JPH0783925A - Fluorescent labeled reagent and fluorescent immunity measurement method - Google Patents

Abstract

PURPOSE:To miniaturize a device and at the same time improve sensitivity by using a fluorescent labeled reagent consisting of a hydrophilic polyfunctional group macromolecule which is labeled by a specific cyanin coloring matter and then performing fluorescent immunity measurement by performing excitation with semiconductor laser at an infrared region. CONSTITUTION:A fluorescent labeled reagent consisting of hydrophilic polyfunctional group macromolecule which is expressed by an equation I or II and is labeled by cyanin coloring matter is used. In the equation I, R indicates a metal ion such as H<+>, Na<+>, K<+>, and L<+> or ammonium compound ion such as N<+>H(C2H5)3, NH4<+>, and pyridinium ion. In the equation II, R indicates a metal ion such as H<+>, Na<+>, K<+>, an Li<+> or ammonium compound ion such as N<+>H(C2H5)3, NH4<+>, and pyridinium ion. By exciting the title fluorescent labeled reagent with a compact, inexpensive, and large-output semiconductor laser at infrared region (750-850nm), a high-sensitivity and inexpensive immunity measurement can be achieved.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescent labeling reagent used for measuring physiologically active substances and the like by a fluorescent immunoassay, a fluorescent immunoassay using the same, and a fluorescent immunoassay kit. More specifically, it relates to a fluorescent labeling reagent and a fluorescent immunoassay method using a semiconductor laser as an excitation light source.

[0002]

2. Description of the Related Art Conventionally, a method of labeling an antigen / antibody with a radioisotope or an enzyme has been used as a method for measuring the concentration of an antigen / antibody.
These methods have problems in terms of sensitivity and safety, and various methods for labeling the antigen antibody with a fluorescent dye and measuring the concentration of the antigen antibody by using this method have been studied instead of these methods.

For example, in Japanese Patent Laid-Open No. 59-501873 (US Pat. No. 4,852,809), an antigen and an antibody are bound to the side surface of an optical fiber, and an antibody and an antigen labeled with a fluorescent dye are immunoreacted on the surface of the optical fiber. , By guiding the excitation light to this optical fiber, the fluorescent dye on the optical fiber is excited, the generated fluorescence is reflected at the exit end face of the optical fiber, the fluorescence and the remaining excitation light are extracted from the entrance end face of the optical fiber, and this is beamed. A method is described in which only the fluorescence is measured by making a spectrum through a splitter.

Conventionally, in such a fluorescent immunoassay, a fluorescently labeled antigen or a fluorescently labeled antibody modified with a fluorescent dye such as fluorescein isothiocyanate or rhodamine isothiocyanate has been used as a labeling substance. However, it is difficult to improve the sensitivity of such a fluorescently labeled antigen or fluorescently labeled antibody because the amount of the fluorescent dye bound to one antigen or antibody is one. In addition, fluorescent dyes such as rhodamine and fluorescein have an excitation wavelength of 4
Since it is around 00 nm, it has a drawback that the excitation light source is large and expensive. Therefore, in place of these fluorescent dyes, by labeling avidin modified with a cyanine dye that is excited in a 630 nm band with a biotinylated antigen or a biotinylated antibody, a fluorescent labeled antibody that can use a He-Ne laser as a light source is obtained. Being developed (WO90 / 1
3029 publication).

However, the He-Ne laser has a problem that the output becomes low when the size is reduced and becomes large and expensive when the output is increased. On the other hand, He-
A semiconductor laser is commercially available as a laser smaller than the Ne laser, but no practical, inexpensive, and high-power semiconductor laser is commercially available in the 630 nm band. Further, conventional fluorescent dyes are difficult to dissolve in an aqueous solvent system, and even if they are dissolved, they are very unstable such as causing hydrolysis, so that it is difficult to prepare a reaction reagent. Therefore, it is necessary to reduce the reaction opportunity (time) in an aqueous solvent system, it is difficult to react in the state where both avidin and the fluorescent dye are dissolved, and the water solubility of the avidin-fluorescent dye conjugate It was pointed out that there were problems such as low cost.

[0006]

SUMMARY OF THE INVENTION Therefore, the present inventors have made extensive studies to solve the above-mentioned problems. As a result, a compact, low-priced, high-output light source that has been put into practical use, can be excited by a semiconductor laser in the infrared region, and has been found to be a fluorescent dye that is easy to handle because it is soluble and stable in an aqueous solvent system. By linking to a hydrophilic polyfunctional polymer such as avidin as a measuring reagent, it is possible to realize a more sensitive and low-cost immunoassay, and it is possible to reduce the size and cost of the measuring device. As a result, they have completed the present invention.

That is, the gist of the present invention is (1) a fluorescent labeling reagent comprising a hydrophilic polyfunctional polymer labeled with a cyanine dye represented by the general formula (1) or (2),

[0008]

[Chemical 7]

(In the formula, R is H ⁺ , Na ⁺ , K ⁺ , Li ⁺
Metal ions such as N ⁺ H (C ₂ H ₅ ) ₃ , N
It represents an ammonium compound ion such as H ₄ ⁺ and pyridinium ion. )

[0010]

[Chemical 8]

(Where R is H ⁺ , Na ⁺ , K ⁺ , Li ⁺
Metal ions such as N ⁺ H (C ₂ H ₅ ) ₃ , N
It represents an ammonium compound ion such as H ₄ ⁺ and pyridinium ion. ) (2) In the fluorescent immunoassay, the fluorescent labeling reagent described in (1) above is used, and 750 to 850 n
(3) A hydrophilic polyfunctional group polymer labeled with a fluorescent dye, and a hydrophilic or polyfunctional group polymer directly or indirectly In a fluorescent immunoassay kit comprising a physiologically active substance that chemically binds, the fluorescent dye is a cyanine dye represented by the general formula (1) or (2), and (4) A conjugate comprising avidin and / or protein A labeled directly with a fluorescent dye represented by the general formula (1) or (2) or indirectly via an aminoglycan and / or a polypeptide, and biotin and / or Alternatively, the immunoglobulin is composed of a conjugate bound to a physiologically active substance directly or indirectly via an aminoglycan and / or a polypeptide. It relates to a fluorescent immunoassay kit.

The fluorescent labeling reagent of the present invention comprises a hydrophilic polyfunctional macromolecule labeled with a cyanine dye represented by the general formula (1) or (2). As the cyanine dye, a dye NK3759 (manufactured by Japan Photosensitive Dye Research Institute) having a structural formula represented by the general formula (1) or a salt thereof, a dye NK3 having a structural formula represented by the general formula (2).
942 (manufactured by Japan Photosensitive Dye Research Institute) or a salt thereof or the like. Since these cyanine dyes used in the present invention are fluorescent dyes that absorb in the infrared region, a compact, high-power and low-cost semiconductor laser can be used as a light source. Therefore, downsizing and cost reduction of the entire device can be realized. The cyanine dye in the present invention has high water solubility, can be easily reacted with a hydrophilic polyfunctional polymer in a water solvent system, and is stable in the solution without hydrolysis.

[0013]

[Chemical 9]

(In the formula, R is H ⁺ , Na ⁺ , K ⁺ , Li ⁺
Metal ions such as N ⁺ H (C ₂ H ₅ ) ₃ , N
It represents an ammonium compound ion such as H ₄ ⁺ and pyridinium ion. )

[0015]

[Chemical 10]

(Where R is H ⁺ , Na ⁺ , K ⁺ , Li ⁺
Metal ions such as N ⁺ H (C ₂ H ₅ ) ₃ , N
It represents an ammonium compound ion such as H ₄ ⁺ and pyridinium ion. )

The hydrophilic polyfunctional polymer to be labeled with a cyanine dye in the fluorescent labeling reagent of the present invention is not particularly limited, and for example, avidin, protein A, aminoglycan, polypeptide or the like is preferable. used. Examples of the aminoglycan include chitosan, polygalactosamine, polyneuraminic acid and the like, and examples of the polypeptide include polylysine and the like.

Labeling of the hydrophilic polyfunctional polymer with a cyanine dye is easily carried out by a known method.
The cyanine dye in the present invention has a strong electrolytic sulfonic acid group in addition to the carboxyl group, and thus has high water solubility and can be reacted with a hydrophilic polyfunctional polymer such as avidin in an aqueous solvent system. That is, the bond between the cyanine dye and the hydrophilic polyfunctional group polymer in the present invention is such that the carboxyl group of the cyanine dye and the amino group of the hydrophilic polyfunctional group polymer are deionized water, triethylamine hydrochloric acid buffer solution, triethanolamine hydrochloric acid. An amide bond can be formed by condensation in a conventional manner using a condensing agent in an aqueous solvent system such as a buffer solution or in an amphipathic solvent such as dimethylformamide. After the reaction between the cyanine dye and the hydrophilic polyfunctional polymer is completed, it is preferable to remove unreacted substances, for example, dialysis method, centrifugation method, gel filtration method or ultrafiltration method, and washing with alcohol, water, etc. It can be excluded by the combined use of. Here, as the condensing agent, 1-ethyl-3- (3'-
Carbodiimides such as dimethylaminopropyl) carbodiimide, di-p-toreoylcarbodiimide, dicyclohexylcarbodiimide, N-cyclohexyl-N '-(2-morpholinoethyl) carbodiimide met-p-toluenesulfonate (CHMC), N-hydroxysuccinimide, N-bromosuccinimide, etc. are used.

For example, when avidin is used as the hydrophilic polyfunctional polymer, a more specific embodiment will be described. Usually, a cyanine dye of 5.0 × 10 ^{−6 to} 0.1 M is dissolved in deionized water. Then, 0.5 to 2.0 mg of avidin is added to 10 ml of the obtained aqueous solution, the condensing agent as described above is added in an amount of 1 to 100 times the molar ratio of the cyanine dye, and the mixture is left at room temperature overnight. After standing overnight, the reaction solution is ultrafiltered, washed with methanol, water, etc. to remove unreacted dye, and the residue on the ultrafiltration membrane is separated using a weak alkaline buffer and dried. The obtained product is referred to as cyanine dye-modified avidin. Although there are various drying methods, the freeze-drying method is preferable. In this case, the greater the number of cyanine dyes bonded to the surface of one molecule of avidin, the better the efficiency, but with the usual cyanine dyes, the water solubility at the time of use becomes a problem when the number of bonded cyanine dyes is large, so the number of molecules should be suppressed to about 4 molecules. However, since the cyanine dye of the present invention has high water solubility, it can be used even if 5 or more molecules are bound.

Next, a method for performing a fluorescent immunoassay using the fluorescent labeling reagent of the present invention will be described by exemplifying an embodiment using the cyanine dye-modified avidin of the present invention. Step A: First, an antibody against the substance to be measured (antigen that is a physiologically active substance) is immobilized on the core surface of the optical fiber. Then, the optical fiber to which the antibody is bound specifically reacts with the substance to be measured (antigen), and an immune complex in which the antibody and the substance to be measured (antigen) are bound to the core surface of the optical fiber (core surface of the optical fiber-antibody). -Substance to be measured) is formed.

Step B: Biotinylated aminoglycan (antibody-aminoglycan-biotin) to which an antibody having a specific binding ability to the substance to be measured (antigen) is bound, and the cyanine dye-modified avidin (avidin) of the present invention. -Cyanine dye) to prepare a cyanine dye-labeled antibody through the biotin-avidin bond (antibody-aminoglycan-biotin-avidin-cyanine dye).

Step C: The immune complex prepared in Step A and the cyanine dye-labeled antibody prepared in Step B are specifically reacted to form a core surface of the optical fiber-antibody-substance to be measured-
Antibody-aminoglycan-biotin-avidin-cyanine dye immunocomplex is formed, and the fluorescence of the cyanine dye immobilized on the core surface of the optical fiber is measured using a fluorescence measuring device using a semiconductor laser of 750 to 850 nm as an excitation light source. To do.

In the above description, avidin is taken as an example of the hydrophilic polyfunctional polymer, but the hydrophilic polyfunctional polymer in the present invention is not limited to avidin, but the protein A and amino are used as described above. Glycan, polypeptide or the like is used. As an intervening compound for fixing the cyanine dye on the core surface of the optical fiber, a compound that specifically binds to the hydrophilic polyfunctional polymer to be used is selected, as in the case of avidin, biotin is used. . Specifically, avidin, biotin, protein A
And an antibody are preferable, and a combination of avidin and biotin is particularly preferable.

As another aspect of the measuring method of the present invention, cyanine dye-modified avidin may be reacted after the immune reaction to bind to the immune complex. That is, an immune complex consisting of the core surface of the optical fiber-antibody-substance to be measured-antibody-aminoglycan-biotin is formed in advance, and this is reacted with a cyanine dye-modified avidin. A cyanine dye can be immobilized and fluorescence is measured as described above.

The immunoassay method of the present invention uses the fluorescent labeling reagent of the present invention in the fluorescent immunoassay as described above.
The method is characterized by using a semiconductor laser of 50 to 850 nm as an excitation light source, and each procedure of immunoassay is not particularly limited, and a known method can be applied as it is. Further, in the above description, an embodiment was shown in which the measurement target substance was bound in a sandwich form with an antibody to fix the cyanine dye on the core surface of the optical fiber, but the measurement sample and the fluorescent labeling reagent of the present invention were allowed to compete with each other, It may be a method of fixing a cyanine dye on the core surface of an optical fiber by binding it with an antigen or antibody bound to the surface of the optical fiber.

The fluorescent immunoassay kit of the present invention has the following two modes. (1) First Aspect As a first aspect of the fluorescent immunoassay kit of the present invention, a hydrophilic polyfunctional group polymer labeled with a cyanine dye as described above, and the hydrophilic polyfunctional group polymer are provided. It is composed of a physiologically active substance that is directly or indirectly bound. The physiologically active substance here means an antibody, an antigen or the like that specifically binds to an antigen, an antibody or the like to be measured. That is, examples of the hydrophilic polyfunctional polymer labeled with a cyanine dye include, for example, cyanine dye-modified avidin (avidin-cyanine dye), protein A modified with a cyanine dye, and amino modified with a cyanine dye as described above. Examples thereof include polypeptides modified with glycan or cyanine dye.

On the other hand, examples of the physiologically active substance that directly or indirectly binds to the hydrophilic polyfunctional polymer include, for example, a physiologically active substance having specific binding ability to the antigen to be measured (eg antibody ) Bound biotinylated aminoglycan (antibody-aminoglycan-biotin) is indirectly bound to a hydrophilic polyfunctional polymer. In this case, the antibody, which is a physiologically active substance, is indirectly bound to avidin, which is a hydrophilic polyfunctional polymer, via aminoglycan-biotin. When directly binding to a hydrophilic polyfunctional polymer, for example, a cyanine dye-modified aminoglycan (aminoglycan-cyanine dye), a cyanine dye-modified polypeptide (polypeptide-cyanine dye), or the like is a measurement target. Physiologically active substance having specific binding ability to the antigen or antibody (for example,
(Antibody and antigen).

(2) Second Aspect As a second aspect of the fluorescence immunoassay kit of the present invention, the above-mentioned cyanine dye is directly or indirectly labeled via an aminoglycan and / or a polypeptide. A conjugate comprising avidin and / or protein A (conjugate a), and a conjugate in which biotin and / or immunoglobulin is directly or indirectly bound to a substance to be measured via an aminoglycan and / or a polypeptide ( It is composed of the conjugate b). For example, examples of the conjugate a include avidin-cyanine dye, protein A-cyanine dye, avidin-aminoglycan-cyanine dye, protein A-aminoglycan-cyanine dye, and the like. Further, examples of the conjugate b include various examples such as biotin-target substance, immunoglobulin-target substance, biotin-aminoglycan-target substance, immunoglobulin-aminoglycan-target substance, and the like.

The optical fiber used in the present invention is a resin optical fiber. The resin constituting the resin-made optical fiber is not particularly limited, but it is necessary that it is a material that does not adsorb an immunological substance and that has a good light-transmitting property. For example, polystyrene, polyacrylic ester, polyester , Polyacrylamide, polyvinyl alcohol, polyethylene terephthalate, polycarbonate, and copolymers thereof. Among them, polyacrylic acid ester is preferable. Polyacrylic acid ester is one having an ester structure among acrylic resins, and is, for example, a synthetic resin composed of a polymer of an ester derivative such as acrylic acid and methacrylic acid, and specifically, methyl acrylate and acrylic acid. Examples thereof include polymers such as ethyl and methyl methacrylate. Of these polyacrylic acid esters, polymethyl methacrylate is particularly preferably used in the present invention. This is because polymethylmethacrylate has particularly good translucency as compared with other resins. The resin optical fiber used in the present invention may be, for example, a copolymer of a monomer such as methyl acrylate, ethyl acrylate, or methyl methacrylate and a monomer such as styrene.

[0030]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples. First, before entering the examples, the solubility of the cyanine dye used in the present invention in an aqueous solvent was compared with the solubility of a conventionally used cyanine dye in an aqueous solvent. In the following experiments, Na salt and triethylamine salt of NK3759 and NK3942 were used as examples of cyanine dyes used in Examples.
In the comparative example, NK1160 (manufactured by Japan Photosensitive Dye Research Laboratory) having the following structural formula (3) was used as a cyanine dye that has been conventionally used.

[0031]

[Chemical 11]

As a result, the NK used in the following examples
3759-Na salt, NK3759-triethylamine salt, NK3942-triethylamine salt, and NK1
The solubility of 160 in water or ethanol at room temperature was as follows. First, regarding the solubility in water, NK3759-Na salt is 2.60 g / 100 ml,
4.78 g / 10 of NK3759-triethylamine salt
0 ml, NK3942-triethylamine salt 10 g /
100 ml, and NK1160 is 0.00002 g /
100 ml, the solubility in ethanol is NK
0.134 g / 100 ml of 3759-Na salt, NK3
759-triethylamine salt 3.04 g / 100 m
1, NK3942-triethylamine salt was 5 mg / 10
0 ml, and NK1160 is 0.5 mg / 100 ml
Met.

Example 1 (1) 12.2 mg of NK3759-Na salt was dissolved in 10 ml of deionized water. (2) 1 mg of avidin was dissolved in 500 μl of water, added to the aqueous solution of (1) above, stirred, and further N-cyclohexyl-N ′-(2-morpholinoethyl) carbodiimide meth-p-toluenesulfonate ( CHMC)
0.1 g was added and left overnight.

(3) The solution of (2) above was washed with methanol 3 to 4 times and then with water 2 to 3 times by ultrafiltration to remove unreacted dye. (4) The obtained precipitate was dissolved in deionized water, and "NK37
59 modified avidin solution "(hereinafter abbreviated as F ₁ avidin)
And

(5) Separately, an anti-human pancreatic amylase antibody derived from rabbit (hereinafter abbreviated as RHAAb) is immobilized on an optical fiber, and this is specifically reacted with human pancreatic amylase (hereinafter abbreviated as HA) to give a rabbit. Derived anti-human pancreatic amylase antibody + human pancreatic amylase ”(hereinafter, abbreviated as RHAAb-HA). (6) Furthermore, sheep-derived anti-human pancreatic amylase antibody (hereinafter, abbreviated as SHAAb) is bound to chitosan (hereinafter, abbreviated as BC) in which a large number of amino groups are biotinylated, and a “biotinylated chitosan + sheep-derived antibody is bound. Human pancreatic amylase antibody "(hereinafter abbreviated as B-C-SHAAb).

(7) Further, the F ₁ avidin of the above (4) is reacted with the B—C-SHAAb of the above (6) to obtain “NK
3759 modified avidin + biotinylated chitosan + sheep-derived anti-human pancreatic amylase antibody "(hereinafter referred to as F ₁ avidin-B
-C-SHAAb). (8) RHAAb-HA of the above (5) and F of the above (7)
₁ specifically reacting avidin-BC-SHAAb,
7 as "rabbit-derived anti-human pancreatic amylase antibody + human pancreatic amylase + sheep-derived anti-human pancreatic amylase antibody + biotinylated chitosan + NK3759-modified avidin"
It was excited with 80 nm LD (30 mW), and its fluorescence was measured. As a result, human pancreatic amylase was found to be 1.2 × 10 ⁻⁴ mg / m 2.
can be detected by 1 and the element of 780 nm LD is 10φ × 10
Therefore, the price was low, and the device was downsized and the price was low.

Comparative Example 1 (1) 0.01M of a large excess of 2.5 mg of NK1160
It was suspended in 10 ml of triethylamine hydrochloric acid buffer solution (pH 8.0). (The actual amount of dissolution is 2.6 μg / 10 ml.)

(2) The precipitate obtained by the same method as in (2) to (3) of Example 1 was suspended in a KOH solution adjusted to pH 8.0. A change was seen and it was not possible to obtain a dye-modified avidin that could be used as a labeling reagent as when using an organic solvent.

Comparative Example 2 (1) 2.0 mg of NK1160 was dissolved in 1 ml of a mixed solution of ethanol: triethylamine = 4: 1.

(2) The same operation as in (2) to (4) of Example 1 was repeated except that dicyclohexylcarbodiimide was used in place of CHMC as the condensing agent.
"NK1160 modified avidin" (hereinafter ^, abbreviated as F ^, avidin) was prepared.

(3) Reaction was carried out in the same manner as in (5) to (6) of Example 1, and the reaction was carried out with the above-mentioned F ^, avidin of (2) and Example 1.
Of NK1160-modified avidin + biotinylated chitosan + sheep-derived anti-human pancreatic amylase antibody (hereinafter, F ^, avidin-BC-SH).
(Abbreviated as AAb). (4) RHAAb-HA of Example 1 and F of the above (3) ^,
By specifically reacting avidin-BC-SHAAb,
"Rabbit-derived anti-human pancreatic amylase antibody + human pancreatic amylase + sheep-derived anti-human pancreatic amylase antibody + biotinylated chitosan + F ^, avidin" was excited with a He-Ne laser (633 nm), and its fluorescence was measured. Human pancreatic amylase could be detected at 1.9 × 10 ^-4 mg / ml, but the He-Ne laser was 30 mW and the length was about 1 m, and it was expensive. Therefore, the device was large and expensive. I got it.

Example 2 (1) 13.2 mg of NK3942-triethylamine salt was dissolved in 10 ml of deionized water. (2) Perform the same operations as (2) to (4) in Example 1,
F ₁ avidin was obtained.

(3) Human chorionic gonadotropin (hereinafter,
(abbreviated as hCG) and a rabbit-derived anti-hCG antibody immobilized on an optical fiber (hereinafter, abbreviated as RhCGAb) are specifically reacted, and “rabbit-derived anti-hCG antibody + hCG” (hereinafter,
RhCGAb-hCG). (4) Polygalactosamine (hereinafter abbreviated as B-pG) in which a large number of amino groups have been biotinylated, and goat-derived anti-hC
G-antibody (hereinafter abbreviated as GhCGAb) is bound, and "biotinylated polygalactosamine + goat-derived anti-hCG antibody"
(Hereinafter, abbreviated as B-pG-GhCGAb).

(5) Further, by reacting the F ₁ avidin of (2) with the B-pG-GhCGAb of (4),
"NK3942 modified avidin + biotinylated polygalactosamine + goat-derived anti-hCG antibody" (hereinafter, abbreviated as F ₁ avidin + B-pG-GhCGAb). (6) The RhCGAb-hCG of the above (3) and the above (5)
Specific reaction of F ₁ avidin + B-pG-GhCGAb of “Rabbit-derived anti-hCG antibody + hCG + Goat-derived anti-hCG antibody + Biotinylated polygalactosamine + NK3”.
942 modified avidin ”. This was excited with a 780 nm LD, and the fluorescence was measured. As a result, hCG was 2.0 ×.
Detectable at 10 ^-4 mg / ml, 780 nm LD element is 1
The price was 0φ × 10 and the price was low.

Example 3 (1) The same operations as (1) to (4) in Example 1 were carried out,
"NK3759 modified avidin solution" (F ₁ avidin) was prepared. (2) Human follicle stimulating hormone (hFSH) and goat-derived anti-hFSH antibody immobilized on an optical fiber (hereinafter referred to as GhFS
(Abbreviated as HAb) specifically reacts with “goat-derived anti-hF
SH antibody + hFSH ”(hereinafter, GhFSHAb-hFS
(Abbreviated as H).

(3) Further, a rabbit-derived anti-hFSH antibody (hereinafter abbreviated as RhFSHAb) was bound to chitosan (BC) in which a large number of amino groups were biotinylated, and a "biotinylated chitosan + rabbit-derived anti-hFSH antibody" ( Below, B-C
-RhFSHAb). (4) Further, F ₁ avidin of (1) above is reacted with B-C-RhFSHAb of (3) above to give “modified avidin + biotinylated chitosan + anti-hFSH antibody derived from rabbit”.
(Hereinafter, abbreviated as RhFSHAb-B-C-F ₁ avidin).

(5) GhFSHAb-hF of the above (2)
SH was specifically reacted with RhFSHAb-B-C-F ₁ avidin of the above (4) to obtain “goat-derived anti-hFSH antibody + hFSH + rabbit-derived anti-hFSH antibody + biotinylated chitosan + NK3759-modified avidin”, which was
When excited with 0 nm LD and measured the fluorescence, hF
SH can be detected at 5.2 × 10 ^-4 mg / ml, 780n
The price of the mLD was 10φ × 10, which was low, and it was possible to reduce the size and cost.

Example 4 (1) 12.2 mg of NK3759-Na salt was dissolved in 10 ml of deionized water. (2) 2 mg of avidin was dissolved in 500 μl of water, added to the aqueous solution of (1) above, 0.1 g of CHMC was further added, and the mixture was allowed to stand overnight. (3) Perform the same operation up to (4) of Example 1, and perform “NK
3759 modified avidin solution ”(F ₁ avidin) was prepared.

(4) An anti-human calcitonin antibody derived from goat (hereinafter, abbreviated as GHCAb) is immobilized on an optical fiber,
This was specifically reacted with human calcitonin (hereinafter abbreviated as HC) to obtain “goat-derived anti-human calcitonin antibody + human calcitonin” (hereinafter abbreviated as GHCAb-HC). (5) In addition, a rabbit-derived anti-human calcitonin antibody was bound to polyneuraminic acid (hereinafter, abbreviated as B-pN) in which a large number of amino groups were biotinylated, and a “rabbit-derived anti-human calcitonin antibody + biotinylated polyneuraminic acid” (hereinafter ,
(Abbreviated as RHCAb-B-pN), and GH of the above (4)
CAb-HC specifically reacts, "goat-derived anti-human calcitonin antibody + human calcitonin + rabbit-derived anti-human calcitonin antibody + biotinylated polyneuraminic acid" (hereinafter abbreviated as GHCAb-HC-RHCAb-B-pN)
And

(6) Further, the F ₁ avidin of the above (3) and the GHCAb-HC-RHCAb-B-pN of the above (5).
Was reacted to give “goat-derived anti-human calcitonin antibody + human calcitonin + rabbit-derived anti-human calcitonin antibody + biotinylated polyneuraminic acid + NK3759-modified avidin”, which was excited with 780 nm LD, and its fluorescence was measured. 4.3 x 10
-Detectable at ^-4 mg / ml, 780 nm LD element is 10φ
The price was low at × 10, and it was possible to realize downsizing and cost reduction.

Example 5 (1) 13.2 mg of NK3759-triethylamine salt was suspended in 10 ml of deionized water. (2) 1 mg of chitosan was dissolved in 0.5 ml of water, added to the suspension of (1) above, stirred, and further added with CHMC 0.1.
g, and left overnight. (3) The solution of (2) above is washed with methanol 3 to 4 times using ultrafiltration, and then washed with water 2 to 3 times to remove unreacted dyes, and NK3759-bound chitosan (hereinafter referred to as “chitosan”). , 3759 chitosan).

(4) The obtained 3759 chitosan was added to 0.0
1M triethylamine hydrochloric acid buffer solution (pH 8.0) 1 ml
Suspended in 0.5 ml of protein A aqueous solution (concentration 2 m
g / ml), and then add 0.1 g of CHMC,
It was left overnight with stirring. (5) The solution of (4) above is washed 3 to 4 times with phosphate buffered saline (PBS) pH 8.0 using ultrafiltration,
Unreacted protein A is removed, and protein A and 3759 are removed.
A conjugate of chitosan (hereinafter abbreviated as PrA-3759 chitosan) was obtained.

(6) The obtained PrA-3759 chitosan was suspended in 1 ml of PBS, and 1 ml of immunoglobulin G against C-reactive protein derived from rabbit (hereinafter abbreviated as CRP) (hereinafter abbreviated as anti-CRP IgG derived from rabbit). ) The mixture was mixed with an aqueous solution (concentration 5 mg / ml) and left overnight with stirring. (7) The solution of (6) above is washed 3 to 4 times with PBS using ultrafiltration to remove unreacted rabbit-derived anti-CRP IgG, and rabbit-derived anti-CRP IgG is conjugated to PrA-3759 chitosan (hereinafter referred to as a conjugate). , Anti-CRP IgG-PrA-37
Abbreviated as 59 Chitosan. Was obtained, and suspended and dissolved in PBS to give an anti-CRP IgG-PrA-3759 chitosan solution.

(8) Separately, a mouse-derived anti-CRP monoclonal antibody was immobilized on an optical fiber, and this was allowed to specifically react with CRP to form a mouse-derived anti-CR on the optical fiber.
P monoclonal antibody-CRP complex (hereinafter referred to as anti-CR
Abbreviated as P-CRP complex. ) Was formed. (9) The optical fiber having the anti-CRP-CRP complex of (8) immobilized thereon is used as the anti-CRP IgG-PrA of (7).
-3759 Chitosan solution was dipped and reacted specifically to form anti-CRP-CRP complex and anti-CRPI on the optical fiber.
As a result of forming a conjugate of gG-PrA-3759 chitosan, exciting it with 780 nm LD and measuring the fluorescence, CRP can be detected at 2.5 × 10 ⁻⁴ mg / ml,
The 780 nm LD element has a low price of 10φ × 10, and it has been possible to realize downsizing and cost reduction.

[0055]

According to the present invention, a semiconductor laser in the infrared region can be used as an excitation light source in fluorescence immunoassay by using a fluorescent labeling reagent labeled with a specific cyanine dye. Further, since the cyanine dye used in the present invention has excellent solubility in an aqueous solvent system, it is easy to prepare a labeling reagent using the cyanine dye. Further, according to the present invention, highly sensitive and low-cost immunoassay can be performed, and the downsizing and cost of the measuring device can be realized.

Claims (6)

[Claims] 1. A fluorescent labeling reagent comprising a hydrophilic polyfunctional group polymer labeled with a cyanine dye represented by the general formula (1) or (2). [Chemical 1] (Wherein, R ^{H +, Na +, K +} , Li + , etc. of the metal ion or _{^{N + H (C 2 H 5}} ) represents a _3, NH ₄ ^+, ammonium compounds such as pyridinium ion ion.) [ Chemical 2] (Wherein, R ^{H +, Na +, K +} , Li + , etc. of the metal ion or _{^{N + H (C 2 H 5}} ) represents a _3, NH ₄ ^+, ammonium compounds such as pyridinium ion ion.) 2. The fluorescent labeling reagent according to claim 1, wherein the hydrophilic polyfunctional polymer is avidin, protein A, aminoglycan, or polypeptide. 3. A fluorescent immunoassay method, which uses the fluorescent labeling reagent according to claim 1 or 2 in a fluorescent immunoassay and uses a semiconductor laser of 750 to 850 nm as an excitation light source. 4. A fluorescent immunoassay kit comprising a hydrophilic polyfunctional macromolecule labeled with a fluorescent dye and a physiologically active substance that directly or indirectly binds to the hydrophilic polyfunctional macromolecule. A fluorescent immunoassay kit, wherein the dye is a cyanine dye represented by the general formula (1) or (2). [Chemical 3] (Wherein, R ^{H +, Na +, K +} , Li + , etc. of the metal ion or _{^{N + H (C 2 H 5}} ) represents a _3, NH ₄ ^+, ammonium compounds such as pyridinium ion ion.) [ Chemical 4] (Wherein, R ^{H +, Na +, K +} , Li + , etc. of the metal ion or _{^{N + H (C 2 H 5}} ) represents a _3, NH ₄ ^+, ammonium compounds such as pyridinium ion ion.) 5. The fluorescent immunoassay kit according to claim 4, wherein the hydrophilic polyfunctional polymer is avidin, protein A, aminoglycan, or polypeptide. 6. A conjugate comprising avidin and / or protein A directly or indirectly labeled with a fluorescent dye represented by the general formula (1) or (2) via an aminoglycan and / or a polypeptide. , And biotin and / or immunoglobulin, which is composed of a conjugate in which a bioactive substance is directly or indirectly bound to a physiologically active substance via an aminoglycan and / or a polypeptide. [Chemical 5] (Wherein, R ^{H +, Na +, K +} , Li + , etc. of the metal ion or _{^{N + H (C 2 H 5}} ) represents a _3, NH ₄ ^+, ammonium compounds such as pyridinium ion ion.) [ Chemical 6] (Wherein, R ^{H +, Na +, K +} , Li + , etc. of the metal ion or _{^{N + H (C 2 H 5}} ) represents a _3, NH ₄ ^+, ammonium compounds such as pyridinium ion ion.)