JP3294415B2 - Method for producing fluorescent labeling reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a fluorescent labeling reagent used in a fluorescent immunoassay. More specifically, a method for producing a fluorescent labeling reagent, wherein a water-soluble cyanine dye having both a sulfone group and a carboxyl group and a polymer compound having an amino group are selectively bonded to an amino group via a carboxyl group. About.

[0002]

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

For example, in Japanese Patent Application Laid-Open No. 59-501873 (US Pat. No. 4,852,809), 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. At the same time, the excitation light is guided to the optical fiber to excite the fluorescent dye on the optical fiber, the generated fluorescence is reflected at the emission end face of the optical fiber, and the fluorescence and the remaining excitation light are taken out from the incidence end face of the optical fiber. A method is described in which spectroscopy is performed by passing through a beam 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 the amount of fluorescent dye that binds to one antigen or antibody is one. In addition, fluorescent dyes such as rhodamine and fluorescein have the disadvantage that the excitation light source is large and expensive because the excitation wavelength is around 400 nm. Therefore, instead of these fluorescent dyes, a biotinylated antigen or a biotinylated antibody is labeled with avidin modified with a cyanine dye excited in a 630 nm band, so that a fluorescent label that can use a He-Ne laser as a light source is used. Antigens and fluorescently labeled antibodies have been developed (WO90 / 13029).

However, conventional fluorescent dyes are hardly soluble in an aqueous solvent system, and are very unstable, such as being hydrolyzed when dissolved, so that it has been difficult to prepare a reaction reagent. In order to solve this problem, a water-soluble cyanine dye having both a sulfone group and a carboxyl group has been developed.
Carbodiimides have been used as condensing agents (J.
C. Seehan, G .; P. Hess, J.M. Am. Ch
em. Soc. , 77 , 1067 (1955)). By the way, when carbodiimides are used as a condensing agent, in the case of a water-soluble cyanine dye having both a sulfone group and a carboxyl group, a sulfone group having a high degree of dissociation has a slightly higher reaction selectivity, so that a sulfone group is selectively used. Is activated to bind to an amino group, and as a result, a low water-soluble conjugate having a free carboxyl group is preferentially generated, and the yield of a conjugate having a high water-soluble free sulfone group is reduced. There's a problem.

[0006]

The present inventors have made intensive studies to solve the above-mentioned problems. As a result, as a condensing agent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (D.Be), which is known as a coupling reagent in the field of peptide synthesis.
lleau, G .; See Malek, J.M. Am. Chem. S
oc. , 90 , 1651 (1968)),
Even with a water-soluble cyanine dye having both a sulfone group and a carboxyl group, it has been found that a highly water-soluble conjugate in which a carboxyl group and an amino group are bonded can be obtained in a high yield, and further research has been carried out. Was completed.

That is, the gist of the present invention is to provide (1) a method for producing a fluorescent labeling reagent by combining a water-soluble cyanine dye having both a sulfone group and a carboxyl group with a polymer compound having an amino group. A method for producing a fluorescent labeling reagent, wherein an alkoxycarbonyl-2-alkoxy-1,2-dihydroquinoline is allowed to act to selectively bind a carboxyl group and an amino group, and (2) a method having both a sulfone group and a carboxyl group. The method according to the above (1), wherein the water-soluble cyanine dye is a cyanine dye represented by the general formula (I), (II) or (III), and

[0008]

Embedded image

(Wherein R is H ⁺ , Na ⁺ , K ⁺ , L
i ⁺ , N ⁺ H (C ₂ H ₅ ) ₃ , NH ₄ ⁺ , or pyridinium ion. )

[0010]

Embedded image

(Where R is H ⁺ , Na ⁺ , K ⁺ , L
i ⁺ , N ⁺ H (C ₂ H ₅ ) ₃ , NH ₄ ⁺ , or pyridinium ion. )

[0012]

Embedded image

(Wherein R is H ⁺ , Na ⁺ , K ⁺ , L
i ⁺ , N ⁺ H (C ₂ H ₅ ) ₃ , NH ₄ ⁺ , or pyridinium ion. (3) The above (1) or (2), wherein the high molecular compound having an amino group is selected from the group consisting of chitosan, sulfonated chitosan, polygalactosamine, polyneuraminic acid, antibody, avidin, and protein A. It relates to a manufacturing method.

The fluorescent labeling reagent used in the method of the present invention is not particularly limited as long as it is a cyanine dye having both a carboxyl group and a sulfone group in the molecule. A dye NK3682 (manufactured by Nippon Kogaku Dye Laboratories) having the structural formula shown below and a salt thereof
Dye NK375 having a structural formula represented by the general formula (II)
9 (manufactured by Japan Photographic Dye Laboratories), its salt, and the general formula (II
Dye NK3942 (manufactured by Japan Photographic Dye Laboratories) having the structural formula shown in I) and salts thereof and the like. 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, an excellent fluorescent immunoassay can be realized.

In the method of the present invention, the reaction between the cyanine dye and the polymer having an amino group is carried out by reacting the sulfone group in the cyanine dye with N-alkoxycarbonyl-2-alkoxy-1,2-dihydroquinoline (AADQ). Of the carboxyl groups, the reaction is performed by activating only the carboxyl group in an aprotic solvent and reacting with the amino group. AADQ (IV) generally reacts with RCOOH, which is an acid component, to produce an intermediate (V) as shown in the following reaction formula, and this intermediate is gradually mixed with an acid anhydride (V).
Decomposes into I) and quinoline (VII). This mixed acid anhydride reacts immediately with the amino group in the reaction with the polymer compound having an amino group to form an acid amide bond. Therefore, when a cyanine dye having both a carboxyl group and a sulfone group as described above is used as the acid component, the sulfone group in which the carboxyl group in the cyanine dye has reacted and dissociated does not participate in the reaction, so that a high yield is obtained. Thus, a reaction bond between a carboxyl group and an amino group can be obtained. AADQ
Examples thereof include various combinations of R ′ and R ″ in the general formula (IV) in addition to the EEDQ described above.

[0016]

Embedded image

(Wherein R ′ and R ″ are selected from methyl, ethyl, propyl, butyl, isobutyl and pentyl.) The cyanine dye thus obtained is combined with a polymer compound having an amino group. The conjugate, that is, the cyanine dye-labeled polymer compound has a free sulfone group and thus has excellent water solubility.

The reaction between a cyanine dye and a polymer having an amino group by AADQ can be carried out by a known method. For example, first, after dissolving a cyanine dye in an organic solvent such as dimethylformamide or dimethylacetamide, and ice-cooling, add 1 to 2 equivalents of AADQ and leave at 0 to 4 ° C. for 0.5 to 4 hours. I do. During this time a mixed anhydride is formed. On the other hand, a polymer compound having an amino group is dissolved in water and cooled with ice. A solution such as dimethylformamide containing a cyanine dye is added to this,
The reaction is allowed to stand at 2 ° C. for 2 to 18 hours. After the reaction,
Unreacted cyanine dye, AADQ and the like are preferably removed, and can be removed by, for example, a combination of dialysis, centrifugation, gel filtration, ultrafiltration, etc., and washing with alcohol, water, or the like.

Examples of the high molecular compound having an amino group used in the present invention include aminoglycans such as chitosan, sulfonated chitosan, polygalactosamine and polyneuraminic acid, and derivatives thereof. The molecular weight of these aminoglycans such as chitosan is usually 10 ⁴
Although it is about 10 ^5, it is preferable to use about 10 ⁵ to 10 ^{6 in} order to increase the number of reactive groups.
In order to increase the water solubility of chitosan, sulfonated chitosan can be advantageously used. In addition to aminoglycans, polypeptides such as antibodies, avidin, protein A, and polylysine can also be advantageously used as the high molecular compound having an amino group used in the present invention.

[0020] The fluorescent labeling reagent produced by the method of the present invention is used in a fluorescent immunoassay by the following steps. For convenience of explanation, a case where a cyanine dye-modified avidin produced by the method of the present invention is used will be described as an example.

Step A: First, an antibody against a substance to be measured (eg, an antigen) is immobilized on the core surface of an optical fiber. Next, the optical fiber to which the antibody is bound and the substance to be measured (antigen) are allowed to react specifically with each other, and an immune complex (the core surface of the optical fiber-antibody) in which the antibody and the substance to be measured (antigen) are bound on the core surface of the optical fiber. -Substance to be measured).

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

Step C: The terminal antigen of the immune complex prepared in Step A is reacted with the cyanine dye-labeled antibody prepared in Step B, and the optical fiber core surface-antibody-substance to be measured-antibody-aminoglycan-biotin- An avidin-cyanine dye complex is formed, and the fluorescence of the cyanine dye fixed on the core surface of the optical fiber is measured using a fluorescence measurement device using a semiconductor laser of 750 to 850 nm as an excitation light source.

In the above description, avidin is taken as an example of a polymer compound having an amino group. However, the polymer compound having an amino group used in the method of the present invention may be an antibody other than avidin as described above. And aminoglycans such as protein A, chitosan, sulfonated chitosan, polygalactosamine and polyneuraminic acid. As the intervening compound for fixing the cyanine dye on the core surface of the optical fiber, a compound that specifically binds to a polymer compound having an amino group to be 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 the case of aminoglycans such as chitosan, polygalactosamine and polyneuraminic acid, these antibodies can be used.However, it is convenient to combine biotin with chitosan or the like and use biotinylated chitosan or the like with avidin. .

[0025]

EXAMPLES Hereinafter, 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 and the like. Example 1 (1) 2.5 g of chitosan was suspended in 500 ml of dimethylacetamide (DMA) containing 5% LiCl, and 18 g of a pyridine-sulfur trioxide complex was dissolved therein and reacted at 60 ° C. for 24 hours. (2) After the reaction, ice water was added while cooling, and pH
The final volume was adjusted to about 1 liter while adjusting to zero. (3) After removing the precipitate by centrifugation, the supernatant was concentrated to about 500 ml by evaporation, and this was dialyzed against distilled water to remove DMA, pyridine and generated Na ₂ SO ₄ . (4) Concentrated HCl was added to the solution of (3) to adjust the final concentration of the solution to 0.08N. This solution is
Heat treatment was performed at 00 ° C. for 2.5 hours. (5) After heating, the mixture was cooled in an ice bath, adjusted to pH 10 with a 6% aqueous NaOH solution, and the resulting precipitate was removed by centrifugation to obtain a supernatant. (6) The supernatant of the above (5) was dialyzed against distilled water overnight, and then concentrated and dried by an evaporator to obtain a sulfonated chitosan (SC) standard.

(7) 27.6 mg of NK3682 was added in 0.1 part.
Dissolve in 5 ml of dimethylformamide (DMF) and add N
After K3682 was dissolved, the solution was cooled in an ice bath. (8) EED was added to the ice-cooled NK3682 solution of (7).
After adding and dissolving 10.4 mg of Q (manufactured by Wako Pure Chemical Industries, Ltd.), the mixture was allowed to stand at 4 ° C. for 2 hours. (9) 0.5 ml of 2 mg of the SC obtained in the above (6)
Was dissolved in water and cooled with ice, and 0.5 ml of the solution of NK3682 activated by EEDQ of (8) was added.
Reaction was performed at 12 ° C. for 12 hours. (10) About 9 ml of methanol was added to the reaction solution of the above (9), and unreacted NK3682 and EEDQ were added by ultrafiltration.
Etc. were removed. (11) After further washing with methanol for 2 to 3 times, the methanol was removed, and 3 ml of water was added thereto.
Thus, an aqueous solution of 82 bonded SC (FSC) was obtained.

(12) The F-S- obtained in the above (11)
Dissolve 3 mg of goat-derived anti-human Ig antibody in C aqueous solution,
Further, 5 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC) was added thereto and reacted at 4 ° C. overnight. (13) Unreacted antibody, FS-
C, WSC and the like were removed to obtain FSC (FSC-Ab) to which a goat-derived anti-human Ig antibody was bound. F
The amount of the antibody recovered as -SC-Ab was 2.8 mg.
(93.3%).

(14) A resin optical fiber made of polymethyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.) of 3 cm
And both ends were polished with a polishing film using ethanol as a lubricant. (15) 10 mg of nickel sulfate was dissolved in 0.5 ml of water, and then 2.5 ml of ethanol was added. The precipitate formed at this time was removed by centrifugation, and the collected supernatant was used as a nickel-ethanol solution. Next, nickel-0.4 ml of 20 mM potassium hydroxide solution of ethanol solvent was added.
0.1 ml of an ethanol solution and 50 μl of 50% glutaraldehyde were added and mixed to obtain a treatment solution. (16) One side of the optical fiber of (14) is
After immersion in the treatment solution of 5) at 50 ° C. for 10 minutes,
Washed with 0 mM hydrochloric acid, then with PBS. Next, this optical fiber is immersed in a 2 mg / ml human pancreatic amylase solution,
Leave at 4 ° C. overnight. (17) The optical fiber is taken out of the solution, immersed in a 1% sodium borohydride aqueous solution for 15 minutes, and then PBS
After washing, a sensor for immobilizing human pancreatic amylase was used as a detection unit.

(18) The detection unit of (17) is immersed in a human pancreatic amylase antibody solution having a known concentration for 20 minutes.
In the solution of FSC-Ab prepared in the above (13), 2
Dipped for 0 minutes. Next, after washing with PBS containing 0.05% Tween 20 (Tween PBS), measurement was performed with a 780 nm semiconductor laser system using the apparatus shown in FIG. 1 which is a fluorescence measurement system using one laser.
It was able to measure up to 3 ng / ml.

Comparative Example 1 (1) S-C by a method similar to (1) to (6) of Example 1
A sample was obtained. (2) 2 mg of the SC of the above (1) was dissolved in 0.5 ml of water, 27.6 mg of NK3682 was dissolved in DMF, and the two solutions were mixed, and water-soluble carbodiimide (WS
10 mg of C) was added and reacted at 4 ° C. for 12 hours. (3) F is obtained in the same manner as in (10) to (13) of the first embodiment.
-SC-Ab was obtained. At this time, the amount of the antibody recovered as FSC-Ab was about 0.4 mg, which was only 13.3% of the antibody used, whereas the amount of the antibody recovered in Example 1 was as described above. And the recovery rate was 90% or more. This difference is due to NK36 activated by EEDQ.
82 and NK36 activated by WSC
This is probably due to the difference in the yield of FSC when 82 was used and the difference in the water solubility of the obtained FSC.

Example 2 (1) 5 mg of NK3942 was dissolved in 100 μl of DMF while cooling, 4 mg of EEDQ was added and dissolved, and the mixture was allowed to stand at 4 ° C. for 2 hours. (2) 1 ml (13 mg / ml) of a goat-derived anti-human CRP antibody solution manufactured by Nippon Biotest Laboratory Co., Ltd. was dialyzed against 5 mM phosphate buffer (pH 7) at 4 ° C. for 4 hours. ) With NK3942 solution and 4 ° C to 16 ° C.
Let stand for hours. (3) Gel filtration was performed using a Sephadex G50 column equilibrated with 5 mM phosphate buffer (pH 7), and NK39 was used.
Forty-two bound anti-human CRP antibodies (F-Abs) were obtained.
At this time, the amount of the antibody recovered as F-Ab was 12.3 m.
g (94.6%). (4) An anti-human CRP antibody-immobilized sensor was prepared in the same manner as in (14) to (17) of Example 1 except that an anti-human CRP antibody was used instead of human pancreatic amylase, and this was used as a detection unit. And (5) The detection unit of (4) is immersed in a CRP solution of known concentration for 20 minutes, and then the F-Ab prepared in (3) above
Immersion in the solution for 20 minutes. After washing with a 5 mM phosphate buffer (pH 7) containing 0.01% Tween 20, measurement was performed with a semiconductor laser system at 780 nm using the apparatus shown in FIG. 1, and it was possible to measure up to 2 ng / ml.

Comparative Example 2 (1) In Example 2 (1) and (2), the EED
5m instead of NK3942 activated by Q
g-NK3942 was directly dissolved in the dialyzed antibody solution, and F-Ab was obtained in the same manner as in (2) and (3) of Example 2 except that 4 mg of WSC was added. At this time, the amount of antibody recovered as F-Ab was 0.11 m
g (recovery rate 8.5%). (2) When measured by the same method as in (4) and (5) of Example 2, the amount of F-Ab obtained was too small.
Only up to 00 ng / ml could be detected. Comparing Example 2 with Comparative Example 2, it is clear that EEDQ is much superior to WSC in binding NK3942 to an anti-human CRP antibody, and the method for producing a fluorescent labeling reagent of the present invention. , EEDQ is extremely suitable.

Example 3 In Example 2, NK375 was used instead of NK3942.
Example 9 An anti-human CRP antibody-immobilized sensor was prepared in exactly the same manner except that CR9 was used.
When P was measured, detection was possible up to 2 ng / ml.

Example 4 The procedure of Example 1 was repeated, except that N-methoxycarbonyl-2-propoxy-1,2-dihydroquinoline (MPDQ) was used instead of EEDQ. % Or more and human pancreatic amylase could be measured up to 0.3 ng / ml.

Example 5 The same procedure as in Example 2 was repeated except that N-butoxycarbonyl-2-butoxy-1,2-dihydroquinoline (BBDQ) was used instead of EEDQ. % Or more and 2 CRP
It was possible to measure up to ng / ml.

[0036]

According to the production method of the present invention, it is possible to produce a highly water-soluble fluorescent labeling reagent in a high yield using a cyanine dye having both a sulfone group and a carboxyl group. Price immunoassays can be facilitated.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a fluorescence measurement system using one laser.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Optical fiber 2 Laser 3 Guide rail for optical axis alignment 4 Detector 5 Filter 6 Fluorescence detector 7 Half mirror

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-57-45455 (JP, A) JP-A-2-191674 (JP, A) JP-A-5-107249 (JP, A) JP-A-7-54 83925 (JP, A) JP-A-6-222059 (JP, A) JP-A-6-82449 (JP, A) JP-T-59-501873 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/48-33/98 CA (STN)

Claims (3)

(57) [Claims] 1. A method for producing a fluorescent labeling reagent by combining a water-soluble cyanine dye having both a sulfone group and a carboxyl group with a polymer compound having an amino group,
N-alkoxycarbonyl-2-alkoxy-1,2-
A method for producing a fluorescent labeling reagent, comprising reacting dihydroquinoline to selectively bind a carboxyl group and an amino group. 2. The water-soluble cyanine dye having both a sulfone group and a carboxyl group is represented by the general formula (I), (II) or (II).
2. The production method according to claim 1, which is a cyanine dye represented by I). Embedded image (Wherein R is H ⁺ , Na ⁺ , K ⁺ , Li ⁺ , N ⁺ H (C
₂ H ₅ ) ₃ , NH ₄ ⁺ , or a pyridinium ion. ) (Wherein R is H ⁺ , Na ⁺ , K ⁺ , Li ⁺ , N ⁺ H (C
₂ H ₅ ) ₃ , NH ₄ ⁺ , or a pyridinium ion. ) (Wherein R is H ⁺ , Na ⁺ , K ⁺ , Li ⁺ , N ⁺ H (C
₂ H ₅ ) ₃ , NH ₄ ⁺ , or a pyridinium ion. ) 3. The high molecular compound having an amino group is selected from the group consisting of chitosan, sulfonated chitosan, polygalactosamine, polyneuraminic acid, antibody, avidin, and protein A.
The manufacturing method as described.