JPH05255264A - Production of labeling agent and labeled ligand - Google Patents

Abstract

PURPOSE:To obtain a labeling agent for a ligand composed of a water-soluble acridinium compound having chemiluminescent properties. CONSTITUTION:The objective ligand is composed of a compound expressed by formula I [R1 is H, 1-10C alkyl, benzyl, etc.; R2 is O, NH, NH-SO2, etc.; R3 to R10 are H or halogen; (n) is 1-5; R11 and R12 are H, 1-10C alkyl, aryl, etc.; X<-> and Y<-> are anionl, e.g. 4-{2-(methylsufonyl-4-phenyldimethylsufonium) oxycarbonylethyl}phenyl 10-acridinium-9-carboxylate fluorosulfonate. This ligand is useful as a labeling agent for immunoassay and hybridization assay. Labeling of a ligand represented by an antigen, an antibody, etc., with the acridinium compound can be carried out in an aqueous medium.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a labeling agent for an immunoassay method or a hybridization assay method and a method for producing a labeled ligand using the labeling agent.
More specifically, it relates to a labeling agent comprising a chemiluminescent acridinium compound and a method for producing a labeled ligand using the labeling agent.

[0002]

As a chemiluminescent acridinium compound, 4- (2-succinimidyloxycarbonylethyl) described in "Ian Weeks et al., Clinical Chemistry, Vol. 29, p. 1474, published in 1983".
Phenyl-10-methylacridinium-9-carboxylic acid ester fluorosulfonate is widely known and used as a labeling agent for a labeled ligand in immunoassays and the like.

[0003]

However, since the above-mentioned acridinium compound is insoluble in water, in the reaction of labeling this compound with a ligand, the compound is added to a water-soluble organic solvent such as DMF, acetonitrile, dioxane, etc. Once dissolved, it must be reacted with an aqueous solution of the ligand, which causes a serious problem that the activity of the ligand is reduced by the organic solvent or the yield of the labeled ligand is reduced.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the use of a water-soluble acridinium compound can solve the above-mentioned problems in labeling. Arrived That is, the present invention provides a labeling agent for an immunoassay or a hybridization assay, which comprises a water-soluble acridinium compound (A) having chemiluminescence; and a label characterized by reacting the labeling agent and a ligand in an aqueous medium. It is a method for producing the prepared ligand.

In the present invention, the water-soluble acridinium compound (A) includes compounds represented by the following general formula (2).

[0006]

[Chemical 2]

[Wherein R ₁ is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, a benzyl group or an aryl group, and -R _2- is-
O -, - NH -, - NH-SO 2 - or -N (SO ₂ -P
h)-{-Ph is a phenyl group which may be substituted with a halogen atom}, R _{3 to} R ₁₀ are each a hydrogen atom or a halogen atom, n is an integer of 1 to 5, R ₁₁ and R ₁₂ are each a hydrogen atom. , An alkyl group having 1 to 10 carbon atoms, a benzyl group or an aryl group, X ⁻ and Y ⁻ each represent an anion. ]

[0008] As the alkyl group having 1 to 10 carbon atoms of wherein R ₁ is exemplified by methyl, ethyl, propyl, butyl, pentyl group, As the aryl group include a phenyl group, as above R ₁ Preferred among these, a hydrogen atom and a benzyl group are a methyl group, an ethyl group, a benzyl group and a phenyl group.

--R ₂ --is --O--, --NH--, --NH--S
O ₂ - or _{-N (SO 2 -Ph) - {} - Ph is is a phenyl group optionally substituted by a halogen atom} but selected from, it is possible to use any of these.

R _{3 to} R ₁₀ are each selected from a hydrogen atom or a halogen atom (fluorine, bromine, chlorine, iodine, etc.), and any of these can be used.

N is an integer of 1 to 5.

Of R ₁₁ and R ₁₂ , alkyl groups having 1 to 10 carbon atoms include methyl, ethyl, propyl, butyl,
Examples of the aryl group include a pentyl group and the like, and a phenyl group and the like are mentioned. Among them, those exemplified as R ₁₁ and R ₁₂ and a hydrogen atom and a benzyl group are preferably methyl group, ethyl group, benzyl group and phenyl group. Is.

The anions X ⁻ and Y ⁻ are not particularly limited, but preferable ones are fluorosulfonate ion, methanesulfonate ion, fluoromethanesulfonate ion, trifluoromethylsulfonate ion and halogen ion (fluorine ion, Bromide, chloride, iodide, etc.). X ⁻ and Y ⁻ may be the same or different.

A specific example of the water-soluble acridinium compound (A) is a compound represented by the following chemical formula 4-
{2- (4-Dimethylsulfoniophenyl) oxycarbonylethyl} phenyl-10-acridinium-9-
Carboxylic acid ester fluorosulfonic acid / methyl sulfonic acid salt (A-1) and N-phenylsulfonyl- [4- {2- (4-dimethylsulfoniophenyl)} oxycarbonylethyl] phenyl-10 represented by the following chemical formula 4. -Methylacridinium-9-carboxamide fluorosulfonic acid / methylsulfonic acid salt (A-2) and the like can be mentioned.

[0015]

[Chemical 3]

[0016]

[Chemical 4]

The water-soluble acridinium compound (A) is usually crystalline powder, and the labeling agent of the present invention is usually (A) itself.

As typical examples of the method for producing the water-soluble acridinium compound (A), the above compounds (A-1) and (A-2)
As an example of the production method of, the reaction steps are as shown in the following Chemical Formulas 5 and 6.

[0019]

[Chemical 5]

That is, acridinecarboxylic acid (a) is reacted with thionyl chloride or the like to give acridinecarboxylic acid chloride (b), which is further reacted with 4-hydroxyphenylpropionic acid benzyl ester to form ester (c). .. This is treated with a hydrogen bromide / acetic acid mixed solution to give a free carboxylic acid (d), and a compound such as 4-hydroxyphenyldimethylsulfonium methylsulfate is dehydrated with DCC (N, N-dicyclohexylcarbodiimide) or the like. Compound (e) is obtained by performing an esterification reaction using a condensing agent. The target compound (A-1) is obtained by reacting this compound (e) with methyl fluorosulfonate.

[0021]

[Chemical 6]

That is, acridinecarboxylic acid chloride (b), which is an intermediate obtained in the synthesis of A-1, is reacted with (N-phenylsulfonyl) benzyloxycarbonylethylaniline to form an amide (f). This is treated with a mixed solution of hydrogen bromide / acetic acid to give a free carboxylic acid (g), and this and a compound such as 4-hydroxyphenyldimethylsulfonium methylsulfate are subjected to an esterification reaction using a dehydration condensing agent such as DCC. Compound (h) is obtained by carrying out. The target compound (A-2) is obtained by reacting this compound (h) with methyl fluorosulfonate.

Ligands that can be labeled by the method of the present invention using the labeling agent of the present invention include antigens, antibodies, haptens, protein A, avidin, biotin, nucleic acids, and molecules containing nucleic acids. The reaction of labeling by the method of the present invention proceeds by a dehydration condensation reaction between an amino group existing in the ligand or introduced by chemically modifying the ligand and the labeling agent of the present invention, and acridinium A labeled ligand can be obtained.

The conditions for the reaction to be labeled by the method of the present invention are usually 1 to 50 mol of (A) per 1 mol of the ligand, which is reacted at room temperature (10 to 30 ° C.) for about 15 minutes. Is obtained. However, without being limited to this condition, it is important to set the optimum conditions depending on the properties of the ligand to be labeled.

In the method of the present invention, the labeling reaction is carried out in an aqueous medium, which may be water itself, or a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a carbonate buffer solution. An aqueous medium such as a liquid is preferably used.

The acridinium-labeled ligand thus obtained can be used in a chemiluminescence immunoassay method and a chemiluminescence hybridization assay method as exemplified in the following <1> to <3>.

<1> “Ian Weeks et al., Clinical Chemistry, Vol. 29, p. 1480, published in 1983” describes that α-fetoprotein (hereinafter referred to as AFP) sandwich immunoassay and 4
A method using an acridinium-labeled anti-AFP antibody obtained by reacting-(2-succinimidyloxycarbonylethyl) phenyl-10-methylacridinium-9-carboxylic acid ester-fluorosulfonate is described. ing.

<2> Regarding competitiveness immunoassay, 4- (2-succinimidyloxy) is also described in “Ian Weeks et al., Methods in Enzymology, Vol. 133, p. 366, published in 1986”. Carbonylethyl)
There is a description regarding the measurement of free T4 using phenyl-10-methylacridinium-9-carboxylic acid ester / fluorosulfonate as a labeling compound.

<3> In the field of gene diagnosis, "J. Lyle et al., Clinical Chemistry Vol. 35,
1588, published in 1989 "by reacting 4- (2-succinimidyloxycarbonylethyl) phenyl-10-methylacridinium-9-carboxylic acid ester fluorosulfonate with oligo DNA. There is a description on leukemia diagnosis using the prepared probe.

[0030]

EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

Example 1 4- {2- (4-dimethylsulfoniophenyl) oxycarbonylethyl} phenyl-10-acridinium-
Synthesis of 9-carboxylic acid ester fluorosulfonic acid / methylsulfonic acid salt (A-1)

The synthesis was carried out as follows. The synthetic scheme is as shown in Chemical formula 5 above. Acridine
9-carboxylic acid (a) (manufactured by Sigma) (5.0 g) was suspended in thionyl chloride (manufactured by Hanai Tesque) (75 ml), and the mixture was stirred under reflux for about 4 hours, and then excess thionyl chloride was depressurized. It was removed to obtain acridine-9-carboxylic acid chloride (b).

(B) (2.4 g, 10 mmol) was suspended in anhydrous pyridine (30 ml) and 4-hydroxyphenylpropionic acid benzyl ester (9.7 g, 10 m) was added.
mol) was added, and the mixture was stirred overnight at room temperature. Then, the reaction solution was added to a 1N aqueous hydrochloric acid solution to obtain a yellow crystalline substance. The crystalline substance was separated by filtration, and the crystalline substance was washed with water on the filter. The crystals were dried under reduced pressure and recrystallized from benzene / cyclohexane to give 4- (2-benzyloxycarbonylethyl) phenyl-9-acridinecarboxylic acid ester (c). Elemental analysis calculated value C: 78.1% H: 5.0%
N: 3.0% Actual value C: 77.5% H: 5.2% N: 3.1%

10 parts of (c) (0.46 g, 1 mmol)
It was dissolved in a mixed solution of hydrogen bromide / acetic acid (45/55 w / w) and stirred at 50 to 55 ° C. 100m of this reaction solution
Yellow crystals were obtained by pouring into 1 l of water. The crystals were separated by filtration, washed with water, and then dried under reduced pressure. Further recrystallized from acetonitrile / chloroform (1/1 v / v) to give 4- (2-carboxyethyl) phenyl-
9-acridinecarboxylic acid ester (d) was obtained. Elemental analysis calculated value C: 74.4% H: 4.6%
N: 3.8% Actual value C: 75.1% H: 4.5% N: 3.6%

(D) (0.37 g, 1 mmol) and Sanserra DSP (4-hydroxyphenyldimethylsulfonium methylsulfate, Sanshin Chemical Co., Ltd.) (0.27
g, 1 mmol) was dissolved in DMF (5 ml), N, N-dicyclohexylcarbodiimide (manufactured by Hanai Tesque) (0.20 g, 1 mmol) was added under ice-cooling, and the mixture was stirred under ice-cooling for 2 hours and further stirred at room temperature overnight. Reacted. The precipitated dicyclohexylurea was filtered off, the filtrate was removed under reduced pressure, the residue was dissolved in ethyl acetate, and this was washed with water. The ethyl acetate layer was taken, anhydrous sodium sulfate was added thereto, and the mixture was allowed to stand overnight.

Anhydrous sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure. Ether / petroleum ether was added to this residue to obtain 4- {2- (methylsulfate.p-dimethylsulfoniophenyl) oxycarbonylethyl} phenyl-9-acridinecarboxylic acid ester (e). Elemental analysis calculated value C: 62.0% H: 4.7%
N: 2.3% Actual value C: 63.1% H: 4.5% N: 2.2%

(E) (0.31 g, 0.5 mmol) was dissolved in anhydrous acetonitrile, 0.5 ml of methyl fluorosulfonate was added thereto, and the mixture was stirred at room temperature for 20 hours. The precipitated crystals were filtered off, washed with anhydrous benzene on the filter, and the crystals were dried under reduced pressure to give 4- {2
-(Methylsulfate / p-dimethylsulfoniophenyl) oxycarbonylethyl} phenyl-10-methylacridinium-9-carboxylic acid ester fluorosulfonic acid / methylsulfonic acid salt (A-1) was obtained. Elemental analysis calculated value C: 53.1% H: 4.3%
N: 1.9% Actual value C: 53.8% H: 4.6% N: 2.0%

Example 2 N-phenylsulfonyl [4- {2- (4-dimethylsulfoniophenyl)} oxycarbonylethyl] phenyl-10-methylacridinium-9-carboxamide / fluorosulfonic acid / methylsulfonic acid salt (A-2)
Synthesis of

The synthesis was carried out as follows. The synthetic scheme is as shown in Chemical formula 6 above.

The intermediate (b) (2.4 g, 10 mmol) obtained by the synthesis of A-1 was converted into anhydrous pyridine (3).
(N-phenylsulfonyl) benzyloxycarbonylethylaniline (3.6 g, 10 g).
mmol) was added, and the mixture was stirred at room temperature for 24 hours. Then, the reaction solution was added to a 1N aqueous hydrochloric acid solution to obtain a yellow crystalline substance.
The crystalline substance was separated by filtration, and the crystalline substance was washed with water on the filter. This crystal was dried under reduced pressure and recrystallized from benzene / cyclohexane to give N-phenylsulfonyl {4- (2-benzyloxycarbonylethyl)} phenyl-9-acridinecarboxamide (f). Elemental analysis calculated value C: 72.0% H: 4.7%
N: 4.7% Actual value C: 71.1% H: 4.6% N: 5.0%

(F) (0.60 g, 1 mmol) was added to 10
It was dissolved in a mixed solution of hydrogen bromide / acetic acid (45/55 w / w) and stirred at 50 to 55 ° C. 100m of this reaction solution
Yellow crystals were obtained by pouring into 1 l of water. The crystals were separated by filtration, washed with water, and then dried under reduced pressure. Further, it was recrystallized from acetonitrile / chloroform (1/1 v / v) to obtain N-phenylsulfonyl {4- (2-carboxyethyl)} phenyl-9-acridinecarboxamide (g). Elemental analysis calculated value C: 68.2% H: 4.3%
N: 5.5% Actual value C: 67.4% H: 4.1% N: 5.6%

(G) (0.51 g, 1 mmol) and Sanserra DSP (0.27 g, 1 mmol) were added to DMF (5
ml), N, N-dicyclohexylcarbodiimide (0.20 g, 1 mmol) was added under ice cooling, and the mixture was stirred and reacted for 2 hours under ice cooling, and at room temperature for 24 hours. The precipitated dicyclohexylurea was filtered off and the filtrate was removed under reduced pressure. The residue was dissolved in ethyl acetate and this was washed with water.
The ethyl acetate layer was taken, anhydrous sodium sulfate was added thereto, and the mixture was allowed to stand overnight.

Anhydrous sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure. By adding ether / petroleum ether to this residue, N-phenylsulfonyl [4- {2- (4-
Dimethylsulfoniophenyl)} oxycarbonylethyl] phenyl-9-carboxamide methylsulfonate (h) was obtained. Elemental analysis calculated value C: 60.2% H: 4.5%
N: 3.7% Actual value C: 61.0% H: 4.5% N: 3.6%

(H) (0.38 g, 0.5 mmol) was dissolved in anhydrous acetonitrile, 0.5 ml of methylfluorosulfonate was added thereto, and the mixture was stirred at room temperature for 20 hours. The precipitated crystals were separated by filtration, washed with anhydrous benzene on the filter, and the crystals were dried under reduced pressure to give N-phenylsulfonyl [4- {2- (4-dimethylsulfoniophenyl)} oxycarbonylethyl]. Phenyl-10
-Methylacridinium-9-carboxamide / fluorosulfonic acid / methylsulfonic acid salt (A-2) was obtained. Elemental analysis calculated value C: 53.7% H: 4.2%
N: 3.2% Actual value C: 54.0% H: 4.4% N: 3.5%

Example 3 Preparation of acridinium-labeled anti-TSH antibody

0.02 mol / l of anti-TSH monoclonal antibody (2 ml, protein amount about 3.7 mg)
(Hereinafter, abbreviated as M), it was dialyzed using a phosphate buffer having a pH of 8.0. In addition, the compound (A-1) synthesized in Example 1
(1 mg) was placed in a test tube and dissolved in a 0.02 M phosphate buffer (1 ml) having a pH of 8.0. The previously prepared solution (80 μl) was added to the dialyzed antibody solution, and the mixture was reacted at room temperature for about 15 minutes. When the reaction is completed, 0.02M,
A Sephadex G-25 column buffered with a phosphate buffer of pH 8.0 was charged with the above reaction mixture for purification.

After completion of the purification, the fractions having both protein activity and chemiluminescence activity of each fraction were collected,
The solution was concentrated, sodium azide was added as a preservative so as to be 0.1%, and the mixture was stored at 2 to 10 ° C until use. This was designated as "marker 1".

Example 4 Example 3 except that (A-2) was used instead of (A-1).
In the same manner as above, A-2 was introduced into the anti-TSH monoclonal antibody to obtain "Labeled product 2".

Comparative Example Similarly, acridinium-I (4- (2-succinimidyloxycarbonylethyl) phenyl-10-methylacridinium-9-carboxylic acid ester / fluorosulfonate, manufactured by Dojindo Laboratories) Was used to introduce an acridinium compound into an anti-TSH monoclonal antibody according to the method of Weeks et al., And this was designated as "marker 3""Clinical Chemistry, Vol. 29, page 1480, 198".
Issued for 3 years ”.

Example of use Comparison of performance between labeled products 1 and 2 and labeled product 3

(1) Comparison of the amount of each luminescence 2 μm as the protein amount of each of Labels 1, 2 and 3
The g portion was dispensed into a 12 × 75 mm glass test tube, and set in a sample holder of Alka-made luminescence reader BLR-201 type. Luminescent reaction liquid A (0.1N hydrochloric acid aqueous solution containing 0.3% hydrogen peroxide) and luminescent reaction liquid B
Each 250 μl of (0.2 N sodium hydroxide aqueous solution containing 0.5% of octylphenol ethylene oxide 10 mol adduct) was sequentially added to carry out a chemiluminescence reaction. The measured value was obtained as an integrated value for 5 seconds. The amount of light emitted from each of the labeled products at that time is shown in Table 1 below. The amount of light emitted from the labeled products 1 and 2 is about 2 to 3 times greater than that from the labeled product 3. From this, it was confirmed that the acridinium compound of the present invention has high luminescence reactivity.

[0052]

[Table 1]

(2) Comparison of respective performances in immunoassay of TSH Immobilization of anti-TSH antibody on inner wall of test tube Carbonate buffer (0.02M) pH 9.5 anti-TSH monoclonal antibody (used in Example 2) Recognizing an epitope different from that of the monoclonal antibody) was dissolved at 50 μg / ml. Nunc polystyrene test tube (1
2 × 75 mm), each containing 5 TSH antibody-containing carbonate buffer solution
The antibody was immobilized on the inner wall of the test tube by dispensing 00 μl each and reacting at 4 ° C. for 48 hours. After the reaction was completed, the liquid in the test tube was removed using an aspirator. The test tube was washed 3 times with 1 ml of physiological saline, and a phosphate buffer solution (0.02 M, p) containing 1% bovine serum albumin (hereinafter abbreviated as BSA).
H7.2) 1 ml was added and stored at 4 ° C. until use.

Preparation of acridinium-labeled antibody solution Labels 1, 2, and 3 obtained in Example 2 were treated with 1% BSA.
Each labeled substance was dissolved in the contained phosphate buffer (0.02M, pH 7.2) so that the protein concentration was 2 μg / ml.

Standard solution Purified human TSH was mixed with 1% BSA-containing phosphate buffer solution (0.0
2M, pH 7.2) 0.5, 1, 3, 5, 10, 2
The dilution was adjusted to be 5, 50 μIU / ml.

The immersion liquid in the antibody-binding test tube prepared in the measuring method (1) was removed using an aspirator, and the inside of the test tube was washed once with 1 ml of physiological saline. Sampling 100 μl of standard solution or sample (serum or plasma) into washed test tube
BSA-containing phosphate buffer (0.02M, pH 7.2) 5
00 μl was added, stirred well and incubated at 37 ° C. for 30 minutes. After the reaction was completed, the reaction solution was removed using an aspirator, 1 ml of physiological saline was added, and the same operation was performed again. This operation was repeated 3 times to remove unreacted substances in the sample.

Add 500 μl of each of the solutions of the labeled substances 1, 2 and 3 prepared in the above test tube, stir well,
Incubated at 37 ° C for 30 minutes. After the reaction was completed, the reaction solution was removed using an aspirator, and the physiological saline solution was 1 m.
1 was added and the same operation was performed again. This operation was repeated 3 times to remove unreacted labeled substances 1 and 2.

The washed test tube was set in a sample holder of luminescence reader BLR-201 type manufactured by Aloka, and 250 μl of each of the luminescence reaction solution A and the luminescence reaction solution B was sequentially added to carry out the chemiluminescence reaction. .. The measured value was obtained as an integrated value for 5 seconds.

Table 2 below shows the amount of luminescence at each standard solution concentration using the standard solution as a sample when each of the labeled substances was used. Also, the amount of luminescence at each standard solution concentration was plotted on a graph paper to obtain a calibration curve as shown in FIG.
According to this, the amount of luminescence in the immunoassay was larger when the labeled substances 1 and 2 were used than when the labeled substance 3 was used, and this result confirmed the high labeling reactivity of the acridinium compound of the present invention.

[0060]

[Table 2]

[0061]

EFFECTS OF THE INVENTION By using the labeling agent of the present invention, acridinium labeling of a ligand represented by an antigen, an antibody, etc. can be carried out in an aqueous medium. As a result, problems such as the activity of the ligand and the decrease in the yield of the labeled ligand can be solved.

[Brief description of drawings]

FIG. 1 is a calibration curve of TSH obtained in a use example by a chemiluminescence immunoassay method.

Claims (4)

[Claims] 1. A labeling agent for a ligand comprising a water-soluble acridinium compound (A) having chemiluminescence. 2. The labeling agent according to claim 1, wherein (A) is a compound represented by the following general formula 1. [Chemical 1] [Wherein R ₁ is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms,
Benzyl or an aryl group, -R ₂ - is -O -, - NH
-, - NH-SO ₂ - or _{-N (SO 2 -Ph) - {} - P
h is a phenyl group which may be substituted with a halogen atom}, R _{3 to} R ₁₀ are each a hydrogen atom or a halogen atom, n is an integer of 1 to 5, R ₁₁ and R ₁₂ are each a hydrogen atom and a carbon number of 1 to 1. The 10 alkyl groups, benzyl groups or aryl groups, X ⁻ and Y ⁻ each represent an anion. ] 3. The labeling agent according to claim 1 or 2 for immunoassay or hybridization assay. 4. A method for producing a labeled ligand, which comprises reacting the labeling agent according to claim 1 with a ligand in an aqueous medium.