JP3130513B2 - Bioactive substance measurement device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention 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 required for using the reagent, and a measurement having a detecting section using the optical fiber. The present invention relates to an apparatus and a method for measuring a bioactive substance using the reagent and the apparatus.

[0002]

2. Description of the Related 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, measurement methods, Alternatively, various technologies have been proposed for devices and the like.

1) Labeling Reagents As labeling reagents, radioactive isotopes, luminescent agents, enzyme-labeled antigens and antibodies have been developed. Among them, the one with the highest sensitivity is labeled with a radioisotope, but handling is not easy. Those labeled with an enzyme are effective only for some substances.
Therefore, it is desired to increase the sensitivity of the luminescent agent labeling reagent.

Japanese Patent Application Laid-Open No. 58-61468 discloses a highly sensitive reagent which can be used in such a luminescence immunoassay, and discloses an immunological reaction in which an organic polymer compound having a plurality of luminescent molecules and either an antibody or an antigen. No. 4,166,105 discloses a first reactant (antibody) capable of specifically reacting with a multifunctional polymer skeleton molecule analyte, and a large number of fluorescent dye molecules. Reagents for detecting bound analytes have been proposed. However, these reagents have a problem in that the detection sensitivity is not practical when a dye having low sensitivity to be excited at a long wavelength is used because the amount of the dye that can be bound is not sufficient.

Japanese Patent Application Laid-Open No. 60-252265 discloses that
A method for measuring a biologically active substance using a water-soluble organic polymer compound to which a luminescent agent is bound and avidin is disclosed. However, in this reagent and method, since the antibody or antigen is bound to avidin via biotin having a small molecular weight, a large amount of biotin binds around the antibody or antigen, and there is a possibility that the immune activity is reduced, There is a problem that it is necessary to take a treatment to prevent this, and that it is difficult to bind the water-soluble polymer to avidin due to steric hindrance.

2) Optical fiber for luminescence immunoassay In luminescence immunoassay, a method of exciting a fluorescent dye (luminescent agent) or transmitting fluorescence using an optical fiber is effective, and various techniques have been disclosed. In order to use an optical fiber as a sensor, it is necessary to immobilize an antigen and an antibody on the optical fiber.
There are a membrane immobilization method in which antigens and antibodies are immobilized on a membrane such as cellophane, and an inclusive method in which antigens and antibodies are enclosed in voids such as acrylamide gel.The former has a decrease in sensitivity due to light scattering, while the latter has There was a problem that response was poor. Therefore, as a method of covalently binding antigens, antibodies, etc. directly to optical fibers, Analytical Chemistry V
ol. 59 No. 8 p1226-1230 (1987) discloses that a silanol group on the surface of a quartz optical fiber is reacted with (3-glycidoxypropyl) trimethoxysilane (GOPS), which is a silane coupling agent. Is treated with HIO ₄ to introduce a formyl group on the surface of a quartz optical fiber, and the formyl group is reacted with an amino group of a protein to immobilize the formyl group. However, this method is effective only for a quartz fiber, and cannot be used for a resin optical fiber. Since resin optical fibers are inexpensive, easily polished, flexible, and easy to handle, a method of binding proteins such as antigens and antibodies to resin fibers is desired.

An apparatus using an optical fiber is disclosed in JP-A-59-501873 (US Pat. No. 4,582,828).
09) discloses an immunoassay apparatus and method.
No. 23358 and Japanese Patent Application Laid-Open No. 62-501102 (Switzerland Patent Application No. 5306 / 84-5) propose an optical fiber type immunosensor. However, these specifications do not describe any technique for performing high-sensitivity labeling of antigens and antibodies, and therefore, as a light source,
It can be used only when a fluorescent dye having high sensitivity, such as being excited by an Hg lamp, a Xe lamp, or an Ar laser, is used. Further, there is a problem that the apparatus is large and expensive.

3) Measuring method Various methods of immunoassay using a luminescent agent have been proposed. For example, JP-A-60-24450 discloses luminescence of a luminescent agent-bound avidin bound to an immune complex by a biotin-avidin bond. A method for measuring a biologically active substance, which measures the amount of light emitted by a reaction, has been proposed. In this method, an avidin-biotin bond is interposed between the luminescent agent and the antibody or antigen. However, since no other organic polymer compound is interposed, the amount of dye that can be bound is small, and a low-sensitivity dye cannot be used. There is. As a reagent using a biotin-avidin bond, a labeled immunoactive substance has been proposed in Japanese Patent Application Laid-Open No. 58-30667 (Switzerland Patent Application No. 6989 / 81-01). However, this technique has a problem that the immunological substance is labeled with an enzyme, and the process for measuring the enzyme activity is troublesome.

[0009]

SUMMARY 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, the protein molecule 1
It is necessary to increase the amount of fluorescent dye bound per unit.To this end, it is necessary to bind a bioactive substance to a compound having a large number of reactive groups, and bind a compound having a large number of fluorescent dyes bound to each reactive group. I thought. In addition, the inventors succeeded in developing a detection unit composed of an optical fiber capable of efficiently collecting and measuring light emitted from a fluorescent dye, a device equipped with the same, and a measurement method using these reagents and devices.

(Reagent for Measuring Biologically Active Substance) The reagent for measuring a biologically active substance according to the present invention is characterized in that a fluorescent label binds to a compound having a plurality of reactive groups and reacts with the reactive group of the compound having a plurality of reactive groups. Is characterized in that a compound modified with a plurality of fluorescent dyes is bound.

The fluorescent dye described in the present invention refers to a dye that is 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 above-mentioned 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 increasing the amount of the fluorescent dye bound to the bioactive substance by binding to the bioactive substance, the detection sensitivity is dramatically improved.

Examples of the reactive group 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.

[0014] The reactive group is preferably 2 per molecule.
Desirably, there are 0 to 100,000. The reason is that if the number is less than 20, no improvement in detection sensitivity can be expected, and if the number is more than 100,000, it is difficult to dissolve such a polymer in a solvent. The number of the reactive groups is more preferably 3000 to 6000 per molecule, more preferably 4000 to 5000.

The compound having a plurality of reactive groups is
Desirably, the compound is an aminoglucan or a polyaminopeptide. This is because these compounds have 4000 to 5000 amino groups.

The aminoglucan includes chitosan,
Polygalactosamine, polyneuraminic acid, and the like can be used, but it is preferable to use chitosan.

The polyaminopeptide is obtained by polymerizing an amino acid having two or more amino groups through a peptide bond, such as polylysine.

The compound to be modified with the fluorescent dye used in the present invention is a semi-hight m (molecular weight: about 1000).
olecule).

The natural high molecular compound is preferably avidin, protein A, antibody, hormone, hormone receptor, etc., which are relatively easily available.

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 appropriately performed. Crosslinking agents 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 compound binds to the compound having a plurality of reactive groups via biotin, an antibody, protein A, a hormone receptor, and a hormone, and particularly binds to avidin. -Biotin "combinations are preferred.

When the above-mentioned "avidin-biotin" combination is used, 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 is changed to protein A, antibody,
Any of them may be bonded.

Avidin is a basic albumin-like crystal protein having a molecular weight of about 68,000, and has a very high affinity for biotin selectively. Avidin is stable against heat, pH, chemical modification, etc.
Avidin is suitable for modification with a fluorescent dye because it has many amino groups on the molecular surface because its isoelectric point is 10.

Avidin has 36 amino groups, some of which contribute to the binding to biotin. Therefore, it is desirable to bind 2 to 10 fluorescent dyes per avidin molecule.

Further, the reagent for measuring a bioactive substance of the present invention can be obtained by labeling biotin, a compound having a plurality of reactive groups and a bioactive substance complex with the modified avidin.

The protein A is a protein having a molecular weight of 42,000 occupying 5% of the cell wall of Staphylococcus aureus and having high affinity for immunoglobulin (antibody protein). , Antibody protein-
By labeling a compound-bioactive substance complex having a plurality of reactive groups, the reagent for measuring a bioactive substance of the present invention can be obtained.

It is necessary that both the protein A and the antibody do not react with the biologically active substance to be measured.

As described above, by binding a compound modified with a plurality of fluorescent dyes, a plurality of fluorescent dyes can be bonded to one reactive group, 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, a much safer measurement can be performed compared to the conventional immunoassay using radioisotopes (radioimmunoassay), and troublesome luminescence processing in chemiluminescence and bioluminescence is omitted. This is because measurement with high accuracy and good reproducibility can be realized in a shorter time.

Preferably, the light is laser light or LED light (light emitting diode).
An e-Ne laser or a semiconductor laser is desirable. The reason for this is that the laser is a Xe lamp or Ar
This is because they are smaller and less expensive than lasers. When the above-mentioned semiconductor laser is used, laser light in a short wavelength region can be emitted by combining it with an SHG element (second harmonic generation element; an element that reduces the wavelength of light to half).

The fluorescent dye used in the present invention is 200 to
It is desirable to be excited by 800 nm light. The reason for this is that if the wavelength is shorter than the above-mentioned wavelength, the energy is too high and the chemical bond is destroyed. If the wavelength is longer than the above range, the quantum yield is too low to be practical.

As the fluorescent dye, coumarin derivatives such as umbelliferone, polycyclic aromatic derivatives, rhodamine isothiocyanate, fluorescein isothiocyanate, cyanine dyes, phycobiliproteins, dansyl derivatives, o-phthalaldehyde and the like are used. Although it is possible, a cyanine dye is particularly preferable.

The cyanine dye has a structure in which a heterocyclic ring containing an azonium ion is bonded by a methine chain, for example,

[0035]

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(Where Y and Y ′ are O, S, Se, −
X represents NH— or —CH ＝ CH—, and R and R ′ represent an alkyl group such as methyl, ethyl, propyl or a carboxyalkyl group such as carboxyethyl;
Represents a halogen atom, and n represents an integer of 0 to 3).

These cyanine dyes can be excited by a He—Ne laser (633 nm) or a semiconductor laser of the shortest wavelength currently transmitted (638 nm).

Accordingly, the size and cost can be reduced as compared with the case where an Ar laser or a Xe lamp is used or the case where a semiconductor laser combined with an expensive SHG element is used.

As the cyanine dye, the compounds represented by the formula (1)
The cyanine dye represented by is preferably used.

[0040]

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In the formula, n represents 0, 1, 2 or 3. Particularly preferably, n is 2.

Labeling with a fluorescent dye is intended for those having a biological activity, so that the reaction can be completed within a short time under mild conditions as much as possible, and can bind to the reactive group without causing side reactions. It is necessary to be. 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.

Further, as the cyanine dye, the following compounds can be used.

[0044]

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The bioactive substance used in the present invention includes saccharides and proteins, and proteins are particularly preferable.

The bioactive substance comprising the protein is desirably an antigen, an antibody, an enzyme, a hapten or an enzyme inhibitor.

It is desirable that the reagent for measuring a biologically active substance of the present invention is coupled to an optical fiber and excited at the time of measurement.

(Resin Optical Fiber) The resin optical fiber of the present invention requires a resin optical fiber having on its core surface a reactive group capable of covalently binding a substance that specifically binds the above-mentioned reagent for measuring a bioactive substance. It is. The reason that the optical fiber is made of resin is that resin optical fiber is inexpensive, easy to be polished compared to glass optical fiber, and can transmit a large amount of light because the core diameter can be increased, and increases the core diameter. However, there is a feature that flexibility can be maintained. In particular, in the case of an optical fiber made of polymethyl methacrylate, the transmission properties in the wavelength region around 560 to 650 nm are excellent.

The density of the reactive groups is, 1.0 × 10 ^{1 0}
6.0 It is desirable that the × 10 ^{1 3} pieces / cm ^2. The reason for this is that, when the density is lower than the above range, the absolute number of reactive groups on the surface of the resin optical fiber is reduced, and the measurement sensitivity is not practical because the sensitivity is lowered. This is because the transmittance of the fluorescent light to the optical fiber becomes very poor. (See FIG. 7: In the case of polymethyl methacrylate)

Further, the density of the reactive groups was 3.0 × 1
0 is preferably ^{1 2 ~4.0} × 10 ^{1 3} pieces / cm ^2,
It is preferred that a ^{1.5 × 10 1 3 ~3.5 × 10} 1 3 pieces / cm ^2. The reason is that when the density is higher than the above range, the distance between the reactive groups becomes short, and the reproducibility starts to decrease due to 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 a bioactive substance and has good translucency. For example, polystyrene, polyacrylate, polyester, polyacrylamide, polyvinyl alcohol, polyethylene Terephthalate, polycarbonate, or a copolymer thereof can be used.

The resin optical fiber preferably contains a resin having a structure that reacts with a crosslinking agent as a main component. The reason for this is that a crosslinking agent for introducing a reactive group to the surface of the optical fiber is easily reacted.

The structure which reacts with the crosslinking agent is preferably an ester bond, an amide bond, an ester group, a carboxyl group, a formyl group, an amino group, a hydroxyl group, an epoxy group or a thiol group. Ester structures such as groups are preferred. The translucent resin having a structure or a functional group that reacts with the crosslinking agent is preferably a polyacrylate or polyester.

The acrylic acid ester polymer has an ester structure among acrylic acid resins,
For example, it is a synthetic resin composed of a polymer of an ester derivative such as acrylic acid and methacrylic acid, and specifically, a polymer such as methyl acrylate, ethyl acrylate, and methyl methacrylate. Further, among the above-mentioned acrylate polymer, one particularly preferably used in the present invention is polymethyl methacrylate. This is because polymethyl methacrylate has a better translucency than other resins.

By the way, on the surface of the resin optical fiber,
Techniques for binding proteins are 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 crosslinking agent. Although not an optical fiber, Japanese Patent Application Laid-Open No. 56-129841 describes a technique for binding a protein to the surface of a cell for measuring absorbance of polymethyl methacrylate or nylon. However, the former technique uses a nylon optical fiber having low translucency, and when the present inventors tried it, the propagation loss of the nylon fiber was large, and the high sensitivity measurement intended by the present inventors was intended. Was difficult. In addition, the latter technique involves binding a protein to an absorbance measurement cell, and is not a technique for immobilization on an optical fiber.

The resin optical fiber used in the present invention may be, for example, a copolymer of a monomer such as methyl acrylate, ethyl acrylate, methyl methacrylate and a monomer such as styrene.

The reactive groups on the surface of the optical fiber include formyl group, carboxyl group, amino group, hydroxyl group, epoxy group, thiol group, isocyanate group,
Examples include an isothiocyanate group, and a formyl group is preferred. The reason for this is that the substance covalently bonded to the fiber of the present invention is a bioactive substance and requires mild reaction conditions that do not reduce its activity, but the formyl group is an amino group of the bioactive substance, particularly a protein. This is because they react easily.

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

When the resin of the resin optical fiber does not have a structure that reacts with the cross-linking agent, a functional group is introduced into the resin, and when the resin has a structure that reacts with the cross-linking agent, the resin has a structure. The functional group is introduced by reacting a suitable crosslinking agent.

As the crosslinking agent, it is desirable to use a polyfunctional compound. For example, dialdehydes such as glutaraldehyde, succindialdehyde and adipaldehyde, N, N'-ethylenebismaleimide, N, N'- o
-Diisocyanate such as phenylenedimaleimide, bisdiazobenzene, or hexamethylene diisocyanate, or diisothiocyanate is used.

As a specific introduction method using the cross-linking agent, for example, when a formyl group is introduced into an optical fiber made of polystyrene, a benzene ring as a side chain is nitrated,
Next, reduction is performed, and this is converted into an amino group, and then formyl group can be introduced by reacting with glutaraldehyde.

In the present invention, the most preferred resin optical fiber for measuring a bioactive substance has a form having a formyl group on the core surface of a resin optical fiber mainly composed of a resin having an ester structure.

In the resin optical fiber for measuring a bioactive substance, a nucleophilic reagent having a formyl group is reacted as a polyfunctional compound on the exposed core surface of the resin optical fiber to introduce a formyl group into the core surface. It is manufactured by

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

[0065]

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(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)

The reagent represented by the above formula (2) includes glutaraldehyde and succinaldehyde.
The reagent of the formula (2) reacts nucleophilically with an ester group of a resin (in the following reaction formula, polymethyl methacrylate) as shown in the following reaction formula.

[0068]

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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 is that the diameter of the optical fiber is usually 1mm
The diameter of the core cross section is about 0.97 mm (the cross-sectional area is 0.73 mm).
This is because, since there is only 9 mm ² ), in order to introduce a large amount of active groups, it is necessary to peel off 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.

The nucleophile having a formyl group is KO
It is desirable to add and dissolve a base such as H, an alcoholic organic solvent such as ethanol, an ethanol solution of a Ni salt such as NiSO ₄ , and a nucleophilic reagent having a formyl group.

The concentration of the base such as KOH is 50 to 1
00 mM is preferred.

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 portion of the resin optical fiber is immersed in the reaction reagent to react with the ester structure. The reaction temperature is preferably adjusted appropriately. After the reaction treatment, washing with water and immersion in an acid such as HCl removes the acetalized alcohol to obtain a resin optical fiber having formyl groups bonded thereto. (See the following reaction formula)

[0075]

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(Detection Unit for Measurement) The detection unit for measurement of a bioactive substance of the present invention needs to covalently bond a substance which specifically binds to a 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 hapten, an enzyme inhibitor, or the like.

The reactive group of the resin optical fiber is
In the case of a formyl group, it is desirable to perform an immobilization treatment after reacting a substance that specifically binds with the substance to be measured. The reason for this is that when the immobilization treatment is not performed, a substance that specifically binds to the substance to be measured is easily released by a reversible reaction.

In the case where a protein is selected as the substance which specifically binds to the substance to be measured,
This 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 an amino group of the protein, and the binding site becomes an imino group.
After washing with water, an appropriate concentration of a reducing agent such as NaB
By treating with H ₄ or the like, the imino group is reduced and inactivated, and the protein is immobilized. (See the following reaction formula. The resin is polymethyl methacrylate.)

[0079]

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The detector of the present invention may be covered with a flow cell 5 as shown in FIG. 1, may be of an opposed type as shown in FIG. 3, or may have a mirror 1 at the tip as shown in FIG.
The reflection type with 2 may be used. In the case of the opposed type, the excitation light is incident from the side facing 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 3 to which the detection unit is attached, and is incident.
2 and propagates through the optical fiber 3.

(Production Method of Reagent for Measuring Bioactive Substance) The reagent for measuring a bioactive substance of the present invention is produced by various organic chemical reactions, but produces a protein modified with a plurality of fluorescent dyes of the present invention. The reaction is carried out by reacting a fluorescent dye with a protein and removing the solvent from the reaction product to obtain a residue having a pH of 2 to 7.
And the unreacted dye is separated and removed. This is because, in the buffer having the pH of 2 to 7, the protein labeled with the fluorescent dye is dissolved, but the unreacted fluorescent dye is not dissolved, so that it can be easily separated. PH of the buffer
When the pH 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 particularly 6.5 ± 0.5 when avidin modified with a cyanine dye is produced.

It is preferable that the fluorescent dye is an acidic fluorescent dye having good solubility in a basic buffer. The fluorescent dye is desirably one that is excited by laser light. Examples of the fluorescent dye include the cyanine dye and fluorescein isothiocyanate.

The protein of the present invention may be various proteins, for example, neocarzinostan, enzymes, hormones, etc., and is preferably a basic protein (a protein having PI ≧ 7). The reason for this is that the basic protein exhibits good solubility in the buffer solution 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 performed by dicyclohexylcarbodiimide, di-p
-Condensing the amino group of the protein with the reactive group of the fluorescent dye by using a carbodiimide reagent such as toluoylcarbodiimide. The reaction solvent may be any as long as it can dissolve the fluorescent dye and the protein. For example, the reaction is carried out in an organic solvent such as methanol, ethanol, methylamine, ethylamine, diethylamine, triethylamine or a solvent such as a basic aqueous solution.

The reaction proceeds too much to cause a reaction between the OH group or SH group of the protein and the fluorescent dye, thereby deactivating the reaction specificity (antigen-antibody reaction or reactivity to biotin when the protein is avidin) possessed by the protein. If necessary, the reaction is stopped with acetic acid or the like to prevent the reaction from occurring. After the reaction,
The solvent was distilled off under reduced pressure and removed to dryness.
Dissolve in buffer. Since the unreacted fluorescent dye does not dissolve, 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, wherein the bioactive substance binds to a compound having a plurality of reactive groups. A compound having a plurality of reactive groups, a compound B bound to the reactive group, and a compound A modified with a plurality of fluorescent dyes bound to the compound B (for the sake of simplicity, hereafter referred to as fluorescent compound). Dye-Compound A-Compound B-Compound having a plurality of reactive groups-Bioactive substance)
It is desirable to manufacture 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-a compound having a plurality of reactive groups-a bioactive substance complex, and then reacting with a compound A modified with a fluorescent dye to obtain a fluorescent dye-a compound A-a compound B-having a plurality of reactive groups A compound-bioactive substance conjugate is produced.

The reason why the above-mentioned production method is desirable is that when the reaction order is changed, side reactions occur and the yield decreases. In the above production method, compound A and compound B
Is preferably a protein and a compound that specifically binds to the protein.Specifically, avidin and biotin, protein A and an antibody, antibody and protein A, and the like are preferable, and a combination of avidin and biotin is particularly preferable. Optimal. These reactions will be described more specifically.

A compound having a plurality of reactive groups, for example, chitosan (I) has a large number of amino groups in the molecule, and biotin (II) is added to chitosan (I) in a basic solution.
When reacted in the presence of a dehydrating agent such as water-soluble carbodiimide (CHMC) and N-hydroxysuccinimide,
Biotin is acid-amide bonded to the amino group of most chitosan to obtain biotinylated chitosan (III). The biotinylated chitosan (III) is reacted with a protein which is a bioactive substance using the same dehydrating agent as described above to obtain biotinylated chitosan (IV) in which the protein is bonded to the remaining free amino groups of chitosan (I). .

On the other hand, avidin (V) modified with a fluorescent dye
Can be obtained by reacting a carboxyl group of a fluorescent dye, for example, a cyanine dye, with an amino group of avidin, which is a protein, in the same manner as described above.

Next, avidin (V) modified with the above fluorescent dye was added to the biotinylated chitosan (IV) to which the above protein was bound.
Avidin selectively binds to biotin with very high affinity and binds to the fluorescently labeled protein (V
I) can be obtained.

[0091]

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The carboxyl group of the cyanine dye can be easily condensed with an amino group of avidin and an organic solvent using a dehydrating condensing agent such as dicyclohexylcarbodiimide by an ordinary method to form an amide bond. After completion of the reaction between the cyanine dye and avidin, unreacted substances are preferably removed as much as possible, for example, dialysis, centrifugation, gel filtration, or filtration using a filtering material.

The acid amide bond formed by binding biotin to chitosan has an excitation wavelength of 225.5 nm at pH 6.
Has characteristic fluorescence peaks around 450 nm, 300 nm and 490 nm. And 458nm and 300nm
Since there is a linear relationship between the difference between the fluorescence intensities and the concentration of acid amide bonds, a calibration curve of chitosan can be prepared by using this characteristic, and the amount of biotinylation can be estimated from the amount of acid amide bonds.

(Measurement Method of Bioactive Substance) A measurement 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 bioactive substance of the present invention is performed by the following method. 1) A complex comprising a fluorescent dye-avidin-biotin-substance to be measured or a substance specifically reacting with the substance to be measured is combined with a substance or a substance to be specifically bound to the substance to be measured on the optical fiber, and After being excited by light,
A measurement method characterized by measuring fluorescence. 2) After specifically reacting the analyte to which biotin is bound or the substance that specifically reacts with the analyte with the substance or the analyte that specifically binds to the analyte on the optical fiber, Reacting avidin modified with a dye,
A measurement method comprising: forming a complex on an optical fiber by avidin-biotin bond; exciting with light; and measuring fluorescence. 3) The substance to be measured or a substance that specifically reacts with the substance to be measured binds to the compound having a plurality of reactive groups, and the reactive group of the compound having the plurality of reactive groups includes a plurality of fluorescent dyes. The reagent to which the compound modified in is bound, the substance or the substance to be specifically bound to the substance to be measured on the optical fiber, and specifically reacted, and then excited by light,
A measurement method characterized by measuring fluorescence. 4) Compound A, which specifically binds to each other,
When the compound B is used, a substance to be measured or a substance that specifically reacts with the substance to be measured is bound to a compound having a plurality of reactive groups, and the reagent having the compound B bound to the reactive group is After reacting specifically with the substance or the substance to be measured specifically bound to the substance to be measured on the optical fiber, the compound A modified with a fluorescent dye is reacted, and the compound A-compound B is bonded on the optical fiber by After forming a complex, a fluorescent dye is excited with light, and the fluorescence is measured.

The above measuring 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-biotin-substance to be measured or a substance that specifically binds to the substance to be measured. The reason for using the reagent is that, when a fluorescent dye is used to label a substance to be measured or a substance that specifically reacts with the substance to be measured, the amount of binding of the fluorescent dye is limited. The binding active site of a substance that specifically reacts with the analyte may be damaged. Therefore, many fluorescent dyes can be bound through the avidin-biotin without damaging the binding active site. The reagent is excited with light after reacting specifically with the substance or the substance to be measured on the optical fiber. The excitation on the optical fiber is because the excitation light and the fluorescence can be propagated by the optical fiber, and efficient measurement is possible. The fluorescent dye,
Desirably, it is a cyanine dye. The reason is that the cyanine dye can be excited by a He-Ne laser (633 nm) or a semiconductor laser (638 nm), and the size and cost of the device can be reduced. In order to bind the cyanine dye to avidin, it is necessary that the reaction be completed in a short time under conditions as mild as possible, and be capable of binding to a reactive group without causing a side reaction. A carbocyanine dye represented by the following formula and having a carboxyl group outside the conjugate system as a functional group is preferably used.

[0096]

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

To obtain avidin modified with a cyanine dye, the carboxyl group of the cyanine dye and the amino group of the protein are formed by forming an amide bond in an organic solvent in the presence of a dehydrating condensing agent such as carbodiimide. Can be At this time, unreacted dye is separated. The light source used in the present invention is desirably laser light or LED light.

The measuring methods are roughly classified into a competitive method and a sandwich method. In the competition method, a sample to be measured and a reagent having a known concentration consisting of a fluorescent dye, avidin, biotin, and a substance to be measured are mixed, and an optical fiber on which a substance that specifically binds to the substance to be measured is immobilized. After immersion and specific reaction, it is excited by light and the fluorescence is measured. In the case of the competition method, a sample to be measured and a reagent composed of a fluorescent dye-avidin-biotin-substance to be measured 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 binding of the reagent consisting of the fluorescent dye-avidin-biotin-substance to be measured relatively decreases, the fluorescence intensity decreases, and the slope of the concentration-fluorescence intensity calibration curve is negative. become.

In the sandwich method, an optical fiber on which a substance which specifically binds to a substance to be measured is immobilized is placed in 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 fluorescent dye-avidin-biotin-a reagent that specifically binds to the substance to be measured. A reagent consisting of a substance that specifically binds to the fluorescent dye-avidin-biotin-substance to be measured is bound to the optical fiber. In the sandwich method, the same number of fluorescent dyes-avidin-biotin-reagents as substances to be specifically bound to the substance to be measured are bound to the optical fiber. Therefore, if the concentration of the sample to be measured is high, the amount of binding of the reagent consisting of the fluorescent dye-avidin-biotin-substance specifically bound to the substance to be measured increases,
The fluorescence intensity increases, and the slope of the concentration-fluorescence intensity calibration curve becomes positive.

Next, measurement method 2) will be described. The measurement method 2) basically has the same effect as the measurement method 1), but in this method, a substance to be measured to which biotin is first bound or a substance that specifically reacts with the substance to be measured is used. After specifically reacting with a substance or a substance to be specifically bound to the substance to be measured on the optical fiber, it is necessary to react avidin modified with a fluorescent dye.
The reason is that since avidin modified with a fluorescent dye is finally bound, a decrease in fluorescence intensity due to hydrolysis and oxidation of the fluorescent dye can be prevented, and measurement with high reproducibility can be performed. The fluorescent dye is desirably a cyanine dye. The light source used in the present invention is desirably laser light or LED light.

The measuring methods are roughly classified into a competitive method and a sandwich method. In the competition method, a sample to be measured and a reagent having a known concentration consisting of biotin and a substance to be measured are mixed, and an optical fiber on which a substance that specifically binds to the substance to be measured is immobilized, and a specific reaction is performed. After that, avidin modified with a fluorescent dye is bound, and is excited and measured by light. In the case of the competition method, a sample to be measured and a reagent composed of a fluorescent dye-avidin-biotin-substance to be measured 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 fluorescent dye
The binding amount of the reagent consisting of avidin-biotin-substance to be measured 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 placed in 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 biotin-a substance that specifically binds to the substance to be measured. A reagent consisting of biotin—a substance that specifically binds to the substance to be measured is bound to the fiber. Avidin modified with a fluorescent dye is bound to an optical fiber to which a reagent composed of the biotin-substance to be measured specifically binds. In the sandwich method, the same number of fluorescent dye-avidin-biotin-substances as substances to be measured are bound to the optical fiber. Therefore, if the concentration of the sample to be measured is high, the binding amount of the substance that specifically binds to the fluorescent dye-avidin-biotin-substance to be measured increases, the fluorescence intensity increases, and the slope of the concentration-fluorescence intensity calibration curve is Become positive.

Next, measurement method 3) will be described. In the measurement method 3), the analyte or a 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 is 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 amount of the fluorescent dye bound per bioactive substance can be increased, and the detection sensitivity can be dramatically improved. The fluorescent dye is desirably a cyanine dye. Further, the compound modified with the plurality of fluorescent dyes is preferably avidin, and it is desirable that the compound is bonded to the reactive group of the compound having a plurality of reactive groups via biotin. Further, the compound having a plurality of reactive groups is desirably selected from aminoglucans, and chitosan is particularly preferred. The light source for exciting the fluorescent dye may be laser light or LED.
Light is desirable.

The measuring methods are roughly classified into a competitive method and a 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 in the reagent. It is a method of measuring. In the case of the competition method, the sample to be measured and the reagent bind 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 above sandwich method, an optical fiber on which a substance that specifically binds to a substance to be measured is immobilized is placed in 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, a substance that specifically binds to the substance to be measured binds to a compound having a plurality of reactive groups, and a plurality of reactive groups of the compound having a plurality of reactive groups include: Into the solution of the reagent to which the compound modified with the fluorescent dye is bound. 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 calibration curve of the concentration-fluorescence intensity becomes positive.

Next, measurement method 4) will be described. In the measurement method, when two types of substances that specifically bind to each other are compound A and compound B, first, the substance to be measured or the substance that specifically reacts with the substance to be measured has a plurality of reactive groups. After reacting specifically with a reagent that binds to a compound having the compound B and binds to the reactive group with a substance or a substance that specifically binds to the substance to be measured on the optical fiber, It is necessary to react the compound A modified with the dye. By using such a method, the amount of fluorescent dye per analyte can be increased,
In addition, a decrease in fluorescence intensity due to hydrolysis or oxidation of the fluorescent dye can be prevented, and measurement with high reproducibility can be performed. The fluorescent dye is desirably a cyanine dye. Compound A and Compound B are avidin-biotin, protein A-antibody, antibody-
A combination of protein A is desirable, and an avidin-biotin combination is particularly preferred. It is necessary that the antibodies used as the compound A and the compound B do not cause a specific reaction with the substance to be measured. The compound having a plurality of reactive groups is preferably selected from aminoglucans, and chitosan is particularly preferable. Also,
The light source for exciting the fluorescent dye is desirably laser light or LED light.

The measuring methods are roughly classified into a competitive method and a sandwich method. 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, and After immersing the optical fiber on which the substance specifically binding to the substance to be measured is immobilized and reacting specifically, the compound A modified with a fluorescent dye is bound, excited with light, and the fluorescence is measured. Is the way. 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 binding amount of the reagent is relatively reduced,
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 immersed in 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 bonded to 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 is combined with a reagent solution to which compound B is bound. Put in. A reagent binds to the fiber. A substance that specifically binds to the analyte is bound to a compound having a plurality of reactive groups, and the reactive fiber is modified with a fluorescent dye to an optical fiber to which a reagent to which compound B is bound is bound. Compound A is bound. In the sandwich method,
The same number of 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 reagent increases, the fluorescence intensity increases, and the slope of the concentration-fluorescence intensity calibration curve becomes positive.

(Bioactive Substance Measuring Apparatus) The apparatus of the present invention, which will be described next, is characterized in that a bioactive substance is bonded to a compound having a plurality of reactive groups, and the compound having the plurality of reactive groups is reacted. The present invention is an apparatus for measuring a bioactive substance using the bioactive substance measurement reagent in which a compound modified with a plurality of fluorescent dyes is bound to an active group. The device 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, and the surface is specifically bound to a substance to be measured. And a photo-counter for measuring the intensity of the fluorescence excited by the detection unit.

The reason for using the optical fiber is that the excitation light and the fluorescent light can be propagated by the optical fiber, and there is no light loss, and efficient measurement can be performed. The optical fiber is
Desirably, it is made of resin. The reason for this is that 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 the bioactive substance can be covalently bonded to form the detection portion by way of the cross-linking agent. The structure that reacts with the crosslinking agent is preferably an ester structure. As the resin, a (meth) acrylate resin such as polymethyl methacrylate or a polyester resin is preferable.

Further, in the detecting section of the present invention, a bioactive substance is bonded to a compound having a plurality of reactive groups, and the reactive group of the compound having a plurality of reactive groups is provided with a plurality of fluorescent dyes. It is necessary that the reagent for measuring a bioactive substance to which the modified compound is bound is bound at the time of measurement. By binding such a reagent, high-sensitivity measurement is possible.

It is desirable that the fluorescent dye is a cyanine dye. The reason is that the cyanine dye is He-
Since it can be excited by Ne laser light (630 nm) or the shortest wavelength semiconductor laser (638 nm) that is currently being transmitted, a large and expensive Xe lamp or Ar laser or an expensive SHG element (the wavelength of the light is used). This is because it is not necessary to use an element), and an inexpensive and small-sized device can be obtained.
It is desirable that the detection unit be detachable from an optical fiber that propagates excitation light or fluorescence by a coupler. As the coupler, a guide rail type as shown in FIG. 3 is preferable.

The light source of the device of the present invention must be small and inexpensive, and may be a He-Ne laser, a semiconductor laser, a laser combining a semiconductor laser and an SHG element,
Or it is desirable to be LED (light emitting diode).
The mechanism for extracting only the fluorescence may be a half mirror, a filter, or the like, but is preferably a filter. The reason for this is that when a half mirror is used, a space for arranging the optical system is required, and it is 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.

[0115]

Next, examples of the present invention will be described.

Example 1 (1) 3 mg of Na ₂ CO ₃ and 4 mg of biotin were dissolved in 100 μl of water. (2) Next, the solution obtained in the above (1) was added to 2 ml of a 1.8 μM chitosan solution. (3) After adding 100 μl of water, 50 mg of CHMC was added.
(Aqueous carbodiimide) was added. The reaction was carried out at room temperature for 5 hours to overnight with further stirring. (4) The reaction was stopped by adding 3 drops of acetic acid. (5) Then, 0.3 g / ml of Na ₂ CO ₃ and NaCl
4 ml of a 0.3 g / ml mixture was added to precipitate the biotinylated chitosan. (6) After collecting the precipitate with a centrifuge, 0.3 g / ml of N
The precipitate was washed with aCl and a 0.1 g / ml Na ₂ CO ₃ buffer. (7) The precipitate obtained in (6) above was dialyzed overnight at 4 ° C. against 10 ml of 10 mM potassium-phosphate buffer (pH = 7) to obtain biotinylated chitosan. (8) An anti-IgG (antibody Y) solution and CHMC were added to the biotinylated chitosan suspension and reacted at 4 ° C. overnight. After completion of the reaction, dialysis was performed for 12 hours, and unreacted substances were removed using an anion exchange column to obtain biotinylated chitosan to which the antibody was bound. (9) 1 mg of avidin and 0.2 ml of triethylamine
Dissolved in ml of ethanol. Then 2 mg of NK11
60 (manufactured by Japan Photographic Dye Laboratories; n in the above formula (1))
= 2 cyanine dye) and sufficiently dissolved to prepare a solution. Further, 14 mg of dicyclohexylcarbodiimide was added to the above solution and reacted at room temperature for 4 hours. (10) After completion of the reaction, ethanol and triethylamine were removed under reduced pressure using an evaporator. (11) The residue generated in the step (10) is reduced to 0.0
After suspending in 2 ml of 1 M acetate buffer (pH = 6.5), the suspension was centrifuged at 5,000 rpm for 10 minutes using a centrifuge, and the supernatant was collected and centrifuged again to obtain NK116.
A solution of avidin modified with 0 was obtained. (12) Diameter 1 mainly composed of polymethyl methacrylate
The tip of an optical fiber made of resin (manufactured by Mitsubishi Rayon, trade name: Esca) having a thickness of 1 mm was immersed in ethyl acetate and wiped off, and the clad 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. (13) 10 mg of NiSO ₄ was dissolved in 0.5 ml of water, and then 2.5 ml of ethanol was added. At this time, since a white precipitate was generated, the precipitate was centrifuged at 3000 rpm to collect a supernatant, which was used as a Ni-ethanol solution.
Ni in 0.4 ml of 50 mM potassium hydroxide-ethanol solution
0.1 ml of an ethanol solution was added, and 50 μl of 50% glutaraldehyde was further added to prepare a reaction solution. (14) The reaction solution prepared in (13) is added to the (1)
The resin optical fiber of 2) was immersed at 50 ° C. for 10 minutes and washed with water. (15) The pair was immersed in a 20 mM hydrochloric acid solution for 5 to 10 minutes, washed with water, and a formyl group was introduced into the surface of the core portion of the resin optical fiber. In FIG. 6B, the above method is used.
FIG. 6A shows the relationship between the processing temperature and the amount of immobilizable enzyme (protein) when a formyl group is introduced into the surface of the optical fiber, and FIG. 6A shows the relationship between the processing temperature and the optical transmittance of the fiber. FIG. 7 shows the relationship between the density of the formyl group on the fiber surface and the light transmission reduction rate. The heat transmission of the optical fiber made of polymethyl methacrylate improves the light transmission rate. However, when the reaction temperature increases, the light transmission rate decreases because the density of formyl groups to be bonded increases. For this reason,
As shown in FIG. 6A, the most preferable temperature is around 50 ° C. (16) Thermostable α- produced by Bacillus 16-3F strain
Mouse I, a monoclonal antibody against amylase
1 mg of gG antigen was added to phosphate buffered saline (pH = 7.
5). A resin optical fiber is added to this solution at 4 ° C.
For 12 hours. (17) The resin optical fiber was taken out of the solution, washed with water, immersed in a 1% NaBH ₄ aqueous solution for 15 minutes, washed with water to immobilize mouse IgG antigen 4, 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
After being immersed in the detection unit 5 prepared in 8), the substrate was washed with phosphate buffered saline. (20) Next, the detection unit 5 was immersed in the biotinylated chitosan solution to which the antibody obtained in the above (8) was bound, and then washed with phosphate buffered saline. (21) Next, the NK1160-modified avidin solution obtained in the above (11) was immersed in the detection unit 5, and then washed with phosphate buffered saline. (22) Next, the device of the present invention shown in FIG.
Fluorescence was measured using a spectrophotometer 8 with a laser optical system. (23) changing the concentration of anti-mouse IgG (Y),
8) to (22) were repeated, and anti-mouse Ig was
The relationship between the concentration of G (Y) and the fluorescence intensity was examined to prepare a calibration curve. This is shown in FIG. FIG. 9 shows the responsiveness of the sensor. The detection limit was measured from the calibration curve and is shown in Table 1.

Example 2 (1) Biotinylated chitosan solution to which an antibody was bound in the same manner as in (1) to (18) of Example 1, NK1160
A modified avidin solution, an antigen-immobilized sensor and a detection unit were prepared. (2) An anti-mouse IgG (Y) solution having a known concentration and a biotinylated chitosan solution to which the antibody prepared in (1) above was mixed at a ratio of 1: 1 and passed through a flow cell 5 shown in FIG. Washed through phosphate buffered saline. (3) Next, the avidin solution modified with NK1160 prepared in the above (1) was passed through the flow cell 5 and 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. The detection limit was measured from the calibration curve and is shown in Table 1.

Example 3 (1) 3 mg of Na ₂ CO ₃ and 4 mg of antibody protein were dissolved in 100 μl of water. (2) Next, the solution obtained in the above (1) was added to 2 ml of a 1.8 μM β-1,4-polygalactosamine solution. (3) After adding 100 μl of water, 50 mg of CHMC was added.
(Aqueous carbodiimide) was added. The reaction was carried out at 4 ° C. overnight with further stirring. (4) Next, 0.3 g / ml of Na ₂ CO ₃ and NaCl
4 ml of a 0.3 g / ml mixture was added, and the antibody protein-bound β
-1,4-polygalactosamine was precipitated. (5) After collecting the precipitate with a centrifuge, 0.3 g / ml of N
The precipitate was washed with aCl and a 0.1 g / ml Na ₂ CO ₃ buffer. (6) The precipitate obtained in the above (5) was suspended in 10 ml of 10 mM potassium-phosphate buffer (pH = 7) and dialyzed overnight at 4 ° C. to obtain β-1, 4-Polygalactosamine was obtained. (7) An anti-mouse IgG (Y) solution and CHMC were added to the antibody protein-bound β-1,4-polygalactosamine suspension and allowed to react at 4 ° C. overnight. After completion of the reaction, dialysis was performed for 12 hours, and unreacted substances were removed using an anion exchange column to obtain antibody protein-bound β-1,4-polygalactosamine. (8) 1 mg of protein A and 0.2 ml of triethylamine
Was dissolved in 1 ml of ethanol. Then 2 mg of NK
1160 (manufactured by Japan Photographic Dye Laboratories) was added and sufficiently dissolved to prepare a solution. Further, 14 mg of dicyclohexylcarbodiimide was added to the above solution and reacted at room temperature for 4 hours. (9) After completion of the reaction, the solvent was removed with an evaporator. (10) The residue generated in the step (9) is treated with 0.01
After suspending in 2 ml of M acetate buffer (pH = 6.5), the suspension was separated at 5,000 rpm for 10 minutes using a centrifuge, the supernatant was collected, and centrifuged again to remove protein A modified with NK1160. A solution was obtained. (11) The antibody protein-bound β-1,4-polygalactosamine obtained in (8) and Protein A modified with NK1160 are reacted at 4 ° C. in a phosphate buffered saline to obtain a cyanine dye-protein. A-antibody protein-β-1,
A complex solution of 4-polygalactosamine-antibody {Y (thick)} was obtained. (12) In Example (13), succindialdehyde was used in place of glutaraldehyde, and an optical fiber containing polyester was used in place of the optical fiber made of polymethyl methacrylate.
The detection unit shown in FIG. 1 was created in the same manner as in 2) to (18). (13) The complex solution of the anti-mouse IgG (Y) solution of known concentration and the complex solution of the cyanine dye-protein A-antibody protein-β-1,4-polygalactosamine-antibody (Y) obtained in the above (12) is , And then washed through a flow cell 5 shown in FIG. 1 and then through a phosphate buffered saline, and then the fluorescence was spectrally analyzed by the He-Ne laser optical system in the apparatus of the present invention shown in FIG. It was measured with a photometer 8. Anti-mouse I
The same measurement was repeated while changing the concentration of gG (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) A biotinylated chitosan solution to which an antibody was bound was prepared in the same manner as in (1) to (8) of Example 1. (2) 1 mg of avidin and 1.8 mg of fluorescein isothiocyanate were added to 5 ml of a basic solvent consisting of a 0.5 M sodium carbonate-sodium hydrogen carbonate buffer (pH = 9.0).
, And the mixture was stirred at 4 ° C. while blocking light, and reacted for 20 hours. (3) Next, the solvent was distilled off from the reaction solution under reduced pressure using an evaporator. (4) This residue, 0.05M phosphate buffer (pH =
4.0) in 5 ml. (5) The mixture was centrifuged at 5000 rpm for 10 minutes to remove unreacted dye, and the supernatant was collected. (6) The above operations (3) and (4) are further repeated twice, and the resulting supernatant is passed through a tube filled with glass wool to remove unreacted dye, and avidin modified with fluorescein isothiocyanate I got (7) An antigen-immobilized sensor and a detection unit 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 biotinylated chitosan solution to which the antibody obtained in (1) was bound was passed through the flow cell 5, and then washed with phosphate buffered saline. (10) Next, the solution of avidin modified with fluorescein isothiocyanate obtained in (6) was passed through the flow cell 5, and washed with phosphate buffered saline. (11) Next, fluorescence was measured using a spectrophotometer 8 and a laser optical system (wavelength 490 nm) in which a semiconductor laser and a thin film waveguide type SHG element were combined with the apparatus of the present invention shown in FIG. (12) The concentration of anti-mouse IgG (Y) was changed, and the same measurement as in the above (8) to (11) was repeated.
The relationship between the concentration of (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.

Embodiment 5 The present invention is basically the same as Embodiment 1, except that (1
After the treatment of 1), a 0.17 g / ml saturated sodium sulfate solution (solvent: 10 mM potassium phosphate buffer, pH =
7) Add 4 ml and centrifuge at 2000 rpm for 10 minutes.
It was left at 0 ° C. for 1 hour. The obtained precipitate was suspended in 10 mM potassium phosphate buffer. The sodium sulfate crystals were removed by centrifugation, and the supernatant was dialyzed and concentrated before use. The detection limit was measured from the calibration curve and is shown in Table 1.

Example 6 The solution obtained in (1) of Example 1 was further added with 50 mg of CHM.
C was added and dissolved, and the mixture was allowed to stand at 12 ° C. for 2 hours. Using this solution, the treatment of Example 1 (2) and thereafter was performed. The detection limit was measured from the calibration curve and is shown in Table 1.

Example 7 In this example, polylysine was used in place of chitosan of Example 1. The reaction conditions are in accordance with the conditions of Example 1. The detection limit was measured from the calibration curve and is shown in Table 1.

Example 8 (1) A solution of avidin modified with NK1160 obtained by the treatments (9) to (11) of Example 1 was obtained. (2) The detection unit of Example 1 was immersed in a mouse IgG (antigen) solution at 4 ° C. for 12 hours. (3) Then, it was washed and treated with 1% NaBH ₄ to immobilize the antigen. (4) immersing the detection part in an anti-mouse IgG solution of known concentration,
Washed with phosphate buffered saline. (5) The detection part was immersed in the biotinylated antibody solution and washed. (6) Then, the detection unit was immersed in the solution of (1), washed, and measured with the apparatus of FIG. (7) The above operation was repeated to prepare a calibration curve shown in FIG. 6 (c), from which the detection limit was measured, which is shown in Table 1.

Example 9 A detection reagent was prepared by reacting the solution of (1) in Example 7 with a biotinylated antibody, and this reagent was mixed with a known concentration of an anti-mouse IgG solution at a ratio of 1: 1. Then, the detection part of Example 7 was immersed and measured to prepare a calibration curve, from which the detection limit was measured, which is shown in Table 1.

Embodiment 10 (1) The processing of (9) to (11) of Embodiment 1 is performed, and NK
A solution of avidin modified at 1160 was obtained. (2) The solution of the above (1) was mixed with the anti-IgG-bound chitosan solution obtained in the same manner as in (8) of Example 1,
Add glutaraldehyde and in the presence of carbodiimide,
By heating at 50 ° C., the amino group of avidin and the amino group of chitosan are cross-linked with glutaraldehyde,
A solution of K1160-avidin- (glutaraldehyde) -chitosan-anti-IgG was obtained. (3) Formic anhydride was reacted with an optical fiber made of polystyrene in the presence of aluminum chloride to introduce a formyl group into a phenyl group of polystyrene, and then reacted with 1,6-hexanediamine in the presence of carbodiimide. Then, it was reduced with 1% NaHB ₄ . Then mouse Ig
G is dehydrated and condensed in the presence of carbodiimide,
It was created. (4) Using the solution of the above (2) and the detection unit of the above (3), measurement was performed by a competition method in the same manner as in Example 2. The detection limit was measured from the calibration curve and is shown in Table 1.

[0126]

[Table 1]

Embodiment 11 FIGS. 1 to 5 show an embodiment of the apparatus of the present invention. FIG.
The detection unit has a structure in which 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 3 is surrounded by the flow cell 5. FIG. 2 shows a reflection type detection unit, and a mirror 12 is provided at the tip. FIG. 3 shows the structure of the opposed-type fluorescence detector. Since the fluorescence detecting section (sensing chip) 9 is fixed by a guide 11 for optical axis alignment and is not adhered, the fluorescence detecting section 9 can be freely attached and detached, which is very convenient in practical use. The fluorescent detector 9 is preferably replaced with a resin optical fiber from the viewpoint of cost since it is replaced every time the measurement is performed. A reactive group is introduced into the resin optical fiber by various methods.
The resin optical fiber preferably has an ester structure such as a polyacrylic acid ester. When the fluorescence detector is formed of a resin optical fiber, a compound having a> CH-CHO structure in the ester group is used as a basic compound. The reaction is carried out under the conditions to introduce a formyl group, and a protein such as an antigen or an antibody is bound to the formyl group. The excitation light is emitted from the fiber facing the detection surface.
In the apparatus shown in FIG.
The laser light is incident on the plastic fiber, and the incident light and the fluorescent light are incident on a plastic fiber having a guide 11 for optical axis alignment, only the fluorescent light is extracted by the filter 7, and the measurement is performed by the spectrophotometer 8. FIG. 5 shows an apparatus using a flow cell type detector.

[0128]

Industrial Applicability The reagent for measuring a bioactive substance of the present invention can be used for immunoassay of 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 active substance is large, the detection sensitivity can be greatly improved.
Further, the optical fiber for measuring a bioactive substance of the present invention can realize small size, low cost, and high sensitivity. These, by the measurement method of the present invention using a bioactive substance measurement reagent and device,
Since simple measurement can be realized in a short time, it can be used for disease diagnosis and the like in the medical field.

[Brief description of the drawings]

FIG. 1 shows a fluorescence detection unit based on a fluorescence labeling method.
Solid arrows indicate laser light, and dotted arrows indicate fluorescence.

FIG. 2 shows a fluorescence detection unit using a fluorescence labeling method.
Solid arrows indicate laser light, and dotted arrows indicate fluorescence.

FIG. 3 shows a fluorescence detection unit using a fluorescence labeling method.
Solid arrows indicate laser light, and dotted arrows indicate fluorescence.

FIG. 4 shows a fluorescence measurement system using a He—Ne laser or a semiconductor laser.

FIG. 5 shows 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 light transmittance of an optical fiber made of poly (methyl methacrylate). FIG.
(B) shows the relationship between the treatment temperature and the amount of enzyme that can be bound. The horizontal axis of (a) and (b) figures is processing temperature (c ⁰ ),
(A) The vertical axis of the figure is the optical transmission rate (%) of the fiber, (b)
The vertical axis in the figure is the amount of immobilized enzyme per area (μg / cm ² ), (c)
The horizontal axis in the figure shows the concentration of biotinylated anti-mouse antibody (mg / ml), and the vertical axis shows the number of counts. FIG. 6C shows a calibration curve of the concentration of the biotinylated anti-mouse antibody and the fluorescence intensity.

FIG. 7 shows the relationship between the optical transmission rate of a polymethyl methacrylate optical fiber and the density of formyl groups. The horizontal axis indicates the number of formyl groups (pieces / cm ² ), and the vertical axis indicates the optical transmission reduction rate (%) of the fiber.

FIG. 8 (a) shows the calibration curve of the sandwich method, and FIG. 8 (b) shows the calibration curve of the competition method. The horizontal axis is anti-mouse IgG (mg / ml),
The vertical axis indicates the count number.

FIG. 9 shows the responsiveness of the sensor. The horizontal axis anti-mouse ^{IgG (10 - 3 mg / ml} ) immersion time in minutes and the vertical axis represents the count number.

[Explanation of symbols]

Reference Signs List 1 optical fiber 2 cladding layer 3 core surface 4 antigen 5 flow cell Y (thick) Fluorescently labeled antibody of the present invention Y antibody in sample 6 He-Ne laser generator or semiconductor laser and SH
Laser generator combined with G element 7 Filter 8 Spectrofluorometer 9 Sensing chip 10 Plate 11 Guide rail for optical axis alignment 12 Mirror 13 Half mirror

──────────────────────────────────────────────────続 き Continuation of the front page (31) Priority claim number Japanese Patent Application No. 1-314404 (32) Priority date December 5, 2001 (12.5 December 1989) (33) Priority claim country Japan (JP) (56) References JP-A-61-271458 (JP, A) JP-A-62-66143 (JP, A) JP-A-59-108009 (JP, A) JP-A-63-198872 (JP, A) JP-A-62-79333 (JP, A) JP-A-62-79334 (JP, A) JP-A-59-81560 (JP, A) JP-T-61-502419 (JP, A) JP-T-62-500736 (JP, A) JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/543 G01N 33/545

Claims (8)

(57) [Claims] 1. An optical fiber made of a resin for measuring a bioactive substance, comprising a formyl group on the core surface of an optical fiber containing an acrylate resin or a methacrylate resin as a main component. 2. The density of the formyl group is 1.0 × 10
¹ is a ⁰ ~6.0 × 10 ^{1 3} pieces / cm ^2, an optical fiber of claim 1. 3. A method for measuring a bioactive substance, wherein a substance specifically reacting with a substance to be measured is covalently bonded to a formyl group of an optical fiber mainly composed of an acrylate resin or a methacrylate resin. Detector. 4. A light source, an optical fiber mainly composed of an acrylate resin or a methacrylate resin, and a core surface at one end face thereof are exposed, a formyl group is introduced into the surface, and the formyl group is measured. A detection unit on which a substance that specifically binds to a substance is immobilized; a mechanism 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. Bioactive substance measuring device. 5. The apparatus according to claim 4, wherein said light source is a semiconductor laser. 6. The device according to claim 4, wherein the detection unit is detachable by a coupler. 7. The detection unit according to claim 4, wherein the detection unit is of a facing type.
An apparatus according to claim 1. 8. The apparatus according to claim 4, wherein the mechanism for extracting only the fluorescence is a filter.