JP3508563B2 - Pyrazine derivative-recognizing antibody and method for measuring 1,2-dicarbonyl derivative using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody which recognizes a pyrazine derivative which is a reaction product of a 1,2-dicarbonyl derivative which is an intermediate of a glycation reaction of a protein in vivo, and an immunity for inducing the antibody. The present invention relates to an immunoassay method for a 1,2-dicarbonyl derivative using a raw material and the antibody.

[0002] Diabetes is a metabolic disorder caused by insulin and is prone to be chronic, and thus causes various complications. The complications include, for example, eye diseases, renal diseases, neuroses, cardiovascular complications, gangrene, etc., and the prognosis of patients is greatly influenced by the presence or absence of complications and the degree thereof.

On the other hand, deoxyglucosone is an intermediate that is considered to be a major cause of the glycation reaction of proteins in diabetes. Deoxyglucosone is a substance that functions as a cross-linker between proteins and has a function of promoting the formation of the final reaction product of the saccharification reaction. This deoxyglucosone is increased in the blood of diabetic patients with persistent hyperglycemia, especially those with nephropathy (Biochem.
Biophys. Res. Commun., Vol196, p837-843, 1993)
And in the blood of patients with diabetic arteriosclerosis (DIABETES, Vol.45, SP3, S81
-83, 1996), it attracted attention as an index for understanding the condition of diabetic complications. Methylgluoxal is also increased in the blood of patients with diabetic arteriosclerosis (DIABETES, Vol.45, SP3, S
81-83, 1996), and attention has been paid to the trend of 1,2-dicarbonyl derivatives in diabetes.

However, since 1,2-dicarbonyl derivatives such as deoxyquiglucosone are very unstable substances, the 1,2-dicarbonyl derivatives have hitherto been derivatives or stable metabolites. It was converted to a substance, extracted, and measured by chromatographic analysis using GS / MS, HPLC, or the like. These methods are 1,2-
Since the extraction operation and the chromatographic analysis operation of the dicarbonyl derivative are complicated and time-consuming, there has been a long-felt demand for a simpler measurement method capable of measuring multiple samples.

[0005]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method for measuring a 1,2-dicarbonyl derivative which is simple and capable of measuring many analytes. Further, it is an object of the present invention to provide an antibody used in the measuring method and an immunogen for inducing the production of the antibody.

[0006]

Means for Solving the Problems As a result of earnest research, the inventors of the present application have used an antibody that recognizes a pyrazine derivative by reacting a 1,2-dicarbonyl derivative with a diamino derivative to convert it into a stable pyrazine derivative. It was found that the 1,2-dicarbonyl derivative in a sample can be measured by measuring the pyrazine derivative by immunoassay,
The present invention was completed by actually providing the above-mentioned antibody.

That is, the present invention provides an antibody that recognizes a region containing at least R ¹ or R ² of a pyrazine derivative represented by the following general formula (I).

[0008]

[Chemical 1]

(Wherein, R ¹ is hydrogen, R ² is a trihydroxybutyl group, A is a 5- or 6-membered aromatic hydrocarbon group or aromatic heterocyclic group bonded to the pyrazine ring, or R ³ is a group that forms an alicyclic hydrocarbon group, R ³ is a crosslinkable residue, and the ring formed by A is, in addition to the above R ³ ,
1 or 2 in case of 5-membered ring, 1-3 in case of 6-membered ring
Optionally further substituted by 1).

The present invention also provides an immunogen comprising a pyrazine derivative represented by the above general formula (I) or an R ³ of the pyrazine derivative bound to an immunogenic carrier.

Furthermore, the present invention is a method for immunoassaying a 1,2-dicarbonyl derivative which is an intermediate of a glycation reaction of a protein in a living body, wherein the 1,2-dicarbonyl derivative in a sample is generally used. Formula (II)

[Chemical 2] (R ³ and A have the same meanings as in the case of the above general formula (I)) to produce a pyrazine derivative represented by the above general formula (I), and the pyrazine derivative and the above An immunoassay method for a 1,2-dicarbonyl derivative, which comprises measuring the pyrazine derivative by an immunoassay utilizing an antigen-antibody reaction with an antibody, and thereby measuring the 1,2-dicarbonyl derivative in a sample. provide.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the antibody of the present invention is
It recognizes the pyrazine derivative represented by the general formula (I).

The definitions of R ¹ and R ^{2 in} the general formula (I) are as described above, wherein R ¹ is hydrogen and R ² is a trihydroxybutyl group.

In the general formula (I), the definition of A is as described above, and the ring formed by A is preferably pyridine, benzene, furan or thiophene. The ring formed by A is 1 in the case of a 5-membered ring in addition to R ^3.
Alternatively, in the case of a 2-membered or 6-membered ring, it may be substituted with 1 to 3 further optional substituents. As the substituent, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, a carbamoyl group, a cyano group, a formyl group, a hydroxy group, a mercapto group, a nitro group, a halogen atom; a carboxy group, a sulfonyl group, an alkoxycarbonyl group,
A carbamoyl group, a cyano group, a formyl group, a hydroxy group, a mercapto group, a nitro group, an aromatic hydrocarbon group which may be substituted with a halogen atom, an aromatic heterocyclic group, or an aliphatic hydrocarbon group (the aliphatic hydrocarbon group). Examples of the hydrogen group include a carbon chain which may be substituted with oxygen or sulfur atom).

In the general formula (I), R ³ is a crosslinkable residue. In the present specification, the crosslinkable residue means a chemical structure capable of binding the pyrazine derivative and another substance. Since R ³ has the purpose of binding the pyrazine moiety to another compound such as an immunogenic carrier or a label, any specific structure thereof can be used as long as this purpose is achieved. Therefore, the means for binding by the crosslinkable residue includes both covalent bond and non-covalent bond. Examples of the non-covalent bond include a hydrophobic bond, a hydrogen bond, an ionic bond, a coordinate bond, and the like.
Long-chain aliphatic hydrocarbon (carbon number is preferably 8 to 1)
8), carboxylate ion, ammonium ion and the like. In the case of a covalent bond, R ³ preferably consists of a spacer moiety and a reactive moiety. The reactive portion means a functional group capable of reacting with another compound to form a covalent bond, and examples thereof include a carboxyl group, a hydroxyl group, a sulfhydryl group, an amino group, a maleimide group, an aldehyde group, a halogen atom, and the like. Mention may be made of derivatives of these functional groups which have been activated to bind to the compounds of The spacer portion means a chemical structure capable of placing the pyrazine portion and the reaction portion at an appropriate distance, and includes, for example, an aliphatic hydrocarbon (carbon number is preferably 2 to
6), aromatic hydrocarbons, or these are ester bonds, amide bonds, ether bonds, thioether bonds,
Examples thereof include a structure linked by a disulfide bond, a Schiff base bond, etc., but R ³ may have a structure that includes only a reactive portion and does not include a spacer portion.

The crosslinkable residue represented by R ³ can be directly bonded to the pyrazine derivative and another substance, or indirectly bonded via a divalent reactive crosslinker.
Here, as specific examples of the divalent reactive crosslinking agent, succinimidyl 3- (2-pyridylthio) propionate (SPDP), N-succinimidyl 4-maleimidobutyric acid (GMBS), 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide hydrochloride (EDC), N,
N'-dicyclohexylcarbodiimide (DCC), 3
-(2-pyridyldithio) propionyl hydrazide (P
DPH), 4- (4-maleimidomethyl) butanoic acid hydrazide hydrochloride (MPBH), and the like.

A preferred structure of the compound represented by formula (I) is that ^one of R ¹ and R ² is hydrogen, the other is 2,3,4-trihydroxybutyl group, A is benzene, and R ³ is 4-carboxamidobutanoyl- (2-mercapto) ethylamide compound N- (4- (3-
(2,3,4-trihydroxybutyl) quinoxaline-
6-carboxamido) butanoyl) -2-mercaptoethylamine or N- (4- (2- (2,3,4-trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -2-mercaptoethylamine is preferred. N- (4- (3- (2,3,4-trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -2-mercaptoethylamine and N- (4- (2
The chemical formula of-(2,3,4-trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -2-mercaptoethylamine is shown below.

[0018]

[Chemical 3]

[0019]

[Chemical 4]

The antibody of the present invention recognizes the above-mentioned pyrazine derivative, and particularly recognizes a region including at least R ¹ or R ² of the pyrazine derivative. That is, as described in more detail below, the antibody of the present invention is used for measuring a 1,2-dicarbonyl derivative in a sample. This 1,2-dicarbonyl derivative reacts with a diamino derivative described below to form a pyrazine derivative represented by the above general formula (I), and the formed pyrazine derivative is measured using the antibody of the present invention. The 1,2-dicarbonyl derivative constitutes the pyrazine ring portion to which R ¹ and R ² in the pyrazine derivative and their substituents are bonded. Therefore, the antibody of the present invention needs to recognize a region including at least R ¹ or R ² of the pyrazine derivative, and for example, an antibody that recognizes only R ³ is not the antibody of the present invention. However, an antibody that recognizes the entire pyrazine derivative including R ³ is, of course, the antibody of the present invention.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred from the viewpoint of obtaining an antibody having uniform reaction specificity with good reproducibility. In the case of a polyclonal antibody, an antibody that recognizes a region other than the region containing at least R ¹ or R ² of the pyrazine derivative may be included.

The antibody of the present invention can be prepared by a conventional method using an immunogen comprising a pyrazine derivative represented by the general formula (I) or an R ³ of the pyrazine derivative bound to an immunogenic carrier. More preferably, the latter immunogen is used. Proteins such as BSA and KLH are preferred as immunogenic carriers.

In the case of a polyclonal antibody, the antibody of the present invention can be obtained by immunizing an animal with the above immunogen and producing the antibody from antiserum by a conventional method. In the case of a monoclonal antibody, antibody-producing cells such as spleen cells of an animal immunized with the above immunogen and tumor cells such as myeloma cells are fused with a fusion agent such as polyethylene glycol. Create hybridomas. Next, the hybridomas are selected using a selective medium such as HAT medium, subjected to monocloning by an appropriate method such as limiting dilution method, and cultured. The culture supernatant is analyzed by an appropriate immunoassay such as enzyme immunoassay to select a clone producing the desired anti-pyrazine derivative antibody. These monoclonal antibody production methods are known methods, for example, Keller and Milstein (Nature 256 495 197).
5) and Scheller (Nature 285 4461980). Further, the monoclonal antibody produced by the above-mentioned method can be recovered from the culture supernatant by analysis / purification means such as salt folding, ion exchange chromatography, gel filtration chromatography and the like. Furthermore, an antibody recognizing a region containing at least R ¹ or R ² of a pyrazine derivative can be prepared by varying the concentration of a 1,2-dicarbonyl derivative according to the method of the present invention described below, as described in detail in the Examples below. It can be obtained by measuring the 1,2-dicarbonyl derivative, writing a calibration curve, and selecting one that changes depending on the concentration of the 1,2-dicarbonyl derivative.

Next, a method for measuring a 1,2-dicarbonyl derivative using the antibody of the present invention will be described.

The 1,2-dicarbonyl derivative to be measured by the immunoassay method of the present invention is a 1,2-dicarbonyl derivative which is an intermediate of protein saccharification reaction in vivo of deoxyglucosone, For example, 2-deoxyglucosone, 3-deoxyglucosone, 4-deoxyglucosone, etc. can be mentioned.

The sample containing the 1,2-dicarbonyl derivative is not particularly limited, but it is usually a body fluid such as serum, plasma, blood, urine or a biological tissue.

In the immunoassay method of the present invention, first, the 1,2-dicarbonyl derivative in the sample is reacted with the diamino derivative represented by the general formula (II) to give the general formula (I).
The pyrazine derivative represented by is produced. This reaction is
Preferably at 4 ° C to 37 ° C, more preferably at room temperature,
It can be carried out preferably by simply mixing for about 12 to 20 hours. The concentration of the diamino derivative to be reacted is usually about 1 to 50 μg / ml, preferably 5 to
It is about 20 μg / ml. The reaction between the 1,2-dicarbonyl derivative and the diamino derivative can be performed before the immune reaction or simultaneously. As an example, a reaction formula of 3-deoxyglucosone, which is one of 1,2-dicarbonyl derivatives, and a diaminobenzene derivative, which is one of diamino derivatives, is shown below.

[0028]

[Chemical 5]

Next, the pyrazine derivative represented by the general formula (I) formed by the above reaction is measured by the immunoassay method using the antibody of the present invention. The immunoassay method itself is well known in this field, and any immunoassay method such as an immunohistological staining method, an immunoturbidimetric method, a radioimmunoassay, an enzyme immunoassay or the like that applies an immunoreaction of the antibody with a pyrazine derivative is adopted. be able to.

As an example of a preferred immunoassay method, R ³ of the above diamino derivative is labeled with a detectable label, and in the immunoassay, the antibody of the present invention is bound to a solid phase,
An example is a method comprising reacting the solid phase-bound antibody with the pyrazine derivative, washing, and measuring the amount of the label bound to the solid phase to measure the pyrazine derivative. As the label in this case, any label commonly used in this field such as biotin, enzyme, radioisotope and the like can be used.

[0031]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Reference Example 1 Nt-butoxycarbonyl-
Synthesis of 4-aminobutyric acid (compound (1))

5.16 g of 4-aminobutyric acid was dissolved in 20 ml of water, and 8.4 ml of N-methylmorpholine and 11.08 g of 2-t-butoxycarbonyloxyimino-2-phenylacetonitrile dissolved in 50 ml of acetone were added. Stir overnight at room temperature. About half of the solvent was distilled off, water and ethyl acetate were added, and the mixture was shaken well and separated. The aqueous layer was washed with ethyl acetate, the organic phases were combined and extracted with a 5% aqueous sodium hydrogen carbonate solution. The aqueous layers were combined, ethyl acetate was added, the pH was adjusted to about 3 with 6N hydrochloric acid, and the layers were separated. The aqueous layer was extracted twice more with ethyl acetate, and the extracts were combined and washed with water. This was dried over anhydrous magnesium sulfate. After the desiccant was filtered off, N, N-dicyclohexylamine was added to crystallize. The crystals were collected by filtration, washed with ether and dried.
Yield of N, N-dicyclohexylamine salt is 15.07.
The yield was 79% in g.

The total amount of N, N-dicyclohexylamine salt was dissolved in ethyl acetate and a 5% aqueous citric acid solution, and the layers were separated.
The aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with a 5% aqueous citric acid solution and water, and dried over anhydrous magnesium sulfate. The solvent was distilled off to obtain 7.57 g of an oily substance as Nt-butoxycarbonyl-4-aminobutyric acid (hereinafter referred to as compound (1) in the present specification). Compound
The chemical formula of (1) is shown below. In the formula, Boc represents a t-butoxycarbonyl group.

[0035]

[Chemical 6]

The NMR measurement results of the compound [1] are as follows. 1 H-NMR (400 MHz, CDCl ₃ , ppm) δ
1.44 (9H, s), 1.82 (2H, m, J = 7.
0, 7.0 Hz), 2.40 (2H, t, J = 7.0H
z), 3.18 (2H, m), 4.72 (1H, s).

Reference Example 2 S- (2-pyridylthio) -2
-Synthesis of mercaptoethylamine hydrochloride (compound (2)) 13.2 g of 2,2'-dipyridyl disulfide was dissolved in 100 ml of methanol, and 3.4 g of 2-mercaptoethylamine hydrochloride dissolved in 30 ml of methanol was stirred with vigorous stirring. Dropped. The reaction solution was stirred for 1 hour and 40 minutes, and then the solvent was distilled off. Ethyl acetate was added to the residue to crystallize and collect by filtration. The obtained crude crystal was recrystallized from methanol-ether. The mother liquor was concentrated, ether was added for further crystallization, and S- (2-pyridylthio) -2-mercaptoethylamine hydrochloride (hereinafter, referred to as compound in the present specification).
(Described as (2)) was obtained. The yield of compound (2) is 5.62.
The yield was 84% in g. The chemical formula of compound (2) is shown below.

[0038]

[Chemical 7]

The NMR measurement results of the compound (2) are as follows. 1 H-NMR (400 MHz, DMSO-D ₆ , pp
m) δ 3.09 (4H, m), 7.30 (1H, m, J
= 1.1, 4.8, 7.4 Hz), 7.75 (1H,
m, J = 0.9, 1.1, 8.0 Hz), 7.84 (1
H, m, J = 1.8, 7.4, 8.0 Hz), 8.10
(3H, s), 8.52 (1H, m, J = 0.9, 1.
8, 4.8 Hz).

Reference Example 3 N- (4-t-butoxycarboxamidobutanoyl) -S- (2-pyridylthio) -2
-Synthesis of mercaptoethylamine (Compound (3)) 0.98 g of the compound (1) synthesized in Reference Example 1, Reference Example 2
1.07 g of the compound (2) synthesized in 1. and 0.65 g of 1-hydroxybenzotriazole were suspended in 20 ml of DMF, and water-soluble carbodiimide 0.8 was added under cooling with an ice / salt bath.
8 ml was added, and the mixture was stirred at room temperature for 6 hours. Next, 0.28 g of water-soluble carbodiimide hydrochloride was added, and the mixture was stirred overnight. The solvent was evaporated, the residue was dissolved in water and ethyl acetate, and the layers were separated. The ethyl acetate phase was washed with water, 5% aqueous sodium hydrogen carbonate solution and water, and dried over anhydrous magnesium sulfate. The solvent was distilled off, and the oily residue was purified by silica gel column chromatography (100 ml, ethyl acetate). N-
(4-t-Butoxycarboxamidobutanoyl) -S-
The yield of (2-pyridylthio) -2-mercaptoethylamine (hereinafter referred to as compound (3) in the present specification) was 1.10 g, and the yield was 56%. The chemical formula of compound (3) is shown below.

[0041]

[Chemical 8]

The NMR measurement results of the compound (3) are as follows. 1 H-NMR (400 MHz, CDCl ₃ , ppm) δ
1.44 (9H, s), 1.82 (2H, m, J = 7.
0Hz), 2.27 (2H, t, J = 7.0Hz),
2.94 (2H, t, J = 6.0Hz), 3.18 (2
H, m), 3.57 (2H, m, J = 6.0, 6.0H
z), 4.86 (1H, s), 7.15 (1H, m, J
= 1.8, 4.9, 6.7 Hz), 7.39 (1H,
s), 7.54 (1H, m, J = 0.5, 0.9, 8.
1 Hz), 7.63 (1 H, m, J = 1.8, 4.9,
6.7 Hz), 8.50 (1 H, m, J = 0.9, 1.
8, 4.9 Hz).

Reference Example 4 Synthesis of 3,4-di-t-butoxycarboxamide benzoic acid (compound (4)) 3.04 g of 3,4-diaminobenzoic acid was dissolved in 25 ml of 1N aqueous sodium hydroxide solution to give di-t. -Butyl dicarbonate 9.6 g dissolved in acetone 30 ml and added,
Stir overnight. Next, 2.0 g of di-t-butyl dicarbonate and 5 ml of a 1N sodium hydroxide aqueous solution were added, and stirring was continued for another 3 days. Acetone was distilled off, water and ethyl acetate were added to the residue, and the mixture was shaken well and then separated. The aqueous layer was extracted with ethyl acetate, the ethyl acetate phases were combined and extracted twice with a 5% aqueous sodium hydrogen carbonate solution. Combine all the aqueous layers, add ethyl acetate, and p with 6N hydrochloric acid.
After H was set to about 3, the layers were separated. The aqueous layer was extracted twice with ethyl acetate, and the ethyl acetate phases were combined and washed with water. The extract was concentrated, and the precipitated crystals were collected by filtration. The crystals were washed with ethyl acetate and dried over phosphorous pentoxide. The yield of 3,4-di-t-butoxycarboxamide benzoic acid (hereinafter referred to as compound (4) in the present specification) was 5.55 g, and the yield was 79.
%Met. The chemical formula of compound (4) is shown below.

[0044]

[Chemical 9]

The NMR measurement results of the compound (4) are as follows. 1 H-NMR (400 MHz, DMSO-D ₆ , pp
m) δ 1.48 (9H, s), 1.49 (9H, s),
7.63 (1H, d, J = 8.6Hz), 7.71 (1
H, d, J = 8.6 Hz), 8.09 (1H, s),
8.68 (1H, s), 8.73 (1H, s), 12.
6-13.0 (1H, breads).

Reference Example 5 Synthesis of 3,4-di-t-butoxycarboxamide benzoic acid N-succinimide ester (Compound (5)) 5.50 g of the compound (4) synthesized in Reference Example 4 and N-hydroxysuccine 2.70 g of acid imide was dissolved in 50 ml of DMF, 4.50 g of water-soluble carbodiimide hydrochloride was added under ice cooling, and the mixture was stirred. After 2 hours, the mixture was returned to room temperature and stirred overnight.
The solvent was evaporated, the residue was dissolved by adding water and ethyl acetate,
The layers were separated. The ethyl acetate phase was washed with water, 5% aqueous sodium hydrogen carbonate solution, 5% aqueous citric acid solution, water and dried over anhydrous magnesium sulfate. The solvent was distilled off, hexane was added to crystallize, and the crystals were collected by filtration. The yield of 3,4-di-t-butoxycarboxamide benzoic acid N-succinimide ester (hereinafter referred to as compound (5) in the present specification) was 6.63 g, and the yield was 95%. . The chemical formula of compound (5) is shown below. In the chemical formula of the compound (5), NSu represents a succinimide group.

[0047]

[Chemical 10]

The NMR measurement results of the compound (5) are as follows. 1 H-NMR (400 MHz, DMSO-D ₆ , pp
m) δ 1.49 (9H, s), 1.50 (9H, s),
2.88 (4H, s), 7.78 (1H, d, J = 8.
6Hz), 7.95 (1H, d, J = 8.6Hz),
8.28 (1H, s), 8.89 (1H, s), 8.9
9 (1H, s).

Reference Example 6 Synthesis of N- (4- (3,4-diaminobenzamido) butanoyl) -S- (2-pyridylthio) -2-mercaptoethylamine (Compound (7)) By the method described in Reference Example 3. Synthesized compound (3) 1.54 g
Is dissolved in 5 ml of chloroform, and 5 m of trifluoroacetic acid is dissolved.
1 was added, the mixture was stirred at room temperature for 30 minutes, and the solvent was distilled off. Chloroform was added to the residue and evaporation was repeated 3 times, then DM
It was dissolved in 20 ml of F. This was ice-cooled and neutralized with triethylamine, 1.86 g of compound (5) was added, and the reaction was carried out overnight while adjusting the pH to the 7-8 position with triethylamine.
The solvent was distilled off, the residue was dissolved in water and ethyl acetate, and the layers were separated. The ethyl acetate phase was washed with 5% aqueous citric acid solution, 5% aqueous sodium hydrogen carbonate solution and water, and dried over anhydrous magnesium sulfate. The solvent was distilled off, and N- (4- (3,4-di-
t-Butoxycarboxamidobenzamido) butanoyl) -S- (2-pyridylthio) -2-mercaptoethylamine (hereinafter referred to as compound (6) in the present specification) was obtained as an oil. The chemical formula of compound (6) is shown below.

[0050]

[Chemical 11]

The NMR measurement results of the compound (6) are as follows. 1H-NMR (300 MHz, CDCl ₃ , ppm) δ
1.51 (9H, s), 1.52 (9H, s), 1.9
7 (2H, m), 2.34 (2H, t, J = 6.9H
z), 2.90 (2H, t, J = 5.5Hz), 3.4.
6-3.57 (4H, m), 6.7-6.9 (1H, b
roads), 7.10-7.17 (2H, m),
7.52-7.70 (4H, m), 7.82 (1H,
s), 8.47 (1H, d, J = 4.9 Hz).

The total amount of the compound (6) is 10 m of trifluoroacetic acid.
It was dissolved in 1 and stirred at room temperature for 1 hour. Trifluoroacetic acid was distilled off, water was added, and the mixture was distilled off again. The residue was dissolved in water, adsorbed on 45 ml of Diaion HP-20 column, washed with 250 ml of water, and then eluted with 300 ml of 40% acetonitrile aqueous solution. Collect and concentrate the eluate,
The separated oily substance was dissolved by adding a small amount of acetonitrile and lyophilized to give N- (4- (3,4-diaminobenzamido) butanoyl) -S- (2-pyridylthio).
2-Mercaptoethylamine (hereinafter referred to as compound (7) in the present specification) was obtained. The yield of compound (7) was 1.30 g. The chemical formula of compound (7) is shown below.

[0053]

[Chemical 12]

The NMR measurement results of the compound (7) are as follows. 1 H-NMR (400 MHz, DMSO-D ₆ , pp
m) δ 1.70 (2H, m), 2.10 (2H, t, J
= 7.3 Hz), 2.89 (2H, t, J = 6.8H)
z), 3.18 (2H, m), 3.33 (2H, m),
5.42 (4H, broads), 6.53 (1H,
d, J = 8.1 Hz) 7.05 (1H, dd, J = 1.
9, 8.1 Hz), 7.15 (1H, d, J = 1.9H)
z), 7.23 (1H, m, J = 1.1, 4.8, 7.
3Hz), 7.76 (1H, m, J = 0.9, 1.1,
8.4 Hz), 7.82 (1H, m, J = 1.8, 7.
3, 8.4Hz), 7.94 (1H, broad
t), 8.06 (1H, broadt), 8.45 (1
H, m, J = 0.9, 1.8, 4.8 Hz).

Example 1 N- (4- (3- (2,3,4
-Trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -S- (2-pyridylthio) -2
-Mercaptoethylamine (Compound (8-1)) and N
-(4- (2- (2,3,4-trihydroxybutyl))
Quinoxaline-6-carboxamido) butanoyl) -S
Synthesis of-(2-pyridylthio) -2-mercaptoethylamine (Compound (8-2)) 28.95 mg of the compound (7) synthesized in Reference Example 6 was dissolved in 1 ml of methanol and the reaction was carried out in the literature (Carbohyd. Res., 17, 17). p183
-192, 1971), 3-deoxy-D-erythrohexose-2-ulose (3-deoxyglucosone) (hereinafter, referred to as 3-DG in the present specification) (36.92 mg) It was dissolved in 1 ml of PBS and mixed. The mixture was stirred overnight at room temperature under an argon atmosphere. The reaction solution was diluted with water, adsorbed on 5 ml of Diaion HP-20 column, washed with water, and eluted with 40% acetonitrile aqueous solution. The eluate was concentrated, adsorbed on another 7 ml of Diaion HP-20 column, eluted with 5%, 10%, 20% acetonitrile aqueous solution in order, and the fractions eluted with 20% acetonitrile aqueous solution were collected and concentrated, This was freeze-dried to obtain the compound (8). Compound (8) is N- (4- (3-
(2,3,4-trihydroxybutyl) quinoxaline-
6-carboxamido) butanoyl) -S- (2-pyridylthio) -2-mercaptoethylamine (hereinafter referred to as compound (8-1) in the present specification) and N- (4-
(2- (2,3,4-trihydroxybutyl) quinoxaline-6-carboxamido) butanoyl) -S- (2-
Pyridylthio) -2-mercaptoethylamine (hereinafter,
In the present specification, it is a mixture with the compound (8-2). The yield of compound (8) was 26.70 mg as a mixture of isomers. Compounds (8-1) and (8-
The chemical formula of 2) is shown below.

[0056]

[Chemical 13]

[0057]

[Chemical 14]

The NMR measurement results of the compound (8) are as follows. 1 H-NMR (400 MHz, DMSO-D ₆ , pp
m) δ1.81 (2H, m), 2.18 (2H, t, J
= 7.3 Hz), 2.90 (2H, t, J = 6.8H)
z), 3.02 (2H, m), 3.34 (4H, m),
3.45 (2H, m), 3.61 (1H, m), 3.9
3 (1H, m), 4.45 (1H, broads),
4.78 (2H, m), 7.22 (1H, m), 7.7
6 (1H, d, J = 8.1Hz), 7.81 (1H, d
d, J = 7.2, 8.1 Hz), 8.08 (2H,
m), 8.17 (0.4H, d, J = 8.8Hz),
8.21 (0.6H, d, J = 8.8Hz), 8.45
(1H, d, J = 4.6Hz), 8.54 (0.4H,
s), 8.56 (0.6H, s), 8.82 (1H, b
roadd, J = 5.1Hz), 8.90 (0.4H,
s), 8.91 (0.6H, s).

Example 2 Synthesis of KLH-Binding Pyrazine Derivative As an immunogen, a conjugate of compound (8) and shell hemocyanin (hereinafter referred to as KLH in the present specification) (manufactured by Calbiochem) was synthesized. That is, KLH 11.16 mg
Was dissolved in 893 μl of 0.1 M phosphate buffer (pH 7.5), and 223 mg of GMBS (4-maleimidoacetic acid N-succinimide ester, manufactured by Dojindo Laboratories) dissolved in 223 μl of DMF (dimethylformamide). In addition,
Stirred at room temperature for 1 hour. 0.1 with 1 mM EDTA
PD-10 equilibrated with M phosphate buffer (pH 7.0)
Add 500μ of reaction solution to two columns (Pharmacia).
Aliquots of 1 were dispensed and eluted with the same buffer. The first 2.5 ml was discarded and the subsequent 2.0 ml was collected, respectively.

On the other hand, the compound [8] synthesized in Example 1
3. 46 mg of 20% acetonitrile aqueous solution 300 μl
Was dissolved in the solution, 11.56 mg of dithiothreitol was added, and the mixture was allowed to stand at room temperature for 30 minutes. 150 μl of this reducing solution was applied to reverse phase HPLC (YMC-PackAP-322, inner diameter 10
mm x length 150 mm), elution separation was performed with a 0.1 M phosphate buffer (pH 7.0) containing 1 mM EDTA and a concentration gradient of 10-20% acetonitrile, and a retention time of 1
A 0 minute peak fraction (A) and a 10.8 minute peak fraction (B) were collected. Fractions (A) and (B) were added to the above maleimidated KLH solution, respectively, and stirred at room temperature. After 40 minutes, 150 μl of the remaining reducing solution was collected and added to each reaction solution. The mixture was reacted for 3 hours in total, transferred to a dialysis tube, and dialyzed overnight against PBS to obtain a KLH-bonded pyrazine derivative. The yield is KL derived from fraction (A)
About 7 ml of H-bonded pyrazine derivative (346 mg / m
1), the KLH-bonded pyrazine derivative derived from fraction (B) was about 6 ml (375 mg / ml).

Example 3 Synthesis of BSA-Bound Pyrazine Derivative In the same manner as in Example 2, 12.88 mg of bovine serum albumin (hereinafter referred to as BSA in this specification) and the compound (8) synthesized in Example 1 were used. Using 3.3 mg, a conjugate of compound (8) and BSA was synthesized as an antigen for immunoassay. The yield was about 8 ml (590 mg / m 2) of the BSA-bonded pyrazine derivative derived from the fraction (A) collected by reverse phase HPLC.
1), the BSA-bonded pyrazine derivative derived from the fraction (B) was about 7 ml (665 mg / ml).

Example 4 Synthesis of biotin-bonded pyrazine derivative (compound (9)) As an antigen for immunoassay, a conjugate of compound (8) and biotin (hereinafter referred to as compound (9) in the present specification) Was synthesized. That is, 21.35 mg of the compound (8) synthesized by the method described in Example 1 containing 0.1 mM of EDTA was added.
Dissolved in 2 ml of 1M phosphate buffer (pH 7.0) and 0.2 ml of methanol, and dithiothreitol 40.53m
g was added, and the mixture was stirred at room temperature under an argon atmosphere. Reverse phase HPLC (Asahi Pack ODP-90, 20 m
mI. D. x300 mmL. Showa Denko KK), 1
0.1 M phosphate buffer (pH 7.
0) and acetonitrile were eluted and separated with a concentration gradient of 10-20%, and a peak fraction (A) with a retention time of about 26 minutes and a peak fraction (B) with a retention time of 27 minutes were fractionated. Biotin-PE-maleimide (manufactured by Dojindo Laboratories) was used in the fraction (A) of 14.0.
mg, 11.3 mg was added to the fraction (B), and the mixture was stirred at room temperature under an argon atmosphere. The reaction solution of the fraction (A) was adsorbed on 7 ml of Diaion HP-20 column, and the reaction liquid of the fraction (B) was adsorbed on 5 ml of Diaion HP-20 column. It was eluted with an aqueous acetonitrile solution and freeze-dried. The yield is
The biotin-bonded pyrazine derivative derived from the fraction (A) was 11.
35 mg, and the biotin-bonded pyrazine derivative derived from the fraction (B) was 4.15 mg. Compounds (9-1) and (9
The chemical formula of -2) is shown below.

[0063]

[Chemical 15]

[0064]

[Chemical 16]

Example 5 Synthesis of biotin-conjugated diaminobenzene derivative (compound (10)) 100 mg of compound (7) synthesized by the method described in Reference Example 6
0.1M phosphate buffer containing 1 mM EDTA (pH
7.0) 4 ml and acetonitrile 1 ml, it melt | dissolved in dithiothreitol 115 mg, and it stirred for 1 hour. Adsorbed on 10 ml of Diaion HP-20 column, water 5
After washing with 0 ml, 20m acetonitrile aqueous solution 50m
Elute with 1. Biotin-PE-maleimide 4 in the eluate
2.6 mg was added and stirred for 5 hours. Diaion HP-
It was adsorbed on 20 columns of 20 ml, washed with 50 ml of water, and then eluted with 50 ml of 40% acetonitrile aqueous solution.
The eluate was freeze-dried to obtain 53.8 mg of a crude product. Purification using reverse phase HPLC gave 8.05 mg of compound (10). The chemical formula of compound (10) is shown below.

[0066]

[Chemical 17]

Reference Example 7 Nα- (9-fluorenylmethoxycarbonyl) -Nε- (3,4-di-t-butoxycarboxamidobenzoyl) -L-lysine (Compound (1
1)) Synthesis Nα- (9-fluorenylmethoxycarbonyl) -Nε
0.54 g of -t-butoxycarbonyl-L-lysine was dissolved in 5 ml of chloroform, and 5 ml of trifluoroacetic acid was dissolved.
Was added and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off, ether was added to cause precipitation, the supernatant was removed, and DMF 5 ml was added.
Dissolved in. Under ice cooling, 322 μl of triethylamine and 3,4-di-t-butoxycarboxamide benzoic acid N-
0.67 g of succinimide ester was added, and the mixture was stirred overnight at room temperature. The solvent was evaporated, the residue was dissolved in ethyl acetate and dilute hydrochloric acid, and the layers were separated. The ethyl acetate phase was washed with water and dried over anhydrous magnesium sulfate. The solvent was distilled off, n-hexane was added to the residue, and the crystals were collected by filtration. The yield of compound (11) is 0.
It was 60 g. The chemical formula of compound (11) is shown below. In the chemical formula of compound (11), Fmoc represents a 9-fluorenylmethoxycarbonyl group.

[0068]

[Chemical 18]

Reference Example 8 Nε- (3,4-di-t-butoxycarboxamidobenzoyl) -L-lysine (Compound
Synthesis of (12)) 0.60 g of the compound (11) synthesized in Reference Example 7 was added to DMF7m
It was dissolved in 1 l, 3 ml of piperidine was added, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was 1.5% trifluoroacetic acid aqueous solution
It was poured into 50 ml and the insoluble matter was filtered off. The aqueous phase was passed through 10 ml of Diaion HP-20 column to adsorb the target product, washed with 50 ml of water, and then eluted with 100 ml of 40% acetonitrile aqueous solution. The eluate was concentrated and freeze-dried. The yield of compound (12) was 101 mg. Compound (1
The chemical formula of 2) is shown below.

[0070]

[Chemical 19]

Example 6 Synthesis of Nα- (5- (biotinylamino) -pentanoyl) -Nε- (3,4-diaminobenzoyl) -L-lysine (Compound (13)) The compound synthesized in Reference Example 8 [12] ] 30.29 mg was suspended in 3 ml of 0.1 M phosphate buffer (pH 7.5),
With vigorous stirring, 4 ml of a DMF solution (10 mg / ml) of biotin-AC5-OSu (manufactured by Dojindo Kagaku) was added in 4 portions, and the mixture was stirred at room temperature overnight. The solvent was evaporated, the residue was dissolved in ethyl acetate and water and acidified with 6N hydrochloric acid.
The deposited precipitate was collected by filtration, washed with water, and dried over diphosphorus pentoxide. The obtained solid was dissolved in 1 ml of trifluoroacetic acid and stirred at room temperature for 1 hour. The solvent was distilled off, the residue was dissolved in water and freeze-dried. The yield of compound (13) was 21.14 mg. The chemical formula of compound (13) is shown below.

[0072]

[Chemical 20]

The NMR measurement results of the compound [13] are as follows. 1 H-NMR (400 MHz, DMSO-D ₆ , pp
m) δ1.2-1.8 (16H, m), 2.05 (2
H, t, J = 7.4 Hz), 2.10 (2H, t, J =
7.3 Hz), 2.58 (1H, d, J = 12.4H
z), 2.82 (1H, dd, J = 5.1, 12.4H
z), 3.00 (2H, m), 3.09 (1H, m),
3.19 (2H, m), 3.2-4.0 (4H, bro
ads), 4.14 (2H, m), 4.31 (1H,
m), 6.36 (1H, broads), 6.42
(1H, broads), 6.71 (1H, d, J =
8.4 Hz), 7.34 (1H, dd, J = 0.7,
8.4 Hz), 7.44 (1 H, s), 7.72 (1
H, m), 8.00 (1H, m), 8.10 (1H,
m).

Reference Example 9 Nα-Biotinyl-Nε-
(3,4-diaminobenzoyl) -L-lysine (compound
(14)) Compound (12) 41.07 mg synthesized in Reference Example 8
Suspended in 4 ml of M phosphate buffer (pH 7.5), and DM with biotin-OSu (manufactured by Dojindo Co., Ltd.) with vigorous stirring.
3 ml of F solution (10 mg / ml) was added, and the mixture was stirred at room temperature overnight. The solvent was evaporated, the residue was dissolved in ethyl acetate and water and acidified with 6N hydrochloric acid. The layers were separated, and the ethyl acetate phase was washed with water. The solvent was distilled off, and the residue was trifluoroacetic acid 1 ml.
And was stirred at room temperature for 1 hour. The solvent was distilled off, the residue was dissolved in water and acetonitrile and freeze-dried. Compound (1
The yield of 4) was 47 mg. The chemical formula of compound (14) is shown below.

[0075]

[Chemical 21]

Example 7 Preparation of anti-pyrazine derivative monoclonal antibody As the anti-pyrazine derivative monoclonal antibody, the KLH-bonded pyrazine derivative derived from the fraction (A) synthesized in Example 2 was used.
It was prepared by immunizing ALB / C mice and fusing their spleen lymphocytes with myeloma cells. That is, B
25-10 of KLH-conjugated pyrazine derivatives emulsified in ALB / C mice with Freund's complete adjuvant
Initial immunization with 0 μg, and 2-3 weeks later, the same antigen 2 emulsified with Freund's incomplete adjuvant
A booster immunization was performed with 5 to 100 μg. The increase in antibody titer is
By a method similar to the screening method described later, the BSA-bonded pyrazine derivative derived from the fraction (A) synthesized in Example 3 was confirmed by ELISA using a solid-phase antigen. After confirming the increase in antibody titer, 25 to 100 μg of KLH-bonded pyrazine derivative was intravenously administered, and 3 to 4 days after that, the spleen was taken out from the mouse to prepare splenocytes. Mouse myeloma cells (P3U1) that had been previously cultured in RPMI-1640 medium were mixed with splenocytes at a ratio of 1: 2 to 1: 5, and cell fusion was performed using polyethylene glycol (Boehringer). The fused cells were suspended in HAT medium, dispensed into a 96-well culture plate, and cultured in a 37 ° C. carbon dioxide incubator.

The screening was carried out by an antigen solid phase ELISA. That is, the BSA-bonded pyrazine derivative derived from the fraction (A) synthesized in Example 3 was added to a 96-well ELISA plate (Pharmacia) at a concentration of 1 μg / ml.
μl / well was poured and left overnight at 4 ° C. for adsorption. After blocking the plate with 1% skim milk, the plate was washed 3 times with a phosphate buffer containing 0.05% Tween 20 (hereinafter referred to as a wash buffer) to perform cell fusion. 50 μl of culture supernatant
Was added and reacted at 37 ° C. for 1 hour. Similarly, after washing three times with a washing buffer, peroxidase (hereinafter, P
Labeled anti-mouse immunoglobulin antibody (denoted as OD) (manufactured by Dako) was added, and the mixture was further reacted at 37 ° C. for 1 hour.
After washing four times with the washing buffer, the substrate ABTS was added to select wells in which color development was observed. Wells showing reactivity to the BSA-bonded pyrazine derivative were further subjected to an inhibition test with a free pyrazine derivative to confirm the specificity. That is,
During the reaction between the solid-phase antigen and the culture supernatant, a fixed concentration (about 10 μm
The pyrazine derivative of M) was allowed to coexist, and the specificity was confirmed by the presence or absence of inhibition. In this case, the well showing suppression is the specific well. The cells in the wells whose specificity was confirmed were cloned by the limiting dilution method to be monocloned. The cells producing the anti-pyrazine derivative monoclonal antibody were cultured in a large amount and then intraperitoneally administered to mice to collect ascites fluid containing the anti-pyrazine derivative monoclonal antibody. The antibody was purified from ascites using protein A-Sepharose to obtain a monoclonal antibody. This anti-pyrazine derivative monoclonal antibody was named 3DG-451 antibody, and was named 3DG-
The hybridoma producing the 451 antibody has been deposited at the Microorganism Depositary Center, Institute of Biotechnology, Institute of Biotechnology, AIST, and the deposit number is FERM P-16179.

Example 8 Confirmation of specificity of anti-pyrazine derivative monoclonal antibody
It was confirmed by an ELISA inhibition test using an A-bonded pyrazine derivative as a solid phase antigen. That is, 75 μl / well of the BSA-bonded pyrazine derivative derived from the fraction (A) or the fraction (B) synthesized in Example 3 was dispensed onto a Nunc ELISA plate (maxisorb) at a concentration of 1 μg / ml, respectively. Fraction (A) -derived B
The SA-bonded pyrazine derivative adsorption plate was used as the plate (A), and the fraction (B) -derived BSA-bonded pyrazine derivative adsorption plate was used as the plate (B). Plate (A) and plate (B) were left in 1% skim milk at 37 ° C. for 3 hours for blocking, and then 1 m as an inhibitor.
The pyrazine derivative (compound (8)) synthesized in Example 1 diluted with M from 5 n, and the diaminobenzene derivative (compound (7)) synthesized in Reference Example 6 diluted from 5 mM to 5 n, 10
40 μl of a solution of quinoxaline (manufactured by Tokyo Chemical Industry Co., Ltd.) diluted with 5 n from mM was added to each well.
Next, 40 μl of 1 μg / ml 3DG-451 antibody was added and reacted at 37 ° C. for 1 hour. After thorough washing with a washing buffer, POD-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) was added, and the mixture was reacted at 37 ° C for 1 hour. Similarly, after thoroughly washing with a washing buffer, the substrate ABTS was added, and the mixture was allowed to stand at room temperature for 20 to 30 minutes, and then with a spectrophotometer at 405 nm.
Was measured. As shown in FIGS. 1 and 2, BSA
The reaction between the bound pyrazine derivative and the 3DG-451 antibody is
It was suppressed by the pyrazine derivative (compound (8)) but not by the diaminobenzene derivative (compound (7)) and quinoxaline. The cross-reactivity of the 3DG-451 antibody with respect to the diaminobenzene derivative (compound (7)) and quinoxaline was 0.1% or less, and it was confirmed that it was specific to the pyrazine derivative (compound (8)).

Example 9 Measurement of biotin-bonded pyrazine derivative (compound (9)) The compound (9) synthesized in Example 4 was sandwiched by ELISA.
It was measured by Method A. The 3DG-451 antibody was dispensed at a concentration of 10 μg / ml in an amount of 75 μl / well onto a Nunc ELISA plate (maxisorb) and left at 4 ° C. overnight for adsorption. Plate with 1% skim milk at 37 ° C for 3
After standing for blocking for a while, the biotin-bonded pyrazine derivative (compound (9)) synthesized in Example 4 was added to 1% BS.
Dilute 5 ng from 40 ng / ml with Tris buffer containing A,
75 μl / well of each diluted solution was added to the antibody adsorption plate and reacted at 37 ° C. for 1 hour. Similarly, 1000 ng
The biotin-conjugated diaminobenzene derivative (compound (10)) synthesized in Example 5 diluted with 5n from the solution / ml was also measured.
After the reaction, the wells were thoroughly washed with a wash buffer, and 75 μl of alkaline phosphatase (hereinafter referred to as ALP) -labeled avidin (manufactured by Dako) was added to each well, and further reacted at 37 ° C. for 1 hour. After sufficiently washing with the washing buffer, 75 μl / well of the substrate pNPP was added and left at room temperature for 30 minutes, and then the wavelength of 405 nm was measured. The results are shown in Fig. 3. Antibody 3DG-4 used as a solid phase as shown in FIG.
51 did not react with the diaminobenzene derivative (compound (10)) at all, but the pyrazine derivative (compound (9)) was measurable up to about 10 pg / ml.

Example 10 3-deoxyglucosone (3
-DG) Measurement-1 3-DG and the biotin-conjugated diaminobenzene derivative (compound (10)) synthesized in Example 5 were reacted in PBS overnight at room temperature, and the reaction product was subjected to 3DG-451 solid phase ELISA. It was measured. That is, 3 diluted from 5 μg / ml to 5 n
Add a biotin-conjugated diaminobenzene derivative solution (compound (10)) to DG to a final concentration of 10 μg / ml,
The reaction was carried out overnight at room temperature. NDG ELISA plate (maxisorb) with 3DG-451 antibody at 10 μg / m
75 μl / well was added at a concentration of 1 and left overnight at 4 ° C. for adsorption, blocking with 1% skim milk, and EL
An ISA plate was made. As a control, a monoclonal antibody PCX22 having no specificity for pyrazine derivatives
An ELISA plate having A4 adsorbed was also prepared. EL
The measurement method of ISA was according to the method shown in Example 9, and using the two kinds of prepared plates, the biotin-bonded pyrazine derivative which is a reaction product of 3-DG and the biotin-bonded diaminobenzene derivative (compound (10)) was used. It was measured. The results are shown in Fig. 4. As shown in FIG. 4, it was confirmed that 3-DG could be measured by this measurement method.

Example 11 Measurement of 3-DG-2 Biotinylated diaminobenzene derivative (compound (13)) synthesized in Example 6 with 3-DG or biotinylated diaminobenzene derivative synthesized in Reference Example 9 (compound (14 )) And each in PBS overnight at room temperature and the reaction product
It was measured by G-451 solid phase ELISA. That is, as in Example 10, 3-n diluted from 200 ng / ml to 3 n was used.
Compound (13) or compound (14) was added to DG at a final concentration of 10 μg /
It was added so that the amount became ml, and the reaction was carried out at room temperature overnight. ELI
The SA measurement method was the same as in Example 10. The results are shown in Figure 5.
Shown in. As shown in FIG. 5, the biotinylated diaminobenzene derivative of compound (14) showed no reaction, but the compound
When the biotinylated diaminobenzene derivative of (13) was used, 3-DG was measurable from about 100 pg / ml.

[0082]

INDUSTRIAL APPLICABILITY According to the present invention, there is provided an antibody capable of measuring a 1,2-dicarbonyl derivative which can be an index of diabetes associated with complications, and a 1,2-dicarbonyl derivative by a measuring method using the antibody.
It has become possible to easily measure dicarbonyl derivatives.

[Brief description of drawings]

FIG. 1 is a diagram showing the specificity of an anti-pyrazine derivative monoclonal antibody using a BSA-bonded pyrazine derivative derived from fraction (A).

FIG. 2 is a diagram showing the specificity of an anti-pyrazine derivative monoclonal antibody using a BSA-bonded pyrazine derivative derived from fraction (B).

FIG. 3 is a diagram showing the measurement results of a biotin-bonded pyrazine derivative with an anti-pyrazine derivative monoclonal antibody.

FIG. 4 shows the measurement results of 3-deoxyglucosone using an anti-pyrazine derivative monoclonal antibody containing compound (10).

FIG. 5 shows the measurement results of 3-deoxyglucosone using an anti-pyrazine derivative monoclonal antibody using Compound (13) and Compound (14).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12N 15/02 C12N 15/00 C C12P 21/08 5/00 B (56) Reference FEBS Lett. , 1997, Vol. 407, No. 3, p. 297-302 J. Clin. Invest. , 1992, Vol. 89, No. 4, p. 1102 -1112 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12P 1/00-41/00 BIOSIS / WPI (DIALOG) CA (STN) REGISTRY (STN) JSTPlus (JOIS)

Claims (14)

(57) [Claims] 1. An antibody which recognizes a region including at least R ¹ or R ² of a pyrazine derivative represented by the following general formula (I). [Chemical 1] (In the formula, R ¹ is hydrogen, R ² is a trihydroxybutyl group, A is a 5- or 6-membered aromatic hydrocarbon group, aromatic heterocyclic group or alicyclic group bonded to a pyrazine ring. R ³ is a group that forms a hydrocarbon group, R ³ is a crosslinkable residue, and
The ring formed by is optionally substituted with 1 or 2 additional substituents in the case of a 5-membered ring and 1 to 3 additional substituents in the case of a 6-membered ring in addition to the above R ³ ). 2. The antibody according to claim 1, wherein the ring formed by A is pyridine, benzene, furan or thiophene. ^3. The antibody according to claim 1 or 2 , wherein R ³ has a reactive portion and a spacer portion. 4. The reactive moiety is a carboxyl group, a hydroxyl group, a sulfhydryl group, an amino group, a maleimide group, an aldehyde group or a halogen atom or a derivative thereof, which can react with another compound to form a covalent bond. The antibody according to claim 3 . 5. The spacer portion is an aliphatic hydrocarbon group or an aromatic hydrocarbon group or a group in which these are bonded to each other through an ester bond, an amide bond, an ether bond, a thioether bond, a disulfide bond or a Schiff base bond. The antibody according to claim 3 or 4 . 6. The ring formed by A is benzene, and R ³
Is 4-carboxamidobutanoyl- (2-mercapto)
The antibody according to claim 1, which is an ethylamide group. 7. The antibody according to any one of claims 1 <br/> to 6 which is a monoclonal antibody. 8. The antibody according to claim 7, which is the monoclonal antibody 3DG-451 produced by the hybridoma deposited under the accession number of FERM P-16179. 9. An immunogen comprising a pyrazine derivative represented by the following general formula (I) in which R ³ is bound to an immunogenic carrier. [Chemical 1] (In the formula, R ¹ is hydrogen, R ² is a trihydroxybutyl group, A is a 5- or 6-membered aromatic hydrocarbon group, aromatic heterocyclic group or alicyclic group bonded to a pyrazine ring. R ³ is a group that forms a hydrocarbon group, R ³ is a crosslinkable residue, and
The ring formed by is optionally substituted with 1 or 2 additional substituents in the case of a 5-membered ring and 1 to 3 additional substituents in the case of a 6-membered ring in addition to the above R ³ ). 10. The immunogen according to claim 9 , wherein the immunogenic carrier is a protein. 11. A method for immunoassaying a 1,2-dicarbonyl derivative which is an intermediate of a glycation reaction of a protein in a living body, wherein the 1,2-dicarbonyl derivative in a sample is represented by the general formula (II): [Chemical 2] (R ³ and A have the same meanings as in the case of the general formula (I) described in claim 1) and are reacted with a diamino derivative represented by the general formula (I) described in claim 1. to generate, claims 1 and said pyrazine derivative 8
The method of claim 1, wherein the pyrazine derivative is measured by an immunoassay utilizing an antigen-antibody reaction with the antibody, and thereby the 1,2-dicarbonyl derivative in the sample is measured. Method for immunoassay of dicarbonyl derivative. 12. The diamino derivative R ³ is labeled with a detectable label, and in the immunoassay, the antibody is bound to a solid phase, and the solid phase-bound antibody is allowed to react with the pyrazine derivative. 12. The method according to claim 11 , comprising measuring the pyrazine derivative by measuring the amount of label bound to the solid phase after washing. Wherein said label claim 12 biotin
The method described. 14. The 1,2-dicarbonyl derivative is
The method according to any one of claims 11 to 13 , which is deoxyglucosone.