WO1990013029A1 - Reactif de dosage de substances biologiquement actives, son procede de production, procede et appareil de dosage - Google Patents

Reactif de dosage de substances biologiquement actives, son procede de production, procede et appareil de dosage Download PDF

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Publication number
WO1990013029A1
WO1990013029A1 PCT/JP1990/000514 JP9000514W WO9013029A1 WO 1990013029 A1 WO1990013029 A1 WO 1990013029A1 JP 9000514 W JP9000514 W JP 9000514W WO 9013029 A1 WO9013029 A1 WO 9013029A1
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WIPO (PCT)
Prior art keywords
compound
substance
optical fiber
protein
fluorescent dye
Prior art date
Application number
PCT/JP1990/000514
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English (en)
Japanese (ja)
Inventor
Takeshi Kobayashi
Hiroyuki Honda
Kenichi Shimada
Original Assignee
Ibiden Co., Ltd.
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Publication date
Application filed by Ibiden Co., Ltd. filed Critical Ibiden Co., Ltd.
Priority to JP2506243A priority Critical patent/JP2951398B2/ja
Publication of WO1990013029A1 publication Critical patent/WO1990013029A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • Bioactive substance measurement reagent its production method, measurement method and
  • the present invention relates to a reagent which can be used for measuring a biologically active substance by an immunoassay, a method for producing the same, a resinous optical fiber necessary for utilizing the same, and a method using the optical fiber.
  • the present invention relates to a measuring device having a detecting section, and a method for measuring a biologically active substance using these reagents and devices.
  • labeling reagents such as radioisotopes, luminescent agents, enzyme-labeled antigens and antibodies have been developed.
  • Japanese Patent Application Laid-Open No. 58-61848 discloses an organic polymer compound to which a plurality of luminescent components are bonded.
  • An immunoreagent conjugated to either an antibody or an antigen and an immunoassay method using the same are described in U.S. Pat.No. 4,166,105.
  • Reagents have been proposed for detecting the first reactant (antibody) capable of reacting with a plurality of fluorescent dye molecules, respectively.
  • these reagents do not have sufficient amount of dye that can be bound, and the problem is that the detection sensitivity is not practical when a dye with low sensitivity that is excited at a long wavelength is used. There is.
  • Japanese Patent Application Laid-Open No. 60-252,265 discloses that a water-soluble organic polymer compound having a luminescent agent bound thereto is bound to avidin, and a biologically active substance is measured using the compound. The method is disclosed.
  • a large amount of bitin is bound around the antibody or antigen because the antibody or antigen is bound to avidin via low molecular weight piotin.
  • the immune activity may be reduced, and it is necessary to take a treatment to prevent this, and it is difficult to bind the water-soluble polymer to avidin due to steric hindrance. problem is there.
  • luminescence immunoassay a method of exciting a fluorescent dye (luminescent agent) using an optical fiber or transmitting fluorescence is effective, and various techniques have been disclosed.
  • P 1226-1230 (1987) describes a silane coupling agent for the silane group on the surface of a quartz optical fiber.
  • Resin light Fibers are inexpensive, easy to polish, flexible, and easy to handle, so it is desirable to have a method for binding proteins such as antigens and antibodies to resinous fibers. It is rare.
  • an immunoassay apparatus and method are disclosed in Japanese Patent Publication No. 59-501873 (US Pat. No. 4,582,809).
  • the optical fan is disclosed in Japanese Patent Application Laid-Open No. S2-123358, and Japanese Patent Application Laid-Open No. Sho 62-501102 (Sheet Patent Application No. 5306-684-5).
  • Each type of immunosensor has been proposed. However, these specifications do not describe any technology for performing high-sensitivity labeling of antigens and antibodies. Therefore, Hg lamps, Xe lamps, and Ae lamps are used as light sources. r There is a problem that only a fluorescent dye having high sensitivity such as that excited by a laser is used, and that the apparatus is large and expensive.
  • Japanese Patent Application Laid-Open No. Sho 60-244550 discloses a method in which a luminescent agent bound to an immune complex by a bitin-avidin bond.
  • Agent binding A method for measuring a biologically active substance that measures the amount of luminescence due to the luminescence reaction of avidin has been proposed. This method binds avidin-piotin between the luminescent agent and the antibody or antigen.
  • the amount of the dye that can be bound is small and a low-sensitivity dye cannot be used because it is not through another organic polymer compound.
  • the present inventor has found that in a method for measuring the concentration of a bioactive substance, in order to further improve the measurement sensitivity, the amount of a fluorescent dye bound per protein molecule must be reduced.
  • the present inventors have found that a bioactive substance is bound to a compound having a large number of reactive groups, and a compound having a large number of fluorescent dyes bound to each reactive group.
  • the development of a detection unit consisting of an optical fiber capable of efficiently condensing and measuring light emitted from a fluorescent dye, a device equipped with the same, and a measurement method using these reagents and devices succeeded in.
  • the fluorescent label binds to a compound having a plurality of reactive groups, and It is noted that a compound modified with a plurality of fluorescent dyes is bonded to the reactive group of the compound having a reactive group.
  • the fluorescent dye described in the present invention refers to a dye that cannot be excited by light, and does not mean a fluorescent dye that emits chemiluminescence or bioluminescence.
  • the light is desirably coherent light such as laser light.
  • the reagent for measuring a biologically active substance comprises a compound having a plurality of reactive groups in which a compound modified with a plurality of fluorescent dyes is bonded to most of the reactive groups of the compound having a plurality of reactive groups.
  • an active substance By binding to an active substance, the amount of fluorescent dye per biologically active substance is increased, thereby greatly improving detection sensitivity.
  • Examples thereof include an amino group, a thiol group, a hydroxyl group, a carboxyl group, and a formyl group, and an amino group is particularly desirable. The reason for this is that the amino group has a relatively high reaction activity and the generated bond is stable.
  • the number of the reactive groups be 20 to 100 000 per molecule.
  • the reason is that if the number is less than 2 ⁇ , improvement in detection sensitivity cannot be expected, and if the number is more than 1 Q 0 This is because it is difficult to dissolve the polymer in the solvent.
  • the number of the reactive groups is 30000 to 600 0 per molecule, which is more preferable than the force s, and is preferably 40000 to 500 000. .
  • the compound having a plurality of reactive groups is desirably a compound such as amimeglucan or polyaminopeptide.
  • the reason for this is that there are 40000 to 5000 amino groups in these compounds.
  • chitosan As the above-mentioned amino glucan, chitosan, polygalactosamine, polyinomyric acid, etc. can be used, but chitosan is used. This is preferred.
  • polyaminopeptidyl is an amino acid having two or more amino groups, which is polymerized by a peptide bond.
  • a resin for example, a resin.
  • the compound to be modified with the fluorescent dye used in the present invention may be a natural polymer compound having a molecular weight of semi-hight molecule or more with a molecular weight of about 1000 or more. Desirable.
  • Naturally occurring high molecular weight compounds such as avidin, protein A, antibodies, hormones, and hormone receptors, which are relatively easily available, are desirable.
  • the bonding between the compound having a plurality of reactive groups and the biologically active substance, and the bonding between the reactive group of the compound having a plurality of reactive groups and the compound modified with a plurality of fluorescent dyes are performed by using a suitable crosslinking agent. Can be used.
  • the compound is bonded to the compound having a plurality of reactive groups through a substance that specifically binds to the compound.
  • the plurality of reactions may be performed via a protein, an antibody, protein A, a hormone receptor, and a hormone, respectively.
  • a combination with a compound having an active group, particularly "avidin-biotin” is preferable.
  • the fluorescent dye may be bound to any of protein A and the antibody.
  • the avidin is a basic albumin-like crystal protein having a molecular weight of about 6800, and has an extremely high affinity for bitin selectively. Avidin is stable against heat, PH, chemical modification, etc. Avidin is suitable for modification with a fluorescent dye because it has a large number of amino groups on the molecular surface due to its electric point of 1 °.
  • Avidin has 36 amino groups, some of which contribute to the binding of biotin, so that 2 to 10 fluorescent dyes can be bound per avidin molecule. And are desirable.
  • the reagent for measuring a bioactive substance of the present invention can be obtained by labeling a complex of one or more reactive groups with a bioactive substance complex with the modified avidin.
  • Protein A is a protein having a molecular weight of 420, which occupies 5% of the cell wall of Staphylococcus aureus, and has a high affinity for immunoglobulin (antibody protein).
  • radioimmunoassay radio-immunoassay
  • the light is desirably laser light or LED light (light emitting diode), and the laser light is desirably a He—Ne laser or a semiconductor laser.
  • the above laser is smaller and less expensive than the Xe lamp or Ar laser.
  • SHG element second harmonic generation element: an element that reduces the wavelength of light
  • the fluorescent dye used in the present invention be excited by light having a wavelength of 200 to 800 nm.
  • fluorescent dye examples include coumarin derivatives such as emperiferon, polycyclic aromatic derivatives, rhodamine isothiocyanate, fluorescein isothiocyanate, cyanine dyes, and fiyne dyes. Copiritanno, ⁇ , dansyl derivatives, o-phthalaldehyde and the like can be used, and cyanine dyes are particularly preferred.
  • a cyanine dye has a structure in which a complex containing azonium ion is bound by a methine chain
  • ⁇ and ⁇ ′ represent 0, S, Se, one NH— or one CH CHCH—, and R and R ′ represent alkyl groups such as methyl, ethyl, and propyl. Or a carboxyalkyl group such as carboxyethyl; X represents a halogen atom; and n represents a natural number of 0 to 3).
  • These cyanine pigments are He—Ne lasers.
  • the size and cost can be reduced as compared with the case where an Ar laser or an Xe lamp is used or the case where a semiconductor laser combined with an expensive SHG element is used.
  • cyanine dye a cyanine dye represented by the formula (1) is particularly preferably used.
  • n 0, 1, 2 or 3. Particularly preferably, n is 2.
  • Labeling with a fluorescent dye targets biologically active substances, so the reaction is completed in a short time under mild conditions as much as possible, and binds to the reactive group without side reaction. It needs to be something like that.
  • a carbocyanine dye represented by the above formula (1) having a carboxyl group outside the conjugate system as a functional group is preferably used.
  • the bioactive substance used in the present invention includes saccharides and proteins, but proteins are particularly desirable.
  • the bioactive substance comprising the protein is preferably an antigen, an antibody, an enzyme, a habutene or an enzyme inhibitor.
  • the reagent for measuring a bioactive substance of the present invention be bound to an optical fiber and excited upon measurement.
  • the resin optical fiber of the present invention is a resin optical fiber having, on a core surface, a reactive active group capable of covalently bonding a substance that specifically binds to the bioactive substance measurement reagent. I need an inverter.
  • the optical fiber is made of resin
  • the resin optical fiber is inexpensive, has a larger polishing diameter than the glass optical fiber, and has a larger core diameter. Therefore, a large amount of light can be transmitted, and the flexibility can be maintained even if the core diameter is increased. This is because an optical fiber made of 'methyl polymethacrylate' has excellent transmission properties in the wavelength range of about 560 to 65 nm.
  • the density of the reactive group be 1.0 X 10 1 Q to 6.0 X 10 13 Zcm 2 .
  • the absolute number of reactive groups on the surface of the resin-made optical fiber decreases, and the measurement sensitivity is lowered, which is not practical, and is higher than the above range. In this case, the transmittance of the fluorescent light emitted from the fluorescent dye to the optical fiber becomes extremely poor. (See Fig. 7: In case of methyl polymethacrylate)
  • the resin constituting the resin optical fiber must be a material that does not adsorb bioactive substances and has good translucency.
  • a material that does not adsorb bioactive substances and has good translucency for example, polystyrene, polyacrylic acid, etc. Esters, polyesters, polyacrylamides, polyvinyl alcohols, polyethylene glycols, polycarbonates, or copolymers thereof. Can be used.
  • the resin optical fiber contains, as a main component, a resin having a structure that reacts with a crosslinking agent.
  • a resin having a structure that reacts with a crosslinking agent is desirable.
  • the surface of the optical fiber has reactive activity. This is because a crosslinking agent for introducing a group is easily reacted.
  • Examples of the structure that reacts with the cross-linking agent include an ester bond, an amide bond, an ester group, a carboxyl group, a formyl group, an amino group, a hydroxyl group, and an epoxy group. And a thiol group are preferred, but an ester structure such as an ester bond or an ester group is preferred.
  • the translucent resin having a structure or a functional group that reacts with the cross-linking agent a polyacrylic acid ester or a polyester is preferable.
  • the acrylate ester polymer has an ester structure among acrylic acid resins, and is, for example, a polymer derived from a polymer of an ester derivative such as acrylic acid or methacrylic acid. Specific examples thereof include polymers such as methyl acrylate, ethyl acrylate, and methyl methacrylate. Further, among the acrylate polymers, one particularly preferably used in the present invention is methyl polymethacrylate. Polymethyl methacrylate has better translucency than other resins.
  • Japanese Patent Application Laid-Open No. 59-501873. (US Pat. No. 4,582,809) describes an example in which an antigen and an antibody are bound to the surface of a nylon optical fiber using a cross-linking agent. Although it is not an optical fiber, Japanese Patent Application Laid-Open No. 56-12841 discloses that the surface of a cell for measuring the absorbance of methyl polymethacrylate or nylon is measured. Techniques for binding proteins are described.
  • the former technique uses an optical fiber made of nylon having low translucency, and the inventors of the present application have found that the propagation loss of the nylon fiber is large. In addition, high-sensitivity measurement as intended by the present inventors was difficult.
  • the latter technique involves binding proteins to the absorbance measurement cell, and is not a technique for immobilizing it on an optical fiber.
  • the resin optical fiber used in the present invention is, for example, a copolymer of a monomer such as methyl acrylate, ethyl acrylate, methyl methacrylate and a monomer such as styrene. It may be.
  • the reactive groups on the surface of the optical fiber include a formyl group, a carboxyl group, an amino group, a hydroxyl group, an epoxy group, a thiol group, an isocyanate group, and Examples thereof include a sothiocyanate group, and a formyl group is preferable.
  • the reason is common to the fiber of the present invention.
  • the substance to be bound is a bioactive substance and requires mild reaction conditions that do not reduce its activity, but the formyl group easily reacts with the bioactive substance, particularly the amino group of the protein. Because you do.
  • the resin of the resin optical fiber does not have a structure that reacts with the cross-linking agent, it has a structure that introduces a functional group into the resin and has a structure that reacts with the cross-linking agent.
  • a functional group is introduced into the part by reacting with an appropriate crosslinking agent.
  • a polyfunctional compound for example, aldehydes such as glutaraldehyde, succinaldehyde, adipoaldehyde, N,, and the like.
  • aldehydes such as glutaraldehyde, succinaldehyde, adipoaldehyde, N,, and the like.
  • cyan or diisothiocinart is used.
  • a formyl group can be introduced by reacting aldehyde.
  • the most preferable resin optical fiber for measuring a bioactive substance has a formyl group on the core surface of the resin optical fiber mainly composed of a resin having an ester structure. It is a form.
  • the optical fiber made of resin for measuring a bioactive substance is a nucleophilic reagent having a formyl group as a polyfunctional compound on the exposed core surface of the optical fiber made of resin. Is produced by introducing a formyl group on the core surface.
  • nucleophilic reagent having a formyl group a reagent represented by the formula (2) is preferable.
  • R 1 and R 2 each represent a hydrogen atom, an alkyl group or a formyl group, and n represents an integer of 0 to 5)
  • Examples of the reagent represented by the formula (2) include glutaraldehyde and succinaldehyde.
  • the reagent of the formula (2) reacts nucleophilically with the ester group of the resin (in the following reaction formula, methyl methacrylate) as shown in the following reaction formula.
  • the diameter of the optical fiber is usually 1 mm and the diameter of the core cross section is only 0.97 (cross-sectional area is 0.739 mm 2 ). This is because, in order to introduce a large amount, it is necessary to separate the cladding layer and increase the core surface area.
  • the polishing is preferably performed using alcohol as a lubricant.
  • the concentration of the base such as KOH is preferably 50 to 100 ntM.
  • the reason for adding the Ni salt is that the Ni salt promotes the reaction and at the same time prevents oxidation of the formyl group and addition of the OH group.
  • the core part of the resin optical fiber is immersed in the reaction reagent to react with the ester structure, and the reaction temperature is preferably adjusted appropriately.
  • the detection unit for measuring a biologically active substance of the present invention needs to covalently bond a substance that specifically binds to the substance to be measured to a reactive group of the resin optical fiber.
  • the substance that specifically binds to the analyte is preferably a protein, and may be an antigen, an antibody, an enzyme, a habutene, an enzyme inhibitor, or the like.
  • the reactive group of the resin optical fiber is a formyl group
  • the resin is methyl methacrylate
  • the detection unit of the present invention may be covered with a flow cell 5 as shown in FIG. 1, may be a facing type as shown in FIG. 3, or may be a type as shown in FIG. It may be a reflection type with a mirror 12 at the tip.
  • the excitation light is incident from the side opposite to the tip, and the fluorescent light propagates through the optical fiber 3 to which the detection unit is attached.
  • the excitation light propagates through the optical fiber 13 to which the detection unit is attached and is incident, and the excitation light is reflected by the mirror 12 at the tip, and Propagate fiber three.
  • the reagent for measuring a bioactive substance of the present invention is produced by various organic chemical reactions.To produce a protein modified with a plurality of fluorescent dyes of the present invention, a fluorescent dye is reacted with a protein. The residue obtained by removing the solvent from the reaction product is suspended in a buffer having a pH of 2 to 7 to separate and remove unreacted dye.
  • the buffer with a pH of 2 to 7 is labeled with a fluorescent dye.
  • the dissolved proteins dissolve, but the unreacted fluorescent dyes do not dissolve, so they can be easily separated.
  • the pH of the buffer is 2 or less, the protein is hydrolyzed, and when the pH is 7 or more, the fluorescent dye is dissolved.
  • the pH of the buffer is desirably 4.9 to 7.0, and is preferably 6.5 ⁇ 0.5, especially when avidin modified with a cyanine dye is produced. It is.
  • the fluorescent dye is desirably an acidic fluorescent dye exhibiting good solubility in a basic buffer.
  • the fluorescent dye be excited by laser light.
  • the fluorescent dye the above-mentioned cyanine dye, full-year-old resin, thiothiosinate, or the like is used.
  • the protein of the present invention may be various proteins, for example, neocarzinostan, an enzyme, a hormone, and the like, and is preferably a basic protein (a protein having PI ⁇ 7).
  • a basic protein a protein having PI ⁇ 7
  • the reason for this is that the basic protein exhibits good solubility in a buffer having a pH of 2 to 7 even when a hydrophobic fluorescent dye is bound thereto.
  • Avidin is preferred as the basic protein.
  • the reaction between the fluorescent dye and the protein is dicyclohexyl
  • a carbodimid reagent such as ruka rubomidide or di-p-toluene rubozimid
  • an amino group of a protein is condensed with a reactive group of a fluorescent dye.
  • the reaction solvent may be any as long as it can dissolve the fluorescent dye and protein.
  • the reaction is performed in a solvent such as an organic solvent or a basic aqueous solution.
  • reaction proceeds too much and the 0H group or SH group of the protein reacts with the fluorescent dye, resulting in the reaction specificity of the protein (antigen-antibody reaction or reactivity with bigtin when the protein is avidin) If necessary, stop the reaction with acetic acid or the like to prevent inactivation.
  • the solvent is distilled off under reduced pressure, removed and evaporated to dryness, and dissolved in a buffer solution having a pH of 2 to 7.
  • Unreacted fluorescent dye does not dissolve, so that the fluorescent dye can be easily separated by an appropriate separation means.
  • the unreacted dye can be removed by centrifugation and passing the supernatant through a tube filled with glass wool.
  • the biologically active substance binds to the compound having a plurality of reactive groups
  • the compound B binds to the reactive group of the compound having the plurality of reactive groups
  • the compound B has a plurality of reactive groups.
  • Compounds to which compound A modified with a fluorescent dye is bound for simplicity, hereinafter, fluorescent dye-compound A-compound B-compound having multiple reactive groups-bioactive substance
  • the compound B is reacted with most of the reactive groups of the compound having a plurality of reactive groups, the compound having the plurality of reactive groups is modified with the compound B, and then the biologically active substance is reacted.
  • Compound B-Compound having multiple reactive groups-Bioactive substance complex and then reacting with Compound A modified with fluorescent dye, Fluorescent dye compound A-Compound B-Multiple reactive activities A compound having a group-a bio-active substance complex is produced.
  • the combination of compound A and compound B be a protein and a compound that specifically binds to the protein.
  • avidin and biotin, and protein A And antibodies, antibodies and protein A is a combination of avidin and piotin.
  • chitosan (I) have a large number of amino groups in the molecule, and chitosan (I) is added with piotin (II) as a basic solution.
  • a dehydrating agent such as water-soluble carbodiimide (CHMC) or N-hydroxysuccinimide
  • bitin is combined with an acid amide to obtain a piotinylated chitosan (m).
  • biotinylated protein is reacted with the biotinylated chitosan ( ⁇ ) using the same dehydrating agent as described above, and the protein is bound to the remaining free amino group of chitosan (I).
  • the obtained chitinated chitosan (IV) is obtained.
  • avidin (V) modified with a fluorescent dye can be obtained by reacting a fluorescent dye, for example, a carboxyl group of a cyanine dye with an amino group of avidin, a protein, in the same manner as described above. And can be done.
  • the carboxyl group of the above cyanine dye can be easily formed by a conventional method using an amino group of avidin and a dehydrating condensing agent such as dicyclohexylcarbodiimide in an organic solvent. It can be condensed to form an amide bond. After completion of the reaction between the cyanine dye and avidin, it is preferable to remove any unreacted substances. For example, dialysis, centrifugation, gel filtration, or use of permeation material? It can be removed by using the law.
  • the acid amide bond formed by binding biotin to chitosan has a wavelength of 450 nm, 300 nm and 490 nm when the excitation wavelength is 25.5 at ⁇ 26. Each region has a characteristic fluorescence peak.
  • the measuring method using the reagent for measuring a biologically active substance of the present invention is performed by the following method.
  • the complex consisting of the fluorescent dye, avidin, and biotin, or the substance to be measured or a substance that specifically reacts with the substance to be measured is converted into a substance that specifically binds to the substance to be measured on the optical fiber.
  • test substance or the substance which specifically reacts with the test substance to which biotin is bound is combined with the substance or the test substance which specifically binds to the test substance on the optical fiber; After specific reaction, avidin modified with a fluorescent dye is reacted, and a complex is formed on the optical fiber by avidin-biotin bond.
  • a measuring method characterized by measuring the following.
  • the analyte or the substance that specifically reacts with the analyte binds to the compound having a plurality of reactive groups, and the reactive group of the compound having the plurality of reactive groups has a plurality of fluorescent light.
  • the reagent to which the compound modified with the dye is bound is specifically bound to the analyte on the optical fiber.
  • the substance to be measured or a substance that specifically reacts with the substance to be measured binds to a compound having a plurality of reactive groups.
  • the fluorescence is measured.
  • the compound A modified with the dye is reacted to form a complex by combining the compound A and the compound B on the optical fiber, and then the fluorescent dye is excited by light to emit a fluorescent light.
  • the measurement method 1) will be described.
  • the measurement method 1) it is necessary to use, as a reagent, a complex composed of a fluorescent dye, avidin, piotin, a substance to be measured, or a substance that specifically binds to the substance to be measured.
  • the reason for using the above reagent is that if a substance to be measured or a substance that specifically reacts with the substance to be measured is labeled with a fluorescent dye, the amount of the fluorescent dye bound is limited, and the binding of the fluorescent dye is limited. There is a risk that the binding site of the analyte or the substance that specifically reacts with the analyte may be damaged. You.
  • the reagent is excited by light after reacting specifically with a substance to be measured or a substance to be measured on the optical fiber.
  • the excitation on the optical fiber is because the optical fiber can transmit the excitation light and the fluorescent light, and can perform efficient measurement.
  • the fluorescent dye is preferably a cyanine dye.
  • cyanine dyes can be excited by a He--Ne laser (633 nm) or a semiconductor laser (636 nm), which reduces the size of the device and reduces the size. Costing is possible.
  • a carbocyanine dye represented by the following formula and having a carboxyl group in addition to a conjugate system as a functional group is preferably used.
  • the carboxyl group of the cyanine dye and the amino group of the protein are dissolved in an organic solvent in the presence of a dehydrating condensing agent such as carposimid. It is obtained by forming a metal bond. At this time, unreacted dye is separated.
  • the light source used in the present invention is desirably laser light or LED light.
  • the measurement methods are broadly classified into a competition method and a sandwich method.
  • a sample to be measured is mixed with a reagent having a known concentration consisting of a fluorescent dye, avidin, a biotin, and a substance to be measured, and a substance that specifically binds to the substance to be measured is immobilized thereon.
  • the immersed optical fiber is allowed to react specifically, and then is excited with light to measure the fluorescence.
  • the sample to be measured and the reagent consisting of the fluorescent dye, avidin, piotin and the substance to be measured are applied to the optical fiber in accordance with the respective concentration ratios. Join You.
  • the concentration of the sample to be measured is high, the binding amount of the reagent consisting of the fluorescent dye, avidin, piotin, and the substance to be measured is relatively reduced, the fluorescence intensity is reduced, and the concentration-fluorescence intensity calibration curve is obtained. The slope becomes negative.
  • an optical fiber on which a substance that specifically binds to a substance to be measured is immobilized on a sample to be measured.
  • a substance to be measured is bound to the optical fiber according to the concentration.
  • the optical fiber to which the substance to be measured is bound is immersed in a solution of a reagent consisting of a fluorescent dye, avidin, biotin, and a substance that specifically binds to the substance to be measured.
  • a reagent consisting of a fluorescent dye, avidin-bitin, and a substance that specifically binds to an analyte is bound to the optical fiber.
  • a reagent consisting of the same number of fluorescent dyes, avidin, and biotin as substances to be measured is bound to the optical fins. I do.
  • the concentration of the sample to be measured is high, the amount of binding of the reagent consisting of the fluorescent dye avidin-bitin and the substance specifically binding to the substance to be measured increases, the fluorescence intensity increases, and the concentration-fluorescence increases.
  • the slope of the intensity calibration curve is positive.
  • the measuring method 2) basically has the same effect as the measuring method 1), but in this method, the substance to which biotin is first bound or a substance which specifically reacts with the substance to be measured is used. Is required to react specifically with a substance or an analyte that specifically binds to the analyte on the optical fiber, and then to react with avidin modified with a fluorescent dye. .
  • the reason for this is that the avidin modified with the fluorescent dye is bound last, so that the decrease in fluorescence intensity due to hydrolysis and oxidation of the fluorescent dye can be prevented, and highly reproducible measurement can be performed. It is.
  • the fluorescent dye is preferably a cyanine dye.
  • the light source used in the present invention is desirably laser light or LED light.
  • the measurement methods are roughly classified into a competitive method and a sandwich method.
  • a sample to be measured is mixed with a reagent of known concentration consisting of pyotin and the analyte, and an optical fiber on which a substance that specifically binds to the analyte is immobilized.
  • This method involves inserting a bar, reacting specifically, binding avidin modified with a fluorescent dye, exciting with light, and measuring.
  • the optical fiber is The reagent consisting of the measurement sample and the fluorescent dye avidin-bitin-substance is bound according to the respective concentration ratios.
  • the concentration of the sample to be measured is high, the binding amount of the reagent consisting of the fluorescent dye, avidin, piotin, and the substance to be measured is relatively reduced, the fluorescence intensity is reduced, and the concentration-fluorescence intensity calibration curve is obtained. Becomes negative.
  • an optical fiber on which a substance that specifically binds to a substance to be measured is immobilized on a sample to be measured.
  • An analyte is bound to the optical fiber according to the concentration.
  • the optical fiber to which the substance to be measured is bound is immersed in a reagent solution composed of piotin and a substance that specifically binds to the substance to be measured.
  • a reagent consisting of a substance that specifically binds to piotin-to-be-measured substance binds to the fiber.
  • the avidin modified with a fluorescent dye is bound to the optical fiber to which the reagent comprising the substance that specifically binds to the above-mentioned piotin-substance to be measured is bound.
  • the optical fiber contains a substance that specifically binds to the same number of fluorescent dyes, avidin, piotin, and the analyte as the number of the measurement sample. Join.
  • the measuring method 3) is characterized in that a substance to be measured or a substance that specifically reacts with the substance to be measured binds to a compound having a plurality of reactive groups, and the reactive group of the compound having a plurality of reactive groups However, it is necessary to use a reagent to which a compound modified with a plurality of fluorescent dyes is bound.
  • the binding amount of the fluorescent dye per bioactive substance can be increased, and the detection sensitivity can be dramatically improved.
  • the fluorescent dye is a cyanine dye.
  • the compound modified with the plurality of fluorescent dyes is preferably avidin, and is bound to the reactive group of the compound having a plurality of reactive groups via piotin. Is desirable.
  • the compound having a plurality of reactive groups is desirably selected from aminoglucan, and chitosan is particularly preferred.
  • the light source for exciting the fluorescent dye may be It is desirable to use the light or LED light.
  • Tsuru-Ki measurement methods are broadly divided into the competitive method and the Sandwich method.
  • the analyte and the analyte bind to a compound having a plurality of reactive groups, and the reactive groups of the compound having the plurality of reactive groups are modified with a plurality of fluorescent dyes.
  • the reagent to which the compound is bound is mixed, and the optical fiber on which the substance that specifically binds to the analyte is immobilized is immersed and allowed to react specifically. This is a method of measuring fluorescence by exciting with light.
  • the sample to be measured and the reagent are bound to the optical fiber according to the respective concentration ratios.
  • the concentration of the sample to be measured is high, the amount of the reagent bound relatively decreases, the fluorescence intensity decreases, and the slope of the concentration-fluorescence intensity calibration curve becomes negative.
  • an optical fiber on which a substance that specifically binds to a substance to be measured is immobilized on a sample to be measured.
  • An analyte is bound to the optical fiber according to the concentration.
  • the optical fiber to which the substance to be measured is bound is combined with a compound in which a substance that specifically binds to the substance to be measured binds to a compound having a plurality of reactive groups, and the reactive group of the compound having the plurality of reactive groups is combined.
  • Has multiple The solution of the reagent to which the compound modified with the fluorescent dye is bound can be used.
  • the same number of measurement reagents as the measurement sample are bound to the optical fiber.
  • the concentration of the sample to be measured is high, the amount of binding of the measurement reagent increases, the fluorescence intensity increases, and the slope of the concentration-fluorescence intensity calibration curve becomes positive.
  • compound A and compound B when two kinds of substances that specifically bind to each other are referred to as compound A and compound B, first, the substance to be measured or a substance that specifically reacts with the substance to be measured is subjected to a plurality of reactions.
  • the amount of the fluorescent dye per substance to be measured can be increased, and a decrease in the fluorescence intensity due to hydrolysis or oxidation of the fluorescent dye can be prevented. This is because measurement with high reproducibility can be performed.
  • the fluorescent dye is not the desire that it is a Shianin dye 0
  • Compounds A and B are preferably a combination of avidin-biotin, protein A-antibody, and antibody-brotin A, and a combination of avidin-biotin is particularly preferred.
  • the antibody used as the compound A or the compound B does not cause a specific reaction with the substance to be measured.
  • the compound having a plurality of reactive groups is preferably selected from aminoglucan, and chitosan is particularly preferable.
  • the light source for exciting the fluorescent dye is laser light or LED light.
  • the measurement methods are broadly classified into a competition method and a sandwich method.
  • the analyte and the analyte bind to a compound having a plurality of reactive groups, and the reactive group is mixed with a reagent having a known concentration to which compound B is bound.
  • a reagent having a known concentration to which compound B is bound.
  • the sample to be measured and the reagent are bound to the optical fiber according to their respective concentration ratios. Therefore, if the concentration of the sample to be measured is high, the amount of reagent bound relatively decreases, the fluorescence intensity decreases, and the slope of the concentration-fluorescence intensity calibration curve becomes negative.
  • an optical fiber on which a substance that specifically binds to a substance to be measured is immobilized is immersed in a sample to be measured.
  • a substance to be measured binds to the optical fiber according to its concentration.
  • the optical fiber to which the substance to be measured is bound is bound to a compound in which a substance that specifically binds to the substance to be measured has a plurality of reactive groups, and compound B is bound to the reactive group.
  • a reagent binds to the fiber.
  • the substance that specifically binds to the substance to be measured binds to a compound having a plurality of reactive groups, and the reactive group is bound to an optical fiber to which a reagent to which compound B is bound is bound.
  • the compound A modified with a fluorescent dye is bound.
  • the bioactive substance has a plurality of reactive activities.
  • the compound having a plurality of reactive groups is bonded to the compound having a group, and the reactive group of the compound having a plurality of reactive groups is determined by using the above-mentioned bioactive substance measurement test in which a compound modified with a plurality of fluorescent dyes is bonded.
  • This is a device for measuring bioactive substances.
  • the apparatus of the present invention has at least the following constitutions: a small light source and an optical fiber for transmitting excitation light or fluorescence, and a core surface at one end surface thereof is exposed.
  • the reason for using the optical fiber which is characterized in that the optical fiber is composed of a counter, is that the optical fiber allows the excitation light and the fluorescent light to propagate, and the optical fiber has no light loss. Efficient measurement can be performed.
  • the optical fiber is made of resin. This is because resin is cheaper and easier to use.
  • the resinous fiber has a structure that reacts with a crosslinking agent.
  • a bioactive substance can be covalently bonded to form a detection portion by using a cross-linking agent.
  • the structure that reacts with the crosslinking agent is preferably an ester structure.
  • a (meth) acrylic ester resin such as methyl polymethacrylate or a polyester resin is preferable.
  • the bioactive substance binds to a compound having a plurality of reactive groups, and the reactive group of the compound having the plurality of reactive groups is modified with a plurality of fluorescent dyes. It is necessary that the biologically active substance measurement reagent to which the obtained compound is bound is bound at the time of measurement.
  • the fluorescent dye is a cyanine dye.
  • the cyanine dye can be pumped by He—Ne laser light (630 nm) or the shortest wavelength semiconductor laser currently transmitting (636 nm), so that it is large in size. It is not necessary to use expensive and expensive Xe lamps or Ar lasers or expensive SHG elements (elements that reduce the wavelength of light), so can a cheap and small device be obtained? It is.
  • the detection section be detachable from an optical fiber that propagates excitation light or fluorescence by a coupler.
  • a guide rail type as shown in FIG. 3 is preferable.
  • the light source of the apparatus of the present invention must be small and inexpensive, and can be a He—Ne laser, a semiconductor laser, a laser combining a semiconductor laser and an SHG element, or an LED (light emitting diode). C) is desirable.
  • the mechanism for extracting only the fluorescent light may be a half mirror, a filter, or the like, but a filter is preferable.
  • the reason for this is that when a half mirror is used, a space for arranging the optical system is required, which makes it difficult to reduce the size.
  • the half mirror is mainly used when the detection unit is of a reflection type, and the filter is mainly used when the detection unit is of a facing type.
  • the detection unit is of a facing type.
  • the optical fiber made of the resin (12) was immersed in the reaction solution prepared in the above (13) at 50 ° C. for 10 minutes and washed with water.
  • the (15) pair was immersed in a 20 mM hydrochloric acid solution for 5 to 10 minutes, washed with water, and a formyl group was introduced on the surface of the core portion of the resin optical fiber.
  • FIG. 6 (b) shows the relationship between the treatment temperature and the amount of immobilizable enzyme (protein) when a formyl group was introduced to the surface of the optical fiber by the above method.
  • Figure (a) shows the relationship between the processing temperature and the optical transmission rate of the fiber.
  • FIG. 7 shows the relationship between the density of the formyl group on the surface of the fiber and the light transmission reduction rate.
  • the optical fiber made of methyl polymethacrylate improves the light transmission rate by heat treatment, but the higher the reaction temperature, the higher the density of the formyl group to be bonded. The transmission rate decreases. For this reason, as shown in FIG. 6 (a), the most preferable temperature is around 50 ° C.
  • the detection unit 5 was immersed in a biotinylated chitosan solution to which the antibody obtained in the above (8) was bound, and then washed with phosphate buffered saline.
  • the detection limit was measured from the calibration curve, and this is shown in Table 1.
  • Binding of the antibody protein] 3-1, 4 An anti-mouse IgG (Y) solution and CHMC were added to the Samin suspension and reacted at 4 ° C overnight. After completion of the reaction, dialysis is performed for 12 hours, and unreacted substances are removed using an anion exchange column.
  • Example 13 (12) In Example 13 (13), succindialdehyde was used in place of glutaraldehyde, and instead of the optical fiber made of methyl methacrylate. Using an optical fiber containing a polyester, the detection unit shown in FIG. 1 was prepared in the same manner as in (12) to (18) of Example 1.
  • the complex solution of (Y) was mixed at a ratio of 1: 1 and passed through a flow cell 5 shown in FIG. 1 and then washed with a phosphate buffered saline, and then the apparatus of the present invention shown in FIG. Fluorescence was measured with a He-Ne laser optics with a spectrofluorometer 8. The same measurement was repeated while changing the concentration of anti-mouse IgG (Y), and the relationship between the concentration of anti-mouse IgG (Y) and the fluorescence intensity was examined to prepare a calibration curve. The detection limit was measured from the calibration curve and is shown in Table 1.
  • the detection limit was measured from the calibration curve and is shown in Table 1.
  • the obtained precipitate is suspended in 10 mM potassium phosphate buffer. did.
  • the sodium sulfate crystals were removed by centrifugation, and the supernatant was dialyzed and concentrated for use.
  • the detection limit was measured from the calibration curve and is shown in Table 1.
  • Example 1 50 mg of CHMC was added and dissolved in the solution obtained in (1) of Example 1, and the solution was allowed to stand at 12 ° C for 2 hours. Using this solution, (2) of Example 1 was used. The following processing was performed. The detection limit was measured from the calibration curve and is shown in Table 1.
  • Example 1 a polylysine was used in place of the chitosan of Example 1.
  • the reaction conditions are the same as those in Example 1.
  • the detection limit was measured from the calibration curve and is shown in Table 1.
  • Example 2 The detection section of Example 1 was immersed in a mouse IgG (antigen) solution at 4 ° C for 12 hours.
  • the detection part was immersed in an anti-mouse IgG solution of known concentration, and washed with phosphate buffered saline.
  • Example 7 The solution of (1) in Example 7 was reacted with the bitinylated antibody to prepare a detection reagent, and this reagent and an anti-mouse IgG solution of known concentration were mixed at a ratio of 1.1.
  • the detection part of Example 7 was immersed, measured, and a calibration curve was prepared. From this, the detection limit was measured, and this is shown in Table 1.
  • Example 1 (1) The treatments (9) to (11) of Example 1 were performed to obtain a solution of avidin modified with NK116.
  • mice IgG was dehydrated and condensed in the presence of carposimid to prepare the detection unit 5.
  • the detection limit was determined from the calibration curve and is shown in Table 1.
  • FIGS. 1-10 Embodiments of the apparatus of the present invention are shown in FIGS.
  • FIG. 1 shows that the cladding layer 2 on the surface of the optical fiber 1 is eliminated, the antigen 4 is bound to the exposed core surface 3, and the core surface is removed.
  • 3 is a detection unit having a structure surrounded by a flow cell 5.
  • FIG. 2 shows a reflection type detection unit, which is provided with a mirror 12 at the tip.
  • Fig. 3 shows the structure of the opposed fluorescence detector. is there.
  • the fluorescence detector (sensing tip) 9 is fixed by the guide 11 for optical axis alignment, and since it is not bonded, the fluorescence detector 9 can be freely attached and detached, which is extremely convenient for practical use. Is good. Since the fluorescence detector 9 is replaced every time the measurement is performed, it is preferable that the fluorescence detector 9 be a resin optical fiber from the viewpoint of cost.
  • a reactive group is introduced into the resin optical fiber by various methods.
  • the above-mentioned resin optical fiber preferably has an ester structure such as a polyacrylic acid ester, and is used when the fluorescent detection section is formed by a resin optical fiber. Reacts a compound having a CH> CH0 structure with this ester group under basic conditions, introduces a formyl group, and binds the formyl group to a protein such as an antigen or an antibody. You.
  • the excitation light is emitted from the fiber facing the detection surface.
  • a laser beam enters the fluorescence detector 9 from the plate side 10, and the incident light and the fluorescence have a guide hole 11 having a guide 11 for optical axis alignment. Then, only the fluorescence is extracted by the filter 7 and measured by the spectrophotometer 8.
  • FIG. 5 shows an apparatus using a flow cell type detector.
  • the reagent for measuring a bioactive substance of the present invention can be used for immunoassay for a bioactive substance such as an antigen, an antibody or an enzyme contained in a very small amount in blood or body fluid in medical diagnosis. Since the amount of fluorescent dye per individual is large, detection sensitivity can be greatly improved.
  • the optical fiber for measuring a bioactive substance of the present invention can realize small size, low cost, and high sensitivity.
  • the measuring method of the present invention using the reagent and the device for measuring a bioactive substance, simple and quick measurement can be realized, so that it can be used for disease diagnosis in the medical field.
  • Figures 1 to 3 show the fluorescence detection unit using the fluorescence labeling method.
  • Figures 4 and 5 show a fluorescence measurement system using a He-Ne laser or a semiconductor laser.
  • Fig. 6 (a) shows the relationship between the processing temperature and the optical transmission rate of an optical fiber made of methyl polymethacrylate.
  • FIG. 6 shows the relationship between the treatment temperature and the amount of enzyme that can be bound.
  • the horizontal axis in the figure is the processing temperature (C °)
  • the vertical axis in the figure is the fiber light transmission rate (%)
  • the vertical axis in the figure is the amount of enzyme immobilization per area (tg / cm 2 )
  • the horizontal axis in the figure shows the concentration of bitinylated anti-mouse antibody (mgZ), and the vertical axis shows the number of counts.
  • FIG. 6 (c) shows a calibration curve of the concentration of the piotinylated anti-mouse antibody and the fluorescence intensity.
  • Figure 7 shows the relationship between the optical transmission rate and the density of the formyl group of an optical fiber made of methyl polymethacrylate.
  • the horizontal axis shows the number of holmyl groups (Zcni 2 ), and the vertical axis shows the fiber transmission reduction rate (%).
  • Fig. 8 (a) shows the calibration curve of the Sandwich method, and (b) shows the calibration curve of the competition method.
  • the horizontal axis indicates the anti-mouse IgG (mg / mg), and the vertical axis indicates the number of counts.
  • Figure 9 shows the responsiveness of the sensor.
  • 1 is an optical fiber
  • 2 is a cladding layer
  • 3 is a core surface
  • 4 is an antigen
  • 5 is a flow cell
  • Y is a fluorescently labeled antibody of the present invention
  • Y is an antibody in a sample
  • 6 is a He-Ne laser generator or a laser generator combining a semiconductor laser and an SHG element
  • 7 is a filter
  • 8 is a spectrofluorometer
  • 9 is a sensing chip
  • 10 is a plate
  • 1 1 is a guide rail for optical axis alignment
  • 1 2 is a mirror

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Abstract

Un réactif de dosage d'une substance biologiquement active comprend une combinaison de composés de (fluorochrome)n et d'avidine-a ayant une pluralité de groupes réactifs, ou un composé dans lequel l'avidine est liée par de la biotine au composé ayant une pluralité de groupes réactifs. L'invention concerne en outre un procédé de production du réactif; une fibre optique composée d'une résine nécessaire pour utiliser le réactif; un appareil de dosage pourvu d'une unité de détection qui utilise ladite fibre optique; et un procédé de dosage de substances biologiquement actives au moyen desdits reactif et appareil. On peut utiliser ce procédé de dosage afin de détecter immunologiquement des traces de substances biologiquement actives contenues dans du sang ou dans une humeur, à des fins de diagnostic médical, telles que des antigènes, des anticorps ou des enzymes. Ce procédé a une sensibilité de détection remarquablement améliorée.
PCT/JP1990/000514 1989-04-19 1990-04-19 Reactif de dosage de substances biologiquement actives, son procede de production, procede et appareil de dosage WO1990013029A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0575973A2 (fr) * 1992-06-23 1993-12-29 Ibiden Co., Ltd. Procédé de production d'une fibre optique avec des groupes formyl sur la surface du coeur
JP2010223846A (ja) * 2009-03-25 2010-10-07 Jasco Corp 検出器および赤外顕微鏡
JP2017128532A (ja) * 2016-01-20 2017-07-27 キヤノン株式会社 光学イメージング用造影剤の製造方法、及び光学イメージング用造影剤

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5981560A (ja) * 1982-08-23 1984-05-11 マイロン・ジエイ・ブロツク 免疫定量方法およびその装置
JPS6036963A (ja) * 1983-06-13 1985-02-26 マイロン・ジエイ・ブロツク 検定の方法及び装置
JPS6188155A (ja) * 1984-08-14 1986-05-06 オ−ソ・ダイアグノステイツク・システムズ・インコ−ポレ−テツド フイコビリタンパク質による螢光エネルギ−転位
JPS61191965A (ja) * 1984-12-10 1986-08-26 プルーテック　リミティド 液状分析物中の種のパラメ−タ−を光学的に確認する方法および装置
JPS61292044A (ja) * 1985-04-12 1986-12-22 ジーイーシー　―　マルコニ　リミテッド 光導波路バイオセンサ
JPS6266143A (ja) * 1985-09-09 1987-03-25 コ−ニング　グラス　ワ−クス 流体試料を検定するための装置
JPS6279333A (ja) * 1985-09-09 1987-04-11 オルド，インコ−ポレ−テツド 免疫検定装置
JPS6279334A (ja) * 1985-09-09 1987-04-11 オルド，インコ−ポレ−テツド 螢光免疫検定装置及び方法
JPS62123358A (ja) * 1985-11-22 1987-06-04 Sumitomo Electric Ind Ltd 光フアイバ型免疫センサ
JPS63289001A (ja) * 1986-11-28 1988-11-25 スカルボ　インコーポレイテッド，ウエスト　コースト 結合分析試薬のための新規な標識デザイン
JPS6447952A (en) * 1987-05-06 1989-02-22 Saibaafuruua Inc Immunoassay, reagent used therefor and making thereof

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5981560A (ja) * 1982-08-23 1984-05-11 マイロン・ジエイ・ブロツク 免疫定量方法およびその装置
JPS6036963A (ja) * 1983-06-13 1985-02-26 マイロン・ジエイ・ブロツク 検定の方法及び装置
JPS6188155A (ja) * 1984-08-14 1986-05-06 オ−ソ・ダイアグノステイツク・システムズ・インコ−ポレ−テツド フイコビリタンパク質による螢光エネルギ−転位
JPS61191965A (ja) * 1984-12-10 1986-08-26 プルーテック　リミティド 液状分析物中の種のパラメ−タ−を光学的に確認する方法および装置
JPS61292044A (ja) * 1985-04-12 1986-12-22 ジーイーシー　―　マルコニ　リミテッド 光導波路バイオセンサ
JPS6266143A (ja) * 1985-09-09 1987-03-25 コ−ニング　グラス　ワ−クス 流体試料を検定するための装置
JPS6279333A (ja) * 1985-09-09 1987-04-11 オルド，インコ−ポレ−テツド 免疫検定装置
JPS6279334A (ja) * 1985-09-09 1987-04-11 オルド，インコ−ポレ−テツド 螢光免疫検定装置及び方法
JPS62123358A (ja) * 1985-11-22 1987-06-04 Sumitomo Electric Ind Ltd 光フアイバ型免疫センサ
JPS63289001A (ja) * 1986-11-28 1988-11-25 スカルボ　インコーポレイテッド，ウエスト　コースト 結合分析試薬のための新規な標識デザイン
JPS6447952A (en) * 1987-05-06 1989-02-22 Saibaafuruua Inc Immunoassay, reagent used therefor and making thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0575973A2 (fr) * 1992-06-23 1993-12-29 Ibiden Co., Ltd. Procédé de production d'une fibre optique avec des groupes formyl sur la surface du coeur
EP0575973A3 (en) * 1992-06-23 1994-09-28 Ibiden Co Ltd Method for producing optical fiber having formyl groups on core surface thereof
US5354574A (en) * 1992-06-23 1994-10-11 Ibiden Co., Ltd. Method for producing optical fiber having formyl groups on core surface thereof
JP2010223846A (ja) * 2009-03-25 2010-10-07 Jasco Corp 検出器および赤外顕微鏡
JP2017128532A (ja) * 2016-01-20 2017-07-27 キヤノン株式会社 光学イメージング用造影剤の製造方法、及び光学イメージング用造影剤

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