Example 2] The first reagent layer (R
Multilayer analytical element-2 was constructed using the same structure except that the anti-glucose oxidase antibody-containing apoglucose (apo-GOD/ant 1-COD) in L-1) was interposed in the porous spreading layer (SL). And so. [Example 3] The second reagent layer (R
A multilayer analytical element-3 was prepared using the same structure except that the coupler X in L-2) was interposed in the first reagent layer (RL-1). [Example 4] The first reagent layer (R
Bpo-COD/ant 1-COD in L-1) is interposed in the porous spreading layer (SL), and coupler X in the second reagent layer (RL-2) is interposed in the first reagent layer (SL-2). RL-
1) The structure was the same except that it was interposed therein, and a multilayer analysis element-4 was prepared. , [Example 5] The second reagent layer (R
L-2) FAD-Theo in porous spreading layer (SL)
The structure was the same except that it was interposed therein, and it was designated as multilayer analysis element-5. [Example 6] The first reagent layer (R
apo-GOD/ant 1-COD in L-1) is interposed in the second reagent layer (RL-2);
coupler X in RL-2) in the first reagent layer (RL-1)
FAD-The interposed therein and in the second reagent layer (RL-2). A multilayer analytical element-6 was prepared with the same structure except that it was interposed in the porous spreading layer (SL). [Example 7] The second reagent layer (R
L-2) FAD-Theo in porous spreading layer (SL)
Coupler X in the second reagent layer (RL-2) was interposed in the first reagent layer (RL-1), and the second reagent layer (RL-2) was removed. Except for this, the structure was the same, and it was designated as multilayer analytical element-7. [Example 8] The second reagent layer (R
FAD-Theo in the first reagent layer (RL-2)
1) First reagent layer (RL-1) interposed therein
apo-GOD/anti-COD and anti-
The same structure was used except that Theo was interposed in the second reagent layer (RL-2), and a multilayer analytical element-8 was prepared. [Comparative Example 1] The first reagent layer (R
apo-GOD/ant 1-GOD and anti-Theo in L-1) are interposed in the second reagent layer (RL-2), and coupler X in the second reagent layer (RL-2) Comparative analytical element-1 was prepared with the same structure except that it was interposed in the first reagent layer (RL-1). [Comparative Example 2] The second reagent layer (R
Same structure except that FAD-Theo and coupler X in L-2> were interposed in the first reagent layer (RL-1), and the second reagent layer (RL-2) was removed. This was designated as Comparative Analysis Element-2. (Comparative Example 3) The first reagent layer (R
The anti-Theo in L-1) is added to the porous expansion layer (SL
), except that it was interposed in the same structure as Comparative Analysis Element-3. [Comparative Example 4] The first reagent layer (R
The anti-Theo in L-1) is added to the porous expansion layer (SL
), and the comparative analysis element had the same structure except that the coupler -4. [Comparative Example 5] The first reagent layer (R
Coupler X in L-2) is TMBZ (3,'3',5
, 5°-tetramethylbenzidine and the porous spreading layer (SL) was omitted, but the same structure was used, and it was designated as Comparative Analytical Element-5. [Comparative Example 6] The first reagent layer (R
a po-GOD/a n t 1-C in L-1)
OD and anti-Theo to TMBZ and FAD-T
heo, FAD-Theo and Gel in the second reagent layer (RL-2) are replaced with apo-〇〇D/an
It was replaced with t 1-COD, ant 1-Theo, and Avicel to provide a comparative analytical element-6. This comparative analysis element-6 is disclosed in Japanese Patent Application Laid-Open No. 1-280253.
It is based on the publication No.

[Characteristics [Measurement of theophylline]] Theophylline (Aldrich Co., Ltd.) at a concentration of 5 to 25 pg/mI dissolved in 50 mM citric acid-dipotassium hydrogen phosphate buffer (pH 5,0) containing 5.0 wt% bovine serum albumin. 10 u+ (manufactured by Manufacturer) was dropped onto the analysis element on each side, and while incubating in a sealed state at 37°C, 546 nm was applied from the Heath side every 30 seconds for 15 minutes.
The reflected light density was measured. Table 1 shows the reflection density after 15 minutes, setting the theophylline concentration of 0 μg/mI as 100. According to this, the analytical elements 1 to 7 of the present invention have markedly improved quantitative sensitivity compared to the comparative analytical elements, and have a remarkable effect. Table 1 In addition, the correlation between the multilayer analytical element of the present invention and the comparative analytical element was investigated using human serum-based samples to which theophylline was added (serum bases are different). The results showed that the analytical element l of the present invention was 0.985,
Analytical element 2 has r of 0.981, and analytical element 3 has r of 0.9.
87, analytical element 4 has r of 0.977, analytical element 5 has r of 0.988, analytical element 6 has r of 0.989, analytical element 7
has r of 0.987, analysis element 8 has r of 0.971, comparison element 5 has r=0.930, comparison element 6 has r=0.
870, which is significantly close to 1, that is, shows a high correlation, and the multilayer analytical element of the present invention
It can be seen that there is a high correlation between the method and the method), indicating that the method has high performance.

[Brief explanation of drawings]

FIG. 1 shows a schematic diagram of a multilayer analytical element according to the present invention.

Claims (5)

[Claims] (1) It has at least one reagent layer on a support and a porous spreading layer above it, and at least one of the reagent layers and/or the spreading layer includes a labeled substance, this labeled substance and a specific component as essential reagents. An analytical element containing a substance that can specifically bind to both the label, a substance that acts on a color reaction due to the action of the label, and a reagent essential for the color reaction, which is specific to both the label and the specific component. A substance that can specifically bind to the label is contained in a reagent layer containing a hydrophilic binder, and the label is colored by the action of the substance and/or label that can specifically bind to both the label and the specific component. A diffusion-resistant coupler is contained in a layer different from the layer containing substances that act on the reaction, and is used as an essential reagent for the coloring reaction, and this coupler is contained in a reagent layer containing a hydrophilic binder. An immunological analysis element characterized by: (2) In the immunological analysis element according to claim 1, the label is contained in a layer above a substance that can specifically bind to both the label and the specific component. (3) In the immunological analysis element according to claim 1, at least two reagent layers are provided on the support, and the reagent layers contain a hydrophilic binder, and
One reagent layer contains a label, the other reagent layer contains a substance that can specifically bind to both the label and a specific component, and a substance that acts on a coloring reaction due to the action of the label; At least one of the layers contains a reagent essential for a color-forming reaction, including a diffusion-resistant coupler. (4) In the immunological analysis element according to claim 1, a first reagent layer is provided on the support in order from the support side;
It has a second reagent layer and a porous spreading layer, the first and second reagent layers contain a hydrophilic binder, and the first reagent layer is specific to both the label and the specific component. The second reagent layer contains a label, and the first and/or second reagent layer has a diffusion-resistant layer. Contains reagents essential for color reaction including couplers. (5) An immunological method characterized in that a fluid sample solution is applied in an amount ranging from 5 μl to 20 μl using the immunological multilayer analysis element according to any one of claims 1 to 5 for analysis of a specific component. Analysis method.