WO1990013029A1

WO1990013029A1 - Reagent for assaying biologically active substance, method of production thereof, and method and apparatus for assaying

Info

Publication number: WO1990013029A1
Application number: PCT/JP1990/000514
Authority: WO
Inventors: Takeshi Kobayashi; Hiroyuki Honda; Kenichi Shimada
Original assignee: Ibiden Co., Ltd.
Priority date: 1989-04-19
Filing date: 1990-04-19
Publication date: 1990-11-01

Abstract

A reagent for assaying a biologically active substance comprising a combination of (fluorochrome)n- avidin-a compound having a number of reactive groups, or one wherein avidin is bound via biotin to the compound having a number of reactive groups; a method of producing the reagent; an optical fiber made of a resin necessary for utilizing the reagent; an apparatus for assaying provided with a detecting unit utilizing the optical fiber; and a method of assaying the biologically active substance with the use of the above reagent and apparatus. This assaying method can be employed in the immunoassay of trace amounts of biologically active substances contained in blood or a humor for medical diagnosis, such as antigens, antibodies or enzymes, with a remarkably improved sensitivity of detection.

Description

Specification

Bioactive substance measurement reagent, its production method, measurement method and

[Technical field ]

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.

[Background Art]

Conventionally, various immunoassays have been known as methods for detecting trace components in research fields such as medical diagnosis and clinical examination, and in this regard, dye-labeled reagents and assays have been known. Various techniques have been proposed for methods or devices.

1) Mark B type 5

Conventionally, labeling reagents such as radioisotopes, luminescent agents, enzyme-labeled antigens and antibodies have been developed.

Of these, the most sensitive ones are labeled with radioisotopes, but are not easy to handle. Those labeled with enzymes are only effective for some substances. For this reason, the sensitivity of luminescent agent labeling reagent Is desired.

As such a highly sensitive reagent that can be used in a luminescence immunoassay, 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. However, 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.

Also, 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. However, in this reagent and method, 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. However, there is a possibility that 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.

2) Optical fiber for luminescence immunoassay

In 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.

In order to use an optical fiber as a sensor, it is necessary to immobilize antigens and antibodies on the optical fiber. Techniques for this purpose include cellophane and the like. There are a membrane immobilization method in which antigens and antibodies are immobilized on the membrane, and an inclusive method in which antigens and antibodies are sealed in pores such as acrylamide gel.The former reduces the sensitivity due to light scattering, but the latter Had a problem of poor responsiveness. For this reason, Analytical Chemistry Vol. 59 No. 8 describes a method for directly covalently binding antigens, antibodies, etc., to optical fibers.

P 1226-1230 (1987) describes a silane coupling agent for the silane group on the surface of a quartz optical fiber.

(3 — Glycidoxypropyl) Trimoxysilane

(G 0 PS) were reacted to which was then processed by HI 0 _4, quartz light off § Lee bar one introduced City, formyl groups on the surface, protein A Mi to the formyl group Roh A method for reacting a group and immobilizing the same is disclosed. However, this method is effective only for quartz fiber, and cannot be used for resin-made 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.

As an apparatus using an optical fiber, 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.

3) Measurement method

Various methods of immunoassay using a luminescent agent have been proposed. For example, 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. However, there is a problem that 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. Also, a reagent using a biotin-avidin bond has been recognized in Japanese Patent Application Laid-Open No. 58-30667 (Swiss Patent Application No. 6989 / 81-01-1). Immunologically active substances have been proposed. However, this technique has a problem in that the immunological substance is labeled with an enzyme, and the process for measuring the enzyme activity is troublesome.

[Disclosure of the Invention]

As a result of intensive studies, 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. For this purpose, 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. In addition, 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.

(Bioactive substance measurement reagent)

In the reagent for measuring a bioactive substance of the present invention, 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. 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.

Further, it is desirable that the number of the reactive groups be 20 to 100 000 per molecule. The reason is that if the number is less than 2Ό, 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.

As the above-mentioned amino glucan, chitosan, polygalactosamine, polyinomyric acid, etc. can be used, but chitosan is used. This is preferred.

Further, the above-mentioned polyaminopeptidyl is an amino acid having two or more amino groups, which is polymerized by a peptide bond. 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.

Further, it is preferable that the compound is bonded to the compound having a plurality of reactive groups through a substance that specifically binds to the compound.

For example, when the compound is avidin, protein A, an antibody, a hormone, or a hormone receptor, 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. —

In the case of using the combination of “avidin-pyotin”, a fluorescent dye is bonded to a large number of amino groups present on the surface of avidin.

When the combination of “protein A—antibody” is used, 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.

Further, 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). By labeling the antibody protein-compound having a plurality of reactive groups-bioactive substance complex with the protein A modified with, the reagent for measuring a bioactive substance of the present invention can be obtained.

It is necessary that neither the protein A nor the antibody react with the biologically active substance to be measured.

In this way, by binding a compound modified with a plurality of fluorescent dyes, a plurality of fluorescent groups can be added to one reactive group. A photopigment can be bound, and the sensitivity can be dramatically improved.

In the present invention, it is necessary to excite the fluorescent dye with light.

The reason for this is that, by excitation with light, it is possible to perform a much safer measurement than radioimmunoassay (radio-immunoassay), which is a conventional technique. In addition, it is possible to omit troublesome luminescence processing in chemiluminescence and bioluminescence, and to achieve highly accurate and reproducible measurement in a shorter time.

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 reason is that the above laser is smaller and less expensive than the Xe lamp or Ar laser. When the above-mentioned semiconductor laser is used, laser light in a short wavelength region can be emitted by combining with an SHG element (second harmonic generation element: an element that reduces the wavelength of light).

It is desirable that the fluorescent dye used in the present invention be excited by light having a wavelength of 200 to 800 nm.

The reason for this is that if the wavelength is shorter than the above wavelength, the energy is too high and the chemical bond is destroyed. If the wavelength is longer than the range, the quantum yield is too low to be practical.

Examples of the fluorescent dye 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,

For example,

(Wherein, 丫 and 丫 ′ 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.

(633 nm) or the shortest wavelength currently being transmitted It can be excited by a semiconductor laser (638 nm).

Therefore, 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.

As the cyanine dye, a cyanine dye represented by the formula (1) is particularly preferably used.

In the formula, n represents 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. For this purpose, a carbocyanine dye represented by the above formula (1) having a carboxyl group outside the conjugate system as a functional group is preferably used.

In addition, the following compounds are used as the cyanine dye. Can use

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.

It is desirable that the reagent for measuring a bioactive substance of the present invention be bound to an optical fiber and excited upon measurement.

(Resin optical fiber)

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 reason that the optical fiber is made of resin is that 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.

It is desirable that the density of the reactive group be 1.0 X 10 ^{1 Q} to 6.0 X 10 ¹³ Zcm ² . The reason If the density is lower than the above range, 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)

3 density of the reactive groups in is ^{found. 0 X 1 0 1 2 ~} 4. 0 X 1 0 1 3 pieces / cm ² Ah Ru and this is good or teeth rather ^{in, 1. 5 X 1 0 1} 3 It is preferable that the ratio be 3.5 × 10 ¹³ pieces / cm ² . The reason is that when the density is higher than the above range, the distance between the reactive groups becomes shorter, and the reproducibility starts to decrease due to the steric hindrance of the reagent for measuring a bioactive substance.

The resin constituting the resin optical fiber must be 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.

Desirably, the resin optical fiber contains, as a main component, a resin having a structure that reacts with a crosslinking agent. The reason for this is that 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.

As 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.

Meanwhile, a technique for binding a protein to the surface of a resin optical fiber is disclosed in 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.

However, 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 method for producing the resin optical fiber of the present invention will be described below.

If 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.

It is desirable to use a polyfunctional compound as the cross-linking agent, for example, aldehydes such as glutaraldehyde, succinaldehyde, adipoaldehyde, N,, and the like. -· N'—Ethylene bismuth resin, N, N'-o-Phenylene dimers, bisdiazobenzene, or hexamethyl benzene For example, cyan or diisothiocinart is used.

As a specific introduction method using the crosslinking agent, for example, when a formyl group is introduced into a polystyrene optical fiber, a benzene ring as a side chain is nitrated, Next, reduction is performed, and this is converted into an amino group. A formyl group can be introduced by reacting aldehyde.

In the present invention, 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.

As the nucleophilic reagent having a formyl group, a reagent represented by the formula (2) is preferable.

Formula R ^1- (CH ₂ ) „-CH-CH0

I (2)

R ²

(Wherein 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. CH: CH- H ₂ -R ^1- (CH ₂ ) nC-CHO-→-| -CH ₂ -C +-

0 = C-0CH ₃ (2)

0 = CC-CHO

When reacting the cross-linking agent, it is desirable that the cladding layer of the optical fiber be peeled off to expose the core surface.

The reason for this is that 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.

It is desirable that the end face of the optical fiber be polished. The polishing is preferably performed using alcohol as a lubricant.

Nucleophile having the formyl group, K 0 base such as H, et motor Roh Lumpur of which A Le co Lumpur based organic solvents, N i ethanol solution of Yo I Do N i salt SO _4, It is desirable to add and dissolve a nucleophilic reagent having a formyl group.

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.

After the above-mentioned reaction treatment, washing with water and immersion in an acid such as HC-6 removes the acetalized alcohol to obtain a resin optical fiber with a formyl group bonded thereto. . (See the following reaction formula)

/

-CH-→ -CH0 + C ₂ H ₅ 0H

\

0C ₂ H ₅

(Measurement detector)

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.

In the case where the reactive group of the resin optical fiber is a formyl group, it is preferable to carry out an immobilization treatment after reacting a substance that specifically binds to the substance to be measured. This reason This is because, if the immobilization treatment is not performed, the substance that specifically binds to the analyte is easily released by a reversible reaction.

The case where a protein is selected as the substance that specifically binds to the substance to be measured in the immobilization treatment will be described below.

First, when a resin optical fiber into which a formyl group is introduced is immersed in a protein solution, the formyl group reacts with the amino block of the protein, and the binding site becomes an imino group. After washing it with water, a reducing agent of a suitable concentration, for example Ri by the and this treatment with etc. N of a BH _4, I Mi cyano group is reduced to inactivated activatable protein is immobilized.

(Refer to the following reaction formula. The resin is methyl methacrylate)

CH: CH;

CH ₂ -C

R ²

I H

0 = C-C-CH0 0 = C-C-C = N-protein

(CH ₂ ) ' _D -R (CH ₂ ) Π -R ¹ CH:

0 = C-C-CH ₂ -NH- protein

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.

In the case of the opposed type, 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. On the other hand, in the reflection type, 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.

(Production method of bioactive substance measurement reagent)

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.

When 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.

It is desirable that the fluorescent dye be excited by laser light.

As 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). 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 By using 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. For example, methanol, ethanol, methylamine, ethylamine, getylamine, triethylamine, etc. The reaction is performed in a solvent such as an organic solvent or a basic aqueous solution.

The 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.

After the completion of the reaction, 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.

Next, among the reagents for measuring a bioactive substance of the present invention, two kinds of compounds that specifically bind, Compound A and Compound B, were used. 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, and 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) However, it is desirable to manufacture it by the following method.

That is, 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 reason why the above production method is desirable is that if the reaction sequence is changed, side reactions occur and the yield decreases. —

In the above production method, it is desirable that the combination of compound A and compound B be a protein and a compound that specifically binds to the protein. Specifically, avidin and biotin, and protein A And antibodies, antibodies and protein A Most preferred is a combination of avidin and piotin.

These reactions will be described more specifically.

Compounds having a plurality of reactive groups, for example, chitosan (I) have a large number of amino groups in the molecule, and chitosan (I) is added with piotin (II) as a basic solution. When reacted in the presence of a dehydrating agent such as water-soluble carbodiimide (CHMC) or N-hydroxysuccinimide, most of the chitosan amino groups Then, bitin is combined with an acid amide to obtain a piotinylated chitosan (m). The biotinylated protein is reacted with the biotinylated chitosan (ΠΙ) 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.

On the other hand, 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.

Next, a biotinylated chitosan bound to the above protein

When avidin (V) modified with the above fluorescent dye is reacted with (IV), avidin selectively binds to piotin with a very high affinity, and the fluorescent label of the present invention Protein

(VI) can be obtained.

Chitosan (I) Piotin (Π)

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 ρ 26. Each region has a characteristic fluorescence peak.

Since there is a linear relationship between the difference between the fluorescence intensities at 458 nm and 300 nm and the concentration of the acid amide bond, a calibration curve for Tosan can be obtained using this characteristic. It can be prepared and the amount of piotinylation can be estimated from the amount of acid amide binding. (Method of measuring bioactive substances)

A measuring method using the reagent for measuring a bioactive substance of the present invention will be described.

The measuring method using the reagent for measuring a biologically active substance of the present invention is performed by the following method.

1) 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. A measurement method characterized by specifically reacting with a substance to be measured, exciting with light, and measuring fluorescence.

2) The 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.

3) 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. A measurement method characterized by specifically reacting with a substance to be combined or a substance to be measured, and then exciting with light and measuring fluorescence.

4) When two types of substances that specifically bind to each other are compound A and compound B, 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. After reacting the reagent to which the compound B is bound with the reactive group with the substance specifically binding to the analyte or the analyte on the optical fiber, 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. A measurement method characterized by measuring

The measurement method 1) will be described.

In 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.

For this reason, a large number of fluorescent dyes can be bound without damaging the binding active site through the avidin-biotin.

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.

The reason for this is that 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.

In order for the cyanine dye to bind to avidin, the reaction is completed in a short time under mild conditions as much as possible, and binds to the reactive group without causing side reactions. This is necessary. For this purpose, 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.

(Where n represents 0, 1, 2, or 3)

In order to obtain avidin modified with a cyanine dye, 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.

In the competition 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. In this method, the immersed optical fiber is allowed to react specifically, and then is excited with light to measure the fluorescence. In the case of the above-mentioned competitive method, 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.

Therefore, when 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.

In the sandwich method, 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.

In the Sandwich method, 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.

Therefore, when 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.

Next, the measurement method 2) will be described.

% 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 measurement methods are roughly classified into a competitive method and a sandwich method.

In the competitive 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. In the case of the competitive method, 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.

Therefore, when 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.

In the sandwich method, 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.

In the above-described sandwich method, 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.

Therefore, if the concentration of the sample to be measured is high, Avidin-bitin-The binding amount of the substance that specifically binds to the analyte increases, the fluorescence intensity increases, and the slope of the concentration-fluorescence intensity calibration curve becomes positive.

Next, the measuring method 3) will be described.

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.

By using the reagent, the binding amount of the fluorescent dye per bioactive substance can be increased, and the detection sensitivity can be dramatically improved.

Preferably, the fluorescent dye is a cyanine dye.

Further, 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.

Further, 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.

In the competition 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. In the case of the competition method, the sample to be measured and the reagent are bound to the optical fiber according to the respective concentration ratios.

Therefore, if 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.

In the sandwich method, 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.

In the sandwich method, the same number of measurement reagents as the measurement sample are bound to the optical fiber.

Therefore, if 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.

Next, the measuring method 4) will be described.

In the above measurement method, 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. A reagent that binds to a compound having an active group, and to which a compound B is bound, is attached to the reaction active group with a substance or an analyte that specifically binds to the analyte on the optical fiber; After specific reaction, it is necessary to react with Compound A modified with a fluorescent dye.

By using such a method, 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.

It is necessary that the antibody used as the compound A or the compound B does not cause a specific reaction with the substance to be measured.

Further, the compound having a plurality of reactive groups is preferably selected from aminoglucan, and chitosan is particularly preferable.

Further, it is desirable that the light source for exciting the fluorescent dye is laser light or LED light.

In the competition 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. After immersing an optical fiber on which a substance that specifically binds to the analyte is immobilized and allowing it to react specifically, bind the compound A modified with a fluorescent dye and excite with light And a method of measuring fluorescence. In the case of the competition method, 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.

In the Sandwich method, 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. Into the reagent solution. 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. Then, the compound A modified with a fluorescent dye is bound.

In the above-mentioned sandwich method, the same number of reagents as the measurement sample are bound to the optical fiber.

Therefore, when the concentration of the sample to be measured is high, the amount of the reagent bound increases, the fluorescence intensity increases, and the slope of the concentration-fluorescence intensity calibration curve becomes positive.

(Bioactive substance measuring device)

Next, the device will be described.

In the device of the present invention, 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. A detector that immobilizes a substance that specifically binds to the substance to be measured; a mechanism for extracting only the fluorescence excited by the detector; and a forme for measuring the intensity of the fluorescence excited by the detector. 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.

Preferably, the optical fiber is made of resin. This is because resin is cheaper and easier to use.

It is desirable that the resinous fiber has a structure that reacts with a crosslinking agent. The reason for this is that 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.

As the resin, a (meth) acrylic ester resin such as methyl polymethacrylate or a polyester resin is preferable.

Further, in the detection unit of the present invention, 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 binding of such a reagent enables high-sensitivity measurement.

Preferably, the fluorescent dye is a cyanine dye. The reason for this is that 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.

It is desirable that the detection section be detachable from an optical fiber that propagates excitation light or fluorescence by a coupler. I

As the coupling, 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.

For this reason, it is preferable that the detection unit is of a facing type.

[Best Mode for Carrying Out the Invention]

Next, examples of the present invention will be described.

Example 1

(1) was dissolved _{1 0 0 N a 2 C 0} 3 of water 3 mg of a 4 mg bicycloalkyl old Chin. (2) Then, the solution obtained in the above (1) was added to 27 ^ of the 1.8 chitosan solution.

(3) After the addition of water 100, 50 mg of CHMC (aqueous carpoimide) was added. The reaction was allowed to proceed for 5 hours at room temperature with further stirring.

(4) The reaction was stopped by adding 3 drops of acetic acid.

(5) Then, Na ₂ C 0 a 0.3 g Z m £ and Na C £ 0.3 g

The mixture was added to precipitate the biotinylated chitosan.

(6) After collecting the precipitate with a centrifugal separator, the precipitate was washed with a NaC £ of 0.3 and a buffer of 0.1 g / m ₂ Na ₂ CO ₃ .

(7) The precipitate obtained in (6) above was dialyzed overnight at 4 ° C. against 10 mM 10 mM calcium-phosphate buffer (pH = 7) to obtain the biotinylated chitosan. Obtained.

(8) An anti-IgG (antibody Y) solution and CHMC were added to the suspension of the biotinylated chitosan, and the mixture was reacted at 4 overnight. After the reaction was completed, dialysis was performed for 12 hours, and unreacted substances were removed using an anion exchange column to obtain antibody-bound biotinylated chitosan.

(9) Avidin (1 mg) and triethylamine (2) were dissolved in 1 of ethanol. Then, 2 mg of NK1160 (manufactured by Nippon Kogaku Dye Laboratories; In the above, a cyanine dye (n = 2) was added and dissolved sufficiently to prepare a solution. Further, to the above solution, 14 mg of dicyclocarbodiimide was added, and the mixture was reacted at room temperature for 4 hours.

(10) After the completion of the reaction, ethanol and triethylamine were removed under reduced pressure using an evaporator.

(11) The residue generated in the step (10) was suspended in 0.01 M acetate buffer (pH = 6.5) 2, and then suspended in a centrifuge. The supernatant was collected by centrifugation at 0 rpm for 10 minutes and centrifuged again to obtain a solution of avidin modified with NK1.160.

(12) The tip of a resin optical fiber (Mitsubishi Rayon, trade name: S-force) with a diameter of 1 mm and a main component consisting of methyl methyl acrylate After immersion in water and wiping, the cladding layer was peeled off by 1 cm and washed with water. Next, the end face of the optical fiber was polished with a polishing film using ethanol as a lubricant.

(1 3) 0.5 water to dissolve 1 0 mg of N i S 0 _4, followed ethanol 2. Was added. At this time, since a white precipitate was formed, the precipitate was centrifuged at 300 rpm to collect a supernatant, which was used as a Ni-ethanol solution. To a solution of 50 mM potassium hydroxide-ethanol solution 0.4 was added Ni-ethanol solution 0.1, and then 50% glutaraldehyde was added 50 0, and the reaction solution was added. It was decided.

(14) 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.

(16) Heat resistance of Bacillus sp. 1 6 — 3 F connection 4 lmg of mouse IgG antigen, a monoclonal antibody against α-amylase, was dissolved in phosphate buffered saline (pH = 7.5). An optical fiber made of resin was immersed in this solution at 4 ° C for 12 hours.

(1 7) a resin light off § Lee bar one and exits Ri taken from the solution, washed with water, 1% N a BH ₄ was immersed for 15 minutes in an aqueous solution, the mouse I g and washed with water G antigen 4 was immobilized and used as an antigen-immobilized sensor.

(18) The resin optical fiber manufactured as described above was used as the detection unit.

(19) An anti-mouse IgG (Y) solution having a known concentration was immersed in the detection section 5 prepared in (18), and then washed with phosphate-buffered saline.

(20) Next, 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.

(21) Next, the avidin solution modified with NK116 obtained in the above (11) was immersed in the detection section 5, and then washed with phosphate buffered saline.

(22) Next, fluorescence was measured with a He-e laser optical system using a spectrophotometer 8 in the apparatus of the present invention shown in FIG.

(23) Change the concentration of anti-mouse IgG (Y) The same measurement as described in (18) to (22) was repeated, and the relationship between the concentration of anti-mouse IgG (Y) and the fluorescence intensity was examined to prepare a calibration curve. This is shown in Fig. 8 (a). Figure 9 shows the responsiveness of the sensor.

The detection limit was measured from the calibration curve, and this is shown in Table 1.

Example 2

(1) Piotinylated chitosan solution to which the antibody is bound, avidin solution modified with NK116, and antigen immobilization by the same method as in (1) to (18) of Example 1 Chemical sensor and detector were created.

(2) An anti-mouse IgG (Y) solution of known concentration and a biotinylated chitosan solution bound with the antibody prepared in (1) above were mixed at a ratio of 1: 1 and shown in Fig. 1. After passing through Sucrose Cell 5, it was washed with phosphate buffered saline.

(3) Next, the avidin solution modified with NK116 prepared in (1) above was passed through the flow cell 5, and then washed with phosphate buffered saline.

(4) Next, the relationship between the concentration of anti-mouse IgG (Y) and the fluorescence intensity was examined in the same manner as in (22) and (23) of Example 1, and a calibration curve was prepared. The calibration curve is shown in Fig. 8 (b). The detection limit was measured from the calibration curve and is shown in Table 1.

Example 3

(1) was dissolved _{1 0 0 N a 2 C 0} 3 of water 3 mg of a 4 mg of antibody protein.

(2) Next, the solution obtained in the above (1) was added to 2) ^ of the 1.8 / ^ 3-, 1,4-polygalactamidine solution.

(3> Water 100 / ^ was added, then 50 mg of CHMC (aqueous carpoimide) was added, and the reaction was allowed to proceed overnight at 4 ° C with stirring.

(4) Next, a mixed solution of Na ₂ C 0.33 g and Na C £ 0.3 g 7 was added, and the antibody protein was bound to 0 — 1,4,1 polysaccharide. Samin was precipitated.

(5) After recovering the precipitate by centrifugation, 0. 3 g / of N a C and 0. Washing the precipitate with lg / of N a ₂ C 0 ₃ buffer.

(6) The precipitate obtained in (5) above was suspended in 10 10 mM calcium phosphate buffer (PH = 7) and dialyzed at 1 透析 4 ° C. to remove antibody protein. The combined 3) -1,4-polysaccharide sumin was obtained.

(7) 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. The antibody protein binding j3—1, 4-polygalactosa Got min.

(8) Protein A 1 mg and triethylamine 0.

Was dissolved in ethanol. Next, 2 mg of NK116 (manufactured by Japan Photographic Dye Laboratories) was added and dissolved sufficiently to prepare a solution. Further, 14 mg of dicyclohexylcarbodiimide was added to the solution, and the mixture was reacted at room temperature for 4 hours.

(9) After completion of the reaction, the solvent was removed by evaporation.

(10) The residue generated in the step (9) was suspended in 27 M acetate buffer (pH = 6.5) 27 ^, and then suspended in a centrifuge at 500 μm. Separation was performed at 0 rpm for 1 minute, and the supernatant was collected and centrifuged again to obtain a solution of Protein A modified with NK116.

(11) The antibody protein binding obtained in the above (8)) 3-1,4 -polysaccharide samin and the protein A modified with the NK116 Reaction at 4 ° C in acid-buffered saline, cyanine dye-protein A-antibody protein A complex solution of -J3-1,41-polysaccharide sumin-antibody (Υ) was obtained.

(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.

(13) An anti-mouse IgG (Y) solution having a known concentration, and the cyanine dye-protein A-antibody protein J3—1,4-polysaccharide obtained in the above (12). One antibody

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.

Example 4

(1) In the same manner as in (1) to (8) in Example 1, a solution of a biotinylated chitosan to which an antibody was bound was prepared. Was.

(2) 1 mg of avidin and 1.8 mg of fluorescein thiothiocyanate were added to 0.5 M sodium carbonate monocarbonate buffer (pH = 9.0) The mixture was dissolved in a basic solvent 5 consisting of, and light was cut off at 4 ° C., stirring was continued, and the mixture was reacted for 20 hours.

(3) Next, the solvent was distilled off from the reaction solution under reduced pressure using an evaporator.

(4) This residue was suspended in 5 of 0.05 M phosphate buffer (pH = 4.0).

(5) The mixture was centrifuged at 500 rpm for 10 minutes to remove unreacted dye, and the supernatant was collected.

(6) Repeat the above operations (3) and (4) twice more, and remove the unreacted dye by passing the obtained supernatant through a tube filled with glass wool. As a result, avidin modified with fluororesin isothiocyanate was obtained.

(7) An antigen-immobilized sensor and a detection section were prepared in the same manner as in (12) to (18) of Example 1.

(8) An anti-mouse IgG (Y) solution of known concentration was passed through the flow cell 5 shown in FIG. 1, and then washed with phosphate buffered saline.

(9) Next, the antibody to which the antibody obtained in (1) was bound After passing the ligchitosan solution through flow cell 5, it was washed with phosphate-buffered saline.

(10) Next, the solution of avidin modified with full-year-old isocyanate obtained in (6) was passed through flow cell 5, and washed with phosphate buffered saline.

(11) Next, in the apparatus of the present invention shown in FIG. 4, a laser optical system (wavelength: 490 nm) combining a semiconductor laser and a thin-film waveguide SHG element and a spectrophotometer 8 were used. The fluorescence was measured.

(12) The concentration of anti-mouse IgG (Y) was changed, and the same measurement as in the above (8) to (11) was repeated, and the relationship between the concentration of anti-IgG (Y) and the fluorescence intensity was examined. Created a line.

The detection limit was measured from the calibration curve and is shown in Table 1.

Example 5

The present invention is basically the same as Example 1, except that after the treatment of (11), a 0.173 硫 saturated sodium sulfate solution (solvent: 10 mM A calcium phosphate buffer (pH = 7) was added, centrifuged at 2000 rpm for 10 minutes, and left at 4 ° C for 1 hour.

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.

Example 6

Further, 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 7

In this example, 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 8

(1) The solution of avidin modified with NK116 obtained by the treatments (9) to (11) in Example 1 was obtained.

(2) The detection section of Example 1 was immersed in a mouse IgG (antigen) solution at 4 ° C for 12 hours.

(3) Next, washed, treated with 1% N a BH ₄ Then, the antigen was immobilized.

(4) The detection part was immersed in an anti-mouse IgG solution of known concentration, and washed with phosphate buffered saline.

(5) The detection part was immersed in the piotinized antibody solution and washed.

(6) Then, the detection part was immersed in the solution of (1), washed, and measured with the apparatus shown in FIG.

(7) These operations were repeated to create the calibration curve shown in Fig. 6 (c), and the detection limit was measured from this, and this is shown in Table 1.

Example 9

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 10

(1) The treatments (9) to (11) of Example 1 were performed to obtain a solution of avidin modified with NK116.

(2) The solution of (1) was mixed with the anti-IgG-bonded chitosan solution obtained in the same manner as in (8) of Example 1, and glutaraldehyde was added. Do By heating at 50 ° C in the presence, the amino group of avidin and the amino group of chitosan were cross-linked with glutaraldehyde, and NK1160 — A solution of avidin- (glutaraldehyde) -chitosan-anti-IgG was obtained.

(3) Formic anhydride is reacted with a polystyrene optical fiber in the presence of aluminum chloride to form holmium on the polystyrene phenyl group. introducing a group, with the following physician, 1, 6 - was reacted in the presence of hexa Njia Mi Noka Rupojii mi de to, was reduced with 1% N a HB _4.

Next, the mouse IgG was dehydrated and condensed in the presence of carposimid to prepare the detection unit 5.

(4) Using the solution of the above (2) and the detection unit of the above (3), measurement was conducted by a competitive method in the same manner as in Example 2.

The detection limit was determined from the calibration curve and is shown in Table 1.

Table 1 Example Detection limit (mgZ £)

1 1.2 X 1 4

o-

2 2.0 X 1 0-4

3 2 .3 X 1 0 1 4

4 3.1 X 1 0-6

5 1.0 X 1 0 1 4

6 1.1 X 1 0-4

7 1.3 X 1 0-4

8 1.0 X 1 0-1

9 1.1 X 1 0-1

1 0 2 .1 X 1 0-4 Example 1 1

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.

In the apparatus shown in FIG. 4, 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. [Industrial applicability]

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.

In addition, the optical fiber for measuring a bioactive substance of the present invention can realize small size, low cost, and high sensitivity.

According to 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.

[Brief description of drawings]

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.

1 to 3, solid arrows indicate laser light and dotted arrows indicate fluorescence.

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 (b) shows the relationship between the treatment temperature and the amount of enzyme that can be bound. (A) and (b) The horizontal axis in the figure is the processing temperature (C °), (a) The vertical axis in the figure is the fiber light transmission rate (%), and (b) The vertical axis in the figure is the amount of enzyme immobilization per area (tg / cm ² ), ( c) 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. The horizontal axis anti-mouse ^{I g G (1 0 - 3} mg ^) immersion time in minutes and the vertical axis represents the mosquito window down betting amount.

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, Is half mirror

Claims

Range of request

1. A bioactive substance is bound to a compound having a plurality of reactive groups, and a compound modified with a plurality of fluorescent dyes is bound to the reactive group of the compound having a plurality of reactive groups. A bioactive substance measuring reagent characterized by the above.

2. The compound modified with the plurality of fluorescent dyes is

The test according to claim 1, which is a protein, protein A or an antibody.

3. The bioactive substance binds to the compound having a plurality of reactive groups, and the reactive group of the compound having the plurality of reactive groups binds to the reactive group, and the biotin binds to a plurality of fluorescent dyes. The reagent according to claim 1, wherein avidin modified with is bound.

4. The reagent according to claim 1, wherein the reactive group is an amino group.

5. The reagent according to claim 1, wherein the compound having a plurality of reactive groups is a polymer having 20 to 100,000 reactive groups per molecule.

6. The reagent according to claim 1, wherein the compound having a plurality of reactive groups is aminoglucan.

7. The reagent according to claim 1, wherein the compound having a plurality of reactive groups is chitosan.

8. The fluorescent dye is a dye that is excited by laser light. The reagent according to claim 1 or 3.

9. The fluorescent dye is 200 nn! 4. The test according to claim 1, wherein the fluorescent dye is a fluorescent dye that is excited by a laser beam having a wavelength of up to 800 nm.

10. The fluorescent dye is a coumarin derivative, a polycyclic aromatic derivative, a dansyl derivative, phycopolyprotein, rhodamin, fluorescein or 0-fluoroaldehyde. The reagent according to claim 1 or 3.

11. The reagent according to claim 1, wherein the fluorescent dye is a cyanine dye.

1 2. The fluorescent dye,

Expression

(Where n represents 0, 1, 2, or 3)

4. The reagent according to claim 1, which is a cyanine dye represented by the formula:

13. The reagent according to claim 1, wherein the bioactive substance is a protein.

14 4. The bioactive substance is an antigen, antibody, enzyme, hapte 4. The reagent according to claim 1, which is an enzyme or an enzyme inhibitor.

15. In a method of producing a protein modified with a fluorescent dye by reacting a fluorescent dye with a protein, the residue obtained by removing the solvent from the reaction product is suspended in a buffer solution having a pH of 2 to 7, A method for producing a fluorescent dye-modified protein characterized by separating and removing a reactive dye.

16. A compound that specifically binds to a protein is reacted with a reactive group of a compound having a plurality of reactive groups, and a compound having a plurality of reactive groups is combined with a compound that specifically binds to a protein. After the reaction, the bioactive substance is reacted to form a complex of a compound that specifically binds to a protein-a compound having multiple reactive groups-a bioactive substance, and then modified with multiple fluorescent dyes A method for producing a reagent for measuring a bioactive substance, which is a complex of a fluorescent dye, a protein, a compound that specifically binds to a protein, a compound having a plurality of reactive groups, and a bioactive substance, characterized in that .

17. The production method according to claim 15 or 16, wherein the fluorescent dye is a dye excited by laser light.

18. The method according to claim 15 or 1 S, wherein the fluorescent dye is a cyanine dye or a full-year resin.

19. The method according to claim 15 or 16, wherein the protein is a basic protein.

20. The method according to claim 15 or 16, wherein the protein is avidin.

21. The method according to claim 16, wherein the compound that specifically binds to the protein-protein is avidin-biotin.

22. A biological material characterized by having a reactive group capable of covalently bonding a substance that specifically binds a reagent for measuring a biologically active substance to the core surface of a resin optical fiber. Resin optical fiber for measuring active substances.

23. The optical fiber according to claim 22, wherein the density of the reactive groups is 1.0 X 10 ¹ ° to 6.0 X 10 ¹³ cm ² .

24. The optical fiber according to claim 22, wherein the reactive group is a formyl group.

25. The optical fiber according to claim 22, wherein the resin optical fiber is a resin containing a resin having an ester structure as a main component.

26. The resin-made optical fiber has a holmium group on the core surface of the optical fiber whose main component is (meth) acrylic acid ester-resin. The optical phono of item 22.

27. Characteristically, a substance that specifically binds to the analyte is covalently bound to the reactive group of the resin optical fiber. Detection unit for measuring bioactive substances.

28. A substance that specifically reacts with the complex consisting of the fluorescent dye, avidin, piotin, the analyte or the substance that specifically reacts with the analyte, with the analyte on the optical fiber Alternatively, after specifically reacting with a substance to be measured, the method is excited with light, and the fluorescence is measured.

29. The analyte to which piotin is bound or the substance that specifically reacts with the analyte is reacted specifically with the analyte or the analyte on the optical fiber. After the reaction, avidin modified with a fluorescent dye is reacted to form a complex on the optical fiber by avidin-biotin bond, and then excited by light. A method for measuring a bioactive substance, comprising measuring fluorescence.

30. The analyte or the substance that specifically reacts with the analyte binds to the compound having a plurality of reactive groups, and the compound having a plurality of reactive groups has a plurality of reactive groups. After reacting the reagent to which the compound modified with the fluorescent dye is bound specifically with the substance or the substance to be measured specifically on the optical fiber, A method for measuring a biologically active substance, characterized by exciting and measuring fluorescence.

3 1. The compound modified with the fluorescent dye is avidin 30. The measuring method according to claim 30.

32. The method according to claim 31, wherein the avidin is bonded to a reactive group via a bitin.

3 3. When two types of substances that specifically bind to each other are Compound A and Compound B, the analyte or the substance that specifically reacts with the analyte has multiple reactive groups. The reagent in which compound B is bound to the reactive group is specifically bound to a substance or a substance to be specifically bound to the substance to be measured on the optical fiber. Then, a compound A modified with a fluorescent dye is reacted, and a complex is formed on the optical fiber by binding of Compound A—Compound B. Then, the fluorescent dye is excited by light, A method for measuring a bioactive substance, which is characterized by measuring fluorescence.

34. The measurement according to claim 33, wherein the combination of compound A and compound B is a combination of avidin and bitin, an antibody and protein A, and a protein A and antibody, respectively. Method.

35. The method according to claim 30 or 33, wherein the compound having a plurality of reactive groups is aminoglucan.

36. The method according to claim 30 or 33, wherein the compound having a plurality of reactive groups is chitosan.

37. The fluorescent dye is a cyanine dye. No. 33 The measurement method according to any one of the items 3 to 3.

38. The measuring method according to any one of claims 28 to 33, wherein the light is laser light.

39. Detection by exposing the light source and the optical fiber for transmitting the excitation light or the fluorescent light, and the core surface on one end surface thereof, and immobilizing a substance that specifically binds to the substance to be measured on the surface. A bioactive substance, comprising: a unit for extracting only the fluorescence excited by the detection unit; and a photocounter for measuring the intensity of the fluorescence excited by the detection unit. measuring device.

40. The apparatus according to claim 39, wherein the optical fiber is made of resin.

41. The apparatus according to claim 39, wherein the light source is a semiconductor laser.

42. The device according to claim 39, wherein the detection unit is detachable by a coupler.

43. The method according to claim 39, wherein the detection unit is of a facing type.

40. The apparatus according to claim 39, wherein the mechanism for extracting only the fluorescence is a filter.