JP3354975B2 - Fluorescent labeling reagent and fluorescent immunoassay - Google Patents

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 a physiologically active substance or the like by a fluorescent immunoassay, a fluorescent immunoassay using the same, and a fluorescent immunoassay kit. More specifically, the present invention relates to a fluorescent labeling reagent and a fluorescent immunoassay using a semiconductor laser as an excitation light source.

[0002]

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

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

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 label. However, with such a fluorescently labeled antigen or fluorescently labeled antibody, it is difficult to improve the sensitivity because only one amount of the fluorescent dye binds to one antigen or one antibody. Fluorescent dyes such as rhodamine and fluorescein have excitation wavelengths of 4
Since the wavelength is around 00 nm, there is a disadvantage that the excitation light source is large and expensive. Therefore, instead of these fluorescent dyes, by labeling avidin modified with a cyanine dye excited in the 630 nm band with a biotinylated antigen or a biotinylated antibody, a fluorescently labeled antibody that can use a He-Ne laser as a light source can be obtained. Developed (WO90 / 1
3029 publication).

[0005] However, there is a problem that the output of the He-Ne laser is reduced when the size is reduced, and the size and the price are increased when the output is increased. On the other hand, He-
Semiconductor lasers are commercially available as smaller lasers than Ne lasers, but practical, inexpensive and high-power semiconductor lasers are not commercially available in the 630 nm band.

[0006]

The present inventors have made intensive studies to solve the above-mentioned problems. As a result, we found a fluorescent dye that can be excited by a semiconductor laser in the infrared region, which is practically used as a compact, low-cost, high-output light source, and binds it to a hydrophilic polyfunctional polymer such as avidin. By using the measurement reagent, it has been found that a more sensitive and inexpensive immunoassay can be realized, and that the measurement device can be reduced in size and cost, and the present invention has been completed.

[0007] Namely, the present invention provides (1) the general formula (1), (2) or fluorescent labeling reagent consisting of a labeled hydrophilic multifunctional polymer by more cyanine dye represented in (3) ,

Embedded image (In the formula, R represents a hydrogen atom or a halogen atom, X represents S or C (CH ₃ ) ₂ , n represents an integer of 0 to 3, and m represents an integer of 0 to 2.) (2) In fluorescence immunoassay A fluorescent immunoassay using the fluorescent labeling reagent according to (1) above and a semiconductor laser having a wavelength of 750 to 850 nm as an excitation light source; and (3) a height of a hydrophilic polyfunctional group labeled with a fluorescent dye. In a fluorescent immunoassay kit comprising a molecule and a physiologically active substance which directly or indirectly binds to the hydrophilic polyfunctional group polymer, the fluorescent dye is represented by the general formula (1), (2) or (3 ) . fluorescence immunoassay kit, characterized in that <br/> a cyanine dye represented is, and (4) the general formula (1), (2) or (3) either directly or by more fluorescent dye represented in Indirectly labeled via aminoglycans and / or polypeptides A conjugate consisting of vidin and / or protein A, and a conjugate wherein biotin and / or immunoglobulin is directly or indirectly bound to a biologically active substance via aminoglycan and / or polypeptide. And a fluorescent immunoassay kit.

The fluorescent labeling reagent of the present invention has the general formula (1):
(2) or those consisting of labeled hydrophilic multifunctional polymer by more cyanine dye represented in (3). As the cyanine dye, a compound represented by the general formula (1), (2) or (3 )
As long as represented by the general formula shown in the present invention is not particularly limited, for example, dye A shown in Table 1 and Table 2
Dye B (manufactured by Japan Photographic Dye Laboratories, NK3792), Dye D, dye
E, dye G, and dye H. Since these cyanine dyes used in the present invention are fluorescent dyes that absorb the infrared region, a small, high-output, and low-cost semiconductor laser can be used as a light source. Therefore, size reduction and price reduction can be realized as the whole apparatus.

[0009]

[Table 1]

[0010]

[Table 2]

[0011] The hydrophilic polyfunctional group polymer 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, or polypeptide is preferably used. used. Examples of the aminoglycan include chitosan, polygalactosamine, and polyneuraminic acid, and examples of the polypeptide include polylysine.

Labeling of a hydrophilic polyfunctional polymer with a cyanine dye is easily carried out by a known method. The bond between the cyanine dye and the hydrophilic polyfunctional polymer is formed by condensing the carboxyl group of the cyanine dye and the amino group of the hydrophilic polyfunctional polymer in an organic solvent using a condensing agent by an ordinary method. Can be combined. After the reaction between the cyanine dye and the hydrophilic polyfunctional polymer is completed, it is preferable to remove unreacted substances as much as possible. For example, dialysis, centrifugation, gel filtration, ultrafiltration, or the like can remove them. Here, 1-ethyl-3-
Carbodiimides such as (3'-dimethylaminopropyl) carbodiimide, di-p-tolyoylcarbodiimide, dicyclohexylcarbodiimide, water-soluble carbodiimide (CHMC), N-hydroxysuccinimide, N-bromosuccinimide and the like are used.

For example, when avidin is used as a hydrophilic polyfunctional group polymer, a more specific embodiment is exemplified. Usually, a cyanine dye of 4.0 × 10 ^{-6 to} 5.0 × 10 ^-3 M is used. Is dissolved in ethanol, and the solution 10 ml
Is added with 500 μl of avidin at 1-2 mg / ml, the condensing agent described above is added at 9.0 × 10 ^{−5 to} 3.0 × 10 ⁻³ M, and the mixture is left overnight at room temperature. After standing overnight, avidin is recovered by centrifugation, and unreacted dye is removed by dissolving in ethanol. After removing the unreacted dye, the precipitate is dissolved in a weakly acidic buffer, and the supernatant is collected as cyanine dye-modified avidin. In this case, the number of cyanine dyes bound to the surface of one molecule of avidin is inferior for one or two molecules, and the solubility is problematic for five or more molecules. Can be

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 a physiologically active substance (for example, an antigen that is a measurement substance) is immobilized on the core surface of an optical fiber. Next, the optical fiber to which the antibody is bound and the physiologically active substance (antigen) are caused to react specifically, and an immunocomplex (the optical fiber core surface-antibody) in which the antibody and the physiologically active substance (antigen) are bound on the core surface of the optical fiber. -Bioactive substances).

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

Step C: The immune complex prepared in step A is specifically reacted with the cyanine dye-labeled antibody prepared in step B, and the core surface of the optical fiber—antibody—bioactive substance (measurement substance) —antibody—amino Form an immune complex of glycan-biotin-avidin-cyanine dye, 750-8
The fluorescence of the cyanine dye immobilized on the core surface of the optical fiber is measured using a fluorescence measurement device using a 50 nm semiconductor laser as an excitation light source.

In the above description, avidin is used as an example of the hydrophilic polyfunctional group polymer. However, the hydrophilic polyfunctional group polymer according to the present invention is not limited to avidin. Glycans or polypeptides are used. As a compound interposed for fixing the cyanine dye on the core surface of the optical fiber, a compound that specifically binds to the hydrophilic polyfunctional polymer used is selected, such as biotin in the case of avidin. . Specifically, avidin and biotin, protein A and an antibody are preferred, and a combination of avidin and biotin is particularly optimal.

In another embodiment of the measurement method of the present invention, a reduction in the fluorescence intensity accompanying hydrolysis and oxidation of the cyanine dye is achieved by reacting the cyanine dye-modified avidin after the immunological reaction and binding to the immune complex. Can be prevented. That is, the core surface of the optical fiber-the antibody-
By forming an immune complex consisting of a physiologically active substance-antibody-aminoglycan-biotin and reacting it with a cyanine dye-modified avidin, a cyanine dye can be immobilized on the core surface of the optical fiber in the same manner as described above. The fluorescence is measured in the same manner as described above.

The immunoassay method of the present invention uses the fluorescent labeling reagent of the present invention in a fluorescent immunoassay as described above.
It is characterized in that a semiconductor laser of 50 to 850 nm is used 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 is shown in which the physiologically active substance is bound in a sandwich manner with an antibody on the core surface of the optical fiber to fix the cyanine dye, but the physiologically active substance as the measurement sample and the fluorescent labeling reagent of the present invention are May be used to fix the cyanine dye on the core surface of the optical fiber by binding to the antigen or antibody bound to the optical fiber surface.

The fluorescent immunoassay kit of the present invention has the following two embodiments. (1) First Aspect As a first aspect of the immunofluorescence assay kit of the present invention, a hydrophilic polyfunctional group polymer labeled with a cyanine dye as described above, and the hydrophilic polyfunctional group polymer It consists of a physiologically active substance that binds directly or indirectly. The term "physiologically active substance" as used herein means not a physiologically active substance as an object to be measured, but an antibody, an antigen or the like which specifically binds to an antigen or an antibody or the like to be measured. That is, as a hydrophilic polyfunctional group polymer labeled with a cyanine dye,
Examples of the above include cyanine dye-modified avidin (avidin-cyanine dye), protein A modified with a cyanine dye, aminoglycan modified with a cyanine dye, and a polypeptide modified with a cyanine dye.

On the other hand, examples of the physiologically active substance which directly or indirectly binds to the hydrophilic polyfunctional group polymer include, for example, a physiologically active substance having a specific binding ability to the antigen to be measured (for example, an antibody) )) To which the biotinylated aminoglycan (antibody-aminoglycan-biotin) is indirectly bonded to the hydrophilic polyfunctional polymer. In this case, the antibody, which is a physiologically active substance, indirectly binds to avidin, which is a hydrophilic polyfunctional polymer, via aminoglycan-biotin. In the case of directly binding to a hydrophilic multifunctional group polymer, for example, a cyanine dye-modified aminoglycan (aminoglycan-cyanine dye), a cyanine dye-modified polypeptide (polypeptide-cyanine dye), etc. Bioactive substances having specific binding ability to antigens and antibodies
(An antibody or an antigen).

(2) Second Embodiment As a second embodiment of the kit for fluorescent immunoassay of the present invention, the kit is directly or indirectly labeled with a cyanine dye as described above via an aminoglycan and / or a polypeptide. (Conjugate a) consisting of avidin and / or protein A, and a conjugate in which biotin and / or immunoglobulin is directly or indirectly bound to a bioactive substance via aminoglycan and / or polypeptide ( Combination b). For example, examples of the conjugate a include various examples such as an avidin-cyanine dye, a protein A-cyanine dye, an avidin-aminoglycan-cyanine dye, and a protein A-aminoglycan-cyanine dye. Examples of the conjugate b include various examples such as biotin-bioactive substance, immunoglobulin-bioactive substance, biotin-aminoglycan-bioactive substance, and immunoglobulin-aminoglycan-bioactive substance.

The optical fiber used in the present invention is a resin optical fiber. The resin constituting the resin optical fiber is not particularly limited, but is required to be a material that does not adsorb the immunity substance and has a good translucency, such as polystyrene, polyacrylate, and polyester. , Polyacrylamide, polyvinyl alcohol, polyethylene terephthalate, polycarbonate, and copolymers thereof. Among them, polyacrylates are preferred. Polyacrylate is an acrylic resin having an ester structure and is, for example, a synthetic resin made of a polymer of an ester derivative such as acrylic acid and methacrylic acid, and specifically, methyl acrylate, acrylic acid Polymers such as ethyl and methyl methacrylate are exemplified. Among these polyacrylates, one particularly preferably used in the present invention is polymethyl methacrylate. This is because polymethyl methacrylate has particularly good translucency compared to 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, methyl methacrylate and a monomer such as styrene.

[0024]

The present invention will be described in more detail with reference to the following Examples and Comparative Examples, but the present invention is not limited to these Examples and the like. The dyes A to H used in the examples have the chemical structural formulas shown in Tables 1 and 2. However, dye C and dye F are used.
The example is a reference example.

Example 1 (1) 2.5 mg of NK3625 (dye A) was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine. (2) 1 mg of avidin was dissolved in 500 μl of water, added to the solution of (1), stirred, further added with 0.3 ml of dicyclohexylcarbodiimide, and left overnight.

(3) The solution of the above (2) was washed with an ethanol: triethylamine mixed solution three to four times using ultrafiltration to remove unreacted dye. (4) The resulting precipitate was washed with 20 mM Buffer Acetate (pH 5.
Were suspended in 5-6), "NK3625 modified avidin '(hereinafter, was referred to as F ₁ avidin.).

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

(7) Further, the F ₁ avidin of the above (4) is reacted with the BC-SHAAb of the above (6) to obtain "NK
3625-modified avidin + biotinylated chitosan + sheep-derived anti-human pancreatic amylase antibody (hereinafter referred to as F ₁ avidin-B
-C-SHAAb). (8) The RHAAb-HA of the above (5) and the FHA of the above (7)
₍₁₎ specifically reacting avidin-BC-SHAAb,
"Rabbit-derived anti-human pancreatic amylase antibody + human pancreatic amylase + sheep-derived anti-human pancreatic amylase antibody + biotinylated chitosan + NK3625-modified avidin"
As a result of excitation with 80 nm LD (30 mW) and measurement of the fluorescence, human pancreatic amylase was found to be 1.2 × 10 ⁻⁴ mg / m 2.
780nm LD device is 10φ × 10
As a result, the cost was reduced, and the size and cost of the device could be reduced.

Comparative Example 1 (1) 2.0 mg of NK1160 (a cyanine dye having the following chemical structural formula, manufactured by Nippon Kogaku Dye Laboratories) was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine.

[0030]

Embedded image

(2) The same operation as in (2) to (4) of Example 1 was carried out, and “NK1160-modified avidin” (hereinafter referred to as F ^,
Avidin).

(3) The reaction was carried out in the same manner as in (5) and (6) of Example 1, and the reaction was carried out with F ^and avidin of (2).
Of NK1160-modified avidin + biotinylated chitosan + sheep-derived anti-human pancreatic amylase antibody (hereinafter referred to as F 2 ^, avidin-BC-SH)
AAb). (4) RHAAb-HA of Example 1 and F 2 ^,
Avidin-BC-SHAAb is specifically reacted,
"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, about 1 m long and expensive, so the equipment was large and expensive. I have.

Example 2 (1) 3.0 mg of Dye D was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine. (2) The same operation as (2) and (3) in Example 1 was performed. (3) The obtained precipitate was washed with 20 mM Buffer Acetate (pH 6).
And suspended in “Dye D-modified avidin” (F ₂ avidin)
And

(4) In the same manner as in (5) and (6) of Example 1, the F ₂ avidin prepared in (3) above was reacted with BC-SHAAb of Example 1 to obtain “Dye D-modified Avidin + biotinylated chitosan + sheep-derived anti-human pancreatic amylase antibody (hereinafter referred to as F ₂ avidin-B-C-SHAab)
Abbreviated). (5) RHAAAb-HA of Example 1 and F _{2 of} (4) above
Avidin-BC-SHAAb is specifically reacted,
810 nm LD as “rabbit-derived anti-human pancreatic amylase antibody + human pancreatic amylase + sheep-derived anti-human pancreatic amylase antibody + biotinylated chitosan + F ₂ avidin”
(30 mW) and the fluorescence was measured.
Human pancreatic amylase can be detected at 1.5 × 10 ⁻⁴ mg / ml, and the 810 nm LD device has a low price of 10φ × 10, and the device can be reduced in size and cost.

Example 3 (1) 2.5 mg of NK3792 (dye B) was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine. (2) The same operation as (2) and (3) in Example 1 was performed. (3) The obtained precipitate was washed with 20 mM Buffer Acetate (pH 5.
5) and suspended in “NK3792 modified avidin” (hereinafter, referred to as “NK3792 modified avidin”).
F ₃ avidin).

(4) Human chorionic gonadotropin (hereinafter, referred to as human chorionic gonadotropin)
hCG) and a rabbit-derived anti-hCG antibody immobilized on an optical fiber (hereinafter abbreviated as RhCGAb) are allowed to react specifically, and “rabbit-derived anti-hCG antibody + hCG” (hereinafter, abbreviated as “hCG”).
RhCGAb-hCG). (5) Goat-derived anti-hC is added to polygalactosamine (hereinafter abbreviated as B-pG) in which many amino groups are biotinylated.
G antibody (hereinafter, abbreviated as GhCGAb) is bound, and “biotinylated polygalactosamine + goat-derived anti-hCG antibody”
(Hereinafter abbreviated as B-pG-GhCGAb).

(6) The F ₃ avidin of the above (3) is further reacted with the B-pG-GhCGAb of the above (5),
"NK3792 modified avidin + biotinylated polygalactosamine + goat anti-hCG antibody" (hereinafter referred to as F ₃ avidin + B-pG-GhCGAb) it was. (7) The RhCGAb-hCG of the above (4) and the above (6)
Specifically by reacting F ₃ avidin + B-pG-GhCGAb the "rabbit anti-hCG antibody + hCG + goat anti-hCG antibody + biotinylated polygalactosamine + NK3
792-modified avidin ". This was excited with an 810 nm LD, and the fluorescence was measured. As a result, hCG was 2.0 ×
It can be detected at 10 ^-4 mg / ml.
The price was low at 0φ × 10, and miniaturization and cost reduction were realized.

Example 4 (1) 2 mg of dye E was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine. (2) The same operation as in (2) to (3) of Example 1 is performed,
The obtained precipitate was washed with 20 mM Buffer Acetate (pH 5.5 to 5.5).
6) to give “Dye E-modified avidin” (hereinafter abbreviated as F ₄ avidin).

(3) The same operation as (6) and (7) in Example 3 was performed, and RhCGAb-hCG and B-pG-G
hCGab was allowed to react specifically, and "rabbit-derived anti-hCG
Antibody + hCG + biotinylated polygalactosamine + goat-derived anti-hCG antibody ”(hereinafter, RhCGAb-hCG-B-
pG-GhCGAb). (4) Further, the F ₄ avidin of the above (2) was reacted with the RhCGGAb-hCG-B-pG-GhCGAb of the above (3), and the following reaction was performed: “rabbit-derived anti-hCG antibody + hCG + goat-derived anti-hCG antibody + biotinylated polygalactosamine + dye E-modified avidin ", which was excited with an 810 nm LD,
As a result of measuring the fluorescence, hCG was 2.3 × 10 ⁻⁴ mg.
/ Ml, 810 nm LD element is 10φ × 10
As a result, the cost was reduced, and miniaturization and cost reduction were realized.

Example 5 (1) 3.0 mg of NK2968 (dye C) was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine. (2) 2 mg of avidin was dissolved in 500 μl of water, added to the solution of (1), further added with 0.2 ml of dicyclohexylcarbodiimide, and left overnight. (3) The same operation was performed up to (3) of Example 1, and the obtained precipitate was suspended in 20 mM Buffer acetate (pH 5.5 to 6), and “NK2968-modified avidin” (hereinafter abbreviated as F ₅ avidin) was obtained. Created.

(4) A goat-derived anti-human calcitonin antibody (hereinafter abbreviated as GHCAb) is immobilized on an optical fiber,
This was allowed to specifically react with human calcitonin (hereinafter abbreviated as HC) to obtain "goat-derived anti-human calcitonin antibody + human calcitonin" (hereinafter abbreviated as GHCAb-HC). (5) Further, a rabbit-derived anti-human calcitonin antibody is bound to polyneuraminic acid (hereinafter abbreviated as B-pN) in which a large number of amino groups have been biotinylated, and a “rabbit-derived anti-human calcitonin antibody + biotinylated polyneuraminic acid” (hereinafter, referred to as B-pN) ,
RHCAb-B-pN), and the GH of the above (4)
CAb-HC is allowed to react specifically, and “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

[0042] (6) further wherein the F ₅ avidin (3) of (5) GHCAb-HC-RHCAb -B-pN
To give "goat-derived anti-human calcitonin antibody + human calcitonin antibody + rabbit-derived anti-human calcitonin antibody + biotinylated polyneuraminic acid + NK2968 modified avidin". 4.3 × 10
It can be detected at ^-4 mg / ml, the 810nmLD element 10φ
The cost was reduced to × 10, and the miniaturization and the price reduction were realized.

Example 6 (1) 2.5 mg of dye F was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine. (2) The same operation as (2) to (4) in Example 1 was performed,
“Dye F-modified avidin” (hereinafter abbreviated as F ₆ avidin)
It was created. (3) Human follicle stimulating hormone (hFSH) and a goat-derived anti-hFSH antibody fixed to an optical fiber (hereinafter, GhFS)
HAb) to specifically react with “goat-derived anti-hF
SH antibody + hFSH "(hereinafter GhFSHAb-hFS
H).

(4) A rabbit-derived anti-hFSH antibody (hereinafter abbreviated as RhFSHAb) is bound to chitosan (BC) in which a large number of amino groups are biotinylated, and "biotinylated chitosan + rabbit-derived anti-hFSH antibody" Hereinafter, BC
-RhFSHAb). (5) Further, the F ₆ avidin of the above (2) is reacted with the BC-RhFSHAb of the above (4) to obtain “dye F-modified avidin + biotinylated chitosan + anti-hFSH antibody derived from rabbit” (hereinafter, RhFSHAb-B). —CF ₆ avidin).

(6) GhFSHAb-hF of the above (3)
Wherein the SH (5) the RhFSHAb-B-C-F ₆ avidin of specifically reacted, as "goat anti-hFSH antibody + hFSH + rabbit anti hFSH antibody + biotinylated chitosan + dye F modified avidin ', this 830 nm
As a result of excitation by LD and measurement of its fluorescence, hFSH can be detected at 5.2 × 10 ⁻⁴ mg / ml, and 830 nm LD
Has a low price of 10φ × 10, realizing miniaturization and low cost.

Example 7 (1) 2 mg of dye G was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine. (2) The same operation as (2) to (4) in Example 1 was performed,
“Dye G-modified avidin” (hereinafter abbreviated as F ₇ avidin)
And

[0047] (3) (5) of Example 1 was reacted in the same manner to (6), further wherein F ₇ avidin and B-C-in (2)
GHAAb is reacted, and “Dye G-modified avidin + biotinylated chitosan + sheep-derived anti-human pancreatic amylase antibody”
(Hereinafter abbreviated as SHAAb-B-C-F ₇ avidin). (4) RHAAb-HA and SHAAb-B of (3) above
And specifically by reacting -C-F ₇ avidin, a "rabbit anti-human pancreatic amylase antibody + human pancreatic amylase + sheep-derived anti-human pancreatic amylase antibody + biotinylated chitosan + dye G modified avidin", it in 810nmLD As a result of excitation and measurement of the fluorescence, human pancreatic amylase can be detected at 2.3 × 10 ⁻⁴ mg / ml, and 810 nm LD
This device was 10φ × 10 and was inexpensive, so that miniaturization and cost reduction could be realized.

Example 8 (1) 2.0 mg of dye H was dissolved in 1 ml of a 4: 1 mixture of ethanol and triethylamine. (2) The same operation as (2) to (4) in Example 1 was performed,
“Dye H-modified avidin” (hereinafter abbreviated as F ₈ avidin)
made.

(3) The reaction was carried out in the same manner as in (5) and (6) of Example 1, and these RHAAb-HA and SHAAb-B-
C specifically reacted, and "rabbit-derived anti-human pancreatic amylase antibody + human pancreatic amylase + sheep-derived anti-human pancreatic amylase antibody + biotinylated chitosan" (hereinafter, RHAAb-
HA-SHAAb-BC). (4) Further reacting the F ₈ avidin of the above (2) with the RHAAb-HA-SHAAb-BC of the above (3),
"Rabbit-derived anti-human pancreatic amylase antibody + human pancreatic amylase + sheep-derived anti-human pancreatic amylase antibody + biotinylated chitosan + dye H-modified avidin"
When excited by LD and the fluorescence was measured, human pancreatic amylase was detected at 2.3 × 10 ⁻⁴ mg / ml, and
The device of the 0 nm LD was 10φ × 10 and was inexpensive, so that miniaturization and cost reduction could be realized.

Example 9 (1) 2.5 mg of NK3792 (dye B) was dissolved in 1 ml of ethanol. (2) 1 mg of chitosan was dissolved in 0.5 ml of water, added to the solution of (1), stirred, further added with 0.3 ml of dicyclohexylcarbodiimide, and left overnight. (3) The solution of the above (2) was washed with ethanol three to four times using ultrafiltration to remove unreacted dye, and NK379
Thus, chitosan to which 2 was bound (hereinafter abbreviated as 3792 chitosan) was obtained.

(4) The obtained 3792 chitosan was suspended in 1 ml of ethanol, mixed with 0.5 ml of an aqueous solution of protein A (concentration: 2 mg / ml), further added with 0.3 ml of dicyclohexylcarbodiimide, and allowed to stand overnight with stirring. . (5) The solution of the above (4) was washed 3 to 4 times with PBS using ultrafiltration to remove unreacted protein A, and a conjugate of protein A and 3792 chitosan (hereinafter PrA-3)
Abbreviated as 792 chitosan. ) Got.

(6) The obtained PrA-3792 chitosan is
Suspended in 1 ml of BS, mixed with 1 ml of an aqueous solution of immunoglobulin G (hereinafter, abbreviated as rabbit-derived anti-CRP IgG) against C-reactive protein (abbreviated as CRP) derived from rabbit (5 mg / ml in concentration) and stirred. , Left overnight. (7) The solution of the above (6) was washed 3 to 4 times with PBS using ultrafiltration to remove unreacted rabbit-derived anti-CRPI IgG, and the rabbit-derived anti-CRPI IgG was converted to a conjugate of PrA-3792 chitosan (hereinafter, referred to as PrA-3792 chitosan) , Anti-CRP IgG-PrA-37
Abbreviated as 92 chitosan. ) Was obtained and suspended and dissolved in PBS to obtain an anti-CRP IgG-PrA-3792 chitosan solution.

(8) Separately, a mouse-derived anti-CRP monoclonal antibody was immobilized on an optical fiber, and the CRP was allowed to react specifically with the monoclonal antibody.
P monoclonal antibody-CRP complex (hereinafter referred to as anti-CR
Abbreviated as P-CRP complex. ) Formed. (9) The optical fiber on which the anti-CRP-CRP complex of (8) is immobilized is replaced with the anti-CRP IgG-PrA of (7).
-3792 immersed in chitosan solution, allowed to react specifically, anti-CRP-CRP complex and anti-CRPI on optical fiber
A conjugate of gG-PrA-3792 chitosan was formed, which was excited with an 810 nm LD, and its fluorescence was measured. As a result, CRP could be detected at 2.5 × 10 ⁻⁴ mg / ml,
The price of the 810 nm LD device was low at 10φ × 10, so that miniaturization and low cost could be realized.

[0054]

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. Therefore, high-sensitivity, low-cost immunoassay can be performed, and the size and cost of the measurement device can be reduced.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-5-287266 (JP, A) JP-A-5-40097 (JP, A) JP-A-63-126833 (JP, A) 238126 (JP, A) JP-A-6-66725 (JP, A) JP-T-59-501873 (JP, A) WO 90/13029 (WO, A1) (58) Fields investigated (Int. Cl. ⁷⁾ G01N 33/533 G01N 21/64 G01N 21/78

Claims (5)

(57) [Claims] 1. A general formula (1), (2) or fluorescent labeling reagent consisting of a labeled hydrophilic multifunctional polymer by more cyanine dye represented in (3). Embedded image (In the formula, R represents a hydrogen atom or a halogen atom, X represents S or C (CH ₃ ) ₂ , n represents an integer of 0 to 3, and m represents an integer of 0 to 2.) 2. The fluorescent labeling reagent according to claim 1, wherein the hydrophilic polyfunctional group polymer is avidin, protein A, aminoglycan, or polypeptide. 3. A fluorescent immunoassay, wherein the fluorescent labeling reagent according to claim 1 or 2 is used as a pumping light source for a semiconductor laser having a wavelength of 750 to 850 nm. 4. A hydrophilic polyfunctional compound labeled with a fluorescent dye.
Direct or between the base polymer and the hydrophilic polyfunctional polymer
Fluorescent immunoassay kit consisting of indirectly binding bioactive substances
Wherein the fluorescent dye has the general formula (1) or (2)Or
(3)Characterized by being a cyanine dye represented by
Fluorescent immunoassay kit. Embedded image(Wherein, R represents a hydrogen atom or a halogen atom, X represents S or
C (CH_Three)_TwoWherein n is an integer of 0 to 3 and m is an integer of 0 to 2
Is shown. ) 5. The general formula (1), (2) or (3) indirectly labeled avidin and / or through direct or amino glycans and / or polypeptides by more fluorescent dye represented Protein A fluorescent immunoassay comprising a conjugate comprising A and a conjugate wherein biotin and / or immunoglobulin is directly or indirectly bound to a bioactive substance via aminoglycan and / or polypeptide. kit. Embedded image (In the formula, R represents a hydrogen atom or a halogen atom, X represents S or C (CH ₃ ) ₂ , n represents an integer of 0 to 3, and m represents an integer of 0 to 2.)