JPH07209296A - Antibody, enzyme-labeled antigen and method and kit for enzyme immunoassay - Google Patents

Abstract

PURPOSE:To realize a high sensitivity quick measurement by covalent-bonding a compound represented by a specified formula and an immunogenic carrier through a carboxyl group thereof. CONSTITUTION:A compound shown by formula II (n is an integer of 0 to 5) is produced by causing reaction between a cyclohexanone derivative and 2- amino-1-cyclopentane-1-carbonitrile in the presence of ethyl polyphosphate and subjecting a compound thus produced to hydrolysis. A protein, polypeptide or sugar protein, is employed as the immunogenic carrier. They are covalent- bonded through a carboxyl group to produce a composite which is employed as an antigen generating antibody. An animal is dispensed by intravenous, hypodermic or intranasal injection. After dosing with small quantity of antigen, the same antigen is given at a predetermined interval and the whole blood is sampled when the value of antibody in the blood serum is maximized. The whole blood is purified by separating the blood serum and used as an antibody thus measuring the quantity of compound shown by formula I in a sample quickly with high sensitivity.

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 an antigen and an enzyme-labeled antibody used therein.

[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 an immunological measurement method. 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 was found that the complex obtained by covalently binding is a preferred antigen (immunogen) for producing an antibody in EIA. The present invention is
It was completed by further research based on such findings.

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

[0007]

[Chemical 3]

An antigen obtained by covalently bonding a compound represented by the formula (n is an integer of 0-5) and an immunogenic carrier via the carboxyl group of the compound [compound (II)] -Immunogenic carrier complex], and (B) an enzyme-labeled antibody obtained by labeling an enzyme with an antibody obtained by administering the above antigen to an animal by a parenteral means, and (C)
For the enzyme-labeled antibody described in (B), an antigen (measurement antigen) immobilized on a solid support, preferably the antigen described in (A) and the formula (I) in the measurement sample.

[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 antigen (A) and an enzyme-labeled antibody (B), which are immobilized on a solid support, are used as essential constituent reagents of the kit. The present invention relates to an enzyme immunoassay kit.

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 0-5 and R represents an alkyl group. In the present invention, n is 1 in the compound (II).
Compounds that are an integer of -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 bonded is used as an EIA antigen and as an immunogen for producing an antibody. The method for producing such an antigen (immunogen) is a conventionally used method, for example, amines such as tri-n-butylamine and isobutyl chloroformate in a solvent such as anhydrous dimethylformamide (DMF). The method of forming a covalent bond between the compound (II) and the immunogenic carrier using the alkyl chloroformate of (a mixed acid anhydride method) is used.

The thus obtained antigen (immunogen) 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 administration, the usual adjuvant (Freund's complete adjuvant Freun
d's complete adjuvant or Freund's incomplete adjuvant)
A method is used in which an emulsion is prepared from and an antigen, and a small amount is administered, and then the same antigen is administered at regular intervals (usually at regular intervals of one week to one month). During administration,
The antibody titer in serum is measured at regular intervals, and when the highest value is obtained, whole blood is collected.

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. The obtained antibody is
An enzyme-labeled antibody can be obtained by labeling with an enzyme such as peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase and the like. Of these, those labeled with peroxidase, particularly horseradish peroxidase (HRP), are preferred in the present invention. As a method of performing enzyme labeling, a method of labeling an enzyme with an antibody conventionally, for example, a periodate method using sodium periodate or an antibody is Fab 'and a maleimide group is introduced into the antibody before enzyme labeling is performed. The maleimide method or the like is used. When the enzyme-labeled antibody is a powder (freeze-dried product), it is dissolved in water to be used as an enzyme-labeled antibody aqueous solution having an appropriate concentration, and when it is an aqueous solution, it is diluted to an appropriate concentration before use. At this time, the enzyme-labeled antibody concentration is usually 0.05-
It is used at a protein concentration of 10 μg / ml, preferably 0.1-3.0 μg / ml. The enzyme-labeled antibody thus obtained can recognize Compound (I) by distinguishing it from its metabolites or other structurally similar compounds, and thus is useful as an antibody for EIA of Compound (I). is there.

In the present invention, when the enzyme-labeled antibody is used for EIA, it is used together with the antigen. Compounds (I) and (II) are used as the antigen (antigen for measurement) at this time.
Alternatively, a compound (II) -immunogenic carrier complex or the like can be used, but in the present invention, it is preferable to use a compound (II) -immunogenic carrier complex. The antigen of the present invention is used as a powder (lyophilized product) by dissolving it in water as an aqueous solution of the antigen having an appropriate concentration, and in the case of an aqueous solution, it is diluted to an appropriate concentration before use.

EIA or EIA of the compound (I) of the present invention
The kit for use essentially comprises an enzyme-labeled antibody having a specific immunoreactivity for compound (I) and the above-mentioned antigen. The EIA or kit for EIA contains these reagents in addition to these reagents. Also required are 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.

The solid support is necessary as a support for immobilizing an antigen. Examples of the shape of such solid support include, for example, plates for immunoassay,
Examples thereof include tubes, beads, and membranes, and examples of the material thereof include polyethylene, polystyrene, polypropylene, nitrocellulose, and glass. When immobilizing an antigen on a solid support,
The antigen is dissolved in an alkaline solution, brought into contact with the solid support (physically) and adsorbed. The concentration of the antigen at this time is
A sufficient excess of the concentration of compound (I) to be measured is required. As the alkaline solution, a sodium carbonate-sodium hydrogen carbonate buffer solution or the like is used.

The washing solution is prepared by immobilizing a fixed amount of the antigen on the solid phase support and then washing and removing those not immobilized on the solid phase support, or by removing the enzyme-labeled antibody from the solid phase support. It is necessary in order to competitively react the antigen immobilized on and the compound (I) in the measurement sample and then wash and remove the unreacted material. The washing solution is also used as a diluent for the measurement sample and a buffer solution in the assay. As the washing solution used for such purpose, for example, water, a buffer solution adjusted to pH during the reaction (phosphate buffer solution, phosphate buffered saline (PBS), buffer solution such as Tris-hydrochloric acid buffer solution), The buffer solution may include 0.01 to 2.0% by volume of a surfactant such as Tween 20. 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 antibody from nonspecifically binding to the surface of the solid support on which the antigen is not immobilized. Blocking solutions used for such purposes include BSA (bovine serum albumin), OVA (ovalbumin), KLH (Kasakai hemocyanin), γ-globulin,
It can be prepared by dissolving proteins such as gelatin and casein in the above-mentioned washing solution, but the concentration of these proteins is preferably 0.5 to 5.0% by weight. 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),
The measurement is carried out by diluting these measurement samples with the above-mentioned washing solution within a range in which compound (I) can be measured.

The enzyme substrate is a colorimetric substrate of the enzyme in the enzyme-labeled antibody, for example, ortho-phenylenediamine (OPD) or 2,2 when the antibody-labeled enzyme is horseradish peroxidase (HRP). '-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 were prepared, and the antigen and the enzyme-labeled antibody were prepared. The compound (I) in the measurement sample can be measured by using each of the following steps.

(1) Immobilizing a fixed amount of antigen on the solid phase support The compound (II) -immunogenic carrier complex or the like is contacted with the solid phase support for a fixed time. The temperature at the time of contact is not particularly limited as long as the antigen solution is not frozen or boiled, but preferably 4 to
37 ℃ is good. (2) Step of removing the antigen that is not immobilized on the solid phase support After immobilizing the antigen on the solid phase support, the antigen solution is removed and the antigen that remains unimmobilized on the solid phase support Are removed by washing several times with a wash solution.

(3) Step of preventing non-specific binding of the enzyme-labeled antibody [enzyme-labeled anti- (I) antibody] to the surface of the solid support on which the compound (I) is not immobilized The antigen is immobilized. A fixed volume of blocking solution is contacted with the solid support for a certain period of time. The temperature at the time of contact is not particularly limited as long as the antigen solution is not frozen or boiled, but preferably 4 to 37 ° C. (4) Step of Competitively Reacting Enzyme-Labeled Antibody with Antigen Immobilized on Solid Support and Compound (I) in Measurement Sample The compound (I) and the enzyme-labeled antibody are brought into contact with each other at the same time or sequentially to cause a competitive reaction for a certain time. The temperature during the reaction is
The reaction mixture is not particularly limited as long as it is not frozen or boiled, but preferably 4 to 37 ° C.

(5) Step of removing the enzyme-labeled antibody that is not bound to the solid phase support through the antigen The reaction mixture is removed, and remains without being bound to the solid phase support through the antigen. Remove the existing measurement sample and enzyme-labeled antibody by washing several times with a washing solution. (6) A step of reacting an enzyme-labeled antibody bound to a solid-phase support via an antigen with an enzyme substrate and hydrogen peroxide. A fixed amount of the enzyme substrate and hydrogen peroxide is solid-phase supported via the antigen. The enzyme-labeled antibody bound to the body is reacted 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 limited as long as it is within the optimum temperature range of the enzyme used at that time, but preferably 15 to 37 ° C.

(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 the present measurement method, a calibration curve was previously prepared from the measurement results using the above-mentioned steps and using a known amount of compound (I) instead of the measurement sample, and the measurement result of the measurement sample was obtained. 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 antigen immobilized on the solid support and the compound (I) in the measurement sample for the enzyme-labeled antibody, so that the compound (I) in the sample The amount can be measured quickly and with high sensitivity.

[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] 2- [9-amino-2,3,5,6,7,8-hexahydro-1
Synthesis of H-cyclopenta (b) quinolin-5-yl] acetec acid [compound (VII)] i) 2- [9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta (b) quinolin-5-yl] acetec Synthesis of 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 (CH2)

[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 ) Was dissolved in dry dimethylformamide (DMF), and tri-n-butylamine was added in a 2-fold molar amount (60 μl), and after cooling to -5 ° C. or lower,
An equimolar amount of isobutyl chloroformate (30 μl) was added, and the mixture was reacted at −5 ° C. or lower for 5 minutes. The above BSA chilled with ice
Added to the 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 the suspension to suspend it, add a small amount of saturated aqueous sodium hydrogen carbonate solution to dissolve it, dialyz it against running water for 6 hours, and lyophilize it to obtain an immunogen, which is a covalent conjugate of compound (VI) and BSA. [Compound (VI) -BSA complex] was obtained. 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 immunosorbent
It was used as an adsorbed antigen in the 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 Enzyme-labeled antibody [HRP-labeled anti- (I) antibody]
Preparation of IgG The IgG obtained in Example 2-iv) is labeled with horseradish peroxidase (HRP) using the periodate method shown below. That is, 4 mg of HRP (TOYOBO Corporation) is dissolved in distilled water, 0.2 ml of 0.1 M sodium periodate solution is added, and the mixture is stirred at room temperature for 20 minutes. This was added to 1 mM acetate buffer (pH 4.
Dialyze against 5) overnight at 4 ° C. 0.2M carbonate buffer (pH 9.5)
0.02 ml was added to adjust the pH of the dialyzed solution to pH 9.0 to 9.5, 1 ml of an anti- (I) IgG solution (8 mg / ml) was immediately added, and the mixture was stirred at room temperature for 2 hours. While stirring under ice cooling, gradually add 0.1 ml of 0.4% sodium borohydride aqueous solution and stir at 4 ° C.
Leave it for 2 hours. After dialysis against 50 mM PBS, HRP-bound antibody and unbound antibody were separated and purified using gel filtration column chromatography (Sephadex G-200), and the enzyme-labeled antibody labeled with HRP [HRP-labeled anti- (I ) Antibody].

[0047]

Example 4 Assay method i) Procedure of EIA using enzyme-labeled antibody 1 μg / minute of the compound (VI) -OVA complex obtained in Example 1-iii)
The solution was dissolved in 20 mM PBS (pH 7.0) so that the amount of the solution became ml, and a solid phase antigen solution was prepared. About 0.5 ml of this solution was dipped with 1 polystyrene bead (1/4 inch, Wako Pure Chemical Industries, Ltd.) and reacted overnight at 4 ° C. while shaking with a shaker. Cleaning liquid
After washing with (0.05% Tween 20-10 mM PBS), add about 0.5 ml of 0.5% gelatin-Tween 20-PBS per bead,
Blocking was performed by shaking overnight at 4 ° C. 250 μl of standard solution or measurement sample of Compound (I) in polystyrene tube (Eiken Tube No. 1), HRP-labeled anti- (I) antibody 250 μl
1 and the above-mentioned antigen-immobilized beads were added, and the mixture was reacted at room temperature for 2 hours while shaking. The reaction solution in the tube was removed by suction and washed 5 times with 3 ml of the washing solution. Transfer the beads to another tube, and use the enzyme substrate solution [ortho-phenylenediamine (OPD) and 0.0034% hydrogen peroxide mixture] 500
μl was added, and the mixture was reacted at room temperature for 30 minutes with gentle shaking. Then, 2 ml of 1N sodium hydroxide was added to stop the reaction, and the absorbance was measured at 492 nm using the reagent blank as a control.

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 examples. From this calibration curve, E of the present invention
The measurable range of IA is 10 pg / ml to 10 ng / ml. The calibration curve is shown in FIG.

Iii) Comparison with other measuring methods In urine (I) of three healthy subjects who were orally administered with compound (I)
The excretion rate was compared between the method of the present invention and the conventional HPLC method. The results are shown in Table 2. There was a high correlation between the two.

[0050]

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 antibody The enzyme-labeled antibody solution obtained in Example 3 was dispensed into polypropylene containers in an amount of 30 ml to prepare an enzyme-labeled antibody for a kit. This product should be diluted before use. ii) Antigen immobilized on solid phase support (antigen-immobilized carrier) Compound (VI) -OVA complex was immobilized on 100 polystyrene beads according to the procedure of EIA of Example 4, and blocked at 1 mg / Immerse in Tween 20-PBS containing ml gelatin and store at 4 ℃ until use. iii) Standard solution of compound (I) A standard solution of compound (I) was prepared by dissolving 10 μl 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. iv) Enzyme substrate Ortho-phenylenediamine 30 mg was added to 50 mM disodium hydrogen phosphate / 12 water (Na2HPO4 / 12H2O) -24 mM citrate buffer (pH
5.0) What was melt | dissolved in 60 ml was freeze-dried. When this product is used 60
Dissolve in ml of purified water and add 120 μl of 1.7% hydrogen peroxide.

[0051]

INDUSTRIAL APPLICABILITY The antigen according to the present invention, that is, the compound (I
The covalent conjugate (complex) of I) and the immunogenic carrier efficiently produces an antibody having high affinity and high titer for compound (I). Further, in the present invention, the antigen (measurement antigen) immobilized on the solid phase support and the compound (I) in the biological fluid competitively react with the enzyme-labeled antibody. 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. Further, in the present invention, a reagent comprising an antigen, an enzyme-labeled antibody and the like can be dispensed and stored, which is also useful as a kit for measuring compound (I).

[0052]

[Brief description of drawings]

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

[Table 2]

Claims (11)

[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 and an immunogenic carrier via the carboxyl group of the compound. 2. The antigen 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 antigen 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 antigen. 5. An enzyme-labeled antibody obtained by labeling an enzyme obtained by administering the antigen according to claim 1, claim 2, claim 3 or claim 4 to an animal by a parenteral means. 6. The enzyme is one selected from the group consisting of peroxidase, alkaline phosphatase, glucose oxidase and β-D-galactosidase.
The enzyme-labeled antibody described. 7. The enzyme-labeled antibody according to claim 5, wherein the enzyme is peroxidase. 8. The enzyme (antibody for measurement) immobilized on a solid support and the formula (I) in the measurement sample for the enzyme-labeled antibody according to claim 5, 6, or 7. 2] 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. An antigen immobilized on a solid support (antigen for measurement), a compound represented by the formula (II) or a compound represented by the formula (I), or claim 1, or 2, The enzyme immunoassay method according to claim 8, which is the antigen according to claim 3 or 4. 10. The enzyme immunoassay method according to claim 8, wherein the antigen (antigen for measurement) immobilized on the solid support is the antigen according to claim 1, claim 2, claim 3 or claim 4. . 11. An antigen according to claim 1, claim 2, claim 3 or claim 4, and an enzyme label according to claim 5, claim 6 or claim 7, which is immobilized on a solid support. An enzyme immunoassay kit for a compound represented by formula (I), which comprises an antibody as an essential constituent reagent of the kit.