JPH06273417A - Antibody for mevalonic acid and determination method of mevalonic acid using the antibody - Google Patents

Abstract

PURPOSE:To make it possible to perform the large amount of measurement easily in a short time by preparing the antibody, which can specifically recognize mevalonic acid, and using the antibody. CONSTITUTION:The antibodies containing polyclonal antibodies, monoclonal antibodies and the like are specifically bonded to mevalonic acid. The cross ratio with glutamic acid, 3-methylglutamic acid and 3-hydroxy-3-methylglutamic acid are very low. The prefarable value is 1.0% or low. For examples, the cross ratio of the polyclonal antibody KLH-(12)R are 0.001% or less, 0.0065% and 0.013%, respectively. It is preferable that the polyclonal antibody is the antibody derived from a rabit and the monoclonal antibody is the antibody derived from a mouse. In preparation of the antibody, e.g. for the polyclonal antibody, mevalon derivative is conjugated with carrier protein, and an adequate sensitive animal is sensitized with the obtained mevalonic acid derivative and protein bond as immunity antigen. The serum fraction is sampled from the sensitive animal, and the object antibody fraction is prepared.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody that specifically recognizes mevalonic acid, and an immunological quantification method of mevalonic acid using the antibody.

[0002]

BACKGROUND OF THE INVENTION Mevalonic acid has the formula

[0003]

[Chemical 1]

The compound represented by the formula (1) is an intermediate substance in the cholesterol biosynthetic pathway. Mevalonic acid is 3-hydroxy-3-methylglutaryl coenzyme A
(Hereinafter, abbreviated as HMG-CoA.) To HMG-C
It is synthesized by the action of oA reductase. Cholesterol in the living body is biosynthesized by the above-mentioned pathway and is also ingested from the diet. Conventionally, the sterol balance method has been used to measure the amount of cholesterol biosynthesis. This method is a complicated method of subtracting the amount of cholesterol contained in the diet from the total amount of cholesterol in feces, and was not applicable to general clinical practice.

In 1982, Parker et al. Showed that the amount of biosynthesis of cholesterol was highly correlated with the mevalonate concentration in plasma, and the blood mevalonate concentration was an index of cholesterol biosynthesis. It revealed that
(J. Clin. Invest., 74 , 795 (1984)). Among cholesterol, low-density lipoprotein is considered to be the main cause of arteriosclerosis. Excessive arteriosclerosis causes myocardial infarction, angina, cerebral infarction, etc., and often leads to death. In treating high cholesterol,
Judging whether the high cholesterol state is due to the enhancement of the biosynthesis system or the excessive intake by diet, appropriate treatment (for example, diet restriction or administration of recently developed HMG-CoA reductase inhibitor etc.) is performed. Or monitoring the biosynthesis system after administration). Therefore, it is considered that knowing the biosynthesis amount of mevalonic acid is clinically very important in understanding the pathological condition of lipid metabolism diseases such as hyperlipidemia.

[0006]

2. Description of the Related Art At present, as a method for measuring mevalonic acid, a mass spectrum method using gas chromatography (GC-MS method) and a radioenzyme method are known. The former requires complicated operations such as sample extraction and derivatization, requires expensive equipment, and requires sophisticated techniques for measurement. In the latter case, radiolabeled [γ- ³² P] ATP was used to phosphorylate mevalonic acid in the sample to 5- [ ³² P] phosphomevalonic acid, which was then treated with ion-exchange column chromatography to measure its radioactivity. However, the operation is still complicated. Therefore, neither method is suitable for mass screening, which requires simple and short-term, large-scale measurement.

[0007]

Means for Solving the Problems The inventors of the present invention have made earnest studies on a method for quantifying mevalonic acid, succeeded in producing an antibody specifically recognizing mevalonic acid, and completed the present invention. I arrived. Antibodies against mevalonic acid, whether polyclonal or monoclonal, have never been reported so far, so the antibody of the present invention is completely novel. Therefore, an immunological quantification method using the antibody was also clarified for the first time in the present invention.

[0008]

The present invention relates to an antibody that specifically recognizes mevalonic acid, and an immunological quantification method of mevalonic acid using the antibody. The present invention also relates to an antibody specific to mevalonic acid used for diagnosis and treatment. The present invention also relates to a conjugate of a mevalonic acid derivative and a protein, which is useful for obtaining an antibody specific to mevalonic acid. The present invention also relates to a mevalonic acid measurement kit for quantifying the amount of mevalonic acid in a sample, which comprises at least an antibody specific to mevalonic acid and a mevalonic acid derivative or a conjugate thereof with a protein.

The antibodies of the present invention include both polyclonal and monoclonal antibodies. The present invention also includes an antibody having an antigen-binding site other than the antigen-binding site of the antibody obtained in the Examples below. The antibody of the present invention specifically binds to mevalonic acid, and has a very low cross rate with glutaric acid, 3-methylglutaric acid, and 3-hydroxy-3-methylglutaric acid.

Among the antibodies of the present invention, preferred are those having a crossing rate of 1.0% or less with glutaric acid, 3-methylglutaric acid and 3-hydroxy-3-methylglutaric acid. For example, the polyclonal antibody KLH- of the present invention
(12) R glutaric acid, 3-methyl glutaric acid and 3-
The crossover rates with hydroxy-3-methylglutaric acid are 0.001% or less, 0.0065% and 0.013%, respectively. The polyclonal antibody of the present invention is preferably a rabbit-derived antibody, and the monoclonal antibody is preferably a mouse-derived antibody, and the immunoglobulin class and subclass are not particularly limited, but are preferable. Is an IgG or IgM class,
Particularly preferred is the IgG class.

The polyclonal antibody of the present invention comprises (1) conjugating a mevalonic acid derivative to a carrier protein,
(2) The mevalonic acid derivative-protein conjugate thus obtained is used as an immunizing antigen to sensitize a suitable sensitized product, and (3) a serum fraction is collected from the sensitized product to obtain the target antibody. It can be prepared as a fraction.

Each step will be described more specifically as follows. The mevalonic acid derivative in the step (1) means a carbon atom at the 2-position, 4-position or 5-position of mevalonic acid, a hydroxyl group substituted at the 3-position or 5-position, or a methyl group substituted at the 3-position, It means a compound in which a reactive functional group is directly or indirectly introduced, and a compound in which a reactive functional group is directly or indirectly introduced instead of a methyl group or a hydroxyl group substituted at the 3-position. Preferably 3
In which a reactive functional group is directly or indirectly introduced into a hydroxyl group or a methyl group substituted at the position or in place of these groups, or a reactive functional group directly or indirectly at the carbon atom at the 5-position Is a compound in which a reactive functional group is directly or indirectly introduced into a methyl group substituted at the 3-position. When the derivative has a free hydroxyl group which is not associated with the bond with the reactive functional group, the hydroxyl group may form an intramolecular lactone with the carboxyl group at the 1-position. The reactive functional group is not particularly limited as long as it can be directly or indirectly bonded to the carrier protein. More specifically, a carboxyl group (-COOH),
Hydroxy (-OH), an formyl group (-CHO), amino group (-NH _2), an azide group (-N _3), a mercapto group (-SH), a sulfonyl group (-SO ₃ H) and the like, A carboxyl group is preferred.

Suitable carrier proteins include albumin, globulin, thyroglobulin, hemocyanin,
Examples include natural proteins such as edestin and synthetic polypeptides such as polylysine, with albumin or hemocyanin being preferred. The reaction of binding the mevalonic acid derivative and the carrier protein is known, for example,
It is described in detail in the document "Enzyme Immunoassay", 2nd edition (1982), edited by Eiji Ishikawa et al.
That is, in a suitable solvent (for example, phosphate buffer), when the mevalonic acid derivative serving as a hapten and the carrier protein can be directly bound, a carbodiimide method, an acid anhydride method, a maleimide method, etc., preferably dicyclohexylcarbodiimide A method using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide as a condensing agent, and when a mevalonic acid derivative and a carrier protein cannot be directly bonded, a crosslinking agent known in the art, for example, 1,5- It is carried out using the method using difluoro-2,4-dinitrobenzene. After the reaction, the desired product is isolated and purified by column chromatography.

The mevalonic acid derivative and the combined product of the mevalonic acid derivative and the cross-linking agent can be easily produced by a known method. Representative synthetic schemes are shown below in Schemes I, II, III, IV, V, VI, VII and VIII.

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[Chemical 2]

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[Chemical 13]

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[Chemical 14]

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[Chemical 20]

In the above reaction scheme, each abbreviation has the following meaning. φ: phenyl group, Et: ethyl group, Ac: acetyl group,
DMF: dimethylformamide, Py: pyridine, DM
SO: dimethyl sulfoxide, THF: tetrahydrofuran, LAH: lithium aluminum halide, LD
A: lithium diisopropylamide, AcOEt: ethyl acetate, MeOH: methanol, Pd-C: palladium carbon, DMAP: 4-dimethylaminopyridine, DC
C: dicyclohexylcarbodiimide, DME: dimethoxyethane, DHP: dihydropyran, CSA: camphorsulfonic acid.

In the immunization step (2), the shape of the sensitizing antigen is not particularly limited, but it is generally administered in combination with a usual adjuvant. Administration is intravenous,
It is performed by intradermal, subcutaneous or intraperitoneal administration. More specifically, at the time of the first sensitization, the conjugate of the mevalonic acid derivative obtained in (1) and the carrier protein was dissolved in a physiological saline-containing phosphate buffer solution (hereinafter abbreviated as PBS). And 1: 1 with Freund's Complete Adjuvant (FCA)
The substance emulsified at the ratio of is subcutaneously administered to the sensitized animal. Furthermore, by subcutaneously administering several times at an interval of 2 to 3 weeks, PBS containing a conjugate of a mevalonic acid derivative and a carrier protein and emulsified with Freund's incomplete adjuvant (FICA) at a ratio of 1: 1. Done. The sensitized animal is not particularly limited as long as it is an animal for which a polyclonal antibody is generally obtained, such as rabbit, guinea pig, rat, donkey, sheep, goat, chicken, etc., but preferably rabbit, especially New Zealand white rabbit is used. . The number of sensitizations is not particularly limited,
It is desirable to carry out until the antibody titer is sufficiently high. The dose of the antigen is not particularly limited, for example, in the case of rabbit,
It is sufficient to administer 1 to 2 mg (as carrier protein) once.

The step (3) is performed by collecting serum and separating and purifying it by affinity chromatography or the like.

Further, the monoclonal antibody of the present invention is
(1) The mevalonic acid derivative was conjugated to a carrier protein, (2) the mevalonic acid derivative-protein conjugate thus obtained was used as an immunizing antigen to sensitize the sensitized animal, and (3) the spleen of the sensitized animal. Cell-fused myeloma cells derived from sensitized animals,
(4) From the resulting hybridoma, screen cells that produce a monoclonal antibody against mevalonic acid, (5) clone the desired antibody-producing hybridoma, (6) grow the cloned antibody-producing hybridoma, 7) It can be produced by separating and purifying the produced antibody.

The steps will be described in more detail below. The step (1) is performed in the same manner as the step (1) of the method for producing a polyclonal antibody of the present invention.
The step (2) is also performed in the same manner as the step (2) of the method for producing a polyclonal antibody of the present invention. However, in the case of producing a monoclonal antibody, it is preferable that the conjugate of the mevalonic acid derivative and the carrier protein is intraperitoneally administered to the sensitized animal. The sensitized animal is not particularly limited as long as it is an animal from which a monoclonal antibody is generally obtained, such as mouse and rat, but is preferably mouse, especially B.
ALB / c is used. In the case of mice, for example, it is sufficient to administer 100 to 200 μg (as a carrier protein) of the antigen once.

In the cell fusion of (3), of the sensitized animals immunized in (2), the spleen of the sensitized animals for which the antibody titer has been sufficiently increased is removed, and the spleen cells are suspended according to a conventional method. A suspension is prepared, and then polyethylene glycol (preferably PEG4000) is added to the resulting mixture of splenocytes and myeloma cells derived from sensitized animals at 37 ° C. P3 × 63Ag8, P3 in mouse myeloma cells
/ NS1 / 1-Ag4-1, SP-2 / 0-Ag-14
There are several known types, and all are easily available. Myeloma cells are HAT medium (hypoxanthine,
A HGPRT (hypoxanthine guanine phosphoribosyl transferase) -deficient cell line that cannot survive in a medium containing aminopterin and thymidine is useful, and it is desirable that the myeloma cell itself does not secrete an antibody. Suitably SP-2 / 0-Ag
-14 is used.

Next, the obtained mixture of cell fusion was dispensed at low cell density into a 96-microwell plate, and HAT was added.
Incubate in medium. After 1 to 2 weeks of culture, unfused myeloma cells, hybridomas of myeloma cells, unfused splenocytes, and hybridomas of splenic cells die because the survival conditions are not satisfied, and only hybridomas of splenocytes and myeloma cells are killed. Will grow. The screening in (4) is carried out by reacting the hybridoma culture supernatant with a labeled mevalonic acid, separating the antibody fraction, and quantifying the amount of the labeled substance in the antibody fraction, or the hybridoma culture supernatant. Whether the hybridoma is producing an antibody against mevalonic acid by quantifying the antibody in the supernatant specifically adsorbed to the antigen by reacting with the immobilized antigen and the labeled second antibody To determine.

In the step (5), the antibody-producing hybridoma is subjected to a soft agar culture method [Monoclonal Antibodies, p. 372 (1980).
(Year) reference]. At this time, it is also possible to use the limiting dilution method.

The step (6) can be obtained by culturing the cloned hybridoma in an ordinary medium and separating and purifying from the culture supernatant. In order to efficiently obtain a large amount of antibody, the hybridoma is used as a mouse. A method in which it is administered intraperitoneally, proliferated, and separated and purified from the ascites is used.

The step (7) is carried out by a conventional method such as salting out,
It can be purified by ion exchange chromatography, gel filtration, hydrophobic chromatography, affinity chromatography, etc., but more effectively protein A-
Affinity chromatography using Sepharose CL-4B (Pharmacia) is used.

Since the antibody of the present invention specifically recognizes mevalonic acid, it can be used for purification and concentration of mevalonic acid, such as affinity chromatography.

However, the most important and important use method of the antibody of the present invention is the application to the immunological quantification method of mevalonic acid which is excellent in accuracy and detection limit. Generally, there are a competitive method and a non-competitive method as an immunological quantification method, but a competitive method is generally used as an immunological quantification method for a low-molecular substance such as mevalonic acid. In the competitive method, a labeled antigen and an unlabeled antigen are competitively reacted with an antibody, and then the labeled antigen bound (or not bound) to the antibody is generally quantified by some method. To know the amount of labeled antigen, quantify without separating antibody-bound and unbound antigen (homogeneous)
And the antibody-bound antigen (abbreviated as Bound, B) and the non-bound antigen (abbreviated as Free, F) (referred to as B / F separation) for measurement (heterogeneous).

In the homogeneous method, a method of measuring how much the unlabeled antigen competes with each other to inhibit the aggregation between the labeled antigen and the antibody (in this case, the labeled antigen is used for the purpose of aggregating the particles on which the antibody is immobilized) In general, particles to which a plurality of antigens are bound, or particles on which an antigen is immobilized for the purpose of aggregating via an antibody are generally used.) Or, when the labeled antigen binds to the antibody, some change in the label, for example, There is also a method of competitively reacting with an unlabeled antigen by utilizing the fact that enzyme activity is activated or inactivated, optical changes (absorption, fluorescence, luminescence, etc.), and electrical changes occur. Furthermore, there is also a method in which a labeled antigen and an unlabeled antigen are spotted on a filter paper on which an antibody is immobilized and developed, and then the degree of competition is checked by the mobility of the labeled antigen.

In the heterogeneous method, a radiolabeled antigen or an enzyme-labeled antigen of a mevalonic acid derivative (that is, one that cross-reacts with an anti-mevalonate antibody) is used as the labeled antigen. The radioisotope used here includes ³ H, ¹⁴ C, ³² P, ¹²⁵ I and the like, and the enzyme may be any enzyme generally used in enzyme immunoassays, such as peroxidase and β. -D-
Galactosidase, alkaline phosphatase, glucose oxidase, glucose-6-phosphate dehydrogenase,
Examples include acetylcholinesterase and alcohol dehydrogenase.

The B / F separation method is also not particularly limited. In the case of a radiolabeled antigen, a precipitation method using polyethylene glycol, an adsorption method using activated carbon, a first antibody solid phase method, a second antibody solid phase method and the like are often used. In the case of an enzyme-labeled antigen, there are a first antibody solid phase method, a second antibody solid phase method and the like.

The first antibody solid phase method is as follows: (1) A sample containing mevalonic acid and a labeled antigen are added to a solid-phased anti-mevalonate antibody (first antibody) to cause a competitive reaction.
(2) A method of measuring the amount of mevalonic acid in a sample by measuring the activity of the labeled substance bound to the solid phase after removing the liquid phase.

The monoclonal antibody of the present invention is used as the first antibody. The solid phase and the immobilization method used for the immunological quantification method are well known [see Immobilized Enzyme (edited by Ichiro Chibata, 1975, Kodansha)]. Examples of the solid phase include polystyrene plates, polystyrene beads, nylon beads, glass beads, protein A agarose beads, protein G agarose beads, polystyrene tubes, and the like. For immobilization, an insolubilization method by physical adsorption or covalent bond is used. Examples of the labeled substance of the labeled antigen include the radioactive substances and enzymes listed above.

The reaction of the first antibody with the sample and the labeled antigen is preferably carried out usually at 4 ° C. for about 10 hours. The activity of the labeled substance of (2) is also measured by a known method. For example, in the case of labeling with peroxidase, it is carried out by reacting with hydrogen peroxide using 3,3 ', 5,5'-tetramethylbenzidine as a substrate and measuring the absorbance of the reaction product. In this case, 3- (4-hydroxyphenyl) propionic acid, orthophenylenediamine, luminol or the like can be used as the substrate. Even when labeled with an enzyme other than this, it is performed using an appropriate substrate.

The second antibody method includes a method in which the reaction between the first antibody and the second antibody is carried out in a liquid phase, and a method in which the second antibody is solid phase. In particular, the second antibody solid phase method is advantageous because B / F separation is facilitated by immobilizing the second antibody.

The second antibody solid phase method is (1) an antibody against an anti-mevalonate antibody (for example, when the anti-mevalonate antibody is mouse-derived IgG, an antibody against mouse IgG).
Immobilize (second antibody), (2) add sample containing mevalonic acid, labeled antigen and anti-mevalonate antibody (first antibody) to them, and after reacting, (3) label solid phase It is done by measuring.

When an antibody against mouse IgG is used as the second antibody, either a polyclonal antibody or a monoclonal antibody may be used, and the animal species to be sensitized is not limited. A polyclonal antibody against mouse IgG can be prepared by administering and sensitizing mouse serum or mouse γ-globulin to another type of animal (for example, rat, guinea pig, rabbit, goat, etc.) by a generally known method [ Clinical Testing, 26 (7), 777 (1982)], but some polyclonal antibodies against mouse IgG are commercially available.

In addition, the solid phase, the immobilization method, the labeled antigen, the labeling method, the first antibody, the activity measuring method of the labeled substance, the reaction temperature and the time, etc. can be arbitrarily selected according to the first antibody solid phase method. As a method other than the above, after the immobilized antibody and the mevalonic acid in the sample are competed with the first antibody, the amount of the first antibody in the solid phase or the liquid phase is labeled with the first antibody in advance. Alternatively, there is also a method of quantifying, for example, by using a labeled second antibody that binds to the first antibody. As a non-competitive method, a method in which an antibody is labeled and a change caused by binding of an antigen is measured (for example, an antibody labeled with a molecule that generates electrochemiluminescence such as luminol is bound to an electrode There is a method of observing reduction of electrochemiluminescence due to complex formation). In the quantification method of the present invention, body fluid (blood, urine) or the like is provided as a sample.

[0056]

INDUSTRIAL APPLICABILITY The antibody of the present invention is an excellent antibody that specifically recognizes mevalonate and has extremely low cross-reactivity with other mevalonate-like compounds. Further, by using the antibody of the present invention, a highly reproducible immunological quantification method of mevalonic acid with high measurement sensitivity, excellent specificity, was established. According to the quantification method of the present invention, the operation is greatly simplified as compared with the conventional methods (GCMS method and radioenzyme method).

[0057]

The present invention will be described in more detail with reference to the following examples, which, however, do not limit the scope of the present invention. In the examples, "TLC", "MS", "I"
“R” and “NMR” represent “thin layer chromatography”, “mass spectrometry”, “infrared absorption spectrum” and “nuclear magnetic resonance spectrum”, respectively. The solvent in the parentheses described at the place of separation by chromatography indicates the elution solvent or developing solvent used, and the ratio indicates the volume ratio.

Example 1: Synthesis of conjugate of mevalonic acid derivative and spacer (a) Succinimide 3- (4-hydroxy-2-oxotetrahydropyran-4-yl) propyl succinate (1) 63% sodium hydride (4.68) g) was suspended in 60 ml of DMF, and 1,4-butanediol (11.0 g) was suspended.
The DMF solution (10 ml) of was slowly added sequentially at 4 ° C., the temperature was raised to room temperature, and the mixture was stirred for 30 minutes. Furthermore, 1
3 ml of benzyl bromide was slowly added dropwise while cooling at 4 ° C, and the temperature was raised to room temperature, followed by stirring at 70 ° C for 22 hours. The reaction solution was extracted with ethyl acetate and subjected to silica gel column chromatography (hexane / ethyl acetate = 8 /
The compound (2) (12.8 g) was obtained after purification by 2). TLC: Rf 0.21 (hexane / ethyl acetate = 6 /
4).

(2) Compound (2) (4.2 g) was added to DMSO.
Dissolve in (20 ml), triethylamine (22 ml)
Was added, pyridine sulfur trioxide (14.20 g) was added little by little with vigorous stirring at room temperature, and the mixture was further stirred for 20 minutes. The reaction solution was extracted with diethyl ether and then purified by silica gel column chromatography (hexane / ethyl acetate = 9/1) to obtain compound (3) (3.18 g). TLC: Rf 0.19 (hexane / ethyl acetate = 9 /
1).

(3) Trimethyl borate (3.42 ml) was added to T
Dissolve in HF (52 ml), add zinc (4.02 g), and
Ethyl bromoacetate (3.42 ml) was slowly added dropwise at 0 ° C, and the mixture was stirred for 10 minutes, compound (3) (2.67 g) was added, and 1
Stir for 0 minutes. The reaction solution was extracted with ethyl acetate and purified by silica gel column chromatography (hexane / ethyl acetate = 8/2) to obtain compound (4) (3.66 g). TLC: Rf 0.52 (hexane / ethyl acetate = 6 /
4).

(4) Lithium aluminum hydride (700 mg) was suspended in THF (25 ml), and a THF solution (5 ml) of the compound (4) (3.26 g) was slowly added dropwise at room temperature. After the reaction, 2N-HCl (3 ml) was added, and the mixture was stirred for 10 minutes. The reaction mixture was filtered through Celite, the filtrate was extracted with ethyl acetate, and the mixture was concentrated to dryness to give compound (5) (2.66 g). TLC: Rf 0.15 (hexane / ethyl acetate = 4 /
6).

(5) Compound (5) (1.657 g) was dissolved in methylene chloride (25 ml), pyridine (3 ml) was added, acetyl chloride (750 μl) was slowly added dropwise at 4 ° C., and the mixture was stirred for 1.5 hours. After that, it was diluted with water and extracted with ethyl acetate, and the crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 7/3),
The compound (6) (1.601 g) was obtained. TLC: Rf 0.30 (hexane / ethyl acetate = 6 /
4).

(6) Compound (6) (1.60 g) was dissolved in 15 ml of acetone, and Jones reagent (3 ml) was added dropwise at 4 ° C. After stirring for 10 minutes, isopropanol (1.5 ml) was added, and the mixture was further stirred at room temperature for 10 minutes. The reaction mixture was extracted with ethyl acetate to give compound (7)
(1.48 g) was obtained. TLC: Rf 0.46 (hexane / ethyl acetate = 6 /
4).

(7) Diisopropylamine (223μ
l) in diethyl ether solution (2 ml) at -20 ° C.
, 1.56 M n-butyllithium in hexane (97
1 μl) was slowly added dropwise, and the mixture was stirred for 1 hr, ethyl acetate (150 μl) was slowly added at −78 ° C., and the mixture was further added to 30
Stir for minutes. Next, a diethyl ether solution (150 μl) of compound (7) (200 mg) was added, and the mixture was stirred for 30 minutes. The compound was diluted with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and then purified by silica gel column chromatography (hexane / ethyl acetate = 8/2) to give the compound
(8) (247 mg) was obtained. TLC: Rf 0.48 (hexane / ethyl acetate = 6 /
4).

(8) To a solution of the compound (8) (30 mg) in methanol (2 ml) was added a 1N sodium hydroxide aqueous solution (21 ml).
3 μl), and the mixture was stirred at room temperature for 4 hours, 1N hydrochloric acid was added, and the mixture was extracted with ethyl acetate and purified by silica gel column chromatography (hexane / ethyl acetate = 6/4) to give compound (9) (22 mg ) Got. TLC: Rf 0.28 (hexane / ethyl acetate = 4 /
6).

(9) 10% Palladium on carbon (20 mg) in a solution of the compound (9) (22 mg) in methanol (2 ml).
Was added, and hydrogenation was carried out at room temperature under atmospheric pressure (1.5 hours), followed by filtration and concentration of the filtrate to obtain the compound (10) (14 mg). TLC: Rf 0.15 (ethyl acetate).

(10) Compound (10) (22 mg) and succinic anhydride (80 mg) were dissolved in THF (2 ml) to prepare pyridine (32 μl) and 4-dimethylaminopyridine (5 m).
g) was added, and the mixture was stirred at room temperature for 1.5 days. The reaction solution was concentrated and purified by silica gel column chromatography (methylene chloride / methanol = 95/5). The obtained crude product (11) and N-hydroxysuccinimide (58 m
g) was dissolved in THF (2 ml), dicyclohexylcarbodiimide (36 mg) was added, and the mixture was stirred at room temperature for 1.5 days. The residue is filtered, the filtrate is concentrated, and silica gel column chromatography (hexane / ethyl acetate = 2 /
The compound (12) (15 mg) was obtained by purification in 8). TLC: Rf 0.41 (ethyl acetate); NMR (CDCl ₃ ): δ 4.70 to 4.25 (2H, m), 4.19 (2H,
t), 3.02 to 2.92 (2H, m), 2.87 (4H, s), 2.8 to 2.73 (2H, m),
2.63 (1H, d), 2.5 (1H, d), 1.90 to 1.60 (6H, m); MS: m / z 372,277,185,93,75.

(B) Succinimide 3- (4-hydroxy-2-oxotetrahydropyran-4-yl) propyl octadienoate (1) 63% sodium hydride (657 mg) was added to DMF.
(20 ml), and adipic acid (3.00
g) was gradually added and stirred for 30 minutes. Next, benzyl bromide (2.00 ml) was gradually added dropwise, and the mixture was stirred at 80 ° C for 1.5 days. The reaction mixture was diluted with 2N hydrochloric acid, extracted with ethyl acetate, and purified by silica gel column chromatography (hexane / ethyl acetate = 1/1) to give compound (14) (1.68).
g) was obtained. TLC: Rf 0.53 (hexane / ethyl acetate = 4 /
6).

(2) Compound (14) (200 mg) was dissolved in thionyl chloride (2 ml) and refluxed for 2 hours.
Distill to remove excess thionyl chloride and remove THF (1 ml)
After being dissolved in THF, it was gradually added dropwise to a THF solution (1.0 ml) of the compound (10) (100 mg) and pyridine (300 μl) at room temperature and stirred for 1.5 hours. Dilute the reaction with water,
Extract with ethyl acetate and purify by silica gel column chromatography (hexane / ethyl acetate = 1/1).
The compound (16) (160 mg) was obtained. TLC: Rf 0.16 (hexane / ethyl acetate = 6 /
4).

(3) 10% Palladium on carbon (50 m) was added to a solution of the compound (16) (160 mg) in methanol (4 ml).
g) was added, hydrogenation was carried out at normal pressure for 1.5 hours, filtered and concentrated to obtain compound (17). Further, N-hydroxysuccinimide (46 mg) was added to the obtained compound, dissolved in DMF (2 ml), dicyclohexylcarbodiimide (76 mg) was added, and the mixture was stirred at room temperature for 18 hours. The reaction solution was filtered, the filtrate was diluted with water, extracted with ethyl acetate, and purified by silica gel column chromatography (hexane / ethyl acetate = 1/4) to give compound (18).
(87 mg) was obtained. TLC: Rf 0.55 (ethyl acetate); NMR (CDCl ₃ ): δ 4.70 to 4.27 (2H, m), 4.20 (2H,
t), 2.85 (4H, s), 2.70 to 2.45 (4H, m), 2.33 (2H, t), 1.95
~ 1.55 (8H, m), 1.55 ~ 1.23 (4H, m).

(C) 3- (4-hydroxy-2-oxotetrahydropyran-4-yl) propyl iodoacetate (1) Compound (10) (24 mg) and iodoacetic anhydride (65)
mg) was dissolved in THF (1.0 ml), 4-dimethylaminopyridine (26 mg) was added at room temperature, the mixture was stirred for 1 hour, diluted with water, extracted with ethyl acetate, and subjected to silica gel column chromatography (hexane / acetic acid). Ethyl = 1 /
It refine | purified in 2) and obtained the compound (19) (22 mg). TLC: Rf 0.57 (ethyl acetate); NMR (CDCl ₃ ): δ 4.70 to 4.30 (2H, m), 4.21 (2H,
t), ~ 3.72 (2H, s), 2.68 (1H, d), 2.53 (1H, d), 1.97 ~ 1.
63 (6H, m).

(D) Succinimide 11- (4-hydroxy-2-oxotetrahydropyran-4-yl) undecanoate (1) Compound (25) [1,12-dodecanediol was used as a starting material, and (a)- From (1) to (a)-
It manufactured by operating in the same manner as (5). ] (3.00 g) in methanol solution (20 ml) was added with 10% palladium carbon (550 mg), and hydrogenated at room temperature and atmospheric pressure (1.5
After filtration, the filtrate was concentrated and the compound (26) (2.
20 g) was obtained. TLC: Rf 0.13 (hexane / ethyl acetate = 7 /
3).

(2) Compound (26) (2.20 g) was dissolved in acetone (30 ml) and Jones reagent (7.0 m) was added at 4 ° C.
l) was added dropwise. After stirring for 10 minutes, isopropanol (2.0 ml) was added, and the mixture was further stirred for 10 minutes. The reaction solution was extracted with ethyl acetate, concentrated, and then concentrated with methanol (20 m
l), treated with diazomethane, concentrated, and purified by silica gel column chromatography (hexane / ethyl acetate = 9/1) to obtain compound (28) (2.04 g). TLC: Rf 0.33 (hexane / ethyl acetate = 7 /
3).

(3) Diisopropylamine (2.67 ml)
In diethyl ether solution (13 ml) at -20 ° C.
56M n-butyllithium hexane solution (10.8 ml)
Was slowly added dropwise, and the mixture was stirred for 1 hour, ethyl acetate (1.69 ml) was slowly added at -78 ° C, and the mixture was further stirred for 30 minutes. Next, a diethyl ether solution (15 ml) of the compound (28) (1.826 g) was added, and the mixture was stirred for 30 minutes. After diluting with saturated aqueous ammonium chloride solution and extracting with ethyl acetate, silica gel column chromatography (hexane /
Purified with ethyl acetate = 9/1) to give compound (29) (1.785
g) was obtained. TLC: Rf 0.47 (hexane / ethyl acetate = 7 /
3).

(4) To a solution of the compound (29) (311 mg) in methanol (20 ml) was added 1N aqueous sodium hydroxide solution (2.9 ml), the mixture was stirred at room temperature for 4 hours, 1N hydrochloric acid was added, and the mixture was extracted with ethyl acetate. Then, the compound (30) (221
mg) was obtained. TLC: Rf 0.72 (ethyl acetate / acetic acid = 99/1).

(5) Compound (30) (220 mg) and N-hydroxysuccinimide (126 mg) were added to THF (5 m).
l) and dissolved in dicyclohexylcarbodiimide (32
6 mg) was added, and the mixture was stirred at room temperature for 1.5 days. After filtration, the filtrate was concentrated and purified by silica gel column chromatography (hexane / ethyl acetate = 1/1) to give compound (31)
(210 mg) was obtained. TLC: Rf 0.24 (hexane / ethyl acetate = 4 /
6); NMR (CDCl ₃ ): δ 4.68 to 4.28 (2H, m), 2.83 (4H,
s), 2.70 to 2.45 (4H, m), 2.0 to 1.2 (14H, m); MS: m / z 283,265,247,237,22.
3,169,115,98.

(E) Succinimide 5- (4-hydroxy-2-oxotetrahydropyran-4-yl) pentanoate (1) Compound (8) (320 mg) was added to methanol (10 m).
l), dissolved in 10% palladium carbon (150 mg), hydrogenated at room temperature and atmospheric pressure (3 hours), filtered, and the filtrate was evaporated to dryness to give compound (32) (290 mg). . TLC: Rf 0.12 (hexane / ethyl acetate = 1 /
1).

(2) DMSO at -78 ° C. in a solution of oxalyl chloride (27 μl) in methylene chloride (1 ml).
A methylene chloride solution (0.5 ml) of (45 μl) was added dropwise, the mixture was stirred for 10 minutes, a methylene chloride solution (1.0 ml) of the compound (32) (50 mg) was added, and the mixture was stirred for 40 minutes.
Next, triethylamine (145 μl) was added, and the temperature was -30 ° C.
The temperature was raised to 30 minutes, diluted with water, and extracted with ethyl acetate. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 2/1) to give the compound
(33) (28 mg) was obtained. TLC: Rf 0.38 (hexane / ethyl acetate = 1 /
1).

(3) Methyl triphenylphosphonoacetate (76 ml) was added to a THF solution (2 ml) of the compound (33) (50 mg).
mg) was added and the mixture was stirred at room temperature for 2.5 days. After concentration, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3/1) to obtain compound (34) (54 mg). TLC: Rf 0.53 (hexane / ethyl acetate = 1 /
1).

(4) To a methanol solution (2 ml) of the compound (34) (54 mg) was added 5% palladium carbon (50 mg), and hydrogenation was carried out at room temperature under atmospheric pressure (30 minutes), followed by filtration and filtration. Was concentrated to obtain compound (35) (48 mg). TLC: Rf 0.53 (hexane / ethyl acetate = 1 /
1).

(5) To a methanol solution (1.5 ml) of the compound (35) (48 mg) was added 1N aqueous sodium hydroxide solution (600 μl) at room temperature, the mixture was stirred for 4.5 hours, and 1N hydrochloric acid (600 μl) was added. I made it Concentrated to dryness and purified by silica gel column chromatography (ethyl acetate / methanol = 95/5) to give compound (38) (35 mg).
Got TLC: Rf 0.10 (ethyl acetate / methanol = 8 /
2).

(6) N-hydroxysuccinimide (16) was added to a THF solution (1.0 ml) of compound (38) (20 mg).
mg) and dicyclohexylcarbodiimide (29 mg)
Was added, and the mixture was stirred for 22 hours at room temperature, filtered, and the filtrate was concentrated. Further, it was purified by silica gel column chromatography (hexane / ethyl acetate = 3/7) to obtain compound (39) (11 mg). TLC: Rf 0.31 (ethyl acetate); NMR (CDCl ₃ ): δ 4.70 to 4.30 (2H, m), 2.85 (4H,
s), 2.75 to 2.48 (4H, m), 1.95 to 1.73 (4H, m), 1.73 to 1.45
(4H, m); MS: m / z 199,181,163,139,11
5,98,85.

(F) Succinimide 4- (4-hydroxy-4-methyl-2-oxotetrahydropyran-6)
-Yl) Benzoate (1) To a solution of the compound (40) (1.36 g) in acetone (4.4 ml) and DME (8.2 ml), 0.03N aqueous sodium hydroxide solution (13.35 ml) was cooled with ice water and vigorously stirred. While adding and stirring for 45 minutes, 1N hydrochloric acid (700
μl) was added. The reaction solution was extracted with ethyl acetate and subjected to column chromatography (hexane: ethyl acetate = 7:
It refine | purified in 3) and the compound (41) (1.252 g) was obtained. TLC: Rf 0.58 (hexane: ethyl acetate = 4:
6).

(2) To a solution of the compound (41) (1.252 g) in acetic anhydride (1.0 ml) was added triethylamine (1.56 ml), and the mixture was stirred at room temperature for 30 minutes, and then 1N hydrochloric acid (20 m) was added.
l) was added. The reaction solution was extracted with ethyl acetate and column chromatography (hexane: ethyl acetate = 8: 2).
The compound (42) (682 mg) was obtained. TLC: Rf 0.61 (hexane: ethyl acetate = 4:
6).

(3) Diisopropylamine (742 μ
l) in diethyl ether (5 ml) solution at -20 ° C
And 1.56M n-butyllithium in hexane (2.89
(ml) was slowly added dropwise, and the mixture was stirred for 1 hr. Ethyl acetate (452 μl) was slowly added at -78 ° C, and the mixture was further added to 30 ml.
Stir for minutes. Next, a solution of the compound (42) (582 mg) in anhydrous THF (5 ml) was added, and the mixture was stirred for 30 minutes, and then diluted with a saturated aqueous ammonium chloride solution. The reaction solution was extracted with ethyl acetate and purified by column chromatography (hexane: ethyl acetate = 7: 3) to give compound (43) (7
03 mg) was obtained. TLC: Rf 0.37 (hexane: ethyl acetate = 6:
4).

(4) A solution of the compound (43) (110 mg) in methanol (4 ml) was cooled with ice water, 1N aqueous sodium hydroxide solution (1.14 ml) was added, and the mixture was stirred at room temperature for 20 hours, then 1N hydrochloric acid ( 2 ml) was added. The reaction solution was extracted with ethyl acetate and purified by column chromatography (ethyl acetate: methanol: acetic acid = 8: 2: 0.1),
Compound (44) (73 mg) was obtained. TLC: Rf 0.36 (ethyl acetate: acetic acid = 98: 2).

(5) Compound (44) (59 mg) and N-hydroxysuccinimide (54 mg) in THF (4 m
l) with dicyclohexylcarbodiimide (97 mg)
Was added and stirred at room temperature for 20 hours. The residue was filtered, the filtrate was concentrated, and purified by column chromatography (hexane: ethyl acetate = 3: 7) to give compound (45) (60 m
g) was obtained. TLC: Rf 0.67 (ethyl acetate); NMR (CDCl ₃ ): δ 8.15 (2H, d), 7.55 (2H, d), 5.83
-5.3 (1H, m), 3.0-2.55 (6H, m), 2.35-1.4 (5H, m).

(G) Succinimide 4-((4-hydroxy-2-oxotetrahydropyran-4-yl) carbonylamino) phenylacetate (1) A solution of the compound (46) (1.84 g) in DMF (17 ml) was added with iodine. Ethyl chloride (1.5 ml) and potassium carbonate (2.0
g) was added and the mixture was stirred at 70 ° C. for 6 hours. The reaction solution was extracted with ethyl acetate and purified by column chromatography (hexane: ethyl acetate = 9: 1) to give compound (47) (3.
32 g) was obtained. TLC: Rf 0.43 (hexane: ethyl acetate = 8:
2).

(2) To a solution of the compound (47) (3.32 g) in methanol (15 ml) was added 10% palladium-carbon (700
mg) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours,
It was filtered and concentrated. 2-hydroxy-γ-
Butyrolactone (4.24 g) and dimethylaminopyridine (388 mg) were added, and the mixture was stirred at 90 ° C. for 3 hours. The reaction solution was purified by column chromatography (hexane: ethyl acetate = 8: 2) to obtain compound (48) (2.49 g). TLC: Rf 0.38 (ethyl acetate).

(3) A solution of the compound (48) (2.49 g) in methylene chloride (25 ml) was cooled with ice water, and pyridine (3.58 g) was added.
ml) was added, acetyl chloride (0.78 ml) was slowly added dropwise, the mixture was stirred for 1 hour, and then diluted with water. The reaction solution was extracted with ethyl acetate and purified by column chromatography (hexane: ethyl acetate = 6: 4) to give compound (49) (1.
74 g) was obtained. TLC: Rf 0.44 (hexane: ethyl acetate = 4:
6).

(4) Jones reagent (5 ml) was added to a solution of the compound (49) (1.74 g) in acetone (25 ml), and the mixture was stirred at room temperature for 20 minutes, and then isopropanol (2 ml) was added.
Was added. The reaction solution was extracted with ethyl acetate and purified by column chromatography (hexane: ethyl acetate = 7: 3) to obtain compound (50) (447 mg). TLC: Rf 0.80 (hexane: ethyl acetate = 4:
6).

(5) Diisopropylamine (109 μ
l) in diethyl ether (1 ml) solution at -20 ° C
And 1.56M n-butyllithium hexane (0.40m
l) The solution was slowly added dropwise, followed by stirring for 1 hour.
Ethyl acetate (62 μl) was slowly added at 0 ° C, and another 3
Stir for 0 minutes. Next, a solution of the compound (50) (50 mg) in anhydrous THF (0.60 ml) was added, and the mixture was stirred for 30 minutes,
Diluted with saturated aqueous ammonium chloride solution. The reaction solution was extracted with ethyl acetate and purified by column chromatography (hexane: ethyl acetate = 6: 4) to give compound (51) (5
0 mg) was obtained. TLC: Rf 0.17 (hexane: ethyl acetate = 6:
4).

(6) A solution of the compound (51) (39 mg) in methanol (1 ml) was cooled with ice water, 1N aqueous sodium hydroxide solution (0.38 ml) was added, and the mixture was stirred at room temperature for 3 hours, and then 1N hydrochloric acid (2 ml) was added. ) Was added. The reaction solution was extracted with ethyl acetate to obtain the compound (52) (25 mg). TLC: Rf 0.72 (ethyl acetate: methanol: acetic acid =
80: 15: 5).

(7) Compound (52) (25 mg) and N-hydroxysuccinimide (20 mg) in THF (1 m
l) with dicyclohexylcarbodiimide (35 mg)
Was added, and the mixture was stirred at room temperature for 18 hours. The residue was filtered, the filtrate was concentrated, and purified by column chromatography (hexane: ethyl acetate = 3: 7) to give compound (53) (19 m
g) was obtained. TLC: Rf 0.58 (ethyl acetate); NMR (acetone-d ₆ ): δ 7.78 (2H, d), 7.38 (2H,
d), 4.6-4.3 (2H, m), 4.02 (2H, s), 3.65-3.58 (1H, m), 3.
2-2.0 (8H, m).

(H) Succinimide 3- (4-hydroxy-4-methyl-2-oxotetrahydropyran-6)
-Yl) propionate (1) Compound (3) (5.4 g) in acetone (180 ml)
The solution was cooled with ice water, 1.44M sodium ethoxide (3.1 ml) was added with vigorous stirring, and 15 seconds later, 1N hydrochloric acid (7.5 ml) was added. The reaction solution was extracted with ethyl acetate and purified by column chromatography (hexane: ethyl acetate = 65: 35) to obtain compound (54) (2.942 g). TLC: Rf 0.31 (hexane: ethyl acetate = 6:
4).

(2) A solution of the compound (54) (2.942 g) in methylene chloride (60 ml) was added with camphorsulfonic acid (12
mg) was added, dihydropyran (2.27 ml) was slowly added dropwise at 0 ° C., the mixture was stirred at room temperature for 2 hr, and triethylamine (0.1 ml) was added. The reaction solution was extracted with ethyl acetate and purified by column chromatography (hexane: ethyl acetate = 9: 1) to obtain compound (55) (4.0 g). Then, to a solution of diisopropylamine (6.03 ml) in diethyl ether (60 ml), at −20 ° C., 1.56M n
A solution of -butyllithium in hexane (22.4 ml) was slowly added dropwise, followed by stirring for 1.5 hours, ethyl acetate (3.49 ml) was slowly added at -78 ° C, and the mixture was further stirred for 55 minutes. Compound (55) (4 g) in diethyl ether (10 m
1) The solution was added, stirred for 40 minutes, and then diluted with a saturated aqueous solution of ammonium chloride. The reaction solution was extracted with ethyl acetate and purified by column chromatography (hexane: ethyl acetate = 7: 3) to obtain compound (56) (5.0 g). TLC: Rf 0.56 (hexane: ethyl acetate = 6:
4).

(3) A solution of the compound (56) (1.517 g) in methanol (6 ml) was cooled with ice water, 0.55N aqueous sodium hydroxide solution (13.7 ml) was added, and the mixture was stirred at room temperature for 4 hours, then 1N hydrochloric acid was added. (5 ml) was added. The reaction solution was extracted with ethyl acetate. A solution of the crude product obtained by concentration in methanol (55 ml) was added with 10% palladium-carbon (600
mg) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 1 hr.
Filtration and concentration gave compound (57) (0.50 g). TLC: Rf 0.08 (ethyl acetate).

(4) A solution of the compound (57) (240 mg) in acetone (24 ml) was added at 4 ° C. to Jones reagent (0.1 ml).
(7 ml) was added, and the mixture was stirred at room temperature for 15 minutes, isopropanol (2 ml) was added, and the mixture was further stirred for 5 minutes. The reaction solution was diluted with chloroform, anhydrous magnesium sulfate was added, the mixture was filtered and concentrated on Celite, and purified by column chromatography (methanol: ethyl acetate = 1: 9) to obtain compound (58) (188 mg). . TLC: Rf 0.67 (ethyl acetate: methanol: acetic acid =
80: 15: 5).

(5) THF (2 m) of compound (58) (171 mg) and N-hydroxysuccinimide (194 mg)
l) solution, dicyclohexylcarbodiimide (279
mg) was added and the mixture was stirred at room temperature for 60 hours. The residue was filtered, the filtrate was concentrated, and purified by column chromatography (hexane: ethyl acetate = 2: 8) to give compound (59) (1
54 mg) was obtained. TLC: Rf 0.36 (ethyl acetate); NMR (CDCl ₃ ): δ 4.9-4.35 (1H, m), 2.9-2.4 (6H,
m), 2.16-1.46 (6H, m), 1.4 (3H, m); MS: m / z 281 (M ⁺ -18), 185, 15
7,129,97.

Example 2 Preparation of Sensitizing Antigen The conjugate of the mevalonic acid derivative synthesized in Example 1 and the spacer (compound (12), 18 μmol) was treated with methanol (71
μL), 0.5 M phosphate buffer (hereinafter abbreviated as PB, pH 7.4, 71 μl) was added, and the mixture was stirred for 10 minutes and then bovine serum albumin (hereinafter abbreviated as BSA).
50 mM PB (pH 7.4, 3.8 m containing 60 mg)
1) was added, and the mixture was stirred at room temperature for 6 hours. The reaction solution is 1 at 4 ℃
After dialysis with 0 mM PB, the target sensitizing antigen (BSA- (1
2)) got.

Next, the same compound (12) (27 μmol) was dissolved in methanol (105 μl) and PBS (pH7.4) was added.
, 105 μl) and stirred for 10 minutes, and then keyhole limpet hemocya
nin: hereinafter abbreviated as KLH. PBS (pH 7.4, 6.4 ml) containing 86 mg (69% content) was added, and the mixture was stirred at room temperature for 6 hours. The reaction solution was PBS (pH7.4) at 4 ℃.
), The target sensitizing antigen (KLH- (12)) was obtained.

Similarly, the sensitizing antigen of interest, BS
A- (18), KLH- (18), BSA- (19), KLH- (1
9), BSA- (31), KLH- (31), BSA- (39), KL
H- (39), BSA- (45), KLH- (45), BSA- (5
3), KLH- (53), BSA- (59) and KLH- (59) were obtained.

Example 3 Preparation of Rabbit Polyclonal Antibody Various sensitizing antigens prepared in Example 2 (1 mg BSA in the case of BSA conjugate and 1 mg KLH in the case of KLH conjugate) were prepared. Including P
An emulsion consisting of BS (500 μl) and FCA (500 μl) was subcutaneously administered to 7-week-old male New Zealand white rabbits. Approximately 4 weeks later, PBS solution (500 μl) containing sensitizing antigen prepared in the same manner as the previous time and FIC
The emulsion consisting of A (500 μl) was sensitized four times every two weeks thereafter. The antibody titer in blood was measured after the first, third, and fifth sensitization with the sensitizing antigen and FICA. The method is as follows.

Blood was collected from a rabbit, water (450 μl), 0.5% γ-globulin (80 μl) and saturated ammonium sulfate (580 μl) were added to serum (50 μl), and the mixture was centrifuged. The precipitate was dissolved in 50 mM PB (200 μl), and R-
[2- ¹⁴ C] 50mM containing mevalonate 400Bq P
After adding B (100 μl) and reacting at 4 ° C. overnight,
0.1M PB (200μ containing 5% bovine γ-globulin
l) is added, and 30% polyethylene glycol 6000 is further added.
(Registered trademark, manufactured by Wako Pure Chemical Industries, 600 μl) was added and centrifuged, and then the radioactivity of the precipitate was measured. After the third sensitization, the serum antibody titers of the sera using KLH- (12) as the sensitizing antigen were significantly increased. Therefore, these sera were used for the polyclonal antibodies KLH- (12) R and BSA-, respectively. (18) R,
KLH- (18) R, BSA- (19) R, KLH- (19) R, B
SA- (31) R, KLH- (31) R, BSA- (39) R, KL
H- (39) R, BSA- (45) R, KLH- (45) R, BSA
-(53) R, KLH- (53) R, BSA- (59) R and KL
It was named H- (59) R.

Example 4: Preparation of mouse polyclonal antibody Various sensitizing antigens prepared in Example 2 (120 μg as BSA amount in the case of BSA conjugate, KLH)
In the case of conjugate, the amount of KLH is 120 μg)
An emulsion consisting of PBS (100 μl) and FCA (100 μl) was intraperitoneally administered to 8-week-old BALB / c male mice. After that, about every 2 weeks, it was sensitized 5 times with an emulsion composed of a PBS solution (100 μl) containing the sensitizing antigen prepared in the same manner as the previous time and FICA (100 μl). Of the sensitization with sensitizing antigen and FICA,
Blood antibody titers were measured after the second and fifth sensitization. The method is as follows.

On a polystyrene 96-well microplate (manufactured by NUNC), 10 mM of 50 mM PB containing a conjugate of a mevalonic acid derivative and a carrier protein (both the derivative used as the sensitizing antigen and the carrier protein are different) was used.
0 μl of each was added, and the mixture was allowed to stand at room temperature for 1.5 hours for immobilization.
Excluding liquid phase, contains 0.5% BSA (when KLH is used as carrier protein for sensitizing antigen) or ovalbumin (hereinafter abbreviated as OVA; when BSA is used as carrier protein for sensitizing antigen) 50 mM PB
(Hereinafter, abbreviated as BSA (OVA) -PB.) (40
0 μl) was added, and the inner wall of the well was blocked at room temperature for 1 hour.

After removing the liquid phase, mevalonic acid (100 μm
g) with or without 50 mM PB (100
μl) was added. 100 μl of a diluted serum solution (diluted with 0.1% BSA (OVA) -PB diluted 100 to 100,000 times) obtained from a mouse was added to each well and reacted at room temperature for 1 hour.
50% Tween 20 except liquid phase 50
It was washed twice with mM PB (300 μl). 0.1% BS
A peroxidase-labeled anti-mouse IgG antibody (manufactured by Vector Co.) diluted 10000 times with A (OVA) -PB was used for 10 times.
0 μl of each was added, and the mixture was reacted at room temperature for 1 hour. After removing the liquid phase and washing twice with 50 mM PB containing 0.05% Tween 20, the substrate solution (dimethyl sulfoxide, water, 20
0.1M citric acid containing mM3,3 ', 5,5'-tetramethylbenzidine, MacIlvain buffer (pH 4.57) and 14mM hydrogen peroxide solution 4: 4.
(Mixed in a ratio of 6: 2: 38: 10, 250 μl)
Was added and reacted at room temperature for 15 minutes. 1N sulfuric acid (50μ
l) was added to stop the reaction, and then the absorbance at 450 nm was measured. Based on the sensitizing antigen, serum in which a difference in absorbance was significantly observed in the presence and absence of mevalonic acid was
Polyclonal antibody KLH- (12) M, KLH- (18) M,
BSA- (19) M, KLH- (31) M, KLH- (39) M, K
It was named LH- (45) M, KLH- (53) M and KLH- (59) M.

Example 5: Preparation of mouse monoclonal antibody (1) Sensitization of mouse Sensitization of mouse was carried out in the same manner as in Example 4 under the following conditions. (i) Sensitization method Sensitization antigen: KLH- (39) Number of sensitizations by FICA: 10 times (ii) Antibody titer measurement Antigen used for immobilization: BSA- (12) (1 as BSA amount)
50 mM PB containing 00 ng, 10 ng or 1 ng
(100 μl)) The 10th sensitization with FICA confirmed that the antibody titer was significantly increased. Therefore, 2 weeks later, PBS (100 μl) containing a sensitizing antigen (300 ng as KLH amount) was added.
1) was administered intraperitoneally.

(2) Cell fusion Three days after the intraperitoneal administration of PBS containing the sensitizing antigen, the spleen was excised from the sensitized mouse prepared in Example 5 to prepare splenocytes. Obtained splenocytes and mouse myeloma cells SP-2 /
It was prepared by the method described in 0-Ag14 [Nature, 276, 269 (1978). ] At a ratio of 5: 1 and polyethylene glycol [PEG4000 (for gas chromatography), manufactured by Merck & Co.] at a concentration of 50% was added, and Goding
The cell fusion was performed according to the method described in [J. Immunol, Methods, 39, 285 (1980)].

[0110] The cell mixture after the fusion operation was treated with 20% fetal bovine serum (FBS), 10% NCTC109 medium (registered trademark, manufactured by MA BioProducts), hypoquinanthine (1
3.6 μg / ml), thymidine (3.9 μg / ml) and glycine (2.0 μg / ml) in Dulbecco's modified Eagle medium (4.5 g / l glucose-containing type, manufactured by Gibco), and suspended at 37 ° C. for 5 Culture was performed in an atmosphere containing% CO ₂ . On days 2, 4 and 7 after the culture, half of the medium was converted to HAT medium (the above Eagle medium containing 0.18 μg / ml of aminopterin) and the culture was continued. From around the 10th day of culture, tuft-shaped colonies of grapes were formed in some wells, and finally hybridoma growth was observed in 801 wells.

Screening of the monoclonal antibody producing strain was carried out by the following method. (3) Screening of Monoclonal Antibody-Producing Strain A polystyrene 96-well microplate was coated with BSA- (1
2) Add 50 mM PB containing 1 ng of BSA (1 ng)
Each 100 μl was added, and the mixture was allowed to stand at room temperature for 1.5 hours for immobilization. 50 mM PB containing 0.5% BSA except liquid phase
(400 μl) was added, and the inner wall of the well was blocked at room temperature for 1 hour.

After removing the liquid phase, mevalonic acid (100 μm
g) with or without 50 mM PB (100
μl) was added. Diluted solution of hybridoma culture supernatant (diluted 50 times with 50 mM PB containing 0.1% BSA)
Was added to each 100 μl and reacted at room temperature for 1 hour. Excluding the liquid phase, 50 mM PB containing 0.05% Tween 20 (3
It was washed twice with 00 μl). 50m containing 0.1% BSA
100 μl of peroxidase-labeled anti-mouse IgG antibody (manufactured by Vector Co.) diluted 10000 times with MPB was added and reacted at room temperature for 1 hour. After removing the liquid phase and washing twice with 50 mM PB containing 0.05% Tween 20, the substrate solution (produced in Example 4, 250 μl) was added and reacted at room temperature for 15 minutes. After stopping the reaction by adding 1N sulfuric acid (50 μl), the absorbance at 450 nm was measured. A culture supernatant in which a difference in absorbance was significantly observed in the presence and absence of mevalonic acid was determined to be a well producing a monoclonal antibody against mevalonic acid.

(4) Culturing of antibody-producing hybridoma cells Cells determined to produce antibodies to mevalonic acid were treated with the method of Kennett [Monoclonal Antibodies, p. 372].
(1980)]. The cloned cell line was named MHM-9H strain. 10 ⁷ cloned cell lines were transplanted into the abdominal cavity of BALB / c female mice which had been previously treated with pristane. After about two weeks, ascites was collected when a large amount of ascites was accumulated. After fractionating the sampled water with 50% saturated ammonium sulfate,
The IgG fraction was obtained by purification by affinity column chromatography using a Protein A-Sepharose CL4B column (Pharmacia). The monoclonal antibody of the present invention, the hybridoma producing MHM-9H, and the MHM-9H strain were designated by the Biotechnology Institute of Technology under the accession number FERM BP-4524, January 6, 1994.
Deposited on the day.

(5) Immunoglobulin subclass of monoclonal antibody Mouse monoclonal antibody MHM-9H
Subclasses were screened using the ono Ab-IDEIA kit (Zymed). As a result, MHM
-9H mouse IgG _2b, was κ.

[0115] Example 6: cross polystyrene tubes with various mevalonate analogues of rabbit polyclonal antibodies, R- [2- ¹⁴ C] 50mM containing mevalonate 400Bq PB (100μl), polyclonal antibody solution (the present invention Antibody KLH- (12) R,
Water: 0.5% γ-globulin and saturated ammonium sulfate 0.5: 4.5
: 0.8: 5.8, and after centrifugation, the resulting precipitate was added to 4 times the amount of 50 mM of the polyclonal antibody used.
PB dissolved in PB, 100 μl) and standard solutions of mevalonic acid analogs shown in the table below (50 mM PB solution,
100 μl) was added and left overnight at 4 ° C. Add 0 to the reaction mixture.
After adding 5% γ-globulin solution (200 μl), 30
% Polyethylene glycol 6000 (600 μl) was added,
It was centrifuged. The precipitate was dissolved in a 0.1 N sodium hydroxide aqueous solution, a liquid scintillator (1 ml) was added, and the radioactivity was measured. The results are shown in Table 1.

[0116]

[Table 1]

From Table 1, it can be seen that the antibody of the present invention specifically binds to a low concentration of mevalonic acid, and the crossover rate with other mevalonate-like compounds is as low as 0.1% or less.

Example 7: Cross-linking of mouse monoclonal antibody with various mevalonate analogues An antibody solution (100 μl prepared by the method described in Example 10 below) was added to a 96-well polystyrene microplate.
Was added and the mixture was allowed to stand at room temperature for 1.5 hours to solidify. Excluding the liquid phase, 50 mM PB (400 μl) containing 0.5% BSA
Was added, and the inner wall of the well was blocked at room temperature for 1 hour.

After removing the liquid phase, 50 mM PB (100 μl) containing various concentrations of mevalonic acid analogs was added. A labeled antigen solution (manufactured by the method described in Example 10 below) was added in an amount of 100 μl / well, and the mixture was left at 4 ° C. overnight. 50 mM containing 0.05% Tween 20 except liquid phase
It was washed twice with PB (300 μl). A substrate solution (250 μl produced by the method described in Example 9 below) was added, and the mixture was reacted at room temperature for 15 minutes. After stopping the reaction by adding 1N sulfuric acid (50 μl), the absorbance at 450 nm was measured. The results are shown in Table 2.

[0120]

[Table 2]

From Table 2, it can be seen that the monoclonal antibody of the present invention specifically binds to a low concentration of mevalonic acid, and the crossover rate with other mevalonate-like compounds is as low as 0.001% or less. .

Example 8: Immunological quantification of mevalonic acid using rabbit polyclonal antibody (radioimmunoassay)

(1) Preparation of immunological assay reagent for mevalonic acid (a) Antibody solution Polyclonal antibody KLH- (12) R, water, 0.5% γ-globulin and saturated ammonium sulfate in a ratio of 0.5: 4.5: 0.8: 5.8. After mixing and centrifuging, the resulting precipitate was mixed with 4 times the amount of 50 mM of the polyclonal antibody KLH- (12) R used.
Dissolved in PB. (b) labeled antigen solution R- [2- ¹⁴ C] 50mM containing mevalonate 400Bq
PB. (c) Standard solution 10000 μg / ml of mevalonic acid in 50 mM PB, 1
00 μg / ml, 10 μg / ml, 1 μg / ml, 0.1
Prepared to be μg / ml and 0.01 μg / ml.

(2) Quantification of mevalonic acid using the reagent prepared in (1) After adding the antibody solution (100 μl), the labeled antigen solution (100 μl) and the standard solution (or sample) (100 μl) to a polypropylene tube, It was left overnight at 4 ° C. After adding 0.5% γ-globulin solution (200 μl) to the reaction solution, 30% polyethylene glycol 6000 (600
μl) was added and centrifuged. Dissolve the precipitate in 0.1 N sodium hydroxide aqueous solution and use a liquid scintillator (1 m
1) was added and the radioactivity was measured. A standard calibration curve prepared by calculating B / Bo from the measured radioactivity is shown in FIG. As is clear from FIG. 1, in this quantitative method,
Mevalonic acid in the range of 20 to 1000 ng / test can be measured.

Example 9: Immunological quantification method of mevalonic acid using mouse polyclonal antibody (enzyme immunoassay, antigen immobilization method)

(1) Preparation of Reagent for Quantification (a) Second Antibody Solution (B or O) Horseradish peroxidase-labeled anti-mouse IgG antibody (manufactured by Vector) was added to 50 mM containing 0.1% BSA.
Diluted 10,000 times with PB (second antibody solution (B)). The antibody was added to 50 mM P containing 0.1% OVA.
Diluted 10000 times with B (second antibody solution (O)). (b) First antibody solution The polyclonal antibody of the present invention shown in Table 3 was added with 0.1% B
Diluted with 50 mM PB containing SA or OVA at a magnification shown in Table 3 (in the case of an antibody obtained by using KLH as a protein for sensitizing antigen, diluted with BSA-containing PB,
In the case of an antibody obtained using BSA, PVA containing OVA
Dilute with. ). (c) Antigen solution A mixture of mevalonic acid and carrier protein shown in Table 3 dissolved in 50 mM PB at a concentration shown in Table 3.

(D) Standard solution Mevalonic acid was dissolved in 50 mM PB, and the concentration was 1 mg /
ml, 100 μg / ml, 10 μg / ml, 1 μg / m
1, 100 ng / ml and 10 ng / ml. (e) Wash solution 50 mM PB containing 0.05% Tween 20. (f) Blocking solution (B or O) 50 mM PB containing 0.5% BSA (blocking solution (B)). 50 mM PB containing 0.5% OVA (blocking solution (O)). (g) Substrate solution dimethyl sulfoxide, distilled water, 20 mM 3,3 ',
0.1 M citric acid containing 5,5'-tetramethylbenzidine, McIlvain buffer (pH 4.57) and 14 m
A mixture of M hydrogen peroxide solution at a ratio of 4: 46: 2: 38: 10. (h) Stop solution 1N sulfuric acid.

(2) Quantification of mevalonic acid using the reagent prepared in (1) 100 μl of the antigen solution was added to each polystyrene 96-well microplate and allowed to stand at room temperature for 1.5 hours for immobilization. The liquid phase was removed, blocking solution (B) or blocking solution (O) was added, and the inner wall of the well was blocked at room temperature for 1 hour.

After removing the liquid phase, the standard solution (100 μl)
Was added. 100 µl of the first antibody solution was added to each, and the mixture was reacted at room temperature for 1 hour. Remove the liquid phase and wash (300 μl)
It was washed twice with. The second antibody solution (B) or the second antibody solution (O) was added in an amount of 100 μl / well and reacted at room temperature for 1 hour. After removing the liquid phase and washing twice with a washing solution, a substrate solution (250 μl) was added and reacted at room temperature for 15 minutes. After adding the reaction stop solution (50 μl) to stop the reaction, 4
Absorbance at 50 nm was measured.

For all mouse polyclonal antibodies, B / B ₀ was determined from the measured values of standard mevalonic acid and plotted to prepare a standard calibration curve. Table 3 shows the measurement conditions and the calibration range of each polyclonal antibody, and FIG. 2 shows the standard calibration curve of the polyclonal antibody, KLH- (39) M.

[0131]

[Table 3]

Example 10: Immunological determination of mevalonic acid using mouse monoclonal antibody (enzyme immunoassay, first antibody solid phase method)

(1) Preparation of quantitative reagent (a) Enzyme-labeled antigen solution Mevalonate derivative-spacer conjugate (18) (Example 1)
PB containing 175 nmol produced in (b) and horseradish peroxidase (produced by Toyobo Co., Ltd., 350 μg)
After adding S (175 μl) and reacting at room temperature for 2 hours and further at 4 ° C. overnight, the reaction product was added with 1,4-dioxane and PB.
It dialyzed with the mixed liquid of S (1: 1), and also dialyzed with PBS. The thus-obtained enzyme-labeled antigen was prepared in 50 mM PB containing 0.1% BSA so as to be 1.25 ng / 100 μl (as a peroxidase protein). (b) Antibody Solution The monoclonal antibody of the present invention, MHM-9H (Example 5)
It was prepared in. ) Was prepared in 50 mM PB to 7 μg / ml. (c) Standard solution Dissolve mevalonic acid in 50 mM PB to a concentration of 500 n
g / ml, 200ng / ml, 80ng / ml, 32n
g / ml, 12.8 ng / ml, 5.12 ng / ml, 2.04 ng
/ Ml and 0.82 ng / ml.

(D) Wash solution 50 mM PB containing 0.05% Tween 20. (e) Blocking solution 50 mM PB containing 0.5% BSA. (f) Substrate solution dimethyl sulfoxide, distilled water, 20 mM 3,3 ',
0.1 M citric acid containing 5,5'-tetramethylbenzidine, McIlvain buffer (pH 4.57) and 14 m
A mixture of M hydrogen peroxide solution at a ratio of 4: 46: 2: 38: 10. (g) Reaction stop solution 1N sulfuric acid.

(2) Quantification of mevalonic acid using the reagent prepared in (1) 1) An antibody solution (100 μl prepared by the above method) was added to a 96-well polystyrene microplate and incubated at room temperature for 1.5 hours. I put it. 2) The liquid phase was removed, a blocking solution (prepared by the above method, 400 μl) was added, and the mixture was left at room temperature for 1 hour. 3) After removing the liquid phase, a standard solution (prepared by the above method) or a sample (100 μl) was added, and then a labeled antigen solution (prepared by the above method, 100 μl) was added,
It was left overnight at 4 ° C. 4) The liquid phase was removed and the product was washed twice with the washing liquid. 5) A substrate solution (prepared by the above method, 250 μl) was added and reacted at room temperature for 15 minutes. 6) A reaction stop solution (prepared by the above method, 50 μl) was added to stop the reaction. 7) The absorbance at 450 nm was measured.

FIG. 3 shows a standard calibration curve obtained by plotting B / B ₀ from the measured values of standard mevalonic acid. As is clear from FIG. 3, in this quantitative method, 0.4 to 20 ng / test
It is possible to measure mevalonic acid in the range of.

Example 11: Immunological quantification of mevalonic acid in human urine Normal human urine and normal human urine supplemented with a known amount of mevalonic acid (2 ml each) were used for Sep-Pak PS-1 column (Waters). Reversed phase column chromatography was carried out using (Production) to obtain a mevalonic acid-containing fraction. The amount of mevalonic acid in this fraction was quantified by the immunological quantification method shown in Example 10. The results are shown in Table 4.

[0138]

[Table 4]

From the results of the dose-increasing experiment, the measured values were almost the same as the conventionally reported mevalonate levels in normal human urine. Moreover, even if the amount of urine used for the measurement changed, the measured values were in good agreement, and the results of the weight increase experiment were good. Similarly,
The result of the addition recovery experiment was also good. From this, it was shown that the immunological assay method for mevalonic acid of the present invention is an extremely simple and practical assay method.

[Brief description of drawings]

FIG. 1 Polyclonal antibody of the present invention, KLH- (12)
It is a calibration curve in the immunological quantification method of mevalonic acid using R.

FIG. 2 Polyclonal antibody of the present invention, KLH- (39)
3 is a calibration curve in an immunological quantification method of mevalonic acid using M.

FIG. 3: Monoclonal antibody of the present invention, MHM-9H
2 is a calibration curve in an immunological quantification method of mevalonic acid using.

Claims (18)

[Claims] 1. An antibody that specifically recognizes mevalonic acid. 2. The antibody according to claim 1, wherein the antibody has a crossing rate of 1.0% or less with respect to glutaric acid, 3-methylglutaric acid and 3-hydroxy-3-methylglutaric acid. 3. A compound in which a reactive functional group is introduced directly or indirectly into a hydroxyl group or a methyl group substituted on the carbon atom at the 3-position of mevalonic acid, or in place of these groups, or of mevalonic acid The antibody according to claim 1, which is obtained by using as a immunogen an conjugate of a protein and a compound in which a reactive functional group is directly or indirectly introduced into the 5-position carbon atom. 4. A compound in which a reactive functional group is directly or indirectly introduced into a hydroxyl group or a methyl group substituted on the carbon atom at the 3-position of mevalonic acid or in place of these groups, and the protein. The antibody according to claim 3, which is obtained by using the conjugate as an immunogen. 5. The antibody according to claim 1, which is a polyclonal antibody. 6. The antibody according to claim 5, which is a rabbit polyclonal antibody obtained by using a conjugate of a mevalonic acid derivative and a protein as an immunogen. 7. Keyhole limpet hemocyanin (K
LH) and succinimide 3- (4-hydroxy-2-)
The antibody according to claim 5, which is a polyclonal antibody obtained by using a conjugate of oxotetrahydropyran-4-yl) propyl succinate as an immunogen. 8. The antibody according to claim 5, which is a mouse polyclonal antibody obtained by using a conjugate of a mevalonic acid derivative and a protein as an immunogen. 9. Keyhole limpet hemocyanin (K
LH) and succinimide 5- (4-hydroxy-2-)
The antibody according to claim 5, which is a polyclonal antibody obtained by using a conjugate of oxotetrahydropyran-4-yl) pentanoate as an immunogen. 10. The antibody according to claim 1, which is a monoclonal antibody. 11. The antibody according to claim 10, which is a mouse monoclonal antibody obtained by using a conjugate of a mevalonic acid derivative and a protein as an immunogen. 12. Keyhole limpet hemocyanin (KLH) and succinimide 5- (4-hydroxy-).
The antibody according to claim 10, which is a monoclonal antibody obtained by using a conjugate of 2-oxotetrahydropyran-4-yl) pentanoate as an immunogen. 13. The antibody according to claim 10, wherein the isotype is IgG _2b , κ. 14. The antibody according to claim 10, which is MHM-9H, which is a monoclonal antibody produced by the MHM-9H strain deposited under accession number FERM BP-4524. 15. A method for quantifying mevalonic acid using the antibody according to claim 1. 16. A method in which a labeled antigen and an unlabeled antigen are competitively reacted with an antibody specific to mevalonic acid, and the labeled antigen which is bound or not bound to the antibody is quantified,
Alternatively, a method for quantifying mevalonic acid by allowing a solid-phased antigen and mevalonic acid in a sample to competitively react with an antibody specific to mevalonic acid, and quantifying the antibody bound or not bound to the antigen. 17. The method according to claim 1, which is used for diagnosis or treatment.
The described antibody. 18. A kit for measuring mevalonic acid, comprising at least an antibody specific to mevalonic acid and a mevalonic acid derivative or a conjugate thereof with a protein for quantifying the amount of mevalonic acid in a sample.