JPH08136496A - Biosensor - Google Patents

Abstract

PURPOSE: To measure a specified component of a testee easily at high precision by bonding a sensing element having a lower bonding function than that of a specific bonding reaction element and a signal forming function with the specific bonding reaction element and fixing the resulting product as a biolayer. CONSTITUTION: A sensing element having a lower bonding function than that of a specific bonding reaction element and a signal forming function is joined with the specific bonding reaction element which reacts with a specified component (an object for analysis) in a sample by a specific bonding reaction. A sample is supplied to a biolayer produced by fixing the specific bonding reaction element in an insoluble-in-water carrier and the sensing element is replaced with the specified component in the sample and thus the sensing element is isolated from the biolayer. The specified component in the testee can be determined by detecting the signal generated from the isolated sensing element. In this case, an antibody. an antigen, lectin, etc., are used for the specific bonding reaction element and a marker, for example a phosphor substance, is joined with a sensing element, e.g. pepsin, an antibody, etc., to provide the sensing element with a signal forming function. Consequently, a specified component of the testee can be measured easily at high precision.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a biosensor for analyzing a specific component in a fluid sample. In particular, the present invention relates to a biosensor using a specific binding reaction, which can easily perform continuous measurement and repeated measurement and can expect high sensitivity.

[0002]

2. Description of the Related Art A measuring method utilizing a specific binding reaction has been developed for rapid and reliable measurement of biological substances, environmental pollutants and the like. A biosensor is an example of a device that can perform such measurement rapidly. The specific binding assay method, particularly the immunological assay method, utilizes an antigen-antibody reaction, which is a specific reaction between an antigen and an antibody against the antigen, but utilizes its high detection sensitivity. It is widely used for the identification and quantitative measurement of antigens, antibodies, and others, and a part of the immunobiosensor based on this principle has been put into practical use and widely used. Thus, biosensors using specific binding are widely used in human clinical tests and disease diagnosis, and also in animals, such clinical tests and disease diagnosis, as well as a wide range of other measurement targets. It is used for analysis, measurement, quantification, and detection.

[0003]

[Means for Solving the Problems] The inventors of the present invention have conducted earnest research to develop a biosensor capable of reliably measuring a specific component in a sample by a quick and simple operation and repeatedly measuring with high sensitivity. As a result, the inventors have succeeded in developing a method and a sensor that can measure a specific component of a specimen with high sensitivity by a simple method by simply adopting a simple apparatus configuration, and completed the present invention.

The present invention provides a biosensor utilizing a specific binding reaction between a specific binding reaction element and a specific component that specifically binds to the specific binding reaction element, and the binding ability of the specific binding reaction element is reduced. And a biolayer on which a specific binding reaction element having a signal-forming ability bound to the specific binding reaction element is immobilized, and a signal-forming ability released from the biolayer It is a biosensor characterized by comprising a detection element capable of detecting a signal obtained from the sensing element. In one specific embodiment according to the present invention, a water-insoluble carrier on which a specific binding reaction element to which a labeled simulated specific component is bound is immobilized is reacted with a sample to be measured to compete. By specifically releasing the simulated specific component, a signal is obtained through the obtained label of the free simulated specific component, and then the signal is detected to measure the specific component in the sample. A measurement method and an apparatus used therefor are provided.

In the present invention, the combination of the specific binding reaction element and the specific component that specifically binds to it includes, for example, an antigen and an antibody against it, an antibody and a hapten, an effector and a receptor, an enzyme and an enzyme inhibitor, and a lectin. Examples include sugar chain-containing substances (for example, polysaccharides, glycoproteins, glycolipids, etc.), enzymes and enzyme substrates, nucleic acids and complementary nucleic acids thereof, and these may be selected from known ones. Representative examples thereof include biologically active molecules such as ionophores, ion exchange resins, enzymes, antibodies, antigens, lectins, nerve receptors, neurotransmitters, oligonucleotides, polynucleotides, polypeptides and DN.
It includes A molecules, RNA molecules, proteins, glycoproteins, metalloproteins, cofactors, active fragments or subunits thereof, and the like.

In the present invention, as the fluid sample, any form of solution or colloidal solution can be used, but preferably a biological fluid sample such as blood, plasma, serum, saliva, amniotic fluid, milk, urine, sweat, tears. , Cerebrospinal fluid, tissue culture medium, cell culture medium and the like. In the present invention, the specific component in the fluid sample that can be measured is one whose presence or absence can be detected or whose amount in the fluid sample can be measured, and which specifically binds to the specific component. A substance or substance group in which a substance can exist. Such specific components include polypeptides, proteins, complex proteins, polysaccharides, lipids, complex lipids, nucleic acids, hormones, drugs, antibiotics, vitamins, pesticides, environmental pollutants and the like. Specific examples thereof include the following substances or substance groups, but are not limited to these.

(A) Drugs and metabolites such as drugs Antihistamines, cholinergic inhibitors, valveronic acid, tegretol, prostaglandins, papaverines, oxazepans, nortriptyline, narcein, methadone, meprobamate, meperidine, L-dopa, imipramine, griseofulvin, Epinephrine, catecholamines, amphetamines, barbiturates, chlorpromazine, caffeine, butyrophenone, atropine, cannabinol, tetrahydrocannabinol, theophylline, propranolol, phenytoin, procainamide, N-acetylprocainamide, methotrexate, lidocaine,
Disopyramide, digoxin, carbamazepine, amitriptyline, acetaminophen, secobarbital, primidone, phenobarbital, ethosuximide,

Diphenylhydantoin, streptomycin, tetracycline, oxytetracycline, chlortetracycline, terramycin, polymyxin,
Penicillin, cephalosporins, erythromycin, chloromycetin, chloramphenicol, kanamycin, actinomycetin, tobramycin, neomycin, gentamicin, amikacin, quinine, quinidine, morphine, lysergic acid, heroin, dextrometrophane, codeine, cocaine, benzoyl. Ecgonine and its metabolites

(B) Vitamins Vitamin A, vitamin B ₂ , vitamin B ₁₂ , vitamin B
₆ , vitamin C, vitamin D, vitamin E, vitamin K, thiamine, biotin, folic acid, etc. (c) hormones and hormone-like substances insulin, glucagon, vasopressin, oxytocin, angiotensin, prolactin, somatostatin, growth hormone, secretin, gastrin, dopamine , Serotonin, thyroxine (T ₄ ), triiodothyronine (T ₃ ), cortisol, aldosterone, catecholamine, estrogen, progesterone, testosterone, prednisolone, somatomedin C, placental gonadotropin, placental lactogen, pituitary hormone releasing factor, follicle Stimulating hormone, luteinizing hormone, thyroid stimulating hormone, adrenocorticotropic hormone, melanin stimulating hormone, β-endorphin, etc.

(D) Cytokine or cytokine-like substance interferon α, interferon β, interferon γ, interleukin 1, interleukin 2,
Interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7,
Interleukin 8, interleukin 11, lymphotoxin, TNF, granulocyte colony stimulating factor, macrophage / colony stimulating factor, granulocyte / macrophage
Colony stimulating factor, erythropoietin, T cell substituting factor (T cell replacing factor)
r), T cell differentiation factor, transforming growth factor β, leukemia inhibitory factor, platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor, epidermal growth factor, nerve growth factor, tissue plasminogen activator ,
Sarcosine oxidase, creatinase, superoxide dismutase, human pancreatic secretory trypsin inhibitor, etc.

(E) Pesticides Phosphate esters, thiophosphates, carbamate-based pesticides, halogenated biphenyls, pyrethrin-based insecticides,
Sulfonylurea drugs, etc. (f) Virus Rubella, paramyxoviruses such as influenza virus, mumps virus, measles virus, pneumonia virus, cytomegalovirus, adenovirus, rotavirus, herpes simplex virus, poliovirus, retrovirus Such as leukemia virus, human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus (HCV)
Such

(G) Microorganisms Mycoplasma, toxoplasma, pathogenic amoeba, salmonella, streptococcus, staphylococcus aureus, chlamydia, treponema, regenera, tubercle bacillus, candida, histoplasma, etc. (h) immunoglobulins, plasma proteins, blood groups Determinants, enzymes, etc. IgG, IgE, IgM, IgA, or heavy or light chains thereof, blood coagulation factor 8, von Willebrand factor, fibrinogen, blood group surface antigen, rheumatism factor, Smith antigen, thrombin, apolipo Protein,
Anti-DNA antibody, immune complex, etc.

(I) Tumor markers and other diagnostic antigens Prostate-specific antigens, Span-1, CA-125, CA-
50, DU-PAN-2, NCC-ST-439, KM
-01, CA-15-3, SCC, atypical alkaline phosphatase, transferrin, ceruloplasmin, α
Examples thereof include 2-antiplasmin, ferritin, α-fetoprotein, CEA, and the like, and JP-A-54-
20134, JP-A-54-136896,
The materials described in JP-A-1-152367 and the like can be included here as a reference together with their measuring methods, labels and the like.

The substance that specifically binds to the specific component in the fluid sample or the sensing element applied to the present invention is an antibody, an antigen, a lectin, a polysaccharide, a glycoprotein, a glycolipid, an enzyme, or an enzyme inhibition depending on the measurement target. Examples include agents, enzyme substrates, receptors, acceptors and nucleic acids. Typically, it includes antibodies, antibody fragments, oligonucleotides, oligopeptides, polypeptides, proteins, glycoproteins, cofactors, active fragments or subunits thereof, and the like. It is particularly preferable that the binding reaction between the specific component and the specific binding reaction element is an antigen-antibody reaction.

The origin of the antibody used in the present invention is not particularly limited, and the antibody can be obtained by a conventional method, for example, Shigeru Muramatsu, et al., Experimental Biology Course 1.
4, Immunobiology, Maruzen Co., Ltd., 1985, edited by the Biochemical Society of Japan, sequel to biochemistry experimental course 5, Immunobiochemistry Research Method, Tokyo Kagaku Dojin, 1986, edited by the Biochemical Society of Japan, new chemistry experimental course 12, According to the method described in Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Kagaku Dojin, 1992, etc., the antigen is administered to mammals such as horses, cows, sheep, rabbits, goats, rats and mice. Then, the antiserum obtained by immunization, the ascites fluid as it is, or a conventionally known method, for example, salting out such as ammonium sulfate precipitation, gel filtration by Sephadex, ion exchange chromatography, electrophoresis, dialysis, Ultrafiltration, affinity
It can be used after being purified by a chromatography method, a high performance liquid chromatography method and the like.

In addition, hybrid cells (hybridomas) are obtained from spleen cells and myeloma cells (myeloma cells) of mammals (eg, mice) immunized with an antigen or the like to prepare a monoclonal antibody, which is specific to a specific component. It is preferable that it is used as a substance that can be chemically bound, or when the specific component is a specific antibody or the like, its monoclonal antibody is modified and used as a simulated specific component to further improve the specificity. Monoclonal antibodies are available from Kohler, G. & Milste.
in, C.I. , Nature, 256, 495, (197
It goes without saying that it may be a monoclonal antibody obtained by utilizing the cell fusion technique using mouse myeloma cells disclosed in 5)) and the like. The monoclonal antibody may be selected from known ones or commercially available ones. The antibody can also be produced by gene recombination technology. Further, as these antibodies, fractions such as IgG, IgM, and IgA can be used. In addition, these enzymes are treated with enzymes such as trypsin, papain, pepsin, etc. to give Fab, Fa
It may be used as an antibody fragment such as b ′ and F (ab ′). Furthermore, even if these antibodies are used alone,
Multiple antibodies may be used in combination.

The antigen or peptide used in the present invention is produced by a gene recombination method or chemically synthesized based on a gene sequence or a peptide sequence determined by gene recombination. You can also For example, a DNA sequence or a modified DNA sequence obtained by applying a gene recombination technique and using a enzyme or the like from a DNA sequence obtained by molecular cloning from a natural virus or cell or a known genome sequence, or by chemical synthesis. To
Recombinant antigens obtained by expression in microorganisms or animals, plants, insects, etc., and peptides or modified peptides obtained by the peptide chemical synthesis method known as the liquid phase method or solid phase method using such information . The solid-phase peptide synthesis method can generally be preferably carried out by an automatic peptide synthesizer, for example, Applied Biosystems model 430A or model 431A, or Migen Bioarch (MilliGAen / Biosearch). Model 9050, Model 9500, or Excel (Ex
cell), DuPont (DuPont) RMPS (RaMPS), Kokusan Kagaku Co., Ltd. "Cock-san", Advanced Chemtech (Advan)
Ced ChemTech) model 350 or the like. The recombinant antigen can be at least one kind of recombinant protein (recombinant protein) expressing different antigen regions of each natural genome. The antigen produced by the gene recombination method may be obtained as a fusion protein. Further, in the present invention, a plurality of antigens can be preferably mixed and used.

Representative antigens include viral antigens, tumor-associated antigens, metabolites, hormones, drugs and the like. Examples of viral antigens include hepatitis C virus (HCV) -related antigen, immunodeficiency syndrome-related virus (H
IV) related antigens, hepatitis B virus (HBV) related antigens and the like. As HCV-related antigens, HCVc
100-3 recombinant antigen, pHCV-31 recombinant antigen, pHCV-34 recombinant antigen and the like can be mentioned, and a mixture thereof can be preferably used. Examples of HIV-related antigens include virus surface antigens, and for example, HIV-I env. gp41 recombinant antigen, HIV-I env. gp120 recombinant antigen, HIV-I gag. p24 recombinant antigen, H
IV-II env. p36 recombinant antigen and the like.

As described above, the antigen used in the present invention can be obtained by applying, for example, a gene recombination technique to obtain natural HCV, H
DNA sequences obtained by molecular cloning from viruses such as IV and HBV, or already known HC
Recombinant antigens obtained by expressing a DNA sequence obtained from a genome sequence of V, HIV, HBV or the like by using an enzyme or by chemical synthesis in a microorganism, animal, plant, insect or the like, and further, for example, HCV and HIV
Can be at least one recombinant protein (recombinant protein) expressing different antigenic regions of the genome. As a typical example, a conservative region of an antigen such as a virus is used.
From the region known as n), a sequence designed to mimic the peptide sequence of the conserved region is designed and, for example, a peptide having 3 to 8 amino acid residues is selected and synthesized. When nucleic acid is used, it can be selected from methods known in the art such as extraction from biological materials, chemical synthesis method, PCR method, site-directed mutagenesis method, or the like. It is possible to use a method in which the above methods are combined or further modified. It can be synthesized or modified using ligase, restriction enzyme, reverse transcriptase, such as Klenow fragment.

The biolayer of the present invention is prepared by immobilizing a specific binding reaction element such as an antigen, a peptide or an antibody on at least a water-insoluble carrier. Examples of the immobilization method include a carrier binding method, a cross-linking method, an entrapping method, and a composite method combining them. A method generally used in the art can be used for the immobilization. The carrier binding method can be carried out by, for example, a method utilizing ionic interaction, hydrophobic interaction, physical adsorption, or the like, or a chemical bond such as covalent bond. When using physical adsorption, for example, activated carbon, acid clay, bleaching earth, kaolinite, alumina, silica gel, bentonite, metal oxides, hydroxyapatite, inorganic substances such as calcium phosphate, starch, chitin, gluten, cellulose, agarose, Examples of the carrier include natural polymers such as tannin, synthetic polymers such as polystyrene, and agarose derivatives having a hydrophobic group. When an ionic bond is used, ion exchangers of polysaccharides such as dextran, cellulose, agarose and starch, such as DEAE group, TEAE group, C
It is possible to use a derivative having an M group, an alkyl sulfonate group, or the like, an ion exchange resin, or the like as a carrier.

As the covalent bond method, a peptide method, a diazo method, an alkylation method, a cyanogen bromide activation method, a binding method using a crosslinking reagent, an immobilization method using a Ugi reaction, and a thiol-disulfide exchange reaction are used. Immobilization method used, Schiff base formation method, chelate binding method, tosyl chloride method,
A biochemical specific binding method and the like can be mentioned, but preferably a more stable bond such as a covalent bond is a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc. Can be used, and a suitable method can be selected from publicly known methods, methods that can be easily performed by those skilled in the art, and methods in which they are modified. Preferably, a chemical binder, a cross-linking agent or the like that can form a more stable bond such as a covalent bond is used.

These include carbodiimide,
Examples thereof include isocyanates, diazo compounds, benzoquinone, aldehydes, periodic acid, maleimide compounds, and pyridyl disulfide compounds. Preferred reagents include, for example, glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate,
N, N'-polymethylenebisiodoacetamide, N,
N'-ethylene bismaleimide, ethylene glycol bissuccinimidyl succinate, bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl)
Carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-sulfosuccinimidyl 4- (N-maleimide) Methyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) aminobenzoate, N-
Succinimidyl 4- (1-maleimidophenyl) butyrate, iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4'-dithiopyridyl)
Examples include propionimidate, methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate, N-succinimidyl-S-acetylmercaptoacetate and the like.

In the peptide method, a peptide bond is formed between the carrier and the specific binding reaction element to be immobilized. For example, a carrier having a carboxyl group is used as a derivative of azide, chloride, isocyanate or the like to form a peptide bond with a free amino group in the specific binding reaction element. A reagent used for peptide synthesis, for example, a carbodiimide reagent, Woodward reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), or the like is used. It is also possible to form a peptide bond between the amino group and the carboxyl group of the carrier and the amino group and the carboxyl group in the specific binding reaction element. In the diazo method, a carrier having an aromatic amino group is a diazonium compound, and this and a specific binding reaction element are diazo-coupled and immobilized. It can be suitably applied to a specific binding reaction device having a free amino group, an imidazole group of histidine, a phenolic hydroxyl group of tyrosine, and the like. As a carrier,
Examples thereof include polysaccharides, amino acid copolymers, polyacrylamides, styrene resins, ethylene / maleic acid copolymers, and porous glass / aromatic amino derivatives.

The alkylation method is a method in which a free amino group, a phenolic hydroxyl group and a sulfhydryl group in a specific binding reaction element are alkylated and immobilized by a carrier having a reactive functional group such as halogen. Examples of the carrier include halogenated acetyl derivatives, triazinyl derivatives, halogenated methacryl derivatives and the like. In the cyanogen bromide activation method, polysaccharides such as dextran, cellulose, agarose and starch, porous glass and the like are activated with cyanogen bromide, and then a specific binding reaction element is immobilized. Among the coupling methods using a cross-linking reagent, particularly when a bifunctional reagent such as glutaraldehyde is used, a natural polymer, a synthetic polymer, or a porous polymer in which an amino group such as cellulose, agarose, albumin, gelatin or chitosan is introduced or has been introduced. Examples include inorganic silane derivatives such as organic glass and porous ceramics. The Yugi reaction utilizes the fact that a condensation reaction occurs when a carboxyl group, an amino group, an aldehyde group and an isonitrile group coexist and are reacted. Examples thereof include one in which a carrier having a carboxyl group or an amino group and a specific binding reaction element are mixed and then reacted by adding acetaldehyde and 3-dimethylaminopropyl isocyanide. Examples of the carrier include polysaccharides, amino derivatives of polyacrylamide, and isonitrile derivatives of nylon.

The biochemical specific binding method utilizes the biochemical specific binding reaction of specific binding reaction pairs, and includes, for example, an antigen and an antibody against it, an antibody and a hapten,
Effectors and receptors, enzymes and enzyme inhibitors,
Enzyme substrates, coenzymes, prosthetic groups in complex proteins,
Examples include lectin and sugar chain-containing substance, enzyme and enzyme substrate, nucleic acid and nucleic acid complementary thereto, and these may be selected from known ones. The encapsulation method encloses the specific binding reaction element in a thin gel matrix of natural polymers or synthetic polymers such as polysaccharides and proteins, and encloses the specific binding reaction element in the space separated by the membrane. It can be roughly divided into things. Examples of the membrane encapsulation include a microcapsule type which is wrapped in a semipermeable solid membrane, a microcapsule type which is enclosed by a semipermeable membrane hollow fiber or an ultrafiltration membrane, and a liposome type which is wrapped in a liquid membrane.

In the method using a polymer gel, a specific binding reaction element is confined and immobilized in a matrix of a polymer gel having a network structure, and the gel is spherical when fixed.
It can be freely formed into a film, tube, or film. The gel is prepared by polymerizing a monomer and a crosslinking agent to form a polymer gel, polymerizing a prepolymer or an oligomer, and changing the polymer from a soluble state to an insoluble state to form a gel. The method of making it etc. is mentioned. Examples of the polymer include synthetic polymers such as polyacrylamide, polyvinyl alcohol, photocurable resin and urethane polymer, and natural polymers such as κ-carrageenan, alginic acid, pectin, chitosan, starch and collagen. In the case of polyacrylamide, acrylamide monomer, cross-linking agent N, N'-methylenebisacrylamide, polymerization accelerator N, N, N ',
It is possible to use N'-tetramethylethylenediamine, a polymerization initiator potassium persulfate for gelation, or to use radiation such as γ-rays or X-rays. In the case of using calcium alginate, sodium alginate is soluble in water, but its calcium salt and aluminum salt are insoluble in water. First, the sodium alginate aqueous solution and the specific binding reaction element are mixed and brought into contact with the calcium chloride aqueous solution.

In the case of κ-carrageenan, κ-carrageenan dissolves in water when heated, but ammonium ions,
Since gelation occurs in the presence of potassium ions, calcium ions, aliphatic amines, etc., the gel thus obtained is crosslinked and stabilized with glutaraldehyde, hexamethylenediamine or the like. As a method using a photo-crosslinkable resin polymer, polyethylene glycol (PEG) or polypropylene glycol (PP) having an appropriate degree of polymerization is used.
An example is one in which G) is used as a main chain, and a prepolymer in which a photosensitive group such as an acryloyl group, a methacryloyl group or a cinnamoyl group is incorporated at the end thereof is used. Such a prepolymer can be mixed with a solution containing a specific binding reaction element in the presence of the photosensitizer benzoin ethyl ether or benzoin isobutyl ether, and irradiated with ultraviolet rays to be gelated. The urethane prepolymer can be gelled simply by mixing it with an aqueous solution containing a specific binding reaction element.

The microcapsule type membrane encapsulation method is, for example, when a hydrophilic monomer and a hydrophobic monomer are polymerized at the interface thereof, by coating and immobilizing a specific binding reaction element, or by a submerged drying method such as benzene or hexane. , A polymer is dissolved in a highly volatile organic solvent such as chloroform, and an aqueous solution containing a specific binding reaction element is dispersed in the polymer to form a primary emulsion, which is then used as gelatin, polyvinyl, or a surfactant. In order to form a capsule, the secondary emulsion is dispersed in an aqueous solution containing the protective colloid substance and the organic solvent is removed from the obtained secondary emulsion. Hollow
In the method of immobilizing a specific binding reaction element on a fiber or an ultrafiltration membrane, it is possible to immobilize a plurality of specific binding reaction elements, and further, it is possible to immobilize in a free state without binding to a membrane. it can. References include, for example, US Pat. No. 4,003,988 and BK Van Weem.
en and AHA Schuurs, Febs Letters, Vol. 15,
No. 15, pp.232-235, (1971, 6); P. Leinikki and Suv
i Passila, J. Clin. Path., 29, pp.116-120, (1976);
BR Brodeur, FE Ashton and BB Diena, The
Journal of Medical Microbiology, Vol. 15, No. 1, p
p.1-9, (1981); J. Clin. Path., 29, pp.150-153, (19
76); Eiji Ishikawa, et al., “Enzyme Immunoassay”, Ikusho Shoin Co., Ltd., 1978.

The water-insoluble carrier refers to a carrier which is substantially insoluble in a liquid medium used for immobilization, storage, measurement and the like. As these carriers, various carriers used for specific binding reactions such as antigen-antibody reactions are known, and in the present invention, these known carriers can be selected and used. Particularly preferably used are:
Examples include those mentioned above, such as cross-linked albumin, collagen, gelatin, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate, cross-linked dextran, polyacrylamide, cross-linked polyacrylamide, polyethylene, polypropylene, polyvinyl chloride. Polyvinyl acetate, polyacrylamide, polymethacrylate, polystyrene, styrene-butadiene copolymer, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer polyester, polyamide such as nylon, polyurethane , Organic polymeric substances such as those obtained by emulsion polymerization of organic polymeric substances such as polyepoxy resin, glass, for example Glass, silica gel, silica - alumina, made of an inorganic material such as alumina, optionally, include those which had been introduced the functional group with a silane coupling agent.

The immobilized biolayer of the specific binding reaction element of the present invention is subjected to a blocking treatment for preventing a nonspecific binding reaction which is commonly used in the art or known to those skilled in the art. You can stay. For example, it can be treated with normal serum proteins of mammals, albumin, skim milk, milk fermented substances, collagen, gelatin and the like. As long as the purpose is to prevent non-specific binding reaction, those methods can be used without particular limitation.

One of the specific binding reaction pairs is a sensing element having a binding ability and a signal forming ability which are reduced with respect to the specific binding reaction element of the present invention.
Mention may be made of those having a reduced binding ability to the specific binding reaction element. The reduced binding ability can be defined from the viewpoint of sufficiently functioning as a sensor. For example, in the case of an antigen / antibody pair, when designing an antigen as a sensing element, an appropriate peptide sequence can be selected by performing epitope mapping. Epitope mapping can be performed by enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, or the like. Since such a sensing element is designed imitating a specific component, it can be called a simulated specific component. Thus, when the specific component is a protein or peptide, the sensing element can be a simulated peptide.

It can be obtained by utilizing the method of synthesizing the above-mentioned antigen or peptide. For example, a conservative region of an antigen such as virus is conserved.
gion), a sequence simulating the peptide sequence of the conserved region is designed, and a peptide sequence region having 3 to 8 amino acid residues is selected and synthesized by the multipin synthesis method. To be Examples of the peptide sequence region to be selected include, but are not limited to, a surface-exposed region, a hydrophilic region, and a region having a small number of Cys residues.

When an antibody is used as the sensing element,
Using a reducing agent to partially form a sulfhydryl group, use it, chemically modify an antibody with a bifunctional cross-linking agent, use cell fusion technology, combine site-directed mutagenesis Antibodies with reduced binding ability can be obtained by using all of the gene recombination methods. Of course, antibodies can be produced by the above-mentioned antibody production methods, and thus those methods can be appropriately combined.
The antibody can also be modified with the affinity for its recognition antigen as an index. The modification can be carried out by utilizing a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc. It can be applied by appropriately selecting from among the methods that can be applied to the above, and methods that have modified them. Further, the method adopted when immobilizing the specific binding reaction element on a water-insoluble carrier can also be applied. Preferably, a chemical binder, a cross-linking agent or the like that can form a more stable bond such as a covalent bond is used. Examples of the bifunctional cross-linking agent include those used for binding between the above-mentioned carrier and the specific binding reaction element. Antibodies include synthetic polymers such as polyethylene glycol, polypropylene glycol, polyamide,
By binding a natural polymer such as dextrin, a protein such as collagen, a peptide, a polysaccharide or the like, the affinity for the specific binding reaction element can be controlled. The hydrophilicity can be controlled in the same manner. When a nucleic acid or an oligonucleotide is used as the sensing element, it can be designed by synthesizing an appropriate sequence as described above. The sensing element can also be used in accordance with the use of a plurality of specific binding reaction elements of various kinds of molecular species.
In other words, a plurality of specific binding reaction element / sensing element pairs are immobilized in the biolayer, and multiple measurements are performed continuously.
Alternatively, it may be possible to do it non-sequentially and simultaneously or separately.

The signal forming ability of the sensing element of the present invention is
The signal is not particularly limited as long as it can form a detectable signal, and examples of the signal include chemical substances, heat, light, and sound. In the case of a chemical substance as a signal, production or consumption of a chemical substance that can be measured electrochemically can be measured by an electrode, a semiconductor element, or the like. As the electrode, a hydrogen ion electrode,
A field effect transistor such as an ammonia electrode, a carbon dioxide electrode, an oxygen electrode, a hydrogen peroxide electrode, or the like can also be used. Furthermore, the thermistor changes the heat due to the chemical reaction.
It can also be detected by a heat measuring device such as an IC chip. Further, the light emission phenomenon associated with the chemical reaction may be detected by a photon counter or the like. A radiation counter or the like can also be used. You may combine these suitably.

Such signal-forming ability can be provided by coupling the label to the sensing element.
As the label, enzymes, enzyme substrates, enzyme inhibitors, prosthetic groups, coenzymes, enzyme precursors, apoenzymes, fluorescent substances,
A radioactive substance etc. can be mentioned. As an enzyme,
Redox enzymes such as dehydrogenase, reductase, oxidase,
For example, a transferase that catalyzes the transfer of an amino group, a carboxyl group, a methyl group, an acyl group, a phosphoric acid group, etc., such as a hydrolase that hydrolyzes an ester bond, a glycoside bond, an ether bond, a peptide bond, or a lyase, Examples thereof include isomerase and ligase. Enzymes can also be used for detection by using multiple enzymes in combination. For example, enzymatic cycling can be utilized. Typical enzyme labels include peroxidase such as horseradish peroxidase, Escherichia coli β-D.
-Galactosidases such as galactosidase, catalase, calf intestinal alkaline phosphatase, and alkaline phosphatase such as Escherichia coli alkaline phosphatase.

When using alkaline phosphatase, 4
-A umbelliferone derivative such as methyl umbelliferyl phosphate, a phosphorylated phenol derivative such as nitrophenyl phosphate, an enzymatic cycling system utilizing NADP, a substrate such as a luciferin derivative or a dioxetane derivative, and the like. It can be measured by light or luminescence. The luciferin and luciferase system can also be used. The detection can also be performed by a photodiode or the like. When catalase is used, it reacts with hydrogen peroxide to generate oxygen, which can be detected by an electrode or the like. The electrode may be a glass electrode, an ion electrode using a poorly soluble salt film, a liquid film type electrode, a polymer film electrode, or the like. The enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin). As the label, a plurality of different types of labels can be used, or a label that can be detected differently depending on a plurality of specific binding reaction element / sensing element pairs can also be used. In such cases, it may be possible to make multiple measurements continuously, or discontinuously, and simultaneously or separately.

In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine, etc., horseradish peroxidase, umbelliferylgalactoside, nitrophenylgalactoside, etc., and β-D-galactosidase are used for signal formation. A combination of enzyme reagents such as glucose-6-phosphate / dehydrogenase can also be used, and quinol compounds such as hydroquinone, hydroxybenzoquinone and hydroxyanthraquinone, thiol compounds such as lipoic acid and glutathione, phenol derivatives, ferrocene derivatives, etc. What can be formed by the action of can be used.

Examples of the fluorescent substance include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate and tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt, rare earth chelate compound. , Coumarin derivatives and the like. The labeling can be carried out by utilizing a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc. It can be applied by appropriately selecting from among the methods that can be applied to the above, and methods that have modified them. Further, the method adopted when immobilizing the specific binding reaction element on a water-insoluble carrier can also be applied. Preferably, a chemical binder, a cross-linking agent or the like that can form a more stable bond such as a covalent bond is used. The measurement operation can be performed using an automated measurement device, and the luminescence detector, photo detector, etc. are used to detect and measure the display signal generated by the conversion of the substrate by the action of the enzyme. You can also The present invention also includes configuring the recorded data to be processed by a computer processing device.

According to the present invention, (a) a sensing element having a binding ability and a signal forming ability which have been reduced in advance with respect to a specific binding reaction element is used as a specific component (analyte) in a sample. A sample is supplied to a biolayer which is bound to a specific binding reaction element capable of performing a specific binding reaction, and (b) is then immobilized on the specific binding reaction element. By replacing the sensing element with, to release the sensing element from the biolayer,
(C) A specific binding analysis method for a specific component in a sample is provided, which is characterized by detecting a signal emitted from the released sensing element. In the present invention, the signal obtained from the sensing element having the signal forming ability released from the biolayer can be continuously detected. The released sensing element can obtain a signal by using the label bound thereto as an index. For example, when the label is catalase, it is made by detecting oxygen produced by the reaction between the hydrogen peroxide-containing liquid as the substrate and the catalase. The substrate can be brought into contact with the sensing element which is continuously released.

In one embodiment of the present invention, a biosensor for detecting AIDS virus using immunoaffinity is provided. For the presence or absence of AIDS virus (HIV) infection, an antibody test by enzyme immunoassay or gelatin agglutination is used as a primary screening, and then Western plotting is carried out as a secondary test for samples that are positive or false positive in this test. By doing so, a definitive diagnosis is made.
On the other hand, although it is essential to test for AIDS virus antigens that appear in blood prior to antibodies at the initial stage of infection, it is hard to say that a sufficient assay system has been developed yet. The present invention also relates to a biosensor for detecting AIDS virus using immunoaffinity, which is one of the AIDS virus-constituting proteins, gp41, which is important for AIDS diagnosis.

The entire nucleic acid sequence of HIV has already been determined
(R. Sanchez-Pescader, MD Power et al., Scienc
e, 227, pp.484 (1985) and M. Guyader, M. Emmerma.
m, L. Montagnier et al., Nature, 326, 662 (1987)).
AIDS virus membrane proteins such as gp41 and gp12
0, p24 and other antigenic determinants, especially the conserved region RG
PDRPEGIEEEGGERDRD (H-Arg-Gly-Pro-Asp-Arg-Pro-Glu-G
ly-Ile-Glu-Glu-Glu-Gly-Gly-Glu-Arg-Asp-Arg-Asp-OH)
(19mer) was synthesized by a peptide synthesizer, and after conjugating with Keyhole Limpet Hemocyanin (KLH), it was used as an immunogen to immunize animals,
Further, if necessary, a monoclonal antibody is prepared by the cell fusion method, and the thus obtained antibody is used to analyze the epitope to obtain a simulated antigen peptide. A mimicking antigen peptide having a weak affinity for the antibody is selected from the antigen-determining portion and labeled with an enzyme such as catalase to obtain a labeled mimicking antigen peptide as a sensing element. This product reacts with the biolayer on which the antibody is immobilized, and becomes a reactive biolayer for a sensor by binding with the immobilized antibody. The reactive biolayer for this sensor is
For example, it is incorporated in the sensor device of the principle as shown in FIG.
When this biolayer is contacted with an AIDS virus membrane protein such as gp41, an enzyme-labeled mimicking antigen peptide such as catalase is released, and when a substrate for a labeling enzyme such as hydrogen peroxide is encountered, an oxygen electrode such as oxygen is used. To form a signal that can be easily detected, and enable detection and measurement of AIDS virus membrane protein antigens such as gp41.

On the other hand, a simulated antigen peptide is immobilized to form a biolayer, and an antibody such as a monoclonal antibody is labeled with an enzyme such as catalase and designed as a sensing element. In this case, a preferable result can be obtained by conjugating with polyethylene glycol, but substantially no anti-H in the sample can be obtained without any special measures for affinity in a specific binding reaction as in the case of the above-mentioned mimicking antigen peptide.
There is no particular limitation as long as it can be replaced by an IV antibody, and a labeled mimic antibody can be used as a sensing element. In a typical example, a labeled mimic antibody modified with polyethylene glycol 500 by a method such as the maleimide method and enzyme-labeled by catalase by the periodate method or the like is used. The labeled mimic antibody is bound to the immobilized mimic antigen peptide to form a reactive biolayer for the sensor. This reactive biolayer for a sensor is incorporated into a sensor device having the principle as shown in FIG.
Upon contact with a sample containing IV antibody, an enzyme-labeled mimetic antibody such as catalase is released, and when it encounters a substrate for a labeling enzyme such as hydrogen peroxide, it produces a signal that can be easily detected by an oxygen electrode such as oxygen. Formed to allow detection and measurement of anti-HIV antibodies, for example. The reactive biolayer for the sensor may be composed of a plurality of layers, which may be the same or different, as required.

[0043]

EXAMPLES Next, the present invention will be described more specifically by showing examples, but it is understood that the present invention is not limited to these examples and can be carried out in various forms as long as the idea thereof is followed. Should be.

Example 1 (1) Preparation of anti-HIV gp41 serum Conserved region of AIDS virus membrane protein gp41 RGPDRPE
GIEEEGGERDRD (H-Arg-Gly-Pro-Asp-Arg-Pro-Glu-Gly-Ile-Glu-Glu-Glu
-Gly-Gly-Glu-Arg-Asp-Arg-Asp-OH) (19mer) with Cys added to the peptide at the end of Science (S
science), vol. 231, pp. 1556-1
559 (1986, March) as described in Peptide Synthesizer Model 431A (manufactured by Applied Biosystems), purified by high performance liquid chromatography, and then Keyhole Limpet.
pet Hemocyanin; KLH) and N-succinimidyl 4-
Conjugated with (m-maleimidobenzoyl) -N-hydroxysuccinimide ester and made into an emulsion with Freund's complete adjuvant,
Immunize the rabbit. Conjugate 100 μg / do
, and rabbits receive intramuscular administration three times every two weeks. After immunization, antiserum against the peptide is obtained from rabbit blood. An antibody is first salted out from the obtained antiserum with 33% ammonium sulfate, and then purified by subjecting it to protein A sepharose affinity column chromatography.

(2) Preparation of anti-HIV gp41 monoclonal antibody Peptide KLH obtained in the same manner as in Example 1 (1)
-About 6-week-old Balb / c mice (male) were immunized with the conjugate. That is, 200 μg of the above conjugate was made into an emulsion using Freund's complete adjuvant and injected into the abdominal cavity of mice. Furthermore, after 2 weeks, 150 μg of the above conjugate was made into an emulsion using Freund's incomplete adjuvant and injected into the abdominal cavity of mice. Further, after 2 weeks, 100 μg of the above conjugate dissolved in 0.15 M saline was intravenously injected into the mouse. Three days later, the spleen was removed from the mouse and splenocytes were collected. This spleen cell (4 × 10 ² )
And azaguanine-resistant mouse myeloma cells X63-Ag8-
6.5.3 (1.2 × 10 ³ cells) was mixed, and cell fusion was performed using 50% polyethylene glycol 4000 (manufactured by Merck).

Spread the fused cells in a 96-well plate, 1
5% fetal calf serum (FCS), penicillin (50 units /
ml) and streptomycin (50 μg / ml) in HAT medium (0.4 μM aminopterin, 16 μM thymidine, RPM1-1 containing 100 μM hypoxanthine)
640 medium). Half of the medium is replaced with fresh HAT medium every 2 days, and after 2 weeks, the culture supernatant of each well is assayed by the enzyme-linked immunosorbent assay (ELISA), and the positive one is cloned by the limiting dilution method. Got Next, this cell was proliferated in the abdominal cavity of a mouse to obtain an anti-HIV gp41 monoclonal antibody. The antibody obtained from the ascites was first salted out with 33% ammonium sulfate and then purified by protein A sepharose affinity column chromatography, and its purity was confirmed by sodium dodecyl sulfate-polyacrylamide electrophoresis. 41 of the anti-gp41 monoclonal antibodies obtained from one of the hybridomas
It was named S-2.

Example 2 Epitope Mapping Conserved region of AIDS virus membrane protein gp41 RGPDRPE
GIEEEGGERDRD (H-Arg-Gly-Pro-Asp-Arg-Pro-Glu-Gly-Ile-Glu-Glu-Glu
-Gly-Gly-Glu-Glu-Arg-Asp-Arg-Asp-OH) (19mer) was synthesized with 15 kinds of 5 mer peptides in which one amino acid was shifted from the N terminus to the C terminus. A multi-pin type peptide synthesizer was used for the synthesis. (See Table 1)

The reactivity of these peptides with the anti-gp41 serum obtained in Example 1 (1) was determined by sandwich type EL.
I checked by SA. As a result, the affinity of the synthesized 5mer peptide for anti-gp41 antibody in serum was GIEEE (H-Gly-Ile-Glu-Glu-Glu-OH) (41-A)> IEEEG (H-Ile-
Glu-Glu-Glu-Gly-OH) (41-B)> EGIEE (H-Glu-Gly-Ile-Glu-
The order is Glu-OH) (41-B). Similarly, anti-gp4 obtained in Example 1 (1)
The reactivity of these peptides against 1 monoclonal antibody, 41S-2, was examined by sandwich type ELSA. Table 1 shows the results.

[0049]

[Table 1]

The 41S-2 antibody was found to loosely recognize a part of the N-terminal side of gp41 and strongly recognize the central part. This is probably because the 19mer peptide is folded so that the central part and the N-terminal side are close to each other.

Example 3 Preparation of Simulated Antigen Peptides 41-A, 41-B and 41 identified above
Peptides each having Cys added to the C-terminal of -B were synthesized by a peptide synthesizer model 431A (manufactured by Applied Biosystems), purified by high performance liquid chromatography, and then labeled with catalase by the maleimide method. That is, 10 mg of catalase (Sigma C-10) was dissolved in 1.2 ml of 0.1 M sodium phosphate buffer (pH = 7.0), and N- (ε
-Maleimidohexanoyloxy) succinimide (E
MCS; Dojindo Co., Ltd.) solution (25 mg / m in DMF)
(Liquid solution) (120 ml) was added, the mixture was stirred at room temperature and reacted for 30 minutes. Next, Sephadex G-
EMCS was removed by gel chromatography using 25 (10 mm x 200 mm, manufactured by Pharmacia). 1 mg of the above synthetic peptide was added to the EMCS-treated catalase solution and reacted at 4 ° C. overnight. The obtained solution is a saline solution buffered with phosphate (PBS; pH = 7.4)
It was dialyzed against.

Example 4 Preparation of Biolayer Immobilized with Anti-gp41 Antibody A Durapore membrane (manufactured by Millipore) was cut into a circle having a diameter of 13 mm and immersed in methanol. The membrane was washed with PBS and the anti-g obtained in Example 1 (1) was obtained overnight at 4 ° C.
It is impregnated with p41 serum and then blocked with 3% skim milk / PBS for 30 minutes at room temperature. The antibody-immobilized membrane was washed with PBS and then placed in the catalase-labeled simulated antigen solution obtained in Example 3 overnight at 4 ° C. to allow the immobilized anti-gp41 antibody to catalase-labeled simulated antigen. Combine. The same treatment is carried out, and Example 1 (2)
Anti-gp41 monoclonal antibody obtained in
The S-2 antibody was also immobilized on the membrane and bound with a catalase-labeled simulated antigen. Thus, a biolayer in which the anti-gp41 monoclonal antibody 41S-2 antibody having the catalase-labeled simulated antigen bound thereto is immobilized is obtained.

Example 5 Measurement of HIV Antigen in Biosensor A biosensor for AIDS is shown by a flow diagram to have the constitution shown in FIG. The membrane, which is the biolayer obtained in Example 4, is attached to the cartridge. Pump 1
PBS at a flow rate of 30 to 60 ml / hr is used throughout the experiment. 200m sample solution containing various concentrations of antigen
1 is set in the pump 1 line with a disposable syringe. Pump 2 with PBS containing 0.6 to 2% hydrogen peroxide. When the hydrogen peroxide is released from the biolayer membrane and encounters catalase conjugated to the mimicking antigen, it is decomposed to form oxygen. Oxygen formed by the enzymatic reaction is transferred to the oxygen electrode (Clark (Cl
ark) type). The resulting current is recorded by an alk ohm resistance measuring device with a recorder.

The results show that the peptide 41-A mimicking antigen has too strong an affinity, so that 3 μg / ml of gp41 was used as the antigen.
Even when introduced (200 μl), the substitution with the simulated antigen did not occur and it did not function as a sensor. Peptide 41-B
The simulated antigen showed a slight electric signal response due to the introduction of high concentration of gp41. The peptide 41-C mimicking antigen showed good performance as a sensor. Similar results were obtained using the 41S-2 antibody-immobilized biolayer.
In this case, with peptide 41-C simulated antigen, 2.5 μg /
flow rate of about 30 ml / hr due to the introduction of ml of gp41,
A signal response as shown in FIG. 2 was obtained under the condition of 2.0% hydrogen peroxide solution. An output of about 130 pA was obtained. The maximum value was obtained about 10 minutes after the introduction of the sample. The antigen concentration dependence at that time is shown in FIG. From this, it was found that 1 μg / ml or less of gp41 can be detected. Similarly, when several biolayers were prepared using the peptide 41-C mimicking antigen, it was confirmed that peptide 41-C was excellent as a device for a senser. 2 in some cases
A sensitivity of up to 3 times was also achieved.

Example 6 (1) Preparation of simulated antibody (a) Method of Mizutani et al. (F. Mizutani and
S. Yabuki, Chemical Sensor
s, 9, Supplement B, 118 (199)
According to 3)), the anti-gp41 antibody obtained in Example 1 (1) is modified with polyethylene glycol. The antibody is labeled with catalase using the periodate oxidation method. Distilled water is added to the antibody, and then 0.1 M NaIO ₄ solution is added, followed by incubation at room temperature for about 30 minutes. After the reaction, this antibody solution is dialyzed against 1 mM sodium acetate buffer (pH 4.4) overnight at about 4 ° C. On the other hand, a phosphate buffered saline (PBS, pH 7.4) and a 10 mM sodium carbonate buffer (pH 9.5) were mixed, and catalase (Sigma C-10) was dissolved therein, and then this catalase was dissolved. The solution was adjusted to 10 mM sodium carbonate buffer (pH 9.
Dialyze against 5) overnight at about 4 ° C.

The pH of the antibody solution treated with NaIO ₄ solution is about pH.
0.2 M sodium carbonate buffer (pH 9.5) was added until the pH was around 9.5, and the catalase solution was added.
React with stirring at room temperature for about 2.5 hours. After completion of the reaction, add 0.4% sodium tetrahydroborate solution,
React with stirring at 4 ° C. for about 2 hours. After the reaction P
Dialyze against BS (pH 7.0) overnight at about 4 ° C. Thus, a catalase-labeled anti-gp41 antibody is obtained. Next, the catalase-labeled antibody solution was added to Molcat L (Millipor
e), UFP2 TMK24, 300,000 cut)
Activated polyethylene glycol (methoxy polyethylene glycol-succinimidyl succinate, Sigma M3152) was added and reacted at about 37 ° C. for about 2 hours with stirring. After the completion of the reaction, the mixture was filtered through Molcut L to obtain a simulated antibody.

(B) Similarly, a mimic antibody was prepared for the anti-gp41 monoclonal antibody 41S-2 antibody obtained in Example 1 (2). 5.19 mg / ml of 41S-2
To 385 μl of antibody was added 616 μl of distilled water and then 0.1
Add 200 μl of MNaIO ₄ solution and incubate at room temperature for about 30 minutes. After the reaction, this antibody solution is dialyzed against 1 mM sodium acetate buffer (pH 4.4) overnight at about 4 ° C. On the other hand, phosphate buffered saline (PBS, pH
7.4) 0.5 ml and 10 mM sodium carbonate buffer solution (pH 9.5) 0.5 ml were mixed, and 16 mg was added thereto.
Catalase (Sigma C-10) is dissolved and the catalase solution is then dialyzed against 10 mM sodium carbonate buffer (pH 9.5) overnight at about 4 ° C.

NaIO ₄ solution treatment To the 41S-2 antibody solution, 0.2M sodium carbonate buffer solution (pH 9.5) was added until the solution was about pH 9.5, the above catalase solution was added, and the solution was allowed to stand at room temperature for about 2 minutes. React for 5 hours with stirring. After completion of the reaction, 0.4% sodium tetrahydroborate solution is added, and the mixture is reacted at 4 ° C. for about 2 hours with stirring. After completion of the reaction, it is dialyzed against PBS (pH 7.0) overnight at about 4 ° C. Thus, the catalase-labeled anti-gp41 monoclonal antibody 41S-2 antibody is obtained. Next, catalase labeled 41S
-2 antibody solution to Molcut L (Millipore), UF
P2 TMK24, 300,000 cut) is filtered and activated polyethylene glycol (methoxy polyethylene glycol-succinimidyl succinate, Sigma (S
igma) M3152) 120 mg was added, and the temperature was about 37 ° C.
The mixture was reacted with stirring for about 2 hours. After the completion of the reaction, the mixture was filtered through Molcut L to obtain a simulated antibody.

(2) Preparation of Biolayer Immobilizing Peptide 41-C Peptide 41-C was immobilized in the same manner as in Example 4. That is, Durapor
e) A membrane (manufactured by Millipore) is cut out into a circle having a diameter of 13 mm and immersed in methanol. The membrane is washed with PBS and impregnated with the mock antigen peptide 41-C obtained in Example 3 at 4 ° C. overnight, and then blocked with 3% skim milk / PBS for 30 minutes at room temperature. The peptide-immobilized membrane was washed with PBS and then overnight at 4 ° C. in Example 6.
It is placed in the catalase-labeled simulated antibody solution obtained in (1), and the catalase-labeled simulated antibody is bound to the immobilized peptide 41-C. Similarly treated, the anti-gp41 monoclonal antibody obtained in Example 1 (2), for example 41S
The -2 antibody was also bound to the above peptide 41-C. Thus, the catalase-labeled simulated antibody was bound 41
Obtain an immobilized biolayer of -C. In the immobilization, the 41-C peptide synthesized in Example 3 can be immobilized by using the maleimide method.

Example 7 Measurement of anti-HIV antibody in biosensor The diagnosis of HIV infection is finally determined by anti-gp41 antibody, anti-g
Diagnosis is made by measuring the presence of p120 antibody and anti-p24 antibody. Then, the biosensor for anti-HIV antibody is shown in the same flow diagram as in Example 5 and has the configuration shown in FIG. 1, and the biolayer membrane obtained in Example 6 is attached to the cartridge. Pump 1 is used to flow PBS at a flow rate of 30 to 60 ml / hr throughout the experiment. Sample liquid 200 containing various concentrations of anti-gp41 antibody
Set ml on the line of pump 1 with a disposable syringe. Pump 2 with PBS containing 0.6 to 2% hydrogen peroxide. The hydrogen peroxide is released from the biolayer membrane and is decomposed when it encounters catalase conjugated to the mimicking antibody to form oxygen. The oxygen formed by the enzymatic reaction is the oxygen electrode (Clark (C
monitor type). The resulting current is recorded by an alk ohm resistance measuring device with a recorder.

As a result, an enzyme reaction was caused by the released mimic antibody conjugate, and an electric signal response generated by oxygen generation generated by decomposition of hydrogen peroxide could be detected. Peptide 4 immobilized with catalase-labeled simulated 41S-2 antibody
Similar results were obtained using the biolayer bound to 1-C. In this case, various concentrations of 41S-2 antibody were added to the anti-gp41 antibody.
When 200 ml of the sample solution containing the antibody was set in the line of the pump 1 with a disposable syringe and allowed to flow, a responsiveness corresponding to the amount of 41S-2 antibody was obtained. The sensitivity is higher than that of the antigen measurement, and it is possible to detect an amount less than 0.2 μg / ml. FIG. 4 shows the results. By repeating the experiment, it was confirmed that about 0.1 μg / ml of the antibody can be detected, and the sensitivity is evaluated to be practically usable. By using the epitope mapping method and peptide synthesis method used in the present invention, it is possible to prepare a sensing element such as a simulated antigen or a simulated antibody having an ideal affinity similarly for the conserved region of various antigens. It was confirmed that it was possible.

[0062]

According to the present invention, it is possible to design a wide range of bisensors capable of quick measurement and detection, and to provide a device having a wide range of applications. Especially until now AIDS
However, there is no suitable bisensor and it takes a lot of time to measure and detect it, but a sensor having excellent sensitivity can be developed according to the method of the present invention.

[Brief description of drawings]

1 shows a flow diagram of a biosensor according to the present invention.

FIG. 2 shows the signal responsiveness obtained by introducing gp41 in a biosensor using a biolayer on which the 41S-2 antibody of the present invention is immobilized.

FIG. 3 shows the antigen concentration dependency when the maximum signal responsiveness is exhibited by the introduction of gp41 in the biosensor using the biolayer on which the 41S-2 antibody of the present invention is immobilized.

FIG. 4 is an anti-gp in a biosensor using a biolayer in which a catalase-labeled simulated 41S-2 antibody of the present invention is bound to immobilized peptide 41-C.
41 shows the sensitivity of antibody measurement.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 33/68

Claims (14)

[Claims] 1. A biosensor using a specific binding reaction between a specific binding reaction element and a specific component that specifically binds to the specific binding reaction element, which has a reduced binding ability with respect to the specific binding reaction element. And a biolayer in which a specific binding reaction element having a sensing element having a signal forming ability bonded to the specific binding reaction element is immobilized.
A biosensor comprising a detection element capable of detecting a signal obtained from a sensing element having a signal forming ability released from the biolayer. 2. A sample to be measured is brought into contact with the biolayer, and the sensing element is released with a specific component that is sensitively bound to the specific binding reaction element, and the signal forming ability released from the biolayer. 2. The biosensor according to claim 1, wherein the sensing element having the above is subjected to a detectable signal forming step, and the signal formed next can be continuously detected by the detecting element. 3. The sensing element is labeled with a specific binding reaction element having a reduced binding ability and a signal forming ability as compared with a specific component which is sensitively bound to the specific binding reaction element. The biosensor according to claim 1, wherein the biosensor comprises a peptide. 4. The sensing element is labeled with a specific binding reaction element having a reduced binding ability and a signal forming ability as compared with a specific component which is sensitively bound to the specific binding reaction element. The biosensor according to claim 1, which comprises an antibody or a derivative thereof. 5. The biosensor according to claim 1, wherein the specific binding reaction element has a specific binding reactivity substantially equal to that of a specific antibody or a specific antibody. 6. The biosensor according to claim 1, wherein the specific binding reaction element has a specific binding reactivity equivalent to that of the specific monoclonal antibody or the substantially specific monoclonal antibody. 7. The biosensor according to claim 1, wherein the specific binding reaction element has a lower affinity for the specific antibody than an antigen-determining site recognized by the specific antibody. 8. The specific binding reaction element is selected by epitope mapping the antigen-determining site and has a lower affinity for the specific antibody. The biosensor described. 9. The biosensor according to claim 1, wherein the specific binding reaction element is a peptide imitating an antigen-determining site recognized by a specific antibody. 10. The biosensor according to claim 1, wherein the biosensor is for detecting acquired immunodeficiency syndrome (AIDS). 11. The biosensor according to claim 1, wherein the signal is formed by an enzymatic reaction. 12. The biosensor according to claim 1, wherein the sensing element is an oxygen electrode. 13. A biolayer in which an enzyme-labeled mimicking antigen peptide is bound to an antibody immobilized by a specific binding reaction or an enzyme-labeled specific antibody is bound by a specific binding reaction. A reactor having a biolayer bound to the immobilized mimicking antigen peptide, a device for supplying a sample to be measured to the reactor, and liberated from the reactor by substitution accompanying a specific binding reaction. The enzyme-labeled mimicking antigen peptide and means for producing a detectable signal by an enzymatic reaction, and means for electrochemically detecting the produced signal are provided. Biosensor. 14. A method for measuring a specific component using a specific binding reaction between a specific binding reaction element and a specific component that specifically binds to the specific binding reaction element, wherein the binding is reduced with respect to the specific binding reaction element. The sensing element having the ability to form a signal and the signal to be measured is supplied to the biolayer on which the specific binding reaction element is immobilized, which is bound to the specific binding reaction element. Is replaced with the specific component to release it, and then the released sensing element is used to generate a signal by utilizing its signal forming ability, and the formed signal is detected by a detecting element capable of detecting the formed signal. And the method of measuring specific components.