JPH04208859A - Immunological analyzing element and analyzing method - Google Patents

Abstract

PURPOSE:To quantify a specific component with high accuracy using a small amount of a sample solution by constituting an analyzing element of three layers and adding a labelled product to the layer far from a support. CONSTITUTION:An analyzing element analyzing the specific component in a liquid sample by the homogenous system competitive reaction of the labelled product formed by the bonding of a labelling substance to either one of the specific component in the liquid sample and the analogue thereof and a substance capable of being specifically bonded to both of the specific component and the labelled product is constituted of three layers. The analyzing element is formed by successively providing a non-porous layer, a non-fibrous porous reaction layer and a fibrous porous developing layer on a support and the labelled product is contained in the farther layer from the support than the non-porous layer. By separating the non-fibrous porous reaction layer from the porous developing layer as mentioned above, even a small amount of the sample can be sufficiently reacted and the specific component can be measured with high sensitivity by specifying the position of the labelled product.

Description

[Detailed description of the invention]

[00011

[Industrial Application Field] The present invention relates to an analytical element for analyzing a specific component in a fluid sample, in particular, a substance and a label that specifically bind to a specific component in a biological fluid sample. The present invention relates to an analytical element and an analytical method for measuring by a homogeneous competitive reaction. BACKGROUND OF THE INVENTION Various analytical methods have been developed as methods for detecting substances contained in minute 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. [0003] That is, in 1958, Berson (Berson)
Radioisotopes have been developed since the success of S. son and Yal 1ow in measuring insulin in serum using bovine insulin labeled with radioisotope I and anti-insulin antibodies in the serum of diabetic patients. Immunoassay methods using Since then, various labeling substances other than radioactive isotopes have been developed. For example, enzymes, enzyme substrates, coenzymes,
Enzyme inhibitors, bacteriophages, cycling reactants, metal and organometallic complexes, organic prosthetic groups, chemiluminescent reactants, fluorescent molecules, and the like. [0004] One of the important technical problems regarding the above-mentioned immunoassay method is the separation (hereinafter, abbreviated as F) of the substance that caused binding (hereinafter abbreviated as B) and the substance that did not (hereinafter abbreviate as F). (abbreviated as B/F separation). Conventionally, in order to solve the problem of B/F separation in immunoassay,
Various measurement methods have been developed. [0005] For example, JP-A-51-146295, JP-A-55-2997, JP-A-58-2116
No. 62, JP-A-54-20134, JP-A-Sho 5
There are homogeneous immunoassay methods described in JP-A No. 5-104896, JP-A-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 to, a detectable modulation response is exhibited compared to the case where no binding occurs, and by using such a label, the step of B/F separation can be omitted. [0006] 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 although reproducibility is sufficient, do not have. 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 with reagent preparation, equipment adjustment, and disposal of reagents and sample solutions after measurement, and a large amount of sample solution is required.
It also requires skill in collection and operation. [0007] By the way, instead of such wet analysis,
Dry analysis methods have been proposed that allow quick measurements with simple operations. For example, in Japanese Patent Application Laid-Open No. 57-103055,
A single-layer analytical element is described in which filter paper or glass fiber cloth is sequentially impregnated with an immune reaction reagent or a coloring reagent and dried. By using this, specific components 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.
Measurement accuracy and reproducibility are not sufficient. [0008] Also, in JP-A-58-18167,
Multilayer analytical elements using homogeneous immunoassays have been described. 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 are not immunogenic in themselves, so they are used as immunogens by covalently bonding them to carrier proteins such as albumin.
We have obtained that antibody. For this reason, the hapten usually shows a stronger affinity for the label to which the labeling substance is attached than to the hapten itself. Therefore, in order to measure a hapten in a fluid sample, it is possible to perform the measurement with high sensitivity by allowing the reaction between the hapten and the antibody to occur first, rather than the reaction between the label and the antibody, during the competitive reaction. However, containing an antibody or a label in a reaction layer made of a porous medium is insufficient to delay the reaction between the label and the antibody. [0009] Furthermore, in the multilayer analytical element described in Japanese Patent Application Laid-Open No. 5L-18167, the sample container has a capacity of 25μI.
A large amount of solution, 200 μl, 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 don't get it. Further, for example, when the sample solution is serum or plasma, a large amount of blood must be collected, which is undesirable in terms of blood collection time, response to the blood sample, etc. [00101

[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. . The purpose of the present invention is to specifically bind a specific component in a fluid sample to a labeled body in which either the specific component or an analog of the specific component is bound to a labeled substance, and to both the specific component and the labeled body. This analytical element performs analysis by a homogeneous competitive reaction with a substance that can react, and this analytical element is made up of a support with (1) a non-porous layer, (2) a non-fibrous porous reaction layer, and (3) a fibrous porous reaction layer. Achieved by an immunological analysis element comprising at least a highly porous spreading layer, and containing the label in a layer farther from the support than the non-porous layer of (1) above. be done. [00111 Also, for analysis of specific components in a fluid sample, at least a support is provided with (1) a non-porous layer, (2) a non-fibrous porous reaction layer, and (3) a fibrous porous spreading layer. and analyzing by a homogeneous competitive reaction between a label in which either the specific component or an analog of the specific component and a labeling substance are bound, and a substance that can specifically bind to both the specific component and the label, And, using an immunological analysis element in which the label is contained in a layer farther from the support side than the non-porous layer of (1), the fluid sample solution 5 is
This is achieved by an immunological analysis method characterized in that amounts ranging from μl to 20 ul are applied. [0012] The measurement method using a homogeneous competitive reaction used in the multilayer analytical element of the present invention is a conventionally known technology in a solution system (wet method), for example, the measurement method described in JP-A-55-2997. can be used to advantage. In the multilayer analytical element of the present invention, the non-fibrous porous reaction layer is a layer for carrying out the above-mentioned homogeneous competitive reaction, and by providing it separately from the fibrous porous development layer, the homogeneous competitive reaction can be carried out efficiently.
It can be done 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. Note that there may be two or more non-fibrous porous reaction layers. [0013] Furthermore, by containing the label in a layer farther from the support side than the non-porous layer, for example, a non-fibrous porous reaction layer or a fibrous porous development layer, the label, the specific component, and the label can be separated. It becomes possible to arbitrarily delay the reaction with a substance that can specifically bind to both, and a 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, spinal fluid, saliva, amniotic fluid, milk, urine, and sweat. , gravy, etc. [00141 A specific component in a fluid sample that can be measured according to the present invention is a substance whose presence or absence or amount in the fluid sample is detected, and in which there may be a substance that specifically binds to the specific component. (substance group). That is, polypeptides, proteins, complex proteins, polysaccharides, lipids, complex lipids,
Examples include nucleic acids, hormones, vitamins, drugs, antibiotics, and agricultural chemicals. Preferably, it is a low molecular compound with a molecular weight of 6000 or less. [0015] Specifically, the following substances (substance groups) can be mentioned, but are not limited to these. [Hormone and hormone-like substances] Follicle-stimulating hormone (FSH), luteinizing hormone (LH)
), growth hormone (GH), thyroid stimulating hormone (T
SH), adrenocorticotropic hormone (ACTH), melanin stimulating hormone (MS, H), vasopressin, oxytocin, insulin, glucagon, asidiotensin,
prolactin, secreti, dopamine, serotonin,
Somatostatin, thyroxine (T4), triiodothyronine (T3), gastrin, cortisol, aldosterone, catecholamines, estrogen, progesterone, testosterone, placental gonadotropin, placental lactogen, pituitary hormone releasing factor (TRH, F
SHRH, CRH, LH-RH, etc.) etc. [Vitamins] Biotin, thiamin, vitamin A, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D
, vitamin E, vitamin K, folic acid, etc. [Various drugs and metabolites] Penzoylecgonine, cocaine, codeine, dextrometrophan, heroin, lysergic acid, morphine, quinidine, quinine, amikacin, gentamicin, kanamycin, neomycin, tobramycin , actinomycetin, caloimycin, chloramphenicol, chloromycetin, chlortetracycline, erythromycin, oxytetracycline, penicillin, cephalosporin, polymyxin B, terramycin, tetracycline, streptomycin, diphenylhydantoin, ethosuximide, phenobarbital, brimidone, Secobarbital, acetaminophene, amitributyline, carbamazepine, digoxin, shisopyramide, lidocaine, methotrexate, N-acetylprocainamide, phenytoin, procainamide, Progra 10-
le, theophylline, canapinol, tetrahydrocanapinol, cholinergic drugs, antihistamines, atropin, butyrophenone, caffeine, chloropromacine, pulpyrate, amphetamines, catecholamines, epinephrine, griseofulvin, imipramine, L-dopa, meperidine, meprobamate, methaton, narcein , nortributyrin, oxazepan, papaverine, prostaglandin, tegretol, valproic acid, etc. and their metabolites [Pesticides] Halogenated piphenyl, phosphoric acid esters, thiophosphates, and analogs of these metabolites and specific components is a substance that has an epitope of 9 specific acids and also has some specific components. [0016] 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. Sodium precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc. (Shunsuke Migita, "Immunochemistry")
(see Chuhaku Shoten pp. 74 to 88). [0017] Alternatively, a monoclonal antibody is produced by obtaining a hybrid cell (hybridoma) from a lung cell or myeloma cell (myeloma) of a mammal, etc. (for example, a mouse) infected with an antigen, and this is used to specifically bind to a specific component. It is preferable to use it as a water absorbing substance because it improves specificity. In addition, these antibodies can be prepared for IgG, IgM, and IgA, or these antibodies can be treated with enzymes to make Fab and Fab.
It may also be used in the form of active antibody fragments such as b' or F(ab')z. Furthermore, these antibodies may be used alone or in combination. [0018] In order to contain these antibodies in the non-fibrous porous reaction layer, it is necessary to contain them while retaining the ability to specifically bind to a specific component of the antibody. This can be carried out by mixing with normal serum proteins of unrelated mammals, albumin, gelatin, their decomposition products, etc., and then freeze-drying the mixture. or,
This can be carried out by physically or chemically bonding and containing the material that forms the porous reaction layer described below, or the water-insoluble carrier and latex described in Chibata et al. [Affinity Chromatography] (Kodansha, 1976). [0019] Examples of the labeling substance used in the present invention include prosthetic groups, coenzymes, etc., but preferably flavin adenine dinucleotide, nicotinamide adenine dinucleotide and its reduced form, nicotinamide adenine dinucleotide phosphate and its reduced form, adenosine phosphate, and flavin mononucleotide. Particularly preferred is flavin adenine dinucleotide (hereinafter abbreviated as FAD). Labels in which the specific component or an analog of the specific component and the above-mentioned labeling substance are bound are D, L, Morr.
1set al;Analytical Che
mistry, Vo153, pp658-665
(1981), Richard, T.J.
C11nical Chemistry Vol.
27, pp1499-1504 (1981) and JP-A-55-2996, JP-A-58-4607
It can be created using the method described in Publication No. 2 and the like. [002O1 In order to detect the signal caused by the label in the homogeneous immunoassay applied to the present invention, the remainder 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 due to the action of the labeled substance, that is, an apoenzyme from which prosthetic groups and coenzymes have been removed, is required. As the apoenzyme, those described in JP-A-55-2997, JP-A-57-103055, etc. can be used. For example, in the form of holoenzyme, glucose oxidase, glutathione reductase, lipoamide dehydrogenase , glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, and the like. Preferably it is glucose oxidase. When using apoglucose oxidase,
In order to stabilize the enzyme, it is desirable to use the anti-glucose oxidase antibody described in JP-A-57-166980. [0,021] Furthermore, it is advantageous to quantify the activity of these enzymes colorimetrically using a color reaction. For example, taking glucose oxidase as an example, using glucose as a substrate, hydrogen peroxide produced by glucose oxidase is converted into peroxidase. Detection methods are widely used in which a detectable dye is formed using a catalyst as a catalyst. The amount of the substance or label that specifically binds to a specific component in a fluid sample used in the present invention is determined by determining the amount of the substance or label that specifically binds to a specific component in a fluid sample so that it can be identified in the measurement area of the specific component. It is determined as appropriate, taking into account the reactivity of the specific component and the sensitivity of the label. As the OO221 coloring reaction reagent, for example, JP-A-64
-35369 publication, Japanese Patent Application Laid-open No. 57-94653,
JP-A-57-94654, JP-A-57-9465
5 and JP-A No. 57-94656, an aromatic primary amine compound or a salt thereof, a diffusion-resistant coupler, a color-forming reagent through a coupling reaction, and the compound shown below. I can do it. [0023] o-Dianisidine 0-1 Aniline derivatives such as p-toluidine O-phenylenediamine, N, N'-dimethyl-p-phenylenediamine, benzidine, 3. 3', 5. 5” Aromatic diamines such as tetramethylbenzidine, catechol,
Phenol derivatives such as guaiacol, orcinol and pyrogallol Aromatic carboxylic acids such as salicylic acid, pyrocatechinic acid and gallic acid Leuco dyes such as leucomalachite green and leucophenolphthalene Combinations of 4-aminoantipyrine and phenol or naphthol derivatives 3 - Combination of methyl-2-benzothiazoline hydrazone and N,N''-dimethylaniline Combination of p-anisidine and 8-hydroxyquinoline 2゜2
°-azinodi(3-ethyl-6-sulfobenzothiazoline)2-(4-hydroxy-3-methoxyphenyl)4
．． These coloring reaction reagents such as 5-bis(p-dimethoxyaminophenyl)imidazole 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. [0024] The signal caused by the label can be detected by an absorbance method (colorimetric method), a fluorescence method, or a luminescence method, and the measurement method is a rate measurement method that measures changes in the signal over time or a fixed time measurement method. It can be measured using an endpoint measurement method that measures the signal afterward. Preferably, the absorbance method is used,
The absorbance method (colorimetric method) can use ultraviolet light, visible light, and near-infrared light. For example, when using serum and plasma as fluid samples, green light is used to reduce the influence of light absorption by serum and plasma. Preferably, red light or near-infrared light is used. [0025] The multilayer analytical element of the present invention is preferably laminated on a light-transmitting support, and such supports include, for example, cellulose acetate, polyethylene terephthalate, polycarbonate, and polyvinyl compounds (such as polystyrene). ) or transparent inorganic compounds such as glass. The fibrous porous spreading layer in the present invention is a layer for spreading a fluid sample solution and uniformly supplying it to the porous reaction layer. Examples of the material include pulp, powdered filter paper, cotton, linen, and silk. , wool, chitin, chitosan, cellulose ester, viscose rayon, copper ammonia rayon, polyamide (
6-nylon, 6-row nylon, 610-nylon, etc.)
Fibers such as polyester (polyethylene terephthalate, etc.), polyolefin (polypropylene, vinylon, etc.), glass, asbestos, etc., such as vegetable, animal, mineral, synthetic, semi-synthetic, or recycled fibers, can be used. They may be mixed or made into woven fabrics, and water-absorbing western paper, Japanese paper, filter paper, plush polymers, or fabrics made from the above-mentioned fibers alone or in combination;
Nonwoven fabric, synthetic paper, etc. can also be used. [00261 The porosity of the fibrous porous spreading layer and the non-fibrous porous reaction layer in the present invention means that each of the above layers formed after coating and/or film formation can freely contact the fluid sample. Means having interconnecting pores, for example, the short axis size is 1 to 300 um, particularly preferably 1 μ
It has a porous structure with interconnecting pores of 1 to 100 tLm. [0027] Also, the non-porous property of the non-porous layer means that the non-porous layer does not have the above-mentioned porous structure. The non-fibrous porous reaction layer of the present invention is a layer that allows a homogeneous competitive reaction to occur,
It contains a substance that can specifically bind to both the specific component and the label, the apoenzyme, and the like. Here, non-fibrous refers not to a fibrous material but to a material having an isotropic shape, and this non-fibrous porous reaction layer is composed of interconnecting pores that can freely contact the fluid sample. For example, it has a porous structure having interconnecting pores with a short axis size of 1 to 300 μl, particularly 1 μl to 100 μl. As long as this condition is satisfied, there is no particular limitation on the material, but examples include a structure made of a material containing 1 μl to 100 μl of granules. 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 self-bonding particles made of compounds having reactive groups described in JP-A-57-101760 and JP-A-57-101,761. Furthermore, several types of these granules can be used in combination. [0028] In order to configure such a fibrous porous spreading layer and a non-fibrous porous reaction layer consisting of granules,
It can be formed by coating and/or forming a film from 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.
No. 8, JP-A-55-90859, JP-A-57
A method such as that disclosed in Japanese Patent No.-67860 can be applied. Organic polymer particles with self-bonding properties are disclosed in Japanese Patent Application Laid-open No. 1983-
Films can be similarly formed by the methods described in JP-A No. 101760, JP-A-57-101761, JP-A-58-70163, and the like. For fibers or fiber-particle mixtures, see JP-A-57-125847 and JP-A-57-1.
A porous layer can be formed by applying a fiber dispersion described in Japanese Patent No. 97466 and the like. At that time, a hydrophobic binder may be used. Preferably, JP-A-6017
It can be formed by applying a dispersion of fibers and/or particles using a water-soluble binder such as gelatin, polyvinylpyrrolidone, or polyvinyl alcohol, as in the method described in Japanese Patent No. 3471. The water-soluble binder can be applied in widely selected amounts, but
or 0.0% based on the weight of the fiber. It is used in an amount of 1 to 25 wt%, preferably 1.0 to 20 wt%. [0029] Many methods can be used alone or in combination to produce such dispersions. For example, one useful method is to add surfactants to the liquid carrier to facilitate dispersion and stabilization of particulates and/or fibers in the dispersion. Examples of typical surfactants that can be used include Triton
100 (manufactured by Rohm and Haas, octylphenoxypolyethoxyethanol), surfactant LOG (
There are nonionic surfactants and ionic surfactants such as Nonylphenoxypolyglycidol (manufactured by Olean). [00301 The above surfactants can be used in widely selected amounts, but range from 20 wt% to 0.005 wt%, preferably from 15 wt% to 0.1 wt%, based on the weight of the granules and/or fibers. Can be used. Furthermore, as another method, ultrasonic treatment, physical mixing, and physical stirring treatment of the fibers and particle units and the liquid carrier,
There is pH adjustment. Combining these with the methods described above is even more useful. [0031] Also, the development layer and reaction layer in the present invention include
If necessary, for the purpose of eliminating non-specific reactions in the immune reaction, proteins that are not involved in the specific reaction to be measured can be included. Typical examples include mammalian normal serum proteins, albumin, gelatin, and their decomposition products. As the material for the non-porous layer of the present invention, at least one type of hydrophilic colloid having film-forming properties is used. [0032] Hydrophilic colloids include gelatin, gelatin derivatives such as phthalated gelatin, polyvinyl alcohol, polyvinylpyrrolidone, polyvinylimidazole,
These include synthetic polymers such as polyacrylamide and sodium polyacrylate, polysaccharides such as cellulose derivatives such as hydroxyethyl cellulose and carboxymethyl cellulose sodium salt, and even sodium alginate. Preferably, gelatin derivatives such as gelatin and phthalated gelatin are used. [0033] A non-porous layer is constructed by applying and/or forming a film of these hydrophilic colloids. The thickness of each layer of the analytical element of the present invention can be selected as appropriate, but the thickness of the fibrous porous spreading layer is preferably 50 to 500 μl, more preferably 100 to 300 μl.
It is. [0034] The thickness of the non-fibrous porous reaction layer is preferably 10 to 200 μl, more preferably 30 to 180 μl. The thickness of the non-porous layer made of a polymeric substance is preferably 1 to 1100 μl, more preferably 5 to 50 μl. Further, in order to improve the physical properties of the film, such as swelling degree and solubility due to heat, the polymeric substance of the detection layer according to the present invention may be partially mixed with other water-dispersible polymers,
That is, it can be replaced with polymer latex. Examples of preferable polymer latex include, for example, Japanese Patent Application Laid-open No. 5
Those described in JP-A No. 7-116258, JP-A-58-99752, etc. are useful. These polymeric latexes can replace up to 70% of the hydrophilic colloid binder, but preferably about 50% or less. [0035] The detection layer may include other additives, such as buffering agents,
Preservatives, surfactants, mordants, etc. can be added depending on the purpose. Further, the film thickness is 3 to 50 μl, preferably 5 to 30 μl. A buffer is included to maintain a pH suitable for specific binding reactions, enzyme reactions, coloring reactions, and the like. As a buffering agent that can be used, Nippon Kagaku Kinsen "Chemical Handbook Basic Edition" (Tokyo, Maruzen Co., Ltd. 19)
66) pp1312-1320, N, E, Go
od etc., Biochemistry Vol 5,
p467 (1966), Kinzai, Saito, Chemistry Area, Vo
130(2), p79 (1976), W, J, F
ergus. n etc. Anal, Biochem, Vol 104,
Examples include those described in literature such as P300 (1980). 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. [0036] Preservatives are included to stabilize the storage of the substrate coloring reagent, and include antioxidants and the like. In order to maintain the activity of the label contained in the layer, the layer contains immobilized enzymes, adsorbents for affinity chromatography, immobilized antibodies, and preservatives used for preserving proteins, enzymes, and the like. The substance is Nihon Seikagaku Kinsen, “Biochemistry Experimental Account 1, Protein Chemistry 1” (Tokyo Kagaku Doujin Co., Ltd. 1976) pp6
6 to 67, Experiments and Applications [Affinity Chromatography Jpp16 to 104, JP-A-60-14992
Examples include those described in Publication No. 7. [0037] Specific examples include gelatin, gelatin decomposition products, albumin, cyclodextrins, non-reducing sugars (sucrose, 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 as the hardening agent, substances commonly used in the photographic industry can be used. , edited by JameSrThe
Theory of the Photo
Examples include those described in ``rapicProcessJ 4th,'' pp. 77 to 87. Specific examples include aldehydes, active olefins,
Examples include active esters. [0038] Surfactants 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 absorbance coefficient when the detection substance is a dye, or shifts the wavelength, and exhibits a strong interaction with the detection substance. Cationic polymers, anionic polymers and latexes of these polymers are used. [0039] 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. [00401 Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. [Comparative example] FAD-labeled theophylline (FAD-Theo) and apoglucose oxidase (apo-C0D)
is disclosed in Japanese Patent Application Laid-Open No. 58-46072 and US Patent No. 4.
238. It was prepared according to the method described in the specification of No. 565. 1 (1) Preparation of monoclonal anti-theophylline antibody The described antibody was prepared according to JP-A-58-46072. [0041] 50 g of 6-chloro-1,3-dimethyluracil and 37 g of 6-aminocaproic acid were added to 250 ml of anhydrous tanol, and 28 g of triethylamine was further added and reacted under heating under reflux for 200 hours. After distilling off the ethanol. A uracil derivative having a (5-carboxypentyl)imino group introduced therein was obtained by recrystallization from water. 36g of this compound
was added to 100 ml of water to suspend it, 14 g of sodium nitrite dissolved in 50 ml of water was added thereto, the mixture was reacted at 50° C. for 1 hour, and after cooling, it was collected by filtration. [0042] 32 g of this filtered material was added to 400 ml of methanol.
and reduced with hydrogen using platinum oxide as a catalyst. After filtering off the catalyst, the mixture was further reacted with methyl formate to obtain a formamidated uracil derivative. Next, put 18 g of this into 1 liter of Q-dichlorobenzene,
By heating under reflux for 4 hours under nitrogen atmosphere, 9-(5
Carboxypentyl)-1,3-dimethylxacytin 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. [00431 Approximately 6 week old Balb/c mice (male) were immunized with the above immunogen. That is, 200 μg of the above immunogen was made into an emulsion using complete Freund's adjuvant and injected into the abdominal cavity of a mouse. After 2 weeks, 150μg
The immunogen was made into an emulsion using incomplete Freund's adjuvant and injected into the peritoneal cavity of a mouse. Furthermore, 1100 u of immunogen dissolved in 0.15 M saline was injected intravenously after 2 weeks. Three days later, the lungs were removed and tile cells were collected. These tile cells (4 x 102 cells) and azaguanine-resistant mouse myeloma cells X63-Ag8-6.5.3
(1.2 x 103 cells) and cell fusion was performed using 50% polyethylene glycol 4000 (manufactured by Merck & Co.). Ta. The fused cells were plated in a 96-well plate and treated with 15% fetal bovine serum, penicillin (50 units/m I) and i-
HA containing leptomycin (50 u g/m I)
T medium (0°RPMI-1640 containing 4 μM aminopterin, 16 μM thymidine, 100 μM hypoxanthine)
cultured in medium). Add half of the medium every 2 days to fresh HA.
After 2 weeks, the medium supernatant of each well was replaced with T medium.
It was assayed by the isa method, and positive cells 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. [00441 200 mg of bovine serum albumin was further added to 0.8 ml of this mouse ascites, freeze-dried, and used in a multilayer analytical element. 1- (2) Preparation of apoglucose oxidase To 50 mg of freeze-dried apoglucose oxidase, add 5 ml of anti-glucose oxidase antibody obtained by immunizing a goat with glucose oxidase and 250 mg of bovine serum albumin, freeze-dry, and perform multilayer analysis. Used in the device. 1-(3) Creation of analytical element for measuring theophylline Coating liquid (1) with the following composition was applied onto a transparent subbed polyethylene terephthalate film with a thickness of 180 μl.
It was dried to form a detection layer. The film thickness after drying is 22μ.
It was l. [0045] Coating liquid (1) Deionized gelatin
4.5g Triton X-100, 5g glucose (manufactured by Tokyo Kasei Co., Ltd.)
540mg3, 3', 5. 5"
-Tetramethylbenzidine (manufactured by Mawashi Kagaku Kenkyusho) 90m
gGantledge ES-225 (manufactured by GAF)
1. 6g peroxidase (manufactured by Boehringer) 3000
uFAD-labeled theophylline (100 u g/ml aqueous solution) 1.20 μl 1.5 M citric acid-dipotassium hydrogen phosphate buffer (pH 7.0)
2.0g1,2bis(vinylsulfonyl)ethane
30mg distilled water
46. 0g
Next, on the layer of coating solution (1), the following composition was applied, in which the anti-theophylline antibody containing bovine serum albumin prepared in 1-(1) and 1-(2) and apoglucose oxidase were uniformly dispersed. Coating liquid (2) was applied and dried to form a non-fibrous porous reaction layer. The film thickness after drying was 150 μl. [0046] Coating liquid (2) Triton X-1000, 4g Anti-theophilic antibody (lyophilized product)
200mg anti-glucose oxidase antibody containing apoglucose oxidase (lyophilized product)

380mg polyvinylpyrrolidone (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 1. 8g Avicel (manufactured by Asahi Kasei)
11.0gn-butanol
34.0g Furthermore, coating liquid (3) with the following composition was applied on this porous reaction layer.
was applied and dried to form a fibrous porous spread layer. The film thickness after drying was 200 μl. [00471 Coating liquid (3) Triton X-1001.8g Polyvinylpyrrolidone
1.8g Powder Paper D (manufactured by Toyo Roshi Co., Ltd.)
18.0gn-
butanol
68.0 g The thus constructed sample was cut into a size of 1.5 cm x 1.5 cm to prepare an analytical element for comparison. [0048] [Example 1] Coating liquid (1)
★★FAD-labeled theophylline (180 μg/ml aqueous solution) was removed from the coating solution (1) of Comparative Example 1, and coating was performed in the same manner. Note that the film thickness was 22 μl. Coating solution (2) Triton X-1000, 4g Apoglucose oxidase containing anti-glucose oxidase antibody (lyophilized product)
380mg polyvinylpyrrolidone (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)
1. 8g Avicel (manufactured by Asahi Kasei)
11.0gn-
butanol
34.0 g of coating liquid (2) having the above composition was applied onto the layer of coating liquid (1) and dried to form a non-fibrous porous layer. Note that the film thickness of ☆☆ after drying was 60 μl. [0049] Coating liquid (3) Triton X-1000, 4g Anti-theophylline antibody (lyophilized product)
200mg polyvinylpyrrolidone (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 1: 8
g Avicel (manufactured by Asahi Kasei)
11.0gn-butanol
34.0
g Coating liquid (3) having the above composition was applied onto the layer of coating liquid (2) and dried to form a non-fibrous porous layer. The film thickness after drying was 60 μl. [00501 Coating solution (4) 500 mg of bovine serum albumin was added to 12 μg of FAD-labeled theophylline, and the lyophilized product was added to the coating solution (3) of Comparative Example 1, and this was coated on the layer of coating solution (3). , dried. The film thickness after drying was 200 μl. The sample configured in this way is 1.5cmX1.5c◆◆
The sample was cut into a size of m to obtain a multilayer analytical element-1 of the present invention. + [00513 [Example 2] Coating liquid (1) The same coating liquid (1) as in Example 1 was applied and dried. Coating Liquid (2) The same coating liquid (2) as in Comparative Example 1 was applied and dried. [0052] Coating liquid (3) Triton X-1000, 4g Polyvinylpyrrolidone (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)
1. 8g Avicel (manufactured by Asahi Kasei)
11.0g
n-butanol
34.0gFAD labeled theophylline 1
A lyophilized product of 2 μg and 500 mg of bovine serum albumin was added, and this was applied onto the coating liquid (2) layer and dried. The film thickness after drying was 60 μl. [0053] Coating liquid (4) The same coating liquid (3) of Comparative Example 1 was applied onto the layer of coating liquid (3), dried, and the sample thus constituted was coated in a 1.5 cm x 1.5 cm layer. It was cut into a size and used as a multilayer analytical element-2 of the present invention. [Example 3] Coating liquid (1) Coating liquid of Example 1 (1) Coating liquid (2) Coating liquid of comparative example (2) Coating liquid (3) Coating liquid of Example 1 (4) The above The sample was coated and dried in this order, and the thus constructed sample was cut into sizes of 1.5 cm, Xl, and 5 cm to obtain a multilayer analytical element-3 of the present invention. [0054] [Example 4] Coating liquid (1) Coating liquid (1) of Example 1 Coating liquid (2) Coating liquid of Comparative Example 1 was obtained by adding 500 mg of bovine serum albumin to 12 μg of FAD-labeled theophylline and freeze-drying the mixture. (2), and this was applied onto the layer of coating liquid (1) and dried. The film thickness after drying was 62 μl. [0055] Coating liquid (3) The same coating liquid (4) of Example 1 was applied onto the layer of coating liquid (2) and dried, and the sample thus constituted was coated with 14
It was cut into sizes of 5 cm, Xl, and 5 cm, and was used as a multilayer analytical element-4 of the present invention. [0056] [Characteristics] [Measurement of theophylline] Theophylline (Aldrich Co., Ltd.) at a concentration of 5 to 45 μg/ml dissolved in 50 mM citric acid-dipotassium hydrogen phosphate buffer (pH 5.0) containing 5.0 wt% bovine serum albumin. 10 µl of the above-mentioned five types of analytical elements was added dropwise to the above-mentioned 5 types of analytical elements, and incubated at 37°C in a sealed state for 30 µl.
The reflection density at 650 nm was measured from the support side every second for 15 minutes. [0057] The reflection density after 15 minutes was as follows, setting the theophylline concentration of 0 μg/ml as 100. That is, when the theophylline concentration is 5 μg/ml, the comparative analytical element has a concentration of 122, and the multilayer analytical element-1 of the present invention has a concentration of 12.
5. 123 in multilayer analytical element-2 of the present invention, 121 in multilayer analytical element-3 of the present invention, multilayer analytical element-4 of the present invention
Then, the theophylline concentration is 15 It
g/m1, 161 for the comparative analytical element, 160 for the multilayer analytical element-1 of the present invention, 164 for the multilayer analytical element-2 of the present invention, and 16 for the multilayer analytical element-3 of the present invention.
2. In the multilayer analytical element-4 of the present invention, it is 160, and
Theophylline concentration is 25 μg/m], 204 for the comparative analytical element, 199 for the multilayer analytical element-1 of the present invention, 208 for the multilayer analytical element-2 of the present invention, 2O2 for the multilayer analytical element 3 of the present invention, and 2O2 for the multilayer analytical element-2 of the present invention. Multilayer analytical element of the invention
4 is 200, and the theophylline concentration is 35ug/
ml, 209 for the comparative analytical element, 230 for the multilayer analytical element-1 of the present invention, and 230 for the multilayer analytical element-2 of the present invention.
In the multilayer analysis element-3 of the present invention, it is 240, in the multilayer analysis element-3 of the present invention it is 231, in the case of the theophylline concentration 45 μg/ml, it is 212 in the comparative analysis element, and in the multilayer analysis element of the present invention it is 240. 261 for element-1, 277 for multilayer analytical element-2 of the present invention, 260 for multilayer analytical element-3 of the present invention, and 27 for multilayer analytical element-4 of the present invention.
It was 0. [0058] From this, the multilayer analytical element of the present invention is as follows:
It can be seen that the concentration of theophylline can be identified with high sensitivity even in a small sample solution of 10 μl, and the analytical element of the present invention has higher discrimination power than the comparative analytical element, especially in the high concentration region. [0059]

[Effects] According to the present invention, specific components in a fluid sample can be detected easily and with a small amount of sample solution, with high sensitivity, accuracy, and
Can be quantified with good accuracy and reproducibility.

Claims (2)

[Claims] Claim 1: A specific component in a fluid sample can be specifically bound to a label in which either the specific component or an analog of the specific component is bound to a labeling substance, and both the specific component and the label. An analytical element that performs analysis by a homogeneous competitive reaction with a substance, and this analytical element has three layers formed on a support in order: (1) a non-porous layer, (2) a non-fibrous porous reaction layer, and (3) a fibrous porous layer. 1. An immunological analysis element comprising at least a sex-developing layer and containing the label in a layer farther from the support than the non-porous layer of (1). 2. For analysis of specific components in a fluid sample, at least (1) a non-porous layer, (2) a non-fibrous porous reaction layer, and (3) a fibrous porous spreading layer are sequentially formed on a support. analysis by a homogeneous competitive reaction between a labeled substance in which either the specific component or an analog of the specific component is bound to a labeled substance, and a substance that can specifically bind to both the specific component and the labeled substance. , and the label is contained in a layer farther from the support side than the non-porous layer of (1) above, using an immunological analysis element, the fluid sample solution 5 is
An immunological analysis method characterized in that an amount in the range of μl to 20 μl is applied.