JPH01203973A - Immunological analyzing element - Google Patents

Abstract

PURPOSE:To obtain an immunological analyzing element which does not degrade enzyme inhibiting power even when a buffer is used by using an org. silver deriv. as a material to modulate the signal occurring in a labeling material by specifically conjugating with the labeling material. CONSTITUTION:The org. silver deriv. is used as the specific modulating material of the analyzing element formed by incorporating either of the bioactive material (biomaterial) which does not conjugate with the specific component in a fluid sample or the material which can conjugate specifically with the biomaterial and the material (specific modulating material) which modulates the signal occurring in the labeling material by specifically conjugating with the labeling material respectively into a part or the whole of a porous reaction layer. This org. silver deriv. reacts specifically with a thiol group with high affinity. The material failing to react directly or indirectly with the material which can conjugate specifically with the specific component in the fluid sample is, therefore, trapped specifically with the high affinity by the org. silver deriv. if the org. silver deriv. is used as the specific modulating material and enzyme having the thiol group as the labeling material.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an analytical element for measuring trace components in a fluid sample, and particularly relates to an analytical element for analyzing a specific trace component in a biological fluid sample. .

[Conventional technology]

Various analytical methods have been developed to detect 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 Yallow 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, batatteriophages, cycling 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 methods have been developed to solve problems in immunoassay methods (for example, Japanese Patent Application Laid-Open No. 53-386
No. 19, No. 53-79024, No. 55-90859, No. 57-67860, No. 57-200862, No. 58-18167, No. 59-77356, and No. 59
-170768 etc.).

However, these methods have incomplete B/F separation.
There are disadvantages such as a lot of noise, problems with signal reliability, and measurable substances are limited to low-molecular substances.

[Problem that the invention seeks to solve]

On the other hand, in wet chemistry, an immunoassay method using a competitive method using a stationary phase has been developed (for example, JP-A-58-209994 and JP-A-59-202064).
.

However, in these measurement methods, competitive reactions are performed between two substances fixed on a carrier in a separate stationary phase in a relatively large amount of aqueous solution, and the substance in the solution, so that the intended binding may not occur. However, since the overall enzyme activity is measured without distinguishing between the two stationary phases, the results may not be satisfactory in terms of sensitivity, accuracy, and reproducibility due to background and noise problems. I can't get it.

On the other hand, in dry chemistry, an immunoassay method using a second antibody has been developed (Japanese Patent Laid-Open No. 57-32776).
No. 57-32767).

However, these methods have room for improvement, such as the complicated operations and the need for techniques for deploying them with good reproducibility. Furthermore, in the invention described in JP-A No. 59-34155,
A method using an unbound material storage sheet is disclosed. However, even with this method, the above-mentioned problems will occur if you try to perform measurements with the reaction sheet and unbound substance storage sheet in close contact with each other. becomes an obstacle when automating.

As a means to solve these conventional B/F separation problems, a method using a substance (hereinafter referred to as a specific modulator) that specifically binds to an unreacted label substance and modulates the signal caused by the label substance has recently been proposed. has been invented.

This technology is disclosed in Japanese Patent Application Laid-Open Nos. 61-292060 and 62-90.
No. 539.

No. 62-90538, No. 62-247256, No. 6
No. 2-249063, No. 62-249060, No. 62
-267666, 62-267667, patent application No. 6
1-280166 etc., Sarabanko,
It has been found that it is preferable to use allylmercury derivatives as the substance, and is disclosed in JP-A-62-249060 and JP-A-62-249060.
No. 247256, No. 62-249063, Patent Application No. 1983
-280166, 62-157002, 62-1
It was proposed in No. 67342 and has a rate of 17%. However, if commonly used buffers such as phosphate, glycine, citric acid, etc. are used as a buffering agent for the purpose of retaining enzyme activity, the enzyme inhibiting ability of the allylmercury derivative may be significantly reduced. found.

[Purpose of the invention]

An object of the present invention is to provide an immunological analytical element whose enzyme inhibiting ability does not decrease even when the buffer is used.

[Means for solving problems]

The gist of the present invention is to provide either one of a biologically active substance that does not bind to a specific component in a fluid sample (hereinafter referred to as biological substance) or a substance that can specifically bind to the biological substance, and a labeling substance. A substance that modulates the signal caused by the labeled substance (specific modulating substance) is used as a specific modulating substance in an analytical element that is contained in a part or all of the porous reaction layer, respectively. This is an analytical element characterized by using a silver derivative.

The organic silver derivative used in the present invention reacts specifically and with high affinity with thiol groups.

On the other hand, many enzymes have a thiol group in their molecular structure, particularly in the sites involved in activity. Such an enzyme binds to the above-mentioned organic silver derivative and undergoes a change in its activity. (
For example, "Enzyme Handbook" supervised by Bunji Maruo and Nobuo Tamiya.
(Shokusho Shoten, 1982)) Therefore, by using an organic silver derivative as a specific modulating substance and an enzyme having a thiol group in the site involved in such activity in the labeling substance, specific components in a fluid sample can be specifically identified. The labeling substance that has not reacted directly or indirectly with the substance that can bind to the organic silver derivative can be specifically and with strong affinity trapped by the organic silver derivative, and the activity of the labeling substance can be changed. Therefore, B/F separation within the analytical element can be performed reliably and efficiently. In addition, as mentioned above, there are many enzymes that have thiol groups in the sites involved in their activity, so a wide range of enzymes can be selected as labeling substances, so we have established a system using -20% organic silver derivatives as specific modulators. For example, even if it is necessary to change the labeling substance due to the addition of an analyte to be measured, it can be immediately applied to this system as long as the enzyme as the changed labeling substance undergoes a change in activity due to the organic silver derivative. , it is quite advantageous for expanding the measurement items.

A method for incorporating the organic silver derivative into an analytical element includes a method in which it is dispersed in a binder made of a hydrophilic colloid and applied.

The hydrophilic colloids used as the binder include gelatin, gelatin derivatives such as heptalized gelatin, agarose, pectin, alginic acid, polyvinyl alcohol, polyvinylpyrrolidone, polyvinylimidazole, polyacrylamide, and synthetic polymers such as sodium polyacrylate. Examples include polysaccharides of cellulose derivatives such as ethyl cellulose and carboxymethyl cellulose sodium salt. Preferred examples include gelatin, gelatin derivatives such as phthalated gelatin, polyvinylpyrrolidone, and agarose.

Furthermore, a portion of the binder according to the present invention can be replaced with another water-dispersible polymer, ie, a polymer latex, in order to improve the physical properties of the film, such as swelling degree and thermal solubility. As preferred examples of polymer latexes, those described in JP-A-57-116258 and JP-A-58-99752 are useful. These polymeric latexes can replace up to 70 percent of the hydrophilic colloid binder, but preferably about 55 percent or less.

Further, as a method for preparing the above-mentioned dispersion, methods known in the art such as ultrasonic dispersion and sand stirrer dispersion can be used.

Furthermore, when using polyvinyl alcohol, polyvinylpyrrolidone, polyvinylimidazole, polyacrylamide, sodium polyacrylate, etc. among the above-mentioned binders, in addition to water, methanol, ethanol, butanol, isoglopanol, tetrahydrofuran, acetone, methyl ethyl ketone, acetic acid can be used as a solvent. An organic solvent such as ethyl can be used depending on the solubility of the binder, and this is a preferred embodiment in terms of preventing undesirable diffusion of water-soluble components contained in the lower layer of the organic silver derivative layer.

Moreover, as another embodiment, it is also possible to disperse the organic silver derivative together in the coating liquid for the porous reaction layer, which will be described later.

Furthermore, although the organic silver derivative-containing layer contains a large amount of highly hydrophobic organic silver, it does not allow the fluid sample to freely permeate.
It is necessary to ensure diffusion.

For this purpose, it is also a preferred embodiment to disperse a surfactant and/or hydrophilic or water-soluble fine particles such as microcrystalline cellulose, Sepharose, Sephacryl, Sephadex agarose, dextrin, dextran, sucrose, mannitol, etc., together with the organic silver derivative. It is.

Further, the particle size of the organic silver derivative contained in the above-mentioned dispersion is preferably o, oot~30μ, more preferably 0.01~30μ.
It is preferable to use a particle size of 32 m.

The organic silver derivative used as a specific modulator in the present invention is an organic compound, Brønsted acid.

It is a silver compound with a base, a Lewis acid, or a base, and preferably has low solubility in water.
It is preferable that 5 g or more of the Toyoki Silver Derivative be dissolved in g, and it is even more preferable that 3 g or more of the Toyoki Silver Derivative should not be dissolved in g.

Organic compounds used in the preparation of organic silver derivatives include substituted or unsubstituted aliphatic hydrocarbon carboxylic acids having 1 to 36 carbon atoms (for example, acetic acid, butyric acid, caproic acid, palmitic acid, linoleic acid, aspartic acid, henic acid, oxalic acid, etc.),
Substituted or unsubstituted aromatic compounds (e.g. benzoic acid, phthalic acid, phenol, etc.) Substituted or unsubstituted heterocyclic compounds (
Examples include pyrrole, pyrimidine, indole, imidazole, nitrobenztriazole, benztriazole, adenine, thymine, etc.), ascorbic acid, and the like.

Specific examples are given below, but the invention is not limited thereto.

1, CHsCOOAg C.I.

3, CICl1s(CH2)acOOA, CHs
(CHx)zcOOAg 5 , CHs(CHz)ac)I= CHCHxCH
-CH(CHx)yCOOAg-6, Ag00CHzC
H(NHz)COOAg7, CHz(CHz)xo
cOOAg'' 80-CI ■ C1,OH The combination of the biological substance and the substance that specifically binds the biological substance to the substance used in the present invention is: enzyme: substrate (product), inhibitor prosthetic group 1: Enzyme 1 Allosteric acid A Lectin: Antigen Protein A Lectin: Polysaccharide Glycoprotein Nucleic acid: Complementary base sequence Histone Nucleic acid Polymerase Hormone: Receptor Biotin: Avidin (streptavidin); (includes, preferably antibodies and antigens, or biotons and avidins, more preferably biotons and avidins.

These biological substances and substances that specifically bind to biological substances are those that undergo binding reactions with specific components of the fluid sample to be measured, substances that specifically bind to the specific components, and labeling substances used for measurement, which will be described later. or substances that do not interact with each other are used.

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:
its presence or absence or its amount in the fluid sample is detected;
A substance or a group of substances in which there may be a substance that specifically binds to that particular component. That is, polypeptides, proteins,
Examples include complex proteins, polysaccharides, lipids, complex lipids, nucleic acids, hormones, vitamins, drugs, antibiotics, agricultural chemicals, and the like. Specifically, the following substances or substance groups can be mentioned, but are not limited to these.

<Protein, complex protein> Prealbumin, albumin, σ1-acid glycoprotein, C
1-antitrypsin, ff1-glycoprotein, transcortin, C1-antichymotrypsin, σ,-lipoprotein, thyroxine-binding globulin, ceruloplasmin, Z
n-σ2-glycoprotein, Qc-globulin, intera
- trypsin inhibitor, C1 - macroglobulin, α
, -H3-glycoprotein, α,-macroglobulin, haptoglobin, α2-lipoprotein, hemovexin, transferrin, β-lipoprotein, β2-glycoprotein, β2-macroglobulin, C-reactive protein, myoglobin, erythromycin , immunoglobulin (IgG, IgMl)
1gA, IgD, IgE), complement system components (C+q, Cl
r%CIss Cx,C1,CiCslC@,C7,C
, CI, etc.), fibrinogen, hemoglobin, glycated hemoglobin, blood coagulation factors, HBs antigen, HBs
antibodies, enzymes (e.g., acid phosphatase, alkaline phosphatase, alkaline phos-7atase isoenzyme, a-amylase, amylase isoenzyme, aldolase, cholinesterase, creatine phosphokinase, talleatine phosphokinase isoenzyme, transaminase (GOT, GPT), Lactate dehydrogenase, lactate dehydrogenase isoenzyme, γ-GTP
, lipase monoamine oxidase, leucine aminopeptidase, glucose hexaphosphate dehydrogenase, etc.).

<Hormones and hormone-like substances> Follicle-stimulating hormone (FSH), luteinizing hormone (LH)
), growth hormone (GH), thyroid stimulating hormone (TS
H), adrenocorticotropic hormone (AcTH), melanin-stimulating hormone (MSH), vanpressin, oxytocin, insulin, glucagon, angiotensin I
and ■, prolactin, secretin, dopamine, serotonin, somatostatin, thyroxine (T4), triiodothyronine (T3), gastrin, flutisol, aldosterone, catecholamines, estrogen, progesterone, testosterone, placental gonadotropin,
Placental lactogen, pituitary hormone releasing factor (TRH)
.

FSH-RH, CRH, LH-RH, etc.) Vitamins> Biotin, thiamin, vitamin A1, vitamin B2, vitamin B6, vitamin B82, vitamin C1, vitamin D
1 Vitamin E1 vitamin and 1 folic acid etc.

Tumor markers> a-fetoprotein, carcinoembryonic antigen, 7-erythin, polyamine, pancreatic carcinoembryonic antigen, basic fetoprotein,
M-protein, prostatic acid phosphatase, carbohydrate antigen (C
A 19-9, CA 125, etc.), ganglio size.

Various drugs and metabolites> Benzoylecgonine, cocaine, codeine, dextrometrophan, heroin, lysergic acid, morphine, quinidine, quinine, amikacin, gentamicin, kanamycin, neomycin, tobramycin, actinomycetin, kaloimycin, chloride ramphenicol, chloromycetin, chlortetracycline, erythromycin, oxytetracycline, penicillin, cephalosporin polymyxin 81 terramycin, tetracycline, streptomycin, diphenylhydantoin, edosuximide, phenobarbital, primidone,
Secobarbital, acetaminophene, amitributyline, carbamazepine, digoxin, diviramide, lidocaine, methotrexate, N-acetylprocainamide, phenytoin, procainamide, progra-10-ol, theophylline, canapinol, tetrahydrocanapinol, cholinergic suppressants, antihistamines, atropin,
Butyrophenone, caffeine, chloropromacine, pulpylate, amphetamine, catecholamine, epinephrine, griseofulvin, imipramine, L-dopa, meperidine, mebrobamate, methatone, narcein, nortributyrin, oxazepan, papaverine, prostaglandin, tegretol, valproic acid, etc. and their metabolites.

<Microbial surface markers> Bacterial antigens, fungal antigens, parasite antigens, viral antigens <Pesticides> Halogenated biphenyls, phosphoric acid esters, thiophosphates, and their metabolites.

and others> Blood type substances, calciolibin, allergens, and substances that specifically bind to specific components in fluid samples that can be used in the present invention include antibodies, antigens, lectins, and inhibitors of protein A1 specific enzymes, depending on the measurement target. However, it is particularly preferable that the binding reaction between the specific component and 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 sodium sulfate obtained by a conventionally known method. It can be purified and used by precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc. (see "Immunochemistry" edited by Shunsuke Migita, pp. 74-88, Nakayama Shoten). can.

Alternatively, monoclonal antibodies can be prepared and used by obtaining hybrid cells (hybridoma) from lung cells of a mammal (eg, mouse) sensitized with an antigen and myeloma cells (myeloma).

In addition, these antibodies are IgG, IgM, IgAIgDl
1gE fractions can be used, or these antibodies can be enzymatically treated to produce FabSFab' or F (ab'
), etc. may be used in the form of active antibody fragments. Furthermore, these antibodies may be used alone or in combination. When an antibody or an antigen is used as a substance that specifically binds to a specific component in a fluid sample, the measurement principle of the analytical element of the present invention belongs to the immunoassay method, and the reaction can be expressed using the competitive method, two-antibody method, or sandwich method. The law is given. Since the analytical element of the present invention can be particularly preferably used in immunoassays, the present invention will be described in detail below using immunoassays as an example, but the present invention is not limited to this explanation and can be applied to various applications. It is clear from what has been described above that this is possible.

The labeling substance that can be used in the present invention is preferably an enzyme, which binds to an organic silver derivative and changes the enzyme activity,
Here are some representative examples.

1.1.1. l Alcohol dehydrogenase1.
1.1.2 Alcohol dehydrogenase (NAD)
1.1.1.8 Glycerol-3-phosphate dehydrogenase 1.1.1.lOL-xylulose reductase 1.1
．． 1.18 myo-inositol-2-dehydrogenase 1.1.1.22 VDP glucose dehydrogenase 1.1.1.26 glyoxylate reductase 1.1.1.27 lactate dehydrogenase 1.1
．． 1.29 Glycerate dehydrogenase 1.1
．． 1.30 3-hydroxybutyrate dehydrogenase 1.1.1.37 Malate dehydrogenase 1.
1.1.40 Malate dehydrogenase (oxaloacetate, decarboxylation) (NADP") 1.1.1.41 Isocitrate dehydrogenase (N A D ") 1.1.1.42 Isocitrate dehydrogenase (NADP") ) 1.1.1.44 Phosphogluconate dehydrogenase (decarboxylation) 1.1.1.50 3α-hydroxysteroid dehydrogenase 1.1.1.95 Phosphoglycerate dehydrogenase 1.1.3.1 Glycolate oxidase 1 ．．
1.3.4 Glucose oxidase 1.2.1
．． l Formaldehyde dehydrogenase 1.2.1.4 Aldehyde dehydrogenase (
NADP”) 1.2.1.5 Aldehyde dehydrogenase (
NAD (P)'') 1.2.1.12 Glyceraldehyde phosphate dehydrogenase 1.4.1.l Alanine dehydrogenase 1.
4.1.3 Glutamate dehydrogenase (N
AD (P) 1.4.1.4 Glutamate dehydrogenase (NADP) 1.4.3.3 D-amino acid oxidase 1.
5.1.5 Methylenetetrahydrofolate dehydrogenase 1.9.6.1 Nitrate reductase (cytochrome) 1.13.11.12 Lipoxygenase 1.14
．． 13.2 4-Hydroxybenzoate-3-monooxygenase 1.14.16.2 Phenylalanine-4-monooxygenase 2.1.1.6 Catechol methyltransferase 3.1.3.2 Aspartate carbamoyltransferase 2,1 .33 Ornithine carbamoyltransferase 2, 3.1.6 Choline acetyltransferase 2, 3.1.7 Carnitine acetyltransferase 2, 4.1.1 Phosphorylase 2, 4.1.2
2 Lactose synthase 2, 6.1.19
Aminobutyric acid aminotransferase 2, 7.1. l Hexokinase 2, 7.1.2
Glucokinase 2, 7.1.12 Gluconokinase 2, 7.1.19 Phospholibrokinase 2, 7.1.32 Choline kinase 2, 7.2-
1 Acetate Kinase 2, 7.2.2. Carbamate kinase 2, 7
．． 3.2 Taleatin Kinase 2, 7.3.3
Arginine Kinase 2, 7.4.3 Adenylate Kinase 2, 7.4.6 Nucleoside Diphosphate Kinase 2, 7.7.7. DNA nucleotidyl transferase 3, 1.1.3 Triadylglycerol lipase 3, 1.1.13 Cholesterol esterase 3, 1.3.2 Acid phosphatase 3, 1.4
．． 4 Phospholipase D3, 1.31. l
Lung enzyme nuclease 3, 2.1. l tr
-Amylase 3, 2.1.2 β-amylase 3
, 2.1.14 Chitinase 3, 2.1.18 Neuramindase 3, 2.1
．． 20 α-D-glucosidase 3, 2.1.2
1 β-D-glucosidase 3, 2.1.23
β-D-galactosidase 3, 2.1.26
β-D-fructofuranosidase 3, 2.1.31
β-D-glucurondase 3, 4.11.8
Pyroglutamyl aminope'butidase 3, 4.14. l Dipeptidyl peptidase■
3, 4.21.1 Chymotrypsin 3, 4.22
．． 2 Papain 3, 4.22.3 Ficin 3, 4.22.4 Promeline 3, 4.22.
6 Chymopapain 3, 4.22.8 Clostripain 3.5.1.5 Urease 3.5.1.24 ``Loylglycine hydrolase 3.5.2.6 β-lactamase 3.5.3
．． 1 Arginase 3.5.4.4 Adenosine deaminase 3.6
．． 1. l Inorganic pyrophosphatase 3.6.1.9 Nucleotide pyrophosphatase 4.1.1.1 Pyruvate decarbonacylase 4.1.1.39 Ribulose phosphate carboxylase 4.1.2.11 Hydroxymanzolonitrile lyase 4.2.1.24 Phosphobilinogen synthase 4.3.1.5 Phenylalanine ammonia lyase 4.3.2.2 Adenylosuccinate lyase 5.1
．． 3.2 VDP Glucose-4-Epimerase 5.3.1.1 Triose Phosphate Imerase 5.3.1.6 Ribose Phosphate Imerase 6.
2.1.1 Acetyl-CoA Synthetase6.
4.1.2 Enzymes such as acetyl-CoA carboxylase as labeling substances include hydrogen peroxide and NADH, N
Oxidation/reductases and hydrolases involving ADHP are preferably used.

The signal caused by the labeled enzyme 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. can be measured using endpoint assays that measure In the absorbance method (colorimetric method), ultraviolet light, visible light, and near-infrared light can be used. For example, when using serum as a fluid sample, green light, It is preferable to use red light or near-infrared light.

Binding of the specific component used in the measurement method of the present invention, an analog of the specific component, and a substance that specifically binds to the specific component, and a labeling substance and biological substance, or a substance that specifically binds to the biological substance The substance can be obtained by directly or indirectly combining both substances by chemical means or the like while retaining the ability of the substance to specifically bind and the ability of the labeling substance to emit a signal. These conjugates can be obtained by combining known reagents and known methods well known to those skilled in the art. (2nd edition)" (Igaku Shoin, 1978) and Japanese Clinical Pathology Lifeline "Clinical Pathology" Special Issue No. 53, "Immunoassays for Clinical Tests - Techniques and Applications" (Clinical Pathology Publishing Association, 1983) ), etc. can be referred to.

As specific methods, various methods described in Japanese Patent Application No. 61-280166 can be used.

The porous reaction layer in the present invention is characterized by a binding reaction between the specific component and a substance that specifically binds to the specific substance, a binding reaction between a labeling substance and a specific modulating substance, and a binding reaction between a biological substance and a substance that specifically binds to the biological substance. The specific modulating substance and the biological substance or the substance that specifically binds to the biological substance must be immobilized on part or all of the reaction layer. In addition, in order to retain the fluid sample during the reaction period, a part or all of the reaction layer is provided with interconnecting pores (minor diameter 1 to 30 mm) that can freely contact the fluid sample.
0 μm is preferred).

The material of the porous reaction layer is not particularly limited as long as the above conditions are met.

Preferred examples include structures made of one or more materials selected from granules with a size of 1 to 350 μm or fibers with a mesh size of 40 to 400.

Materials for the granules 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, and various synthetic resins (such as polystyrene). ), etc., as well as patent applications 1986-28.
Examples include self-bonding particles made of a compound having a reactive group as described in No. 0166.

Furthermore, several types of these granules can also be used in combination.

In addition, the fibers used in the porous reaction layer of the present invention include pulp, powder filter paper, cotton, hemp, silk, wool, chitin, chitosan, cellulose ester, viscose rayon, cuprammonium rayon, polyamide (6-nylon, 6 Vegetable, animal, and mineral 9 synthetic, semi-synthetic, and recycled fibers such as raw nylon, 610-nylon, polyester (polyethylene terephthalate, etc.), polyolefins (polypropylene, vinylon, etc.), glass fiber, and asbestos can be used. or a mixture of these may be used. Alternatively, water-absorbing Western paper, Japanese paper, filter paper, plush polymer, glass fiber, mineral fiber (asbestos, etc.), vegetable fiber (cotton, hemp, pulp, etc.),
Woven fabrics, non-woven fabrics, and synthetic papers made from animal fibers (wool, silk, etc.), synthetic fibers (various types of nylon, vinylon, polyethylene terephthalate, polypropylene, etc.), recycled fibers (rayon, cellulose ester, etc.), singly or in combination, etc. etc. can also be used for the porous reaction layer.

By coating and/or film-forming such granules, fibers, or a mixture of granules and fibers, a porous reaction layer is created in which a porous structure with interconnecting pores that can freely contact the fluid sample is present. form. 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, JP-A-55-90859, and JP-A-57-67860. method can be applied. Organic polymer particles having self-bonding properties are disclosed in JP-A-57-101760 and JP-A-57-101.
Films can be similarly formed by the methods described in No. 761, No. 58-70163, and the like. For fibers or mixtures of fibers and particles, see JP-A-57-125847 and JP-A-57-1974.
A porous reaction layer can be formed by applying the fiber dispersion described in No. 66. Also, JP-A-60-17347
It is also possible to apply a fiber and/or particulate dispersion using a water-soluble binder such as gelatin or polyvinylpyrrolidone as in the method described in No. 1. A number of 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 the particulates and/or fibers in the dispersion.

Examples of typical surfactants that can be used include Triton ■
There are nonionic surfactants such as 1□o60 (manufactured by O,-A Co., Ltd.; NiK7xnoxypolyglycidol).

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.
It can be used from wt% to 0-005wt%, preferably from 15wt% to 0.05wt%. Still other methods include ultrasonication, physical mixing, and physical stirring of the particle unit and the liquid carrier, and pH adjustment. These are even more useful in combination with the methods described above.

Also, biological substances immobilized on fibers, granules, etc., substances that specifically bind to the biological substances, specific modulators, specific components, analogs of the specific components, and substances that specifically bind to the specific components. In order to retain the ability of a biological substance or a substance that specifically binds to the biological substance and a conjugate of a labeling substance to specifically bind and the ability of the labeling substance to emit a signal, and to contain the substance in a porous reaction layer or a layer described below. In, Japanese Patent Publication No. 1986-17
The method described in No. 7997 can be used. A biological substance, a substance that specifically binds to the biological substance, and a specific modulating substance can be immobilized on the porous reaction layer by physically attaching the substance to the surface of the porous reaction layer using various known methods. This is achieved by adsorption or direct or indirect bonding through chemical reactions. At that time, care must be taken not to lose the specific binding property of the substance to the specific component. Shoin, 1
(Published in 1978) and "Experimental and Applied Affinity Chromatography" by Part Chibata, Tetsuya Tosa, and Yushi Matsuo (Kodansha: 19
The method described in 1976) can be cited as an example of a preferred method. In addition, the immobilization of these substances into the porous reaction layer retains the specific binding site,
And it does not need to be free or dissolved in the fluid sample,
It may also be dispersed in an insoluble state in the fluid sample. Also, methods used for dispersing couplers used in color photography (for example, "Fundamentals of Photographic Engineering, Silver Salt Edition" published by the Photographic Society of Japan)
(Corona Publishing, 1978), a method of incorporating it into a lipid bilayer membrane, etc. can also be used. In addition, after immobilizing the substance that can specifically bind to the specific component, proteins that are not involved in the specific reaction to be measured may be supported, if necessary, in order to eliminate non-specific reactions in the immune reaction. It is possible.

Typical examples thereof include mammalian normal serum proteins, albumin, gelatin, and decomposition products thereof.

These immobilization operations may be performed on the above-mentioned granules or fibers in advance to form a porous reaction layer, or the immobilization operations may be performed after forming a porous reaction layer. It is. In the former case, in addition to the particles or fibers on which the above substance is immobilized, it is also possible to add particles or fibers on which a protein not involved in the above-mentioned specific reaction is immobilized for the purpose of adjustment.

The mixing ratio of the two types of immobilized substances is determined depending on the amount and binding constant of the specific binding substance immobilized on each. If necessary, granules or fibers that do not immobilize the specific binding substance may be added. May be added for adjustment.

The combination of immobilized substances of the present invention is a combination of a biological substance and a specific modulating substance, and a combination of a substance that can specifically bind to the biological substance and a specific modulating substance. The reason why two immobilized substances are used in this way is to perform B/F separation in the device and to modulate the signal caused by the labeling substance.

The form of the analytical element of the present invention is not particularly limited as long as it can perform analysis, but it is preferably in the form of a film or a sheet from the viewpoint of manufacturing, operation, and measurement.

An aspect of the present invention is a case where two types of immobilized substances are fixed and contained in the same porous reaction layer ρ, and a case where the two types of immobilized substances are contained and immobilized in two or more porous reaction layers. Including cases where

In order to aid in understanding the analytical element of the invention, the principle will be explained by way of example.

FIG. 1 is an example of a cross-sectional view of the simplest embodiment of the analytical element according to the present invention. In this case, the analytical element is composed only of the porous reaction layer 0, and the two types of immobilized substances are immobilized and contained in the porous reaction layer.

FIG. 2 is an enlarged view of FIG. 1. In Fig. 2, A is a granule on which a biological substance or a substance that specifically binds to the biological substance is immobilized, B is a granule on which a specific modulating substance is immobilized, and C is a granule for adjustment. It is a granular material added to.

FIG. 2 shows that the porous reaction layer is constructed by uniformly mixing the granules A, B, and C and adhering them at points.

FIG. 3 is a schematic diagram for explaining the measurement principle of the present invention, and the reaction type will be explained using a competitive method as an example.

In FIG. 3, l is a specific component, 2 is a labeling substance, 3 is a combination of a specific component and a labeling substance (abbreviated as a label), 4 is a substance that specifically binds to a specific component, and 5 is a biological substance (or 6 is a combination of a substance that specifically binds to a specific component and a biological substance, 7 is a substance that specifically binds to a biological substance (or biological substance), 8 is a specific modulation substance, 9 is a binding reaction product of a specific component and binding substance 6;
1O is the binding reaction product of label 3 and conjugate 6, A is 7
B represents the immobilized product of 8.

A certain amount of the fluid sample, a solution containing the label 3, and a solution containing the bond 6 of a substance that specifically binds to a specific component in the fluid sample and a biological substance are mixed or sequentially dropped.

Since the specific component 11 label 3 and conjugate 6 exist in the liquid phase, these binding reactions (1 and 6 or 3 and 6) become a liquid phase reaction system, and the binding of immobilized substance A and conjugate 6 reaction (binding sites 5 and 7), binding reaction of immobilized substance B and labeled substance 3 (
Since the binding site occurs sufficiently faster than 2 and 8), it is thought that the liquid phase binding reaction occurs preferentially. Therefore, as a result, before the solid-liquid reaction described above occurs, a sufficient amount of the liquid phase reaction occurs, and then the solid-liquid reaction occurs. As a result of the binding reaction in the liquid phase system, unreacted bound substances 3 present in the liquid phase
, the binding reaction product 9 of the specific component 1 and the bound substance 6, the unreacted labeled body 3 is bound to the immobilized substance B (binding sites are 2 and 8), and the binding reaction product 9 is It undergoes a binding reaction with compound A (binding sites are 5 and 7). Furthermore, the binding reaction product IO between the label 3 and the bound substance 6 can undergo a binding reaction with the immobilized substance A or B, but the bonding force between 5 and 7 is 2 and 8.
It is possible to preferentially bind to the immobilized substance A by selecting a combination stronger than the binding force with the immobilized substance A.

In this way, the binding reaction products 9 and 10 of the bound substance 6 to the specific component 1 and the labeled body 3 consisting of the specific component and the labeling substance become the immobilized substance A, and the unreacted labeled substance 3 becomes the immobilized substance B. Separation can be performed within each layer (B/F separation). Since the signal caused by the labeling substance 2 of the labeling body 3 is modulated by binding with 8 of the immobilized substance B, there is a difference between the concentration of a specific component in the fluid sample and the signal intensity of the entire labeling substance. A functional relationship is established. Therefore, by creating a calibration curve using several types of fluid samples (standard samples) in which the concentration of the specific component I is known in advance, it is possible to know the concentration of the specific component in an unknown liquid sample.

A similar explanation can be given to measurement by the Sanderuch method, in which a label is a combination of a substance that specifically binds to a specific component and a labeling substance.

In FIG. 4, the porous reaction layer has two layers, and a biological substance, a substance that specifically binds to the biological substance, and a specific modulation substance are immobilized and contained in separate layers, respectively. These layers can be obtained by coating, casting or laminating materials used for similar or different porous reaction layers.

Although the above-mentioned porous reaction layer is the minimum necessary component of the analytical element of the present invention, various auxiliary layers may be provided to further exhibit the effects of the invention.

4 to 12 are schematic cross-sectional views showing embodiments of the analytical element of the present invention.

The analytical element of the present invention shown in FIG.
A porous reaction layer 0 is laminated on top of the substrate, and the presence of the support improves the handling of the device.

Supports that can be used for this purpose include, for example, polymeric compounds such as cellulose acetate, polyethylene terephthalate, polycarbonate, and polyvinyl compounds (eg, polystyrene), or transparent inorganic compounds such as glass. The porous reaction layer may be directly coated and/or formed into a film on such a support, or the porous reaction layer may be formed separately and then attached to the support. In the embodiment shown in FIG. 5, the fluid sample must be dropped from the reaction layer side, but signals can be measured from both sides.

In another embodiment of the invention shown in FIG. 6, a reactive layer is provided on the light reflective support 12. In this embodiment, both the dropping of the sample and the signal measurement are performed from the porous reaction layer side, and the light reflective support facilitates the measurement of the signal in terms of reflection density.

Support materials that can be used for this purpose include, in addition to the above-mentioned support materials, ceramics, metals, and paper coated with resin to make it waterproof. This purpose can be achieved by coating or containing white pigments such as Tie, Ba5O, mica, etc.

The embodiment shown in FIG. 7 has a similar purpose, and a porous reaction layer 01 and a light reflection layer 13 are laminated in this order on a light-transmitting support 11. In this embodiment, the sample or the like is dropped from the light-reflecting layer side, and the signal measurement is performed from the light-transmitting support side. For the light reflection layer, any of those used in known analytical elements and similar products can be used, but preferably, the same particles and fibers as those used in the porous reaction layer contain the above-mentioned white pigment, etc. It can be coated or filmed or pasted. More preferably, a biological substance, a substance that specifically reacts with the biological substance, and a specific modulation substance are immobilized on the surface of particles containing a white pigment inside, as in the porous reaction layer, and the porous reaction layer in the lower layer is immobilized. A light reflecting layer having the same function as the layer can be provided. By using such a special light-reflecting layer, it is possible to prevent a large amount of unreacted labeling substance from remaining in the light-reflecting layer.

In the embodiment shown in FIG. 12, a coloring reagent layer 15 is provided on a light-transmitting support. This coloring reagent layer consists of at least one layer of hydrophilic colloid containing a substrate and a coloring reagent necessary for enzyme activity measurement.

The substrate and coloring reagent can be dissolved or dispersed in a binder made of hydrophilic colloid to form a coating solution. In particular, for dispersing hydrophobic compounds, various known dispersion methods such as oil protect dispersion method and direct dispersion method, which are frequently used in the photographic industry, can be used.

Further, the hydrophilic colloid used in the coloring reagent layer according to the present invention may be the hydrophilic colloid and polymer latex used in the porous reaction layer containing the organic silver derivative described above, but under the same conditions and under the same consideration. used for.

The coloring reagent layer contains other additives such as buffers, preservatives,
Surfactants, mordants, etc. can be added depending on the purpose. In addition, the film thickness is 3 to 50 μm, preferably 5 to 50 μm.
It is 30 μm. A buffer is contained to adjust the pH to a level suitable for specific binding reactions, enzymatic reactions, coloring reactions, and the like. Buffers that can be used include Nippon Kagaku Yosen, "Chemical Handbook Basic Edition" (Tokyo, Maruzen Co., Ltd. 1966), pp1312.
~1320, N, E, Good et al.; Biochemistry, VoQ5.
p467 (1966), Wakumura, Saito: Chemistry Domain voQ
30(2) p79 (1976), W. J7 Ferguson et al.: ^na1. Biochem,
.

vo12104. Examples include those described in literature such as p.300 (1980). Specific examples include borates, phosphates, carbonates, citrates, trisbarbyl, glycine, Gud buffer, and the like.

These buffers may be contained in layers other than the coloring reagent layer, if necessary.

Preservatives are included to stabilize the storage of the substrate coloring reagent, and include antioxidants and the like. In addition, in order to maintain activity when two types of immobilized substances, bound substances, and labeled substances are contained in the layer, immobilized enzymes, adsorbents for affinity chromatography, immobilized antibodies, and preservation of proteins, enzymes, etc. It may also contain a preservative used in. The substance is described in "Biochemistry Experiment Course I, Protein Chemistry IJ (Tokyo Kagaku Dojin Co., Ltd. 1976) PP66-67, the above-mentioned experiments and applications [Affinity Chromatography J PP10]"
3 to 104, and those described in JP-A-60-149927.

Specific examples include gelatin, gelatin decomposition products, albumin BSA, cyclodextrins, non-reducing sugars (
Examples include sucrose, trehalose), polyethylene glycol, amino acids, various ions, and sodium azide. These preservatives are preferably present in the vicinity of the two immobilized substances, the conjugate, and the enzyme label.

As the hardening agent, substances that are widely used in the photographic industry can be used.
S) Edition [The Theory of the Photographic Process J]
photographyProcess) (4th edition) p.
Examples include those described on pages 77-87. Specific examples include aldehydes, active olefins, and active esters.

Examples of the surfactant include those mentioned above.

Other reagents contained in the layer include a solubilizing agent and a blocker reagent. 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 coloring reagent layer. When the detection substance is a dye, it is a substance that increases the absorbance coefficient or shifts the wavelength. Show interaction. Cationic polymers, anionic polymers and latexes of these polymers are used.

The embodiments of the present invention described above are all about a biological substance for a substance that specifically binds to a specific component in an element, or a bound substance (hereinafter abbreviated as a bound substance) with a substance that specifically binds to the biological substance, and a label. Contains no body. The embodiment of the present invention, which does not contain a conjugate or a label in the device, as represented by these embodiments, is a free embodiment free from the constraints of both of the above, and this embodiment has the following surprising effects in particular. occurs.

In other words, the immunological analytical elements that have been developed so far have all been created to analyze a specific component, and the analytical element created to measure the specific component M1 is called the specific component MI. This method has the disadvantage that it cannot be used to measure a specific component M2 that is different from the above method.

For this reason, it is necessary for the measurer to always keep the same number of analytical elements as the measurement items on hand, and to select the analytical element that meets the purpose each time he or she performs a measurement.

In general, the effort and economic burden of keeping a large variety of analytical elements with expiration dates on hand and managing inventory is enormous, and the work of selecting the type of analytical element that corresponds to the measurement item each time a measurement is performed reduces processing capacity. This also increases the risk of erroneous measurements due to incorrect selection of analytical elements. Moreover, when automating analysis operations, it is necessary to prepare cartridges of analytical elements as many as the number of measurement items and to provide a mechanism for automatically switching between them, resulting in an increase in the size and cost of the measuring device.

These drawbacks of conventional analytical elements halve the ease of measurement inherent in dry chemistry.

However, in an analytical element that is free from the conjugate and label as in the present invention, even if the same analytical element is used, the conjugate and label dropped together with the fluid sample during analysis can be used for analysis purposes. If appropriately selected according to the above, it is possible to construct a multipurpose mode capable of measuring any specific component Mi, and thus a multipurpose analytical element. Furthermore, it is also possible to change the sensitivity and measurement range of the analytical element by adjusting the amounts of the conjugate and label. Such a versatile analytical element is only made possible by the versatile aspect of the present invention.

However, the present invention is not limited to this multi-purpose embodiment, and it is also possible to incorporate the conjugate and the label into the analytical element with the intention of simplifying the analytical operation.

In this embodiment of the present invention (hereinafter referred to as the built-in embodiment), the types of specific components that can be measured with the same analytical element are limited, but the conjugate and/or the label is The trouble of dripping can be saved. As described above, one of the features of the present invention is that it is possible to provide analytical elements in a multi-purpose type mode and a built-in type mode depending on a consumer's purpose of use.

Examples of built-in embodiments of the present invention are shown below.

In the embodiment shown in FIG. 8, the coloring reagent layer 1
5 is provided, on which a substance that specifically binds to a specific component, a binding containing layer 14 of a biological substance or a substance that specifically binds to the biological substance, and a porous reaction layer 14 are laminated in this order. There is. A binding-containing layer is a porous medium containing bound substances so that the area concentration is constant, and when a fixed amount of a fluid sample and a solution containing a label are dropped, a fixed amount of bound substances is eluted. , which diffuses into the porous reaction layer together with the sample. As for the material, the same material as the porous reaction layer may be coated or pasted, or water-absorbing western paper,
Japanese paper, filter paper, plush polymer, or glass fiber,
Mineral fibers (asbestos, etc.), vegetable fibers (cotton, hemp, pulp, etc.), animal fibers (wool, rope, etc.), synthetic fibers (various types of nylon, vinylon, polyethylene terephthalate,
A bond-containing layer made of woven fabric, non-woven fabric, synthetic paper, etc. made of polypropylene (polypropylene, etc.), recycled fiber (rayon, cellulose ester, etc.) alone or in combination may be attached.

The position of the binding-containing layer can be selected from various positions as required. When the binding-containing layer is located in a portion other than the top layer as in this embodiment, materials used for the binding-containing layer include gelatin, Gelatin derivatives, polysaccharides (agarose, etc.)
There is a method of applying a homogeneous binder such as a hydrophilic polymer substance such as , carboxymethyl cellulose, hydroxyethyl cellulose, or a homopolymer or copolymer containing monomers such as vinyl pyrrolidone, acrylic acid derivatives, methacrylic acid derivatives, vinyl alcohol, and sulfonylstyrene. .

FIG. 9 shows an example in which a label is also contained in the analytical element. The layer structure is the same as that shown in FIG. 8, but the marker is contained in the porous reaction layer.

Since this porous reaction layer contains an immobilized substance of a specific modulation substance, in order to prevent a binding reaction with a label during the manufacture and storage of an analytical element, for example, clinical chemistry (Clinical Chemistry) vo.
ff27. The method described in p. 1499 (1981), etc. can be used.

Another method of incorporating a bound substance or a label into an analytical element is a method in which a porous reaction layer contains a bound substance and a binding-containing layer contains a label. The label-containing layer can be formed by the same method as for the binding-containing layer, and the same method as for the label can be used when the porous reaction layer contains a binder.

Still another embodiment is a case where the porous reaction layer contains a binder and a label.

FIG. 1θ and FIG. 11 show a cross-sectional view and a perspective view of one example of a preferred embodiment of the present invention.

In order to facilitate handling of the analytical element, the whole is covered with a plastic mount 17, with a sample injection hole in the upper part of the mount and a signal measurement hole in the lower part.

The analytical element of the present invention further includes a development layer that assists in the development of a fluid sample when it is applied to the element, a blood cell separation layer that may be necessary when the fluid sample is blood (whole blood), and a blood cell separation layer that may be necessary when the fluid sample is blood (whole blood). Auxiliary layers such as adhesive layers, protective layers, and timing layers may be provided.

These auxiliary layers, the above-mentioned coloring reagent layer, binder- or label-containing layer, and timing layer may be provided independently, or may be provided as a layer having multiple functions.

The positions where these layers should be provided can be easily determined depending on their functions.

〔Example〕

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited to these Examples.

Example 1 l-(1) 1.2.3-Benzotriazole Preparation of silver 1.2.3-benzotriazole (manufactured by Wako Junkei Kogyo) 5
Dissolve 0g in 300mff of acetone and add silver nitrate (
A solution of 75 g (manufactured by Wako Junkei Kogyo) dissolved in 200 m0 of deionized water was gradually added dropwise to precipitate 1,2,3-benzotriazole silver. After thoroughly washing the precipitate several times with deionized distilled water (hereinafter referred to as pure water),
It was dried to produce 1.2.3-benzotriazole silver.

1-(2) Preparation of β-galactosidase-labeled human IgG Human IgG (manufactured by Kappel, USA) 20119 at 0.1
Dissolved in M phosphate buffer (pH 6,5) 2.0rxQ,
To this was added 77 μQ of a 2.511 g/IIQ dimethylformamide solution of N-(g-maleimidocaproyloxy)succinimide (manufactured by Isotope Kagaku Kenkyusho) and
After reaction for 0 minutes, Sephadex G-2 equilibrated with 0.1M phosphate buffer (pt16.0) containing 5mM EDTA.
5 column to obtain maleimidated human IgG. Next, β-galactosidase (manufactured by Toyobo Co., Ltd.) at 10.5
s/mff O, 3.2 raQ of the solution containing the maleimidized human IgG 13°61119 was added to 1.8 mQ of 1M phosphate buffer, and after reacting at 4°C for 45 hours, 0.
Add 175 μg of 1M 2-mercaptoethylamine and
The reaction was carried out for 0'020 minutes, and β- Galactosidase labeled human Ig
I got a G.

1-(3) Preparation of p-aminophenyl mercuric acetate (enzyme inhibitor) immobilized Avicel 80 g of Avicel (manufactured by Asahi Kasei Corporation) was suspended in 800 2.5 M phosphate buffer (pH 1 pH 12-0) and placed on ice. Cyanogen bromide 80.0 was dissolved in 800 mQ of pure water under water cooling.
After reaction for 20 minutes, the mixture was collected by filtration and thoroughly washed with water.

This Avicel 809 was suspended in 950 mg of a 25% aqueous dimethyl sulfoxide solution containing 4.89 g of p-aminophenyl mercuric acid (f-), and stirred at room temperature for 20 hours. This was collected by filtration, washed with dimethyl sulfoxide and pure water, and then mixed with IMI-Lys-HCl buffer (pH 8,5) for 10 minutes.
00m<2 and stirred at room temperature for 20 hours to block unreacted groups. This was collected by filtration, thoroughly washed with water, collected by filtration, thoroughly washed with water, washed with acetone, and dried.
Avicel fixed with aminophenyl mercuric acetate was prepared.

1-(4) Preparation of Sample 11 Comparative Samples (1) and (2) Sample 11 Comparative Sample (1) ) to (2) were created.

Sample-1 Ossein gelatin 1.5g 20% Triton X-100 (manufactured by Rohm and Haas)
0.759NeoTB (manufactured by Isotope Kagaku Kenkyusho) 751190.25% 1.2-bis(vinylsulfonyl)ethane aqueous solution 1.0
9 Pure water 14.09 Avicel 3.0 g Polyvinylpyrrolidone 0.2591.2.3-benzotriazole silver 1.095-7' Romo-4-chloro-3-indolyl β-logalactopyranoside 200 mg
7 X-1000,19 n-butanol 5.0g fiber powder 36.09 7
X-1003,69 Polyvinylpyrrolidone 1.8 gn-butanol 140 g Comparative sample - (1) In sample 1, instead of Avicel and silver benzotriazole, 3.0 g of para-aminophenylmercuric acetate-immobilized Avicel prepared in 1-(3) Comparative sample-(1) was prepared in the same manner as sample-1 except that .

Comparative Sample = (2) A sample obtained by excluding only benzotriazole silver from Sample 1 was prepared as Comparative Sample-(2).

Next, for this sample-1 and comparative samples-(1) to (2),
1-1g of β-galactosidase labeled prepared from (2)
G Add BSA to 10μy/laQ of 0.3M sodium phosphate buffer (pH 7.4) and add the solution to a final concentration of 6% v/w dropwise in portions of 1θμα and heat at 37°C in a sealed state.
After 110 minutes of incubation, 5 minutes from the support side.
The reflection density of 4on- was measured.

The results are shown in Table 1.

Table = 1 Samples - 10, 31 Comparative sample - (1) 0.58 Comparative sample - (2) 1.30 As is clear from these results, 1.2 .3-benzotriazole silver has a higher enzyme inhibition rate.

Example 2 2- (1) Preparation of avidin-immobilized Avicel Bovine serum albumin (Fraction v1 manufactured by Arma, USA) 1
．． 09 was dissolved in 0.01M phosphate buffer (pH 7,4) containing O, 15M sodium chloride, 330mff4::, dimethylformamide solution containing 3.0+ml of Collenibiotin-N-Hydroxysuccinimide ester (manufactured by Boehringa) IO,4+1g. After reaction at room temperature for 2.5 hours, the mixture was thoroughly dialyzed against the buffer and lyophilized to obtain biotinylated bovine serum albumin.

Next, 90 g of Avicel (manufactured by Asahi Kasei Co., Ltd.) was added to 1800 ml of 2.5 M phosphate buffer (pH 12,0) in a 124:1 suspension ML.
To this was added 45.09 g of cyanogen bromide dissolved in 5501 IIQ of pure water under ice-water cooling, and after reaction for 20 minutes, it was collected by filtration and thoroughly washed with water. This Avicel 909 was dissolved in a 1.1M aqueous sodium bicarbonate solution (containing 0.15M sodium chloride) containing the biotinylated bovine serum albumin 500119.
The mixture was suspended in 900 mQ and stirred at 4°C for 20 hours. This was collected by filtration, purified water, 0.1% containing 0.15M sodium chloride
M sodium bicarbonate aqueous solution, 0.1M sodium acetate buffer containing 0.15M sodium chloride (pH 4,1)
After washing alternately with 1M Tris-HCl buffer (pH
8,5) It was suspended in 1200 mQ and stirred in a room for 20 hours to block unreacted groups.

After filtering this and thoroughly washing with water, a 0.05M phosphate buffer (pH 7.4) containing 0.15M sodium chloride in which avidin (manufactured by Oriental Yeast Co., Ltd.) 270119 was dissolved.
The suspension was suspended at 450mffi, reacted at 4°C for 200 hours, collected by filtration, and washed with water to prepare Avicel with avidin immobilized thereon. In addition, bovine serum albumin 15.49 and sucrose 9.9
The avidin-immobilized Avicel 809 was suspended in 240 va (l) of pure water in which 19 was dissolved and lyophilized to prepare avidin-immobilized Avicel containing bovine serum albumin sucrose.

2-(2) Preparation of biotinylated goat anti-human IgG antibody IgG antibody (manufactured by Kappel, USA) 5.8
119 in 0.1M phosphate buffer containing 0.15M sodium chloride (pH 7-4)2. To this was added 500 μa of a dimethylformamide solution containing 0.32119 biotin-N-hydroxysuccinimide ester (manufactured by Boehringa), and after reacting at room temperature for 3 hours, 0.1
A biotinylated goat anti-human IgG antibody was obtained by thorough dialysis against a 0.01M phosphate buffer containing 5M sodium chloride.

2-(3) Creation of immunological analysis element Thickness: 180 μm
A coating liquid (1) having the following composition was applied onto a transparent subbed polyethylene terephthalate film and dried to form a gelatin layer.

Coating liquid - (1) Deionized gelatin 6.0° Triton Avicel containing 1,2,3-benzotriazole silver prepared in 1) was dispersed, and a coating liquid (2) containing a coloring reagent and having the following composition was applied onto the gelatin layer and dried.

Coating liquid - (2) Avicel 14.0g1.2
．． 3-benzotriazole silver 6.0g Dryde 7 X-1001,49 Polyvinylpyrrolidone (manufactured by Wako Junkei) 1.2g5
Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Behringa) 0.909 NeoTB * (manufactured by Isotope Kagaku Kenkyusho) 0.3
59n-butanol 34.
09* NeoTB; 3.3'-(4,4'-biphenylene)bis(2,5-diphenyl-2B tetrazolium chloride) Next, the avidin-immobilized Avicel prepared in 2-(1) was dispersed, and the following composition was prepared. The coating liquid-(3) of 1.2.
It was coated on top of the 3-benzotriazole silver-containing Avicel layer and dried.

Coating liquid - (3) Bovine serum albumin, sucrose-containing avidin-immobilized Avicel 22.59 Dryde 7
-1002,509 Polyvinylpyrrolidone 3.509 n-butanol 52.0 g Furthermore, a coating solution (4) having the following composition was coated on the avidin-immobilized Avicel layer and dried to form a spreading layer.

Coating liquid - (4) Powder filter paper - D (manufactured by Toyo Roshi Co., Ltd.) 30.09 Triton , was used as an analytical element.

2-(4) Calculation of human IgG using 0,3M bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Eis) containing 1mM magnesium chloride and 6wt% bovine serum albumin.
A human IgG solution (0-640/J 9/IIQ) dissolved in a hydrochloric acid buffer (pH 7, 2) was prepared using β-galactosidase-labeled human I prepared in 1-(2).
gG (10/J y/sQ) above buffer solution) and 2- (
2)-c Prepared biotinylated anti-human IgG antibody (20μ
g/laQ of the above buffer solution), drop 10 μa of this mixed solution onto the analytical element prepared in 2-(3), and
After incubation in a closed state for 7°010 minutes,
The reflection density of 546n+a was measured from the support side. The results are shown in FIG. 13 (1).

As is clear from this result, by using this device, B/F separation can be performed within the device, and human Ig
G could be measured with high sensitivity.

Example 3 3-(1) Preparation of avidin-immobilized cellulose fiber In place of the avidin-immobilized Avicel prepared in 2.(1) above, a cow was prepared using powdered filter paper D (manufactured by Toyo Roshi Co., Ltd.) in the same manner. Avidin-immobilized cellulose fibers containing serum albumin and sucrose were prepared.

3-(2) Creation of immunological analysis element Thickness: 180 μm
A coating liquid (5) having the following composition was applied onto a transparent subbed polyethylene terephthalate film and dried to form a gelatin layer.

Coating liquid - (5) Deionized gelatin 4.5g Dryde 7 X-1000,109 1,2-His(hinylsulfonyl)ethane 0.007g NeoTB O,23
9 Pure water 40.59Next, apply the coating liquid described in 2-(3)-(2) to NeoTB
A dispersion having a composition other than the following was applied onto the gelatin layer and dried to form an Avicel layer containing 1,2,3-benzotriazole silver.

Further, on this layer, a coating liquid (6) having the following composition was applied, dried, and then cut into a size of 1.5 x 1.5 cmR to obtain an analytical element.

Coating liquid - (6) Bovine serum albumin, sucrose-containing avidin-immobilized powder filter paper D 30.0 g Dryde 7 X-1003,09 Polyvinylpyrrolidone 1.5 gn-butanol 55.0 g
3) Measurement of human IgG Human IgG was measured in the same manner as in 2-(4). The results are shown in Figure 13 (2).

As shown in FIG. 13 (2), by using this device, B/F separation can be performed within the device, and human I
gG could be measured with high sensitivity.

Example 4 4- (1) Creation of immunological analysis element Thickness: 180μ
3-(2) Description f) Coating liquid-(5) was applied onto the transparent undercoated polyethylene terephthalate film and dried to form a gelatin layer. Next, 2-(
3) The dispersion obtained by removing NeoTB from the coating solution described in (2) is coated on the gelatin layer and dried to form 1.2.3.
-A benzotriazole silver-containing Avicel layer was formed.

Further, on this layer, a coating solution with the following composition containing β-galactosidase-labeled human IgG prepared in 1-(2) and biotinylated goat anti-human IgG antibody prepared in 2-(2) -(7 ) applied.

After drying, coat the coating liquid (6) described in 3-(2) on top of this layer, and after drying, 1.5 x 1.5 cm"
It was cut to a size of 100 ml and used as an analytical element.

Coating liquid - (7) Bovine serum albumin, sucrose-containing avidin-immobilized Avicel 22.5g) 5 a) 7 X
-1002,509 polyvinylpyrrolidone
3.309β-galactosidase labeled human IgG
O,16119 biotinylated goat anti-human IgG antibody
0.60119 bovine serum albumin
38.01+9n-butanol
55.0g4-(2) Measurement of human IgG A human IgG solution (0-
640 tt g/mff) was dropped onto this analytical element,
After incubation in a sealed state for 37°C 10 minutes,
The reflection density at 546 nm was measured from the support side. The results are shown in Figure 13 (3).

As shown in Figure 13 (3), by using this device, B/F separation can be performed within the device without complicated operations, and human 1gG can be measured with high sensitivity. Ta.

Example 5 5- (1) Preparation of β-galactosidase-labeled rabbit anti-human IgG antibody Rabbit anti-human IgG antibody (manufactured by Kappel, USA) 20m
A β-galactosidase-labeled rabbit anti-human IgG antibody was obtained using 9 and in the same manner as in 1-(2).

5-(2) Measurement of human IgG Human IgG solution (0-640) dissolved in 0.3M old 5-tris-HCl buffer (pH 7.2) containing mM magnesium chloride and 6% bovine serum albumin
tt y/raQ”) 4- β- created in (1)
Galactosidase-labeled heron anti-human gG antibody (15μ
g/mQ of the above buffer solution) and the biotinylated goat anti-human IgG antibody prepared in 2-(2) (100 μg/mQ of the above buffer solution), 10 μa of this mixed solution was mixed with 2-
After dropping it onto the analytical element prepared in (3) and incubating it in a sealed state for 37°C 10 minutes,
The reflection density at 6 nm was measured. The results are shown in Figure 13 (4
)It was shown to.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 and 4 to 9 and 12 are cross-sectional views showing examples of the layer structure of the analytical element of the present invention. FIG. 2 is an enlarged view of the porous reaction layer. FIG. 3 is a schematic explanatory diagram of the measurement principle of the analytical element of the present invention. FIG. 1O and FIG. 11 are a sectional view and a perspective view, respectively, of the analytical element of the present invention. FIGS. 13(1), (2), (3) and (4) are graphs showing measurement results using the analytical element of the present invention. 1. Specific component 2. Labeling substance (or a substance that specifically binds to the labeling substance and modulating the signal caused by the labeling substance) 3. Conjugate of specific component and labeling substance 4. Binding specifically to the specific component 5. Biological substances (or substances that specifically bind to biologically active substances) 6. Conjugations of biological substances and substances that specifically bind to specific components 7. Substances that specifically bind to biological substances (or Biological substance) 8. Substance (or labeling substance) that specifically binds to the labeled substance and modulates the signal caused by the labeled substance 9. Binding reaction product 10 of specific component and bound substance 6, bound to bound substance 3 Binding reaction product A with substance 6, immobilized product of 7 8. immobilized product of 8

Claims (1)

[Claims] (1) A substance that specifically binds to a labeling substance and modulates the signal caused by the labeling substance, and a biologically active substance that is immobilized on a carrier and does not bind to a specific component in a fluid sample, or the biologically active substance. In an immunological analysis element that contains either one of the substances capable of specifically binding in part or all of the porous reaction layer, it specifically binds to the labeling substance and emit a signal caused by the labeling substance. An immunological analysis element characterized by using an organic silver derivative as a modulating substance.