JPH07209295A - Antibody, enzyme-labeled antigen, enzyme immunoassay using the same, and assay kit - Google Patents

Abstract

(57) [Abstract] [Objective] An enzyme immunoassay for 9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinoline (I), which has a therapeutic effect on Alzheimer's disease, and Provided are a measurement kit, an enzyme-labeled antigen and an antibody used therein. [Structure] (A) Formula (II) (Wherein, n represents an integer of 0-5) and an immunogenic carrier are covalently bonded via a carboxyl group of the compound, and the obtained antigen is administered to an animal. The antibody, (B) an enzyme-labeled antigen obtained by covalently bonding a compound represented by the formula (II) and an enzyme via a carboxyl group of the compound, (C) immobilized on a solid phase support ( The enzyme immunoassay of (I) using the antibody of (A) and the enzyme-labeled antigen of (B), and (D) the antibody of (A) and the enzyme-labeled antigen of (B) are used as essential components of the kit. The enzyme immunoassay kit of (I).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a compound represented by the following formula (I) having a therapeutic effect on Alzheimer's disease [hereinafter,
Sometimes referred to as compound (I) or simply (I)] enzyme immunoassay (enzyme immunoassay).
oassay, hereinafter also referred to as EIA), a measurement kit, and enzyme-labeled antigens and antibodies used therefor.

[0002]

2. Description of the Related Art Conventionally, liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC-MS) have been generally used as a method for measuring the concentration of a drug or the like in a biological fluid.

[0003]

However, when the compound (I) is measured by these methods, it is always satisfactory in terms of sensitivity (10 ng / ml) in HPLC and rapidity in GC-MS. Not a thing. Therefore, it has been desired to develop a method with high sensitivity and capable of rapidly measuring many samples.

[0004]

The present invention provides compound (I)
This method was completed as a result of intensive research on this method, focusing on immunological measurement methods. Generally, a low molecular weight compound such as compound (I) in the present invention is called a hapten and has antigenicity but no immunogenicity. Therefore, in order to produce an antibody against these haptens, a method of immunizing an animal after covalently bonding an amino group, a carboxyl group, etc. in the hapten and an immunogenic carrier such as a protein is generally performed. There is. However, with respect to compound (I), even if a protein was directly covalently bonded to its amino group, the amino group of compound (I) was poor in reactivity and a covalent bond with a protein was difficult to obtain. Immunization of animals with covalent conjugates does not yield the preferred antibody.

The present inventors have conducted various studies on the immunological assay method of compound (I), and as a result, a compound obtained by binding a carboxypropyl group or the like to the 5-position of compound (I) was labeled with a protein by a conventional method. It has been found that the covalently bound complex provides a preferred immunogen for raising antibodies in EIA. The present invention has been completed by further research based on such findings.

That is, the present invention provides (A) the following formula (II)

[0007]

[Chemical 3]

An antigen (immunogen) obtained by covalently bonding a compound represented by the formula (n represents an integer of 0-5) and an immunogenic carrier via the carboxyl group of the compound.
And an enzyme obtained by covalently bonding a compound represented by the above formula (II) and an enzyme through a carboxyl group of the compound. For the labeled antigen and (C) the antibody (A) immobilized on a solid phase support, the enzyme-labeled antigen (B) and the formula (I) in the measurement sample are used.

[0009]

[Chemical 4]

The compound represented by [9-amino-2, 3, 5,
6,7,8-hexahydro-1H-cyclopenta (b) quinoline] in a competitive manner, and an enzyme immunoassay method for a compound represented by the formula (I), and (D)
At least an antibody according to (A) and an enzyme-labeled antigen according to (B) are used as essential constituent reagents of the kit, and a kit for enzyme immunoassay of a compound represented by formula (I),
It is about.

The present invention will be described in detail below. Formula (II)
A compound represented by (hereinafter, compound (II) or simply (II)
Is a novel substance, and as shown in the following reaction formula [Chemical formula 5], the cyclohexanone derivative of the formula (III) and the formula (I
It can be produced by reacting 2-amino-1-cyclopentene-1-carbonitrile of V) in the presence of ethyl polyphosphate and hydrolyzing the obtained compound of the formula (V). (JP-A-63-297367) When the cyclohexanone derivative of formula (III) is a novel substance, the substance is 1-pyrrolidino-1-cyclohexene of formula (III-1) and formula (III-2). The compound of formula (III-3) can be produced by reacting the compound of formula (3) with an organic solvent, and water can be added to the compound to react. In the reaction formula, n represents an integer of 0 to 5 and R represents an alkyl group. In the present invention, n is 1 in the compound (II).
Compounds that are integers from -4 are preferred.

[0012]

[Chemical 5]

Examples of immunogenic carriers include proteins, polypeptides and glycoproteins. In particular,
Examples of proteins include bovine serum albumin, human serum albumin, ovalbumin, bovine gamma globulin,
Examples of the polypeptide include polylysine, and examples of the glycoprotein include lipopolysaccharide, keyhole limpet hemocyanin, and the like. Of these, proteins are preferable.

In the present invention, a complex in which compound (II) and an immunogenic carrier are covalently bound is used as an immunogen for producing an antibody. As a method for producing such an immunogen, conventionally used methods, for example, amines such as tri-n-butylamine and alkylchloroforms such as isobutylchloroformate in a solvent such as anhydrous dimethylformamide (DMF). A method of forming a covalent bond between the compound (II) and the immunogenic carrier using a formate (mixed acid anhydride method) is used.

The thus obtained antigen for preparing an antibody for immunochemical assay is used for immunizing a host animal and producing an antibody. Examples of host animals include rabbits, rats, guinea pigs, mice, goats and the like. Immunization of such animals is performed by administering the antigen by parenteral means such as intravenous administration, intradermal administration or subcutaneous administration. For the administration, an ordinary adjuvant (Freund's complete adjuvant or Freund's complete adjuvant) is used.
A method is used in which an emulsion prepared from a lete adjuvant) and an antigen is used, and a small amount is administered, and then the same antigen is administered at regular intervals (usually at regular intervals of 1 week to 1 month). During administration, antibody titers in serum are measured at regular intervals, and whole blood is collected when the highest value is obtained.

Blood can be separated into serum and used as it is as an antiserum (antibody), but in order to enhance the measurement sensitivity of compound (I), ammonium sulfate fractionation, gel filtration, DEAE anion exchange column chromatography, protein A is used. It is preferable to use the antibody purified to IgG by affinity chromatography or the like as the antibody. When this antibody is a powder (freeze-dried product), it is dissolved in water and used as an aqueous solution of the antibody having an appropriate concentration, and when the antibody is an aqueous solution, it is diluted to an appropriate concentration before use. The antibody concentration at this time is usually 0.
It is used at a protein concentration of 01 to 10 μg / ml, preferably 0.05 to 1.0 μg / ml. The thus-obtained antibody is useful as an antibody for EIA or radioimmunoassay of compound (I), since compound (I) can be recognized by distinguishing it from its metabolites or other structurally similar compounds. Is.

When such an antibody is used for EIA, it will be used together with an enzyme-labeled antigen as described later. The enzyme-labeled antigen can be obtained by covalently bonding the compound (II) and an enzyme such as peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase and the like. Among these, in the present invention, a peroxidase, particularly a horseradish peroxidase (HRP) covalently bonded thereto is preferable. As the method for covalently bonding, the above-mentioned method for covalently bonding the immunogenic carrier and the compound (II) is similarly used. The enzyme-labeled antigen of the present invention is a powder (lyophilized product), which is dissolved in water to be used as an aqueous solution having an appropriate concentration.
Usually, it is diluted with water to an appropriate concentration before use.

EIA or EIA of the compound (I) of the present invention
The kit for use essentially comprises an antibody having a specific immunoreactivity to compound (I) and an enzyme-labeled antigen of compound (II). Such an EIA or EIA kit contains these reagents. In addition to the above, a solid phase support, a washing solution, a blocking solution, a compound (I) solution for preparing a calibration curve of compound (I), an enzyme substrate, hydrogen peroxide and the like are also required.

The solid-phase support is necessary as a support for immobilizing an antibody or an antigen. Examples of the shape of such a solid phase support include plates for immunoassays, tubes, beads, membranes, and the like, and examples of the material thereof include polyethylene, polystyrene, polypropylene, nitrocellulose, and glass. I can give you. When immobilizing an antibody on a solid support, the antibody is dissolved in an alkaline solution and brought into contact with (physically) adsorbed on the solid support. The concentration of the antibody at this time is usually 0.1 to 20 μg / ml, preferably 1 to 10 μg / ml.
Used with a protein amount of ml. As the alkaline solution, a sodium carbonate-sodium hydrogen carbonate buffer solution or the like is used.

The washing solution comprises a fixed amount of anti-I (I) on the solid support.
After immobilizing an antibody such as an antibody, those not immobilized on the solid phase support are washed and removed, or the compound (I) in the measurement sample is added to the antibody immobilized on the solid phase support. It is necessary to wash and remove unreacted substances after competitively reacting with the enzyme-labeled antigen. The washing solution is also used as a diluent for the measurement sample and a buffer solution in the assay. As a cleaning liquid used for such a purpose, for example,
Water, buffer solution adjusted to pH at the time of reaction (phosphate buffer solution, phosphate buffered saline (PBS), buffer solution such as Tris-HCl buffer solution), a surfactant such as Tween 20 in the above buffer solution It is possible to cite, for example, those containing 0.01 to 2.0% by volume. Above all, it is preferable to use a buffer solution containing the above-mentioned surfactant in an amount of 0.02 to 0.50% by volume and adjusted to the pH during the reaction.

The blocking solution is necessary to prevent the enzyme-labeled antigen from nonspecifically binding to the surface of the solid support on which the anti- (I) antibody is not immobilized. The blocking solution used for such purpose is BSA (bovine serum albumin),
OVA (ovalbumin), KLH (mussel hemocyanin), γ-globulin, gelatin, and casein can be prepared by dissolving them in the above washing solution, and the concentration of these proteins is 0.5 to 5.0% by weight. It is preferable that If necessary, a protein preservative such as sodium azide can be added to the blocking solution in a required amount.

As the measurement sample, human body fluids such as human urine, serum and plasma as well as animal body fluids such as rats, mice and dogs can be used. In the measurement of compound (I),
These measurement samples are diluted with the above-mentioned washing solution within a range in which compound (I) can be measured, and then measured.

As a substrate for the enzyme, a colorimetric substrate for the enzyme in the enzyme-labeled antigen, for example, when the enzyme labeled with the antigen is horseradish peroxidase (HRP), ortho-
Phenylenediamine (OPD) or 2,2'-aminobis (3
-Ethylbenzothiazoline-6-sulfonic acid) (ABTS) and the like are used. Further, p-hydroxyphenylpropionic acid (HPPA), which is a fluorescent substrate of HRP, or luminol, which is a luminescent substrate, can also be used. Hydrogen peroxide is required for these enzymatic reactions using HRP.

As described above, the solid phase support, the washing solution, the blocking solution, the compound (I) solution for preparing the calibration curve of compound (I), the substrate, and hydrogen peroxide are prepared, and the anti- (I) antibody, By using the enzyme-labeled antigen and the following steps, the compound (I) in the measurement sample can be measured.

(1) Immobilizing a fixed amount of anti- (I) antibody or the like on a solid support: An anti- (I) antibody or an antibody against an anti- (I) antibody (donkey or goat anti-rabbit) on the solid support A second antibody such as IgG) is contacted for a certain period of time. The temperature at the time of contact is not particularly limited as long as the antibody solution does not freeze or boil, but is preferably 4
~ 37 ℃ is good. (2) Step of removing anti- (I) antibody or the like not immobilized on the solid phase support After immobilizing the anti- (I) antibody or the like on the solid phase support, the antibody solution is removed to form a solid phase support. The anti- (I) antibody or the like remaining unimmobilized is removed by washing with a washing solution several times.

(3) Step of preventing non-specific binding of the enzyme-labeled antigen to the surface of the solid support on which the anti- (I) antibody or the like is not immobilized. Solid-phase support to which the anti- (I) antibody or the like is bound. The body is contacted with a certain volume of blocking solution for a certain period of time. The temperature at the time of contact is not particularly limited unless the antibody solution is frozen or boiled,
It is preferably 4 to 37 ° C. (4) A step of competitively reacting the compound (I) in the measurement sample with the enzyme-labeled antigen for the anti- (I) antibody or the like immobilized on the solid support Anti- (I) antibody for the solid support When is immobilized, the measurement sample and the enzyme-labeled antigen are brought into contact with each other simultaneously or sequentially to cause a competitive reaction for a certain period of time. When anti-rabbit IgG is immobilized on the solid support, a measurement sample,
The anti- (I) antibody and the enzyme-labeled antigen are simultaneously or sequentially contacted with each other to cause a competitive reaction for a certain period of time. The temperature during the reaction is not particularly limited as long as the reaction mixture does not freeze or boil, but is preferably 4 to 37 ° C.

(5) Step of removing the enzyme-labeled antigen not bound to the solid phase support through the anti- (I) antibody, etc. The reaction mixture is removed, and the solid phase is removed through the anti- (I) antibody, etc. The measurement sample remaining without being bound to the support, the enzyme-labeled antigen and the like are removed by washing several times with a washing solution. (6) A step of reacting an enzyme-labeled antigen bound to a solid phase support through an anti- (I) antibody, etc., with an enzyme substrate and hydrogen peroxide. I) React with an enzyme-labeled antigen bound to a solid support through an antibody or the like for a certain period of time. Preferably, the enzymatic reaction should then be stopped with an acid such as sulfuric acid, an alkali such as sodium hydroxide or an enzyme inhibitor such as sodium azide. The reaction temperature is not particularly problematic as long as it is within the optimum temperature range of the enzyme used at that time, but preferably 15 to 37 ° C.
Is good.

(7) Absorbance of the reaction solution after the enzyme reaction,
Step of measuring fluorescence intensity or luminescence intensity The absorbance, fluorescence intensity or luminescence intensity of the reaction solution is measured at the wavelength showing the maximum absorbance of coloration of the reaction solution after the enzyme reaction or at the maximum excitation wavelength. In addition, in the present measurement method, the calibration curve is prepared in advance by using the operations of the above-mentioned respective steps, and a calibration curve is prepared from the measurement result using a known amount of compound (I) instead of the measurement sample. By comparing with, the concentration of compound (I) in the measurement sample can be known. As described in (4) above, a competitive reaction occurs between the enzyme-labeled antigen and the compound (I) in the measurement sample for the anti- (I) antibody, whereby the amount of compound (I) in the sample can be rapidly and increased. The sensitivity can be measured.

[0029]

EXAMPLES Hereinafter, the present invention will be specifically described by showing Reference Examples and Examples of the present invention. It should be noted that these examples are for illustrating the present invention and do not limit the scope of the present invention.

[Reference Example 1] 4- [9-amino-2,3,5,6,7,8-hexahydro-1
Synthesis of H-cyclopenta (b) quinolin-5-yl] butyric acid [compound (VI)] i) Synthesis of ethyl-2-cyclohexenone butyrate 1-Pyrrolidino 1-cyclohexenone 25.0 g, 4-bromobutyric acid in 70 ml of ethanol Ethyl ester 3
3.6 g was added, the mixture was refluxed for 24 hours, 60 ml of water was further added, and the mixture was refluxed for 5 hours. After the reaction was completed, add 60 ml of water and add ether.
Extracted twice. The ether layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was purified by vacuum distillation. Yield: 5.1g (15%) Boiling point: 136-141 ℃ / 3mmHg

Ii) Synthesis of 4- [9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinolin-5-yl] butyric acid ethyl ester 57 g of ethyl polyphosphate was dissolved in 50 ml of anhydrous chloroform, To this was added 5.07 g (23.9 mM) of ethyl-2-cyclohexenone butyrate and 2.58 g (23.9 mM) of 2-amino-1-cyclopentene-1-carbonitrile obtained in i), and the mixture was added at 55 ° C.
The reaction was carried out for 10 hours. After cooling, the mixture was added dropwise to 28% ammonia water containing ice to make it alkaline, and extracted twice with chloroform. The chloroform layer was washed with water, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was crystallized from ethyl acetate to give 3.28 g of crude crystals. Also, from the mother liquor,
Silica gel column chromatography gave 1.50 g of crude crystals. These were combined and 4.20 g (58.0%) of white powdery crystals were obtained from an ethyl acetate-ether mixed solution. Melting point: 112-114 ° C

Iii) 4- [9-amino-2,3,5,6,7,8
-Hexahydro-1H-cyclopenta (b) quinoline-5
Synthesis of butyl] butyric acid [compound (VI)] 4- [9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinolin-5-yl] obtained in ii)
Butyric acid ethyl ester compound 0.337g (1.10m
13 ml of 0.1N sodium hydroxide was added to M), and the mixture was heated and reacted at 90 ° C. for 2 hours. After cooling, the mixture was made acidic with 15 ml of 0.1N hydrochloric acid, a small amount of saturated saline was added, and the mixture was washed with chloroform. The crystals precipitated from the aqueous layer were collected by filtration and washed with a small amount of water. The crude crystals were recrystallized from water to obtain 0.21 g (68.6%) of white powdery crystals.

Melting point: 242.0-243.5 ° C. (decomposition) IR: νKBrmaxcm ^-1 3400, 3350 (NH ₂ ) 3000-2500 (OH) 1710 (C = O) ¹ H-NMR: CDCl ₃ , TMS, PPM 1.51-1.75 (6H, m) 2.05 to 2.12 (2H, m) 2.18 to 2.29 (3H, m) 2.41 to 2.49 (3H, m) 2.68 to 2.72 (2H, t) 2.85 (1H, br) 2.95 to 2.99 (2H, t) ) 7.43 (2H, br) ¹³ C-NMR: CDCl ₃ , TMS, PPM 174.2 (C = O) 154.3, 153.0 (pyridine ring) 149.0 (pyridine ring) 118.8, 113.6 (pyridine ring) 35.1, 33.5, 33.2, 30.7 , 27.6, 23.8, 22.2, 22.0, 21.8, 16.6
_{(CH 2) MS: m /} Z 246 (M +)

[0033]

Reference Example 2 Synthesis of 2- [9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinolin-5-yl] acetec acid [compound (VII)] i) 2- [9-amino-2 Synthesis of 3,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinolin-5-yl] acetec acid ethyl ester 40 g of ethyl polyphosphate was dissolved in 100 ml of anhydrous chloroform, and ethyl-2-cyclohexenone acetate 5.
63 g (30.6 mM) and 2-amino-1-cyclopentene-1-carbonitrile (3.25 g, 30.1 mM) were added, and the mixture was reacted at 40 ° C. for 20 hours. After cooling, the mixture was added dropwise to 28% ammonia water containing ice to make it alkaline, and extracted twice with chloroform.
The chloroform layer was washed with water, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was crystallized with ethyl acetate to obtain 10.55 g of crude crystals. Recrystallize with ethyl acetate, 6.
312 g (yield 76.5%) of white powdery crystals were obtained. Melting point: 11
7-120 ° C

Ii) Synthesis of 2- [9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinolin-5-yl] acetec acid [compound (VII)] 9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinolin-5-yl] acetec acid ethyl ester (500mg, 1.82mM) was added with 1N sodium hydroxide (2.3ml) and heated at 90 ° C for 4 hours. It was made to react. After cooling, it was neutralized with 2.3 ml of 1N hydrochloric acid, and the precipitated crystals were collected by filtration and washed with water and ethyl acetate to give 407 mg (yield 91.1
%) White powdery crystals were obtained.

IR: νKBrmaxcm ^-1 3375, 3200 (NH) 2650 (NH ⁺ ) 1670, 1625 (C = N, C = C) 1565,
1390 (COO ^{-) 1} H over NMR: DMSO over _{d 6, PPM 1.34~1.46 (1H,} m) 1.55~1.69 (1H, m,) 1.85~1.98 (2H, m) 2.02~2.12 (2H, dt,) 2.22-2.35 (2H, m) 2.40-2.50 (1H, m) 2.58-2.66 (1H, m) 2.69 (2H, t, J = 7.1Hz) 2.83 (2H, t, J = 7.3Hz) 2.96-3.09 ( 1H, m) ¹³ C-NMR: DMSO-d ₆ , PPM 35.8 (CH) 43.4 (CH ₂ ) 112.9, 117.7, 151.2, 153.5, 156.4 (pyridine ring) 174.3 (COOH) 20.8, 22.1, 23.0, 27.4, 30.3 , 32.0 (CH ₂ )

[0036]

Example 1 Preparation of Immunogen and Antigen for Measuring Antibody Titer i) Compound (VI) (21 mg) in a molar amount about 50 times that of bovine serum albumin (BSA, manufactured by Sigma) (100 mg) which is an immunogenic carrier ) Is dissolved in dry dimethylformamide (DMF), tri-n-butylamine is added in a double molar amount (60 μl), and after cooling to -5 ° C. or lower, an equimolar amount of isobutyl chloroformate (30 μl) is added, -5 The reaction was carried out at a temperature of ℃ or below for 5 minutes. This is ice-cooled above B
It was added to the SA solution all at once. Further, the solution was stirred under ice cooling for 6 hours while adjusting the pH of the solution to around 9.0 with 1N sodium hydroxide. Further, it was left in a cool dark place (4 ° C.) for 12 hours, dialyzed with running water for 12 hours, and the pH of the inner solution was adjusted to 4.5 with 1N hydrochloric acid to obtain a cloudy precipitate. After adding water to this and suspending it, a small amount of saturated aqueous sodium hydrogen carbonate solution was added to dissolve it,
After dialyzing against running water for 6 hours, freeze-drying the compound (VI) and BSA
Immunogen [Compound (VI) -BSA complex]
Got The yield was 111.9 mg, and the binding ratio of compound (VI) per mol of BSA was about 6 mol.

Ii) 100 mg of the compound (VII) obtained in Reference Example 2
Was dissolved in dry DMF to prepare an immunogen [compound (VII) -BSA complex] by the same method as i). The yield in the case of compound (VII) was 62.6 mg, and the binding ratio per mol of BSA was about 18
It was a mole.

Iii) An antibody titer-determining antigen [Compound (VI) -OVA complex] treated in the same manner as in i) except that ovalbumin (OVA, manufactured by Sigma) is used instead of BSA as an immunogenic carrier.
Was prepared. The yield based on 21 mg of compound (VI) was 62.9 mg, and the binding ratio of compound (VI) per mol of OVA was about 2 mol.

[0039]

Example 2 Preparation of Antibody [Anti- (I) Antibody] i) Immunization of Rabbits 2 mg of compound (VI) -BSA complex or 2 mg of compound (VII) -BSA complex were respectively added to Freund's complete adjuvant.
(FCA, DIFCO Laboratories) 0.5 ml and Japanese Pharmacopoeia physiological saline solution (Fuso Yakuhin Kogyo) 0.5 ml were sufficiently suspended and administered to more than 20 sites on the back skin of rabbits (male New Zealand White) And immunized. For both complexes, 5 rabbits were boosted once a month after the first immunization by measuring the antibody titer by partial blood sampling.

Ii) Measurement of antibody titer The antibody titer of the antiserum collected at the time of each partial blood sampling is the compound (V
I) -OVA complex by ELISA (Enzyme-linked immunosorben
It was used as an adsorbed antigen in the t assay). 96-hole flat bottom ELISA plate (Non-static-charge Microplate 9
Compound (VI) -OV in each well of 6F-RN, Biotech
The A-complex was added and left overnight at 4 ° C to solidify, then the washing solution (0.
After washing with 05% Tween 20-phosphate buffer) 0.5% BSA-0.05%
Tween 20-phosphate buffer was left overnight at 4 ° C for blocking. After washing with the same washing solution, antiserum diluted 2-fold serially was added to each well and left at room temperature for 2 hours. After washing with the same washing solution, HRP-labeled anti-rabbit IgG goat IgG (manufactured by YE Laboratories) was added, and the mixture was allowed to stand at room temperature for 1 hour. After washing with the same washing solution, color was developed with ortho-phenylenediamine-0.01% hydrogen peroxide at room temperature for 15 minutes, and the enzymatic reaction was stopped with 0.1% sodium sulfite-1N sulfuric acid. Immediately after that, the absorbance at 492 nm of each well was measured, and the highest dilution factor at which the absorbance of each antiserum was at least twice that of the control normal untreated rabbit serum was taken as the antibody titer of the antiserum.

Iii) Evaluation of antiserum Immunization was carried out according to Example 2-i), and 8 times for the compound (VI) -BSA complex and 11 times for the compound (VII) -BSA complex.
Whole blood was collected after the second immunization. In order to select an antiserum to be used for EIA, an antiserum purified into IgG according to individual rabbits according to Example 2-ii) is used to prepare a compound (I) and a compound that is a metabolite preparation of compound (I). (VIII), (IX),
(X) (Japanese Patent Laid-Open No. 3-275672) was measured, and the antiserum was evaluated at a concentration that competitively inhibits each 50%. These metabolite preparations are shown in Structural Formulas (VIII) to (X), and the evaluation results are shown in Table 1.

[0042]

[Chemical 6]

[0043]

[Table 1]

The 50% competitive inhibitory concentration of Compound (I) was obtained by immunization with Compound (VI) -BSA complex (Lot.
B-5) is 0.1-0.3 ng / ml, whereas the antibody (Lot.A-1, obtained by immunizing with the compound (VII) -BSA complex is
In 2, 3, 5), it was 0.3-0.7 ng / ml. In addition, the cross-reactivity rates with metabolite preparations varied from 0.2 to 4.0% and 0.3 to 5.7% for compounds (VIII) and (IX), which are monohydroxides, and to compounds that are one of the dihydroxides. 0.2% for (X)
It was below. From these 9 antibodies, the compound (I)
Lot.B-1, B-3 and B-4 were selected from the 50% competitive inhibitory concentration against E.
It was decided to use it for IA. The mixed antibodies are believed to have minimal effect of cross-reactivity on the accurate quantification of compound (I). Thus, the antibody is a reagent that distinguishes Compound (I) from closely related metabolites and provides accurate quantitation of Compound (I).

Iv) Purification of antiserum Among the antisera obtained by immunizing with the compound (VI) -BSA complex, 264 ml of the antisera of rabbit Nos. 1, 3, and 4 were subjected to ammonium sulfate fractionation and DEAE anion. Purification by exchange column chromatography was performed. 264 ml of 50 mM phosphate buffered saline (PBS) (pH 7.0) is added to 264 ml of antiserum. 203.3 g of solid ammonium sulfate was gradually added with stirring (50% saturation), and the mixture was slowly stirred for 20 minutes and then centrifuged at 8000 rpm for 20 minutes. The obtained precipitate is dissolved in PBS to make the total volume 50 ml. 33 ml of saturated ammonium sulfate was added with stirring (40% saturation), and the mixture was stirred for 20 minutes and then centrifuged. The precipitate is again dissolved in PBS to make the total volume 50 ml, 12.5 ml of saturated ammonium sulfate is added (20% saturation), and the mixture is stirred for 20 minutes. The precipitate was removed by centrifugation and the supernatant was taken, and 20.5 ml of saturated ammonium sulfate was added (40% saturation). Dissolve the precipitate in 25 mM PBS (pH 8.0),
It was dialyzed against the same buffer. The sample after dialysis is centrifuged at 8,000 rpm for 10 minutes, and the supernatant is charged on a column packed with DE-52 (manufactured by Whatman) equilibrated with 25 mM PBS (pH 8.0) and eluted with 25 mM PBS. The flow-through fraction is collected and 280n
The absorbance at m was measured. Fractions in which highly pure IgG was fractionated were collected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Yield: IgG fraction 2811 mg.

[0046]

Example 3 Preparation of Enzyme-Labeled Antigen Horseradish peroxidase (HRP) was used as a labeling enzyme.
Compound (VI) or compound (VII) by the mixed acid anhydride method
A covalent bond (complex) with was prepared. That is, compound (VI) 2.8 mg (0.01 mM) or compound (VII) 6.7 mg (0.024 m)
M) was dissolved in 500 μl of anhydrous dimethylformamide (DMF),
20 μl (0.001 mM) of tri-n-butylamine and 10 μl (0.0005 mM) of isobutyl chloroformate were added, and the mixture was stirred under ice cooling for 5 minutes. HRP (TOYOBO Corporation) 20mg (0.0005mM)
Was added, and the mixture was reacted for 6 hours under ice cooling while adjusting the pH to 9.0.
After the reaction, dialysis against 50 mM PBS for about 17 hours, and then Toyopearl HW55F
Fractionation and purification were performed by gel chromatography using (TOSOH Co., Ltd.). The immunoreactivity of the fractions at which absorption was observed at 415, 260, and 280 nm was examined, and the fraction with the highest activity was used as the enzyme-labeled antigen [compound (VI) -HRP-labeled antigen 1.3 ml and compound (VII) -HRP-labeled antigen 1.3 ml. ml].

[0047]

Example 4 Assay Method i) Procedure for EIA Using Enzyme-Labeled Antigen Secondary antibody (goat anti-rabbit IgG, H + L chain, Kirkegaard & Perry Laboratories) 0.015M sodium carbonate-0.035M sodium bicarbonate buffer Dissolve the solution (pH 9.7) to 10 μg / ml, and dissolve this in a 96-well microtiter plate (Mxiso
rb F96, manufactured by Nunc) (250 μl)
It was left overnight at 0 ° C. The second antibody was discarded, and 300 μl of Block Ace (blocking solution, Snow Brand Milk Products Co., Ltd.) diluted 4-fold was put into each well to stand at 4 ° C. overnight for blocking. After discarding the blocking solution, washing 4 times with a washing solution (0.05% Tween 20-10 mM PBS), 100 μl of the compound (I) standard solution or measurement sample, anti- (I) antibody (IgG, 0.25 μg / ml)
50 µl of compound (VI) HRP-labeled antigen and 50 µl of buffer solution (having the same composition as the washing solution) were added and reacted overnight at 4 ° C. After washing, 0.128% 2,2'-aminobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABT
S, Boehringer Mannheim Yamanouchi) 100 μl, 0.01%
50 μl of hydrogen peroxide and 100 μl of buffer solution (having the same composition as the washing solution) were added, and the mixture was reacted at room temperature for 2 hours. Then
Stop the reaction by adding 50 μl of 0.1% sodium azide.
The absorbance was measured at 5 nm.

Ii) Preparation of calibration curve using EIA of the present invention A calibration curve for measuring the concentration of compound (I) in human serum was prepared based on the above-mentioned example. From this calibration curve, the measurable range of EIA of the present invention is 10 pg / ml to 10 ng / ml. The calibration curve is shown in FIG.

[0049]

Example 5 Preparation of Compound (I) Measuring Kit Each reagent was prepared by the method shown below to prepare a compound (I) measuring kit. i) Enzyme-labeled antigen The enzyme-labeled antigen [Compound (VI) -HRP-labeled antigen] obtained in Example 3 was diluted 2-fold with 0.2% BSA-10 mM PBS, and 10 µl of the diluted enzyme was dispensed into a 5 ml polystyrene tube. Labeled antigen was prepared. This product should be diluted before use. ii) Anti- (I) antibody 5 mg of anti- (I) antibody (IgG fraction) obtained in Example 2-iv) was used.
An anti- (I) antibody was prepared by dispensing 10 μl each into a ml polystyrene tube. This product should be diluted before use. iii) Second antibody-immobilized carrier Rabbits were prepared in the same manner as described in the operation procedure of the measurement method above.
A goat second antibody against IgG was adsorbed on a polystyrene microtiter plate to prepare a second antibody-immobilized carrier. This product is used after the blocking operation shown in Example 4-i) at the time of use. iv) Standard solution of compound (I) A standard solution of compound (I) was prepared by dissolving 10% of compound (I) dissolved in distilled water to a concentration of 1 mg / ml into a 5 ml polystyrene tube. This product should be diluted before use.

[0050]

INDUSTRIAL APPLICABILITY The covalent conjugate (complex) of the compound (II) and the immunogenic carrier according to the present invention efficiently produces an antibody having a high affinity and a high titer for the compound (I). . Further, the enzyme-labeled antigen of compound (II) reacts with the antibody in a competitive manner with compound (I) in the biological fluid. Therefore, according to the present invention, the concentration of compound (I) in a sample can be measured with extremely high sensitivity and reproducibility, and the operation method is simple, and a large number of samples can be processed quickly. can do. Furthermore, in the present invention, an enzyme-labeled antigen,
A reagent composed of an antibody or the like can be dispensed and stored, and is also useful as a kit for measuring compound (I).

[0051]

[Brief description of drawings]

FIG. 1 shows a calibration curve when compound (I) is added to human serum.

Claims (9)

[Claims] 1. The following formula (II): (In the formula, n represents an integer of 0-5.) An antigen obtained by covalently bonding a compound represented by the formula (5) and an immunogenic carrier via a carboxyl group of the compound is parenterally administered to animals. An antibody obtained by administration in. 2. The antibody according to claim 1, wherein the compound represented by the above formula (II) is a compound in which n in the formula represents an integer of 1-4. 3. The antibody according to claim 1, wherein the immunogenic carrier is one selected from the group consisting of proteins, polypeptides and glycoproteins. 4. The immunogenic carrier is a protein.
The described antibody. 5. An enzyme-labeled antigen obtained by covalently bonding a compound represented by the formula (II) of claim 1 and an enzyme through a carboxyl group of the compound. 6. The enzyme is one selected from the group consisting of peroxidase, alkaline phosphatase, glucose oxidase and β-D-galactosidase.
The enzyme-labeled antigen as described. 7. The enzyme-labeled antigen according to claim 5, wherein the enzyme is peroxidase. 8. The enzyme according to claim 5, claim 6, or claim 7 against the antibody according to claim 1, claim 2, claim 3, or claim 4 immobilized on a solid support. Formula (I) in the labeled antigen and the measurement sample A compound represented by the formula (I), which is characterized by reacting 9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinoline represented by Enzyme immunoassay. 9. At least the antibody according to claim 1, claim 2, claim 3 or claim 4 and the enzyme-labeled antigen according to claim 5, claim 6 or claim 7 as an essential constituent reagent of the kit. A kit for enzyme immunoassay of a compound represented by formula (I), which comprises: