JPH04164254A - Immunological multilayer analyzing element and analyzing method - Google Patents

Abstract

PURPOSE:To obtain optimum reactivity within a limited time, and to enable detection sensitivity to be heightened by providing an analyzing element with a hydrophilic polymeric nonporous reaction layer containing a substance that can be peculiarly combined with both a specific component and a labeled body. CONSTITUTION:A hydrophilic polymeric nonporous reaction layer is a layer in which homogeneous competitive reaction is carried out, and is provided separately from a fibrous porous developing layer. As applicable labeled materials, flavin adenine dinucleotide, nicotinamide adenine dinucleotide and its reductant, nicotinamide adenine dinucleotide phosphate and its reductant, adenosine phosphate, and flavin mononucleotide are preferably given. By impregnating a labeled body into a nonporous layer consisting of polymeric substances, the reaction of the labeled body with a substance that can be peculiarly combined with both a specific component and the labeled body can be arbitrarily delayed, so that the specific component can be caught and measured in a high- sensitive time zone.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an analytical element for analyzing a specific component in a fluid sample, and in particular to an analytical element for analyzing a specific component in a biological fluid sample. The present invention relates to an analytical method and an analytical element for measuring by a homogeneous competitive reaction between a label and a substance capable of binding to a substance.

[Conventional technology]

Various analytical methods have been developed as methods for detecting extremely small amounts of substances contained in biological fluid samples. The analytical method is mainly based on the immune reaction. Although various measurement methods have been developed using the above principle, immunoassay is known as the most accurate method.

The immunoassay method was developed in 1958 by Berson.
) and Yallov succeeded in measuring insulin in serum using radioactive iodine-labeled bovine insulin and anti-insulin antibodies in the serum of diabetic patients. Radioimmunoassay has been widely used since then. It is being

Since then, various labeling substances other than radioactive isotopes have been developed. Other labeling substances include, for example, enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, circulating reactants, metal and organometallic complexes, organic prosthetic groups, chemiluminescent reactants, and fluorescent molecules. Can be mentioned.

One of the important technical problems regarding the above immunoassay is the separation of substances that have bound (hereinafter abbreviated as B) and substances that have not bound (hereinafter abbreviated as F) (hereinafter referred to as B/
(abbreviated as F separation).

Conventionally, various assay methods have been developed to solve the problems of B/F separation in immunoassays.

For example, JP-A-51-446295, JP-A-55-299
There are homogeneous immunoassay methods described in No. 7, No. 58-211662, No. 54-20134, No. 55-104896, No. 57-150681, etc. That 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. , which shows a detectable modulation response in the case of no binding, 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 reliable measurement results, and reproducibility is not sufficient. 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 the complexity of the liquid delivery mechanism and the prevention of sample contamination. This requires a special mechanism and is a very expensive device. In addition, 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, and requires skill in collection and operation.

[Problem that the invention seeks to solve]

On the other hand, in place of such wet analysis, a dry analysis method has been devised that allows quick measurement with simple operations.

For example, JP-A-57-103055 describes a single-layer analytical element 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 method, 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 effects of large amounts of stray light and noise are unavoidable, resulting in poor measurement accuracy. Reproducibility is not sufficient.

For example, if only one of the two reagent layers is a non-fibrous porous reaction layer, the maximum sensitivity has already been reached within the time required for measurement, which is unavoidably determined due to operational and other constraints, e.g. 3.5 to 7 minutes. On the other hand, due to the layer structure, sensitivity may not yet be achieved at high concentrations of the label.

Another problem is that when proteins such as enzymes and antibodies are brought into contact with organic solvents, denaturation and activity deterioration may occur, and the operation for finely dispersing them is also complicated.

Furthermore, the analytical multilayer element described in JP-A-58-18167 requires a large amount of sample solution ranging from 25 .mu.a to 200 .mu..rho. In order to drip and measure such a large amount of sample solution onto an analytical element, the analytical element must contain a large amount of immune reaction reagent in proportion to the amount, which makes the analytical element expensive. I don't get it.

Furthermore, if the sample solution or liquid is serum or plasma, for example, a large amount of blood must be collected, which may be undesirable in terms of blood collection time, response to the blood sample, etc.

[Purpose of the invention]

The present invention eliminates the above-mentioned drawbacks of the prior art, and an object of the present invention is to achieve the highest sensitivity with a simple operation and a small amount of sample solution, to adapt to the measurement time, and to provide a fluid solution with excellent accuracy and reproducibility. An object of the present invention is to provide a multilayer analysis element for quantifying a specific component in a sample, and an analysis method using the multilayer analysis element.

[Means for solving problems]

The gist of the present invention is to target a specific component in a fluid sample by applying a label in which a labeling substance is bound to either the specific component or an analogue of the specific component, thereby making it specific for both the specific component and the label. A specific multilayer analytical element is used for measurement by a homogeneous competitive reaction for a substance that can specifically bind to a substance, and the multilayer analytical element is capable of specifically binding to both the specific component and the label. This is a multilayer analytical element characterized by having a hydrophilic polymer non-porous reaction layer containing a substance.

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 No. 997 can be advantageously used.

In the multilayer analytical element of the present invention, the hydrophilic polymer non-porous reaction layer is a layer that allows the above-mentioned homogeneous competitive reaction to occur, 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. In addition, by incorporating the label into a non-porous layer made of a polymeric substance,
It is possible to arbitrarily delay the reaction between the labeled body and a substance that can specifically bind to both the specific component and the labeled body, making it possible to capture and measure the specific component in a highly sensitive time period.

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, amniotic fluid, milk, urine, etc. Examples include sweat and meat juices.

The specific components in a fluid sample that can be measured by the present invention are:
A substance or group of substances whose presence or absence or amount in a fluid sample is detected, and for which there may be a substance that specifically binds to that particular component. 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 or substance groups can be mentioned, but are not limited to these.

Hormones 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 (MSH), vasopressin, oxytocin, insulin, glucagon, angiotensin ■ and ■, prolactin, secretin, dopamine, serotonin, somatostatin, thyroxine (T1)
, triiodothyronine (T3), gastrin, cortisol, aldosterone, catecholamines, estrogen, progesterone, testosterone, placental gonadotropins, placental lactogen, pituitary hormone releasing factor (TRH).

FSH-RH, CRH, LH-RH, etc.) etc. (vitamins) Biotin, thiamin, vitamin A5, vitamin B2, vitamin B6, vitamin B1□, vitamin C1 vitamin D
1. Vitamin E, 1 hitamine, 1 folic acid, etc.

-9 = Various drugs and metabolites> pensoylecgonine, cocaine, coteine, textrometrophan, heroin, lysergic acid, morphine, quinidine, quinine, amikacin, gentamicin, kanamycin, neomycin, tobramycin, actinomycetin, Caloimycin, chloramphenicol, chloromycetin, chlortetracycline, erythromycin, oxytetracycline, penicillin, cephalosporin, polymyxin B1 terramycin, tetrazycline, streptomycin, diphenylhydantoin, ethosuximide, phenobarbicur, brimidone, secobarbiquor , acetaminophen, amitributyrin, carbamazepine, digoxin, dinviramide, lidocaine, methotrexate, N-acetylprocainamide, phenytoin, procainamide, Progra 10-
le, theophylline, canapinol, tetrahydrocanapinol, cholinergic drugs, antihistamines, atropine, butyrophenone, caffeine, chlorolO-promacine, pulpyrate, amphetamines, catecholamines, epinephrine, griseofulvin, imipramine, L-dopa, meperidine, mesolobamate, Metatone, narcein, nortributyrin, oxazepan, pababerin, prostaglandin, tegretol, valproic acid, etc. and their metabolites.

<Pesticides> Halogenated biphenyls, phosphoric esters, thiophosphates, and their metabolites.

Substances that can be used in the present invention that specifically bind to a specific component in a fluid sample include antibodies, antigens, lectins, protein A, etc., depending on the target to be measured. Particularly preferred 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 sodium sulfate obtained by a conventionally known method. Precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chroma l-clay method, electrophoresis method, etc. (Shunsuke Migita ed. "Immunochemistry" Nakahaku Shoten pp74-8)
8).

Alternatively, a monoclonal antibody is created by obtaining miscellaneous tumor cells (hybridoma) from lung cells of a mammal (e.g., mouse) sensitized with an antigen and myeloma cells (myeloma), and a substance that can specifically bind to the specific component. It is preferable to use it as the specificity improves.

In addition, these antibodies can be used as IgG, IgM, and IgA antibodies, or they can be treated with enzymes to produce active antibody fragments such as Fab, Fab', or F(ab')2. You can.

Furthermore, these antibodies may be used alone or in combination.

In order to contain these antibodies in a non-porous reaction layer, it is necessary to contain them while retaining the ability to specifically bind to a specific component of the antibody. After mixing with normal serum proteins, albumin, gelatin and their decomposition products, etc., lyophilize and
It can be included.

Labeling substances that can be applied to the present invention include, for example, prosthetic groups and coenzymes, 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).

A labeled substance in which a specific component or an analog of a specific component is bound to the above-mentioned labeling substance is described by D. L. Morris et al.
, ;Analytical chemistry, v
o (153, p658-665 (1981), T, J
, Richard at a(1,; C11nic
al Chemistry.

voo, 27, p1499-1504 (1981
) and JP-A-55-2996, JP-A No. 58-46072
It can be created using the method described in No.

In order to detect a signal caused by a labeling substance in a homogeneous immunoassay applicable to the present invention, an apoenzyme that is combined with a prosthetic group or a coenzyme is required. As the apoenzyme, those described in JP-A-55-2997 and JP-A-57-103055 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, etc. can be mentioned. Preferably it is glucose oxidase. When using apoglucose oxidase, in order to stabilize the enzyme, JP-A-57-166980
It is desirable to use the anti-glucose oxidase antibodies described in No.

Furthermore, it is advantageous to quantify the activity of these enzymes colorimetrically using a color reaction. For example, to explain glucose oxidase as an example, if glucose is M'Jt,
A widely used method is to detect hydrogen peroxide produced by glucose oxidase by using peroxidase as a catalyst to form a detectable dye.

As a coloring reaction reagent, the following compound I4- can be used.

Aniline derivatives such as 0-dianindine 0- and p-toluidine, 0-7 enylene diamine, N, N'-dimethyl-p-phenylene diamine, benzidine, 3.3', 5.5'-tetramethylbenzidine, etc. Aromatic diamines Phenol derivatives such as catechol, guaiacol, orcinol, and pyrogallol Aromatic carboxylic acids such as salicylic acid, pyrocatechinic acid, and gallic acid Leuco dyes such as leucomalachite green and leucophenolphthalene 4-aminoantipyrine and phenol Combination of 3-methyl-2-benzothiazoline hydrazone with naphthol derivatives and N,N
'-The combination of p-anisidine and 8-dimethylaniline
Combination with hydroxyquinoline 2.2'-azinodi(3-ethyl-6-sulfobenzothiazoline) 2-(4-hydroxy-3-methoxyphenyl) -4
, 5=his(p-dimethoxyaminophenyl)imidazole, etc.

These color-forming reaction reagents can be dissolved in water or an organic solvent and easily incorporated into the detection layer.

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 signal caused by the label can be detected using an absorbance method (colorimetric method), a fluorescence method, or a luminescence method.Measurement methods include a rate measurement method that measures changes in the signal over time, or a signal after a certain period of time. Endpoint assays can be used to measure . Preferred is an absorbance method. In the absorbance method (colorimetric method), ultraviolet light, visible light, and near-infrared light can be used. For example, when serum and plasma are used as fluid samples, serum and plasma In order to reduce the influence of light absorption, it is preferable to use green light, red light, or near-infrared light.

The multilayer analytical element according to the present invention is preferably manufactured by laminating it on a light-transmitting support. Examples of the support that can be used in the present invention include cellulose acetate, polyethylene terephthalate, polycarbonate, and polyvinylimida (e.g. Polymer compounds such as polystyrene (polystyrene) or transparent inorganic compounds such as glass may be used.

The fibrous porous spreading layer according to the present invention is a layer for spreading a fluid sample solution and uniformly supplying it to the reaction layer, and its materials include, for example, pulp, powder filter paper, cotton, hemp, rope, etc.
Wool, chitin, chitosan, cellulose ester, viscose rayon, copper ammonia rayon, polyamide (6
- nylon, 6-row nylon, 610-nylon, etc.)
, polyester (such as polyethylene terephthalate),
Polyolefins (polypropylene, vinylon, etc.), glass.

Fibers such as asbestos, for example, vegetable, animal, mineral, synthetic, semi-synthetic, and recycled fibers can be used, and a mixture of these may also be used. In addition, absorbent Western paper, Japanese paper,
It is also possible to use filter paper, plush polymers, or woven fabrics, nonwoven fabrics, synthetic papers, etc. made of the above-mentioned fibers alone or in combination.

The non-porous reaction layer of the present invention is a layer that allows a homogeneous competitive reaction to occur, and contains a substance capable of specifically binding to both a specific component and a label, the apoenzyme, and the like.

Furthermore, the development layer and reaction layer according to the present invention may contain proteins that are not involved in the specific reaction to be measured in order to eliminate non-specific reactions in the immune reaction, if necessary. Typical examples include mammalian normal serum proteins, albumin, gelatin, and their decomposition products.

At least one type of hydrophilic colloid having film-forming properties is used as a material for the non-porous layer made of a polymeric substance containing the label of the present invention.

Examples of hydrophilic colloids include gelatin, gelatin derivatives such as phthalated geladine, synthetic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinylimidazole, polyacrylamide, and storium polyacrylate, and cellulose such as hydroxyethyl cellulose and carboxyl methylcellulose sodium salt. This includes derivatives such as polysaccharides and even sodium alginate. Preferably, gelatin derivatives such as gelatin and phthalated gelatin are used.

By coating and/or forming a film with these hydrophilic colloids, a non-porous layer made of the polymeric substance according to the present invention can be formed. To contain the labeled substance,
It can be easily incorporated by dissolving it in water or an organic solvent.

Further, in the multilayer analytical element according to the present invention, a detection layer may be provided in which the non-porous layer made of a polymer substance contains the coloring reagent for detecting a signal caused by a label.

Of course, the coloring reagent can be contained in the non-porous layer made of a polymeric substance containing a label.

Furthermore, the polymer substance of the detection layer according to the present invention has film physical properties,
For example, in order to improve the degree of swelling and thermal solubility, a part of the polymer can be replaced with another water-dispersible polymer, that is, a polymer latex. Examples of preferable polymer latexes include those disclosed in JP-A-57-116258 and JP-A-57-116258;
-99752 is useful. These polymer latexes contain up to 70% of the hydrophilic colloid binder
It is possible to substitute, but preferably about 50% or less substitution.

Other additives such as buffers, preservatives, surfactants, mordants, etc. can be added to the non-porous layer or the detection layer according to the present invention depending on the purpose.

A buffer is included to maintain a pH suitable for specific binding reactions, enzymatic reactions, coloring reactions, and the like. As a buffering agent that can be used, Nippon Kagaku Kinpan "Chemical Handbook Basic Edition" (Tokyo,
Maruzen Co., Ltd. 1966) p1312-1320, N.

E., Good et al.; Biochemistry (Beoch
emistry), v.

Q5. p467 (1966), Imamura, Saito; Chemistry Area, vo1230(2), p79 (1976), W.
J, Ferguson et al., Anal
,Biochem,,vo(2104,p300(19
Examples include those described in documents such as 80). Specific examples include citrate, borate, phosphate, carbonate, trisbarbyl, glycine, and Gud 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. 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 Nippon Biochemistry Kinpei [Biochemistry Experiment Course I, Protein Chemistry IJ (Tokyo Kagaku Dojin Co., Ltd. 1976) p66-67,
Experiments and Applications "Affinity Chromatography" p16~
104, and those described in JP-A-60-149927.

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, it is possible to use substances commonly used in the photographic industry. Deki, T, H, James (T, H
, James) ed. “The Theory of the Photographic Process J”
thePhotographic Process) (
4th), p77-87. 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 labeled substance, the elution and diffusion of the labeled substance into the porous reaction layer can be arbitrarily controlled. It is useful to delay reactions with substances that can bind to.

Typical examples of surfactants that can be used as surfactants include Triton X-100 (Octylphenoxypolyethoxyethanol manufactured by Mu Haas) and Surfactant IOG (manufactured by Olean). There are nonionic surfactants such as glycidol and ionic surfactants.

The surfactants can be used in widely selected amounts, ranging from 2Qwt% to 0.5Qwt% based on the weight of the granules.
0.05 wt%, preferably 15 wt% to 0.1 wt%. Still other methods include ultrasonication, physical mixing, physical stirring, and pH adjustment of the particle units and the liquid carrier. These are even more useful in combination with the methods described above.

Other reagents contained in the layer include a solubilizing agent, a pro-7ka reagent, and the like. These additives are added in appropriate amounts as necessary. A mordant is a substance that concentrates the detection substance in the detection layer for enzyme activity measurement, increases the absorbance coefficient, or shifts the wavelength in the case of the detection substance or dye, and has a strong interaction with the detection substance. The following 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 can be easily 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.

FAD-labeled theophylline and apoglucose oxidase are described in Japanese Patent Application Laid-Open No. 58-46072 and US Pat.
It was prepared according to the method described in No. 238,565.

Example 1 l-(1) Preparation of monoclonal anti-theophylline antibody 6-chlor-1 in absolute ethanol 250+1112
, 50 g of 3-dimethyluracil and 37 g of 6-aminocaproic acid were added, followed by 28 g of triethylamine, and the reaction was carried out under heating under reflux for 200 hours. After distilling off the ethanol, it was recrystallized from water to obtain the (5-carboxypentyl)imino group. The introduced uracil derivative was obtained. 36 g of this compound was suspended in 100 ml of water, and 14 g of sodium nitrite dissolved in 50 ml of water was added thereto, reacted at 50° 0 for 1 hour, cooled, and collected by filtration.

32 g of this filtered material was dissolved in 400+110 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, this 18g
9-(5-carboxypentyl)-1,3-dimethylxanthine was obtained by placing the mixture in e o-dichlorobenzene 112 and heating under reflux for 4 hours under a nitrogen atmosphere. This compound was bound to bovine serum albumin and used as an immunogen according to the method described in JP-A-58-46072.

BaQb/c mice (female), approximately 6 weeks old, were immunized with the above immunogen. That is, 200 μg of the above immunogen was made into an emulsion using complete 70% indoor 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 incomplete 70-indoor adjuvant and injected into the peritoneal cavity of the mouse. After approximately 2 weeks, mice were injected intravenously with 100 μ9 of immunogen dissolved in 0.15 M saline. Three days later, the spleen was removed and the spleen cells were collected. These tile cells (4 x 1.0" cells) and azaguanine-resistant mouse myeloma cells X63-Ag8-6.5.3 (1.2 x 108
) and 50% polyethylene glycol 400
Cell fusion was performed using 0 (manufactured by Merck & Co.). 9 fused cells
Spread on a 6-well plate and add 15% fetal bovine serum, penicillin (50 units/mQ) and streptomycin (50 μg).
/mL) in HAT medium (RPMI-1640 medium containing 0.4 μM aminosterin, 16 μM thymidine, and 100 μM hypoxanthine). Half of the medium was replaced with fresh HAT medium every two days, and after two weeks, the medium supernatant of each well was assayed by the EQisa 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 mO of this mouse ascites was used in a multilayer analysis element.

1-(2) Anti-glucose oxidase antibody-containing 26
1. Preparation of apoglucose oxidase 25 mg of lyophilized apoglucose oxidase,
4ml (l) of anti-glucose oxidase antibody obtained by immunizing a goat with glucose oxidase was added 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 22μ
It was m. ) Coating liquid (1) Deionized gelatin 4.0g Triton X
-], 00 (Rohm-Haas M) 2.0 g Anti-theophylline antibody 3.2 mff Apoglucose oxidase containing anti-glucose oxidase antibody 4.0 mm Glucose (manufactured by Tokyo Kasei) 600 m1 + Peroxidase (manufactured by Boehringa) 700 U 1.2- Bis(vinylsulfonyl)ethane 7 mg Distilled water 21.0 g Next, apply coating liquid (2) with the following composition on this detection layer and dry it.
A non-porous layer consisting of a gelatin binder containing AD-labeled theophylline was formed. (The film thickness after drying is 10μ
It was m. ) Coating liquid (2) Deionized gelatin 2.0g Dryde 7
X-1001,0g FAD-labeled theophylline (JOOug/m (l aqueous solution)
600μα 1.2-bis(vinylsulfonyl)ethane 4m9 1.5M <Epic acid-dipotassium hydrogen phosphate buffer (pH 5,0) 3.6g3.
3', 5.5'-Tetramethylbenzidine 5 mg Gantrette ES-225 (manufactured by GA F) 1.8 g = 28- Distilled water 28.0 g Next, on top of this layer, a coating solution with the following composition was applied as an adhesive layer. (3) was applied and dried.

Coating liquid (3) N-vinylpyrrolidone-vinyl acetate copolymer (weight cost 2:8) 1.0 g Butanol 38.0 g Furthermore, coating liquid (4) having the following composition was applied onto this adhesive layer and dried. to form a fibrous porous spread layer. (The film thickness after drying was 280 μm.) Coating liquid (4) Triton x-100 (manufactured by Rohm-Haas) 9.1 g Styrene-glycidyl methacrylate copolymer (9: I) 23.0 g For filter paper raw material Fiber D (manufactured by Toyopo Paper Co., Ltd.) 90.0 g Xylene 280 g The sample coated in this way was cut into a size of 1-5 cm x 1.5 cm, and a plastic mount (2, 5 cm x 3.0 cm) and used as analytical element-1.

Comparative Example (1) (1)-(1) Creation of monoclonal anti-theophylline antibody Mouse ascites 0°8mQ obtained in the same manner as in 1-(1)
Further, 200 mg of bovine serum albumin was added to the mixture, lyophilized, and used in a multilayer analytical element.

(1)-(2) Preparation of apoglucose oxidase 250 mg of bovine serum albumin was added to 5 mff of anti-glucose oxidase antibody obtained in the same manner as in 1-(2), and the mixture was lyophilized and used in a multilayer analytical element.

(1) = (3) Creation of analytical element for theophylline measurement Coating solution (5) with the following composition was applied onto a transparent subbed polyethylene terephthalate film with a thickness of 180 μm, dried, and labeled body and coloring reagent were applied. A non-porous layer was formed containing the material. (The film thickness after drying was 25 μm.

) Coating liquid (5) Deionized gelatin 4.5g Triton X-100 (Rohm Haas & H) 0.5g Glucose (manufactured by Tokyo Kasei Co., Ltd.) 540mg3.
3', 5,5'-tetramethylbenzidine (manufactured by Anabolic Chemical Research Institute) 90mg Gauntlets ES
-225 (manufactured by GAF) 1.6g peroxidase (manufactured by Boehringa) 000U FAD-labeled theophylline (100μg 7mfl aqueous solution)
120p (1 1,5M < citric acid-dipotassium hydrogen phosphate buffer (pH 5
,0) 2.0g1.2-bis(vinylsulfonyl)ethane 0my Distilled water 46.0gNext,
Above this layer (1)-(1) and (1)-(2)
A coating solution (6) with the following composition in which anti-theophylline antibody containing bovine serum albumin and apoglucose oxidase prepared in step 1 was uniformly dispersed was applied and dried to form a non-fibrous porous reaction layer (after drying) The film thickness was 150 μm).

Coating solution (6) Triton
Wako Tsumugi Co., Ltd.) 1.8 g Avicel (Asahi Kasei Co., Ltd.) 11.0 g Butanol 34.0 g Furthermore, coating liquid (7) having the following composition was applied onto the porous reaction layer and dried to form a fibrous porous layer. A sexual development layer was formed. (The film thickness after drying is 200 μm.) Coating liquid (7) ) 7X-'100'
1.8g polyvinylpyrrolidone 1.
8 μ filter paper raw material fiber D (manufactured by Toyo Roshi Co., Ltd.) 18.0 g Butanol 68.0 g The sample coated in this manner was cut into a size of 1.5 cm x 1.5 cm to form an analytical element of comparative example (1). Toshitoko.

(1)-(4) Measurement of theophylline 5QmlJ containing 5.3 wt% bovine serum albumin
10 μl of theophylline (manufactured by Aldrich) with a concentration of 25 μg 1 mα was added dropwise to the analytical elements-■ and (1) prepared in 1(3) and (1)-(3) above, and incubated at 37°C in a sealed state for 30 The reflection density at 650 nm was measured from the support side every second for 7 minutes, and the reflection density at 1 minute, 4 minutes, and 7 minutes (Dr);
The concentration difference between Dr+, Dr, and between Dr,, 017 (
△Dr); △1) r(1-t+ and △DrL4-+,
Table 1 As is clear from Table 1, there is no big difference in the measurement results between analytical elements 1 and (1) in △DI'' (+-al).
There is a clear difference in the practical measurement time of 4 to 7 minutes,
When converted into sensitivity, it is about 3 times higher.

Examples 2 to 8 Analytical elements 2 to 8 using the same reagent as in Example 1 and covering a two-layer stack of non-porous upper and lower reagent layers with a fibrous porous spreading layer.
8 was prepared with the configuration shown in Table 2. For reference, the configuration of analytical element 1 was also described using the same notation method.

Table 2 (Note) apo-GOD; apoglucose oxidase containing anti-glucose oxidase antibody; anti-Theo; anti-theophylline antibody, FAD-T
heo; FAD-labeled theophylline, TMBZ; 3,3'
, 5,5'-Tetramethylbenzidine As a result of carrying out the same measurements as in Example 1 using these analytical elements, the results were almost the same as those of analytical element-1.

〔Effect of the invention〕

By providing at least one reagent layer and using a hydrophilic polymer as a binder in the core layer, it is possible to achieve the optimal reactivity for reaction detection within the measurement time range forced by external constraints. By increasing the detection concentration, the detection sensitivity can be tripled.

In addition, the hindrance caused by organic solvents to enzymes and antibodies is avoided, and simple ultrasonic dispersion is sufficient for fine dispersion, and freeze-drying is not necessary. Therefore, the activity of the reagent is stable and reproducibility is improved.
In order to utilize competitive reactions, the near-ideal multilayer analytical device and analytical method must be changed.

Claims (4)

[Claims] (1) A specific component in a fluid sample is combined with a label in which a labeling substance is bound to either the specific component or an analog of the specific component, and a substance that can specifically bind to both the specific component and the label. An analytical element for analysis by a homogeneous competitive reaction, characterized in that the analytical element has a hydrophilic polymer non-porous reaction layer containing a substance capable of specifically binding to both the specific component and the label. Immunological multilayer analysis element. (2) The immunological multilayer analysis element according to claim 1, wherein the labeling substance is a substance selected from the group consisting of prosthetic groups and coenzymes. (3) The immunological multilayer analysis element according to claim 1 or 2, wherein the labeling substance is flavin adenine dinucleotide. (4) An immunological analysis method in which 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 3 for analysis of a specific component.