JPH07234222A - Quantitative analysis method for 1,5-anhydroglucitol and antibody - Google Patents

Abstract

PURPOSE:To provide a quantitative analysis method for 1,5-anhydroglucitol used for diagnosis of diabetes and an antibody used for the analysis. CONSTITUTION:Immunoreaction is carried out by adding an antibody having specific reactivity with 1,5-anhydroglucitol and labeled 1,5-anhydroglucitol in a test solution containing 1,5-anhydroglucitol, and then, a bounded compound, which is generated by the immunoreaction, of the labeled 1,5-anhydroglucitol and the antibody is separated from the unbound labeled 1,5-anhydroglucitol, and subsequently, labels of either of the separated 1,5-anhydroglucitol are measured, and as a result, 1,5-anhydroglucitol contained in the test solution is quantitatively determined.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for quantifying 1,5-anhydroglucitol (hereinafter referred to as "1,5-AG"), which is expected as a diagnostic marker for diabetes, and an antibody used for the measurement.

[0002]

2. Description of the Related Art 1,5-AG is a compound which is present in human cerebrospinal fluid and plasma and has been reported to be reduced in plasma level in certain diseases, particularly in diabetes. This one
It has not been known that antibodies capable of reacting with 5-AG can be prepared, and conventionally, 1,5-AG has been mainly measured by gas chromatography.

[0003]

However, the conventional methods require advanced techniques for pretreatment of samples and maintenance and management of analytical instruments, and there has been a demand for a simple method for measuring 1,5-AG.

[0004]

The present inventors have found that 1,5-A
As a result of intensive studies on a simple method for measuring G, it is possible to prepare a low-molecular compound which is generally difficult to prepare a hapten antibody and which specifically reacts with 1,5-AG which is a serum component. I found out that,
The present invention has been completed by applying it to the determination of 5-AG.

That is, according to the present invention, (1) a test liquid containing 1,5-anhydroglucitol is labeled with an antibody having a specific reactivity with 1,5-anhydroglucitol and labeled with 1,5
-Anhydroglucitol is added to cause an immunoreaction, and then a conjugate of labeled 1,5-anhydroglucitol produced by the immunoreaction with the antibody and unbound labeled 1,5-an After separating from hydroglucitol, 1,5-anhydroglucitol in the test solution is quantified by measuring the label of either one of the separated ones. The present invention relates to a method for quantifying hydroglucitol, and (2) an antibody having specific reactivity with 1,5-anhydroglucitol.

The test liquid used in the present invention is 1,
There is no particular limitation as long as the concentration of 5-AG is desired to be measured. Can be given.

Antibodies with specific reactivity used in the present invention
With the body , the reactivity with sugars known to be present in serum is negligible compared to 1,5-AG,
Or, it does not react at all. Especially in the immunoassay method for 1,5-AG, a large amount is present in serum,
Glucose, which has a structure similar to AG, becomes a problem. In the case of glucose, its reactivity to 1,5-
An antibody that is 10% or less, more preferably 1% or less of AG is desirable.

Labeled 1,5-AG used in the present invention
Examples thereof include 1,5-AG labeled by a labeling method as used in a usual hapten immunoassay. Up to now, a number of methods have been proposed as immunoassays using a labeled substance depending on the type of labeling substance (tracer), but any of these methods may be used. As an example, radioimmunoassay tracer is a radioactive substance
In the measurement method (RIA) , a substance in which 1,5-AG itself is replaced with a radioactive isotope can be used. Also included are 1,5-AG derivatives having a side chain in which radioactive iodine has been introduced into the 3-, 4-, or 6-position hydroxyl groups of 1,5-AG. Specific examples of the introduction of the tracer include the introduction of tritium ( ³ H) at the 1-position of 1,5-AG and the substitution of carbon atoms of 1,5-AG with ¹⁴ C.

Enzyme immunoassay in which the tracer is an enzyme
In (EIA) , labeling enzyme and 1,5-AG
There is a problem in the selection of the coupling method with The enzyme that can be used in the EIA of the present invention may be any enzyme generally used in EIA, and horseradish peroxidase, alkaline phosphatase, β-galactosidase and the like can be used. Regarding the binding method with 1,5-AG, a compound of the following general formula (I), which is a 1,5-AG derivative, is used, directly or indirectly via the amino group or carboxyl group of the functional group A of the side chain. It can bind to enzyme proteins. The conditions required for these EIA enzymes and the binding method are described in detail in published books such as the enzyme immunoassay method and Eiji Ishikawa et al.

[0010] Fluorescence immunity in which the tracer is a fluorescence substance
In the measurement method (FIA) , a compound of the following general formula (I ) which is a 1,5-AG derivative is used, and the compound can be bound via the amino group or carboxyl group of the functional group A of the side chain, and the fluorescence intensity is strong Any one may be used as long as it is stable.
Further, in order to increase the labeling density of the fluorescent substance, a large number of fluorescent substances may be bound to one molecule of the 1,5-AG derivative via a suitable carrier. As specific fluorescent substances, fluorescein isothiocyanate, rhodamine, dansyl chloride, coumarin and the like can be used.

Luminescent immunoassay in which the tracer is a luminescent substance
In the method (LIA) , a compound of the following general formula (I ) , which is a 1,5-AG derivative, was used to chemically bond an isoluminol compound or the like through the amino group or carboxyl group of the functional group A of the side chain. Things can be used. In addition, the binding of 1,5-AG and the labeled compound in each of the above-mentioned immunoassays,
Various methods via a binding reaction between avidin and biotin can also be used.

Labeled 1,5-AG used in the present invention
And the antibody-bound product and unbound labeled 1,5-AG
As a method for separation, a BF separation operation used in a usual immunoassay method is appropriately used. For example, when utilizing what is called a “solid phase method” in which an antibody is immobilized on a solid phase carrier, the surface of the solid phase carrier is physically, chemically or immunologically bound, After the 1,5-AG (antigen) and 1,5-AG labeled with 1,5-AG in the test solution are competitively immunoreacted (competitive method) with an antibody that specifically reacts with 5-AG, By washing the phase carrier, labeled 1,5-AG bound to the antibody can be obtained.

On the other hand, unbound labeled 1,5-AG is
It is sufficient to collect the liquid that has undergone an immune reaction. As the commonly used solid-phase carrier, beads, microtiter plates, latexes and filters formed by using various synthetic resins are known, but any of them can be used.

The method for quantifying the label used in the present invention may be any method as long as it can measure various labeling substances with high sensitivity, and is limited by the measuring principle and the apparatus. Not something.

The antigen prepared from the 1,5-AG derivative used for preparing the antibody used in the present invention will be described. The term "antigen" as used herein refers to an antigen having an animal-sensing action necessary for producing an antibody that specifically reacts with 1,5-AG, that is, an immunogen, which is represented by general formula (I) below. It is an AG derivative chemically bound to an antigenic carrier substance. The antigenic carrier material can be selected from any protein or polypeptide known in the art for this purpose. Most antigenic proteins and polypeptides have a molecular weight of 5,000 to 10,000,0.
00, preferably 15,000 or more, more preferably 5
It has a molecular weight of 10,000 or more.

In general, a protein collected from one animal species becomes an antigen when introduced into the bloodstream of another species. Particularly useful proteins include albumin, globulins, enzymes, hemocyanins, glutelins, proteins with significant non-protein components (eg glycoproteins) and the like. Molecular weight 30,000
Particularly preferred is 0 to 200,000 albumin and globulin. Synthetic polypeptides may be used. Other references showing techniques relating to conventional antigenic carrier substances include:

Parker, Radioimmunoassay of Biological
ly Active Compounds, Prentice-Hall (Englewood Clif
fs, New Jersey USA, 1976); Butler, J. Immunol. Met
h., 7, 1-24 (1975); Weinryb and Shoroff, Drug Meta
b. Rev., 10, 271-283 (1975); Broughton and Strong,
Clin. Chem., 22, 726-732 (1976) and Playfair eta
l., Br. Med. Bull., 30, 24-31 (1974).

The antigenic determinant (epitope) density, ie the average number of hapten moieties attached to the carrier, is theoretically limited only by the number of available binding sites on the selected carrier molecule. However, in the usual case where the carrier is a naturally occurring protein such as albumin, it will average 1 to about 50, more usually 2 to about 20. Regarding the method of conjugation, the above-mentioned enzyme label for EIA 1,
The method can be carried out in exactly the same manner as in the method for producing 5-AG.

The method of producing an antibody using an antigen derived from a 1,5-AG derivative may be according to any conventional technique (for example, Parker Radioimm).
unoassay of Biologically Active Compounds, Prentic
e-Hall (Englewood Cliffs, New Jersey USA, 1976)) Normally, host animals such as rabbits, goats, mice, guinea pigs and horses are usually mixed with an immunogenic complex with an adjuvant. Then, one or more various sites are injected. In addition, injections may be made at the same or different sites at regular or irregular intervals, after which blood is drawn and the antibody titer determined until an optimal titer is determined. Blood is drawn from the host animal to obtain the appropriate volume of the specific antiserum. If desired, undesired substances such as non-specific antibodies may be removed during the purification step before being judged as suitable as an antiserum to be used in the actual assay.

An antibody (for example, an antibody which is usually called a monoclonal antibody) is a somatic cell mating (fusion) technique (somati c).
ell hybridization techniques). (Lymphocyte Hybridomas, ed. Melchers et al, S
pringer-Verlag, New York, (1978), Nature 266, 495
(1977), Science, 208, 692 (1980), and Methodsin
Enzymology, 73 (Part B), 3-46 (1981))

1,5-used to generate antigen
The AG derivative is a novel compound in which a side chain is introduced via the —OH group at the 3-position, 4-position or 6-position of 1,5-AG represented by the general formula (I).

[0022]

[Chemical 1]

(In the formula, one of X, Y and Z is the formula A
-Alkylene-B-, the other two being -OH.
Here, A represents —NH ₂ or —COOH, and B represents —O.
-Or-COO- is shown, and the alkylene has 1 to 12 carbon atoms. )

In the general formula (I), examples of the alkylene include those having about 1 to 12 carbon atoms such as methylene, ethylene, propylene and butylene. These may be branched and preferably have about 2 to 10 carbon atoms, more preferably ethylene, propylene, butylene and the like. Examples of the compound of the general formula (I) of the present invention include the compounds shown in Table 1 below.

[0025]

[Table 1] Table 1 Compound No. XYZ 1 H ₂ NCH ₂ COO- -OH -OH 2 HOOC (CH ₂ ) ₂ COO- -OH -OH 3 HOOCCH ₂ O- -OH -OH 4 HOOC (CH ₂ ) ₂ CH ₂ O- -OH -OH 5 -OH HOOC (CH ₂ ) ₂ CH ₂ O- -OH 6 -OH -OH HOOC (CH ₂ ) ₂ CH ₂ O- 7 H ₂ N (CH ₂ ) ₅ COO --OH -OH 8 H ₂ N (CH ₂ ) ₁₀ COO- -OH -OH

These compounds of the present invention are synthesized as follows. That is, the general formula (II)

[0027]

[Chemical 2]

(Wherein any one of R ₁ , R ₂ and R _{3 is} the formula A-alkylene-B-, the other two and R ₄ are -OBn, -OTr, -O-THP, -O. -C (CH ₃₎
Indicates _2- O-. Where A is -NH / CBZ, -COO
H or -COOBn is shown and B is -O- or -COO-
And the alkylene has 1 to 12 carbon atoms. CBZ is benzyloxycarbonyl, Bn
Represents benzyl, Tr represents triphenylmethyl, and THP represents tetrahydropyranyl. If desired, the compound represented by the formula (1) is subjected to acid hydrolysis or catalytic reduction to remove the protecting group, whereby the 1,5-AG derivative of the general formula (I) can be obtained.

Acid hydrolysis is carried out in the presence of acid in a water solvent in the presence of 20
C. to 100.degree. C., preferably 25.degree. C. to 60.degree. C., for about 1 to 5 hours. As the acid, mineral acids such as hydrochloric acid and sulfuric acid,
C ₁ -C ₄ lower fatty acids such as formic acid, acetic acid and trifluoroacetic acid can be used, and hydrochloric acid and acetic acid are preferred. The amount of the acid added is such that when a mineral acid is used, the concentration in the reaction solution is 0.01 to 5N, preferably 0.1 to 2N, and when a fatty acid is used, it is added in the reaction solution. It may be added so that the concentration is 50% or more, preferably about 60 to 80%.

The catalytic reduction is usually carried out in a solvent in the presence of a catalyst at 20 ° C to 80 ° C, preferably 25 ° C to 35 ° C for 1 to 12 ° C.
It only has to be done for about an hour. As the solvent, alcohols such as methanol and ethanol, ethers such as tetrahydrofuran and dioxane, water, dimethylformamide, acetic acid and the like can be used alone or as a mixture as required, and preferred are methanol and ethanol.

As the catalyst, palladium carbon, palladium black, palladium chloride, palladium hydroxide or the like can be used, preferably palladium black or palladium chloride. About 10 to 20% of the amount of the raw material compound may be added as the amount of the catalyst. Representative compounds of the general formula (II) used as a raw material are shown below.

[0032]

[Chemical 3]

The compound of the general formula (II) has the following formula (II
I), (IV), or (V) is used for the synthesis.

[0034]

[Chemical 4]

The compound of the general formula (I) of the present invention is isolated and purified from the reaction solution by a conventional method to obtain a free acid or free base.

[0036]

EXAMPLES The present invention will now be described with reference to examples. Example 1 Preparation of calibration curve A test tube was charged with 0.1 of 1,5-AG or glucose standard.
0.1m sample serially diluted with PBS containing% BSA
1, PBS containing 0.1% BSA was added to 1,5-AG antiserum prepared by immunizing a rabbit with a conjugate of compound 4 and BSA.
0.1 ml of a 1000-fold solution diluted with 1000 times, 0.1 ml of a 1000-fold diluted solution of the conjugate of compound 2 and horseradish peroxidase (HRP) with PBS containing 0.1% BSA, and 0.3% BSA described above. One DASP bead blocked with PBS was added and immunoreacted overnight at 4 ° C.

After the reaction, the beads were mixed with P containing 0.1% BSA.
After washing 4 times with BS, the solution containing 0.05% p-hydroxyphenylpropionic acid and 0.001% hydrogen peroxide was added to 0.1%.
The sample was transferred to a new test tube containing 1 ml of 05M phosphate buffer (pH 7.0) (gas liquid), and the enzyme reaction was carried out at 37 ° C for 1 hour. This reaction solution contained 1.5% sodium azide,
25N sodium hydroxide solution (stopper) 0.1 ml
After stopping the reaction by adding, a fluorescence measuring device (manufactured by Fujirebio Co.,
Auto FP-1) was used to measure the fluorescence intensity of the reaction solution at an excitation wavelength of 313 nm and a detection wavelength of 405 nm to prepare a calibration curve as shown in FIG.

As is apparent from the calibration curve of 1,5-AG in FIG. 1, the detection limit of 1,5-AG in this measuring method is 0.1 μm.
It was g / ml, and had sufficient sensitivity to be applied to clinical sample measurement. The cross-reactivity of the antibody with glucose was only 0.2%. Therefore, the assay method of the present invention can be used as a practical assay method for 1,5-AG.

Compound 2 and HR used in this Example
The conjugate of P was prepared by the following method. Compound 2, 6.
7 mg, N-hydroxysuccinimide 7.3 mg and DCC 8.4 mg were dissolved in 0.4 ml dioxane,
The reaction was carried out at room temperature for 5 hours with stirring. The ureide generated by the active esterification reaction was removed by filtration, and the filtration residue was washed with 0.05 ml of dioxane to obtain a dioxane solution of the active ester of compound 2. Next, HRP (manufactured by Toyobo, RZ = 3.41) 25.
4.2 mg of 0.1 M phosphate buffer (pH 7.5) 4.2 m
The active ester solution was added to the solution dissolved in 1 and reacted at room temperature for 5 hours with stirring. After removing the precipitate in the reaction solution by centrifugation, it was dialyzed against 0.01 M phosphate buffer (pH 7.0) containing 0.9% sodium chloride (hereinafter referred to as PBS) to obtain the desired bound product. Obtained.
Next, the solution of the bound product was concentrated using a concentrator equipped with an ultrafiltration membrane PM10, and 0.2 μm of filter sterilization was performed to obtain 4.4 ml of HRP-labeled 1,5-AG (HRP concentration 6. 1 mg / ml, protein recovery rate 100%, HRP
The activity recovery rate was 100%).

DASP beads were prepared by the following method. Polystyrene beads for EIA (made by Sekisui, 6.5 mm
300 pieces of φ) were immersed in 2% concentration score roll 900 (manufactured by Kao Corporation) at room temperature overnight. Next, it was washed with water until no bubbles appeared, drained sufficiently, spread on filter paper and air dried. Affinity purified goat anti-rabbit IgG (H & L)
2 mg of antibody (manufactured by Jackson ImmunoResearch)
0.05M phosphate buffer containing 1% sodium azide (pH
7.5) The above washing beads were dipped in a solution dissolved in 100 ml and left at room temperature for 2 days to physically adsorb the antibody. The second antibody-immobilized beads (hereinafter referred to as DASP beads) thus prepared are soaked in a PBS solution containing 0.3% BSA before use and incubated at 45 ° C. for 10 hours to block nonspecific adsorption. It was subjected to EIA.

The present invention will be described in detail below with reference to specific examples in the order of preparation of 1,5-AG derivative, preparation of antigen, preparation of antibody, and 1,5-AG immunoassay.

Synthesis Example 1. 1,5-anhydro-6-
Production of O-glycyl-D-glucitol (Compound 1 in Table 1) 1,5-AG derivative (III obtained by the method shown in Reference Example 1
) 434 mg and 397 mg of benzyloxycarbonylglycine chloride in the presence of dioxane solvent and pyridine,
The mixture was stirred at room temperature for 20 hours. The reaction mixture was concentrated under reduced pressure and purified by silica gel chromatography to give 266 mg.
Of oily substance was obtained. In ¹ H-NMR using deuterated chloroform, a proton corresponding to 20 H component derived from aromatic hydrogen was observed at 7.3 ppm. Positive and negative ion FAB-MS was measured using a mixture of glycerin and DMSO as the matrix, 626 corresponding to (M + H) ⁺ and (M−H)
624 corresponding to ⁻ was observed as each standard peak.

Next, 250 mg of the above compound was subjected to catalytic reduction using palladium black as a catalyst in a mixed solution of methanol, acetic acid and water, and the fragrance derived from benzyl ether and benzyloxycarbonyl group was measured by ¹ H-NMR using heavy water. 104 mg of the title compound free of group hydrogen were obtained. Compound 1 was a positive ion FAB-MS measured using a mixture of glycerin and DMSO as a matrix.
A reference peak was observed at 222 corresponding to (M + H) ⁺ . Also, in an IR by a thin film method, absorption based on ester 1750 cm ^-1, absorption of amine 1630 cm ^-1, otherwise, 1510,1420,1240,1100,
Absorption was observed at 1050 cm ⁻¹ .

Synthesis Example 2. 1,5-anhydro-6-
Preparation of O- (3-carboxy-propionyl) -D-glucitol (Compound 2 in Table 1) 3.17 g of benzyl succinic acid chloride, 3.5 g of 1,
A solution of 5-AG in DMF (10 ml) was added at 0 ° C., then 1.7 g of dimethylaminopyridine was added, and the mixture was stirred at room temperature for 1 hour.
Stir for 5 hours. Dilute the reaction mixture with ice water and add ethyl acetate.
The mixture was extracted twice with 5 ml, washed with 1N hydrochloric acid, washed with water, and dried over sodium sulfate. Then, the product was purified by silica gel chromatography to obtain 680 mg of an oily substance.

650 mg of the above compound was catalytically reduced in a mixed solution of methanol, acetic acid and water using palladium black as a catalyst to obtain 480 mg of the title compound. Compound 2 had a reference peak at 265 corresponding to (M + H) ⁺ in the positive ion FAB-MS as in Synthesis Example 1, and a negative ion FAB-MS.
A standard peak was observed at 263 corresponding to (M−H) ⁻ . Also, in IR by the thin film method, 2930, 173
Absorption was observed at 0 (-CO-O-), 1400 and 1080 cm ^-1 , respectively.

Synthesis Example 3. 1,5-anhydro-6-
Preparation of O-carboxymethyl-D-glucitol (Compound 3 in Table 1) Sodium hydride (53 mg) was placed in a two-necked flask, DMF (10 ml) was added under a nitrogen stream, and the mixture was obtained by the method of Reference Example 1 while stirring. , 5-AG derivative (III) 430
Add mg. After stirring for 30 minutes, 113 mg of chloroacetic acid was added, and the mixture was stirred at room temperature for 15 hours. Water 75 in the reaction solution
After adding ml, the mixture was extracted twice with ethyl acetate, washed with saturated brine and then with water, and purified by silica gel chromatography to obtain 170 mg of an oily substance. In this product, a standard peak was observed at 493 corresponding to (M + H) ⁺ in the positive ion FAB-MS, and a standard peak was observed at 491 corresponding to (M−H) ⁻ in the negative ion FAB-MS.

140 mg of the above compound was subjected to catalytic reduction in a mixed solution of methanol, acetic acid and water using palladium black as a catalyst to obtain 55 mg of the title compound. For compound 3, a standard peak was observed at 223 corresponding to (M + H) ⁺ by positive ion FAB-MS, and a standard peak at 221 corresponding to (M−H) ⁻ by negative ion FAB-MS. Also, in IR by the thin film method, 3350, 2930, 1740, 12
Absorption was observed at 50, 1100 and 1060 cm ^-1 , respectively.

Synthesis Example 4. 1,5-anhydro-6-
Production of O- (3-carboxy-propyl) -D-glucitol (Compound 4 in Table 1) Sodium hydride (180 mg) was placed in a two-necked flask, and DMF (20 ml) was added under a nitrogen stream. 1,5-AG derivative (III) obtained by the method
1.3 g is added dropwise. After stirring for 30 minutes, 671 mg of 5-bromo-1-pentene was added, and the mixture was stirred at room temperature for 15 hours.
After adding 75 ml of water to the reaction solution and extracting twice with 75 ml of ethyl acetate, the extract was washed with saturated saline and then with water, and purified by silica gel chromatography to give 1.01 g.
Of oily substance was obtained.

980 mg of the above compound was dissolved in 8 ml of acetone, and while stirring, 10 ml of water and 8 m of water were added.
2.4 g NaIO ₄ dissolved in 1 l acetone are added dropwise. Next, while cooling to 0 ° C., 80 mg of KMnO ₄ dissolved in 4 ml of water was added, and after stirring at room temperature for 1 hour, 600 mg of NaIO ₄ was further added and stirred for 1 hour. 100 ml of water was added to the reaction solution, and ethyl acetate was 75 m.
After being extracted twice with l, washed with saturated saline and then with water,
Purify by silica gel chromatography, 660
Obtained mg of oily substance.

630 mg of the above compound was subjected to catalytic reduction in a mixed solution of methanol, acetic acid and water using palladium black as a catalyst to obtain 290 mg of the title compound. For compound 4, a standard peak was observed at 251 corresponding to (M + H) ⁺ by a positive ion FAB-MS, and a standard peak was observed at 249 corresponding to (M−H) ⁻ by a negative ion FAB-MS. Moreover, in IR by the thin film method, 2930, 1720, 1420, 1
Absorption was observed at 370, 1260, and 1080 cm ⁻¹ , respectively.

Synthesis Example 5. 1,5-anhydro-4-
Production of O- (3-carboxy-propyl) -D-glucitol (Compound 5 in Table 1) Sodium hydride (169 mg) was placed in a two-necked flask, DMF (10 ml) was added under a nitrogen stream, and the compound (IV) 1 was stirred. .24 g is added dropwise. After stirring for 30 minutes, 630 mg of 5-bromo-1-pentene was added, and the mixture was stirred at room temperature for 15 hours. Cold water (50 ml) was added to the reaction solution, and the mixture was extracted twice with 75 ml of ethyl acetate and then purified by silica gel chromatography to obtain 850 mg of an oily substance.

820 mg of the above compound was dissolved in 8 ml of acetone, and while stirring, 10 ml of water and 8 m of water were added.
1.4 g NaIO ₄ dissolved in 1 l acetone are added dropwise. Then, while cooling to 0 ° C., 50 mg of KMnO ₄ dissolved in 4 ml of water was added, and after stirring at room temperature for 1 hour, 350 mg of NaIO ₄ was further added and stirred for 1 hour. The reaction solution was acidified with hydrochloric acid, extracted with ethyl acetate, and then subjected to silica gel chromatography to obtain 600 mg.
Of oily substance was obtained. 80% acetic acid was added to 560 mg of the above compound, and the mixture was stirred at 65 ° C. for 2 hours. 50m in the reaction solution
After adding 1 liter of water and extracting with ethyl acetate, purification was performed by silica gel chromatography to obtain 320 mg of an oily substance.

280 mg of the above compound was subjected to catalytic reduction in a mixed solution of methanol, acetic acid and water using palladium black as a catalyst to obtain 150 mg of the title compound. Compound 5 is
251 corresponding to (M + H) ⁺ by positive ion FAB-MS
The reference peak, a negative ion FAB-MS (M-H) to ^-
A reference peak was observed at 249 corresponding to. In the IR by the thin film method, 2930, 1720, 1460,
Absorption was observed at 1380 cm ^-1 .

Synthesis Example 6. 1,5-anhydro-3-
Production of O- (3-carboxy-propyl) -D-glucitol (Compound 6 in Table 1) Sodium hydride (200 mg) was placed in a two-necked flask, DMF (10 ml) was added under a nitrogen stream, and the mixture (V) was stirred at 720 mg. Is dripped. After stirring for 30 minutes, 744 mg of 5-bromo-1-pentene was added, and the mixture was stirred at room temperature for 15 hours. 50 ml of cold water was added to the reaction solution, and the mixture was extracted twice with 75 ml of ethyl acetate and then purified by silica gel chromatography to obtain 560 mg of an oily substance.

1.3 g of the above compound, which was repeatedly synthesized,
It was dissolved in ethanol and stirred at 65 ° C. for 1 hour in the presence of a catalytic amount of p-toluenesulfonic acid to remove the protective group. Then, in the same manner as in Reference Example 2, the 2nd place, 4th place, 6th place
After benzylating the hydroxyl group at the position, treatment with NaIO ₄ and KMnO ₄ was conducted in the same manner as in Examples 4 and 5, and then catalytic reduction was conducted using a palladium black catalyst to give 90 m of the title compound.
g was obtained. Compound 6 was analyzed by negative ion FAB-MS (M-
A reference peak was observed at 249 corresponding to (H) ⁻ . or,
2950, 1720, 12 in IR by thin film method
Absorption was observed at 50 cm ⁻¹ .

Synthesis Example 7. 1,5-anhydro-6-
Production of O- (6-amino-hexanoyl) -D-glucitol (Compound 7 in Table 1) 434 mg of the compound (III) shown in Reference Example 1 was dissolved in 10 ml of pyridine, and benzyloxycarbonyl-ε- was obtained at room temperature. Aminocaproic acid chloride 535 mg is added dropwise. After stirring at room temperature for 15 hours, 75 ml of water was added to the reaction solution.
Was added, extracted twice with ethyl acetate, and purified by silica gel chromatography to obtain 366 mg of an oily substance.

The obtained compound was catalytically reduced in a mixed solution of methanol, acetic acid and water using palladium black as a catalyst to obtain 104 mg of the title compound. For compound 7, a reference peak was observed at 278 corresponding to (M + H) ⁺ by positive ion FAB-MS. In the IR by the thin film method, 1
Absorption was observed at 740, 1460 and 1100 cm ^-1 , respectively.

Synthesis Example 8. 1,5-anhydro-6-
Preparation of O- (11-amino-undecanoyl) -D-glucitol (Compound 8 in Table 1) The benzyloxycarbonyl-ε-aminocaproic acid chloride of Example 7 is changed to 637 mg of benzyloxycarbonyl-11-amino-undecanoic acid chloride. Except
The reaction was carried out in exactly the same manner to obtain 400 mg of the title compound. For compound 8, a standard peak was observed at 348 corresponding to (M + H) ⁺ by positive ion FAB-MS. Further, in IR by the thin film method, absorption was observed at 2925, 1740, and 1460 cm ⁻¹ , respectively.

Preparation of Antigen Although various methods for preparing a hapten antigen are known as described above, here, as an example, bovine serum albumin (hereinafter abbreviated as BSA) is used as an antigenic carrier substance. An example in which the 1,5-AG derivative (I) is chemically bonded is shown. The method of chemical bonding differs depending on the introduced side chain terminal group of the 1,5-AG derivative (I) [A of the general formula (I)], but when A is an amino group, a water-soluble carbodiimide method is used, and a carboxyl group is used. In this case, the active ester method was used to directly bind to BSA. However, the method of chemical bonding is not limited thereto. In addition, a spacer of a low molecular weight compound may be used for binding the terminal group A and BSA.

Reference Example 1. 1,5-anhydro-6-O
-Preparation of Glycyl-D-glucitol (Compound 1) and BSA conjugate 1.6 ml of Compound 1, 16 mg and BSA 23.9 mg
1 / 15M phosphate buffer (pH 5.6).
208 mg of water-soluble carbodiimide hydrochloride was added to this while stirring at room temperature in several batches, and the reaction was carried out while maintaining pH 5-6. Furthermore, after reacting overnight at room temperature,
The reaction product was dialyzed against physiological saline to obtain the desired bound product. Next, this is used as an ultrafiltration membrane PM10 (manufactured by Amicon).
Was concentrated using a concentrating device set with, and 0.2 μm filter sterilization (manufactured by Millipore) was performed to obtain an immunizing antigen.
The protein recovery rate in all steps was 88%. BSA conjugates of compound 7 and compound 8 were also prepared using the same method.

Reference Example 2. 1,5-anhydro-6-O
Preparation of BSA conjugate with-(3-carboxy-propyl) -D-glucitol (Compound 4) Compound 4, 145.8 mg, N-hydroxysuccinimide 134 mg, and N, N'-dicyclohexylcarbodiimide (DCC) 180.3 mg It was dissolved in 3.5 ml of dioxane and reacted at room temperature with stirring for 3 to 4 hours. The ureide generated by the active esterification reaction was removed by filtration, and the filtration residue was washed with 2 ml of dioxane to obtain a dioxane solution of the active ester of compound 4. Next, the above active ester solution was added to 25.7 ml of a BSA solution prepared by dissolving BSA in a concentration of 15 mg / ml with a 0.1 M phosphate buffer (pH 7.5), and the mixture was stirred overnight at room temperature overnight. Reacted The precipitate in the reaction solution was removed by centrifugation and then dialyzed against physiological saline to obtain the desired bound product. Next, the solution of the bound product was concentrated using a concentrating device equipped with an ultrafiltration membrane PM10, and 0.2 μm of filter sterilization was performed to obtain an immunizing antigen. The protein recovery rate through the whole process is 98
%Met. In addition, compounds 2, 3,
BSA conjugates of 5 and 6 were also made.

Preparation of Antibody Using BSA conjugates of the general formula (I) shown in Table 1 prepared according to Reference Examples 1 and 2 and their methods as immunogens, a method known from rabbits was used. The 1,5-AG antibody was prepared.

Example 2 No. The BSA conjugate of the compound of 4 was diluted with physiological saline and adjusted to a concentration of 2 mg / ml. To 1.5 ml of this solution, add 1.5 ml of Freunds complete Adjuvan
Add 1 t and mix well, and inject this into rabbits (2-3 birds for each antigen) 1 ml / wing intradermally (1 week interval, 4 doses, then 2 week intervals, 3 doses), and thoroughly After sensitization, blood was collected 2 weeks later, and 3000 rpm x 1
The serum was obtained by centrifugation for 5 minutes, heat-treated at 60 ° C. for 30 minutes and stored at −20 ° C. to obtain 1,5-AG antiserum corresponding to each immunogen. The presence of an antibody against 1,5-AG in this serum was confirmed by a dot immunobinding assay method after absorbing BSA-immobilized resin. No. BSA of compounds 1-3, 5-8
A 1,5-AG antiserum was obtained in the same manner as above using the conjugate.

1,5-AG Immunoassay As an immunoassay, as described above, RIA, EIA,
Although it can be measured by various methods such as FIA and LIA, an example of applying it to EIA is shown here as an example, and the so-called second antibody solid-phase method is used as a measuring method in EIA. An example of what is called a law is shown.

Example 3 Glucose and 1,5-AG were each added at 10 μg / ml,
A model test solution containing 1 μg / ml was prepared. This is shown in Example 1 as "EI of 1,5-AG by competitive method.
When measured according to the method of "A" and quantified using the calibration curve of Fig. 1, the concentration of 1,5-AG was 1.1 µg / ml.

Compounds (III), (IV) and (V) necessary for the synthesis of the general formula (II) which is a starting material for the compound of the general formula (I)
Was synthesized, for example, according to the reaction formula shown below.
As a specific example, a synthesis example of the compound (III) is shown.

[0067]

[Chemical 5]

[0068] THP: Tetrahydropyranyl x: Isopropyl Bn: Benzyl Tr: Trityl

Reference Example 3. 1,5-anhydro-2,
3,4-tri-O-benzyl-D-glucitol (III
3.28 g of 1,5-AG was dissolved in 20 ml of pyridine, and 6.12 g of trityl chloride was added dropwise with stirring under ice cooling. The mixture was stirred under ice cooling for 1 hour and further at room temperature for 15 hours. The reaction solution was diluted with 200 ml of water, extracted twice with 150 ml of ethyl acetate and then purified by silica gel chromatography to obtain 7.44 g of 6-trityl-.
1,5-AG was obtained. This was added to the 1,5-AG signal by ¹ H-NMR using deuterated chloroform,
Protons of 15H corresponding to aromatic hydrogen were observed at 7.3 to 7.9 ppm.

900 m of sodium hydride in a two-necked flask
g, 40 ml of nitrogen gasified DMF is added, and 4.06 g of the above compound is added dropwise with stirring. After stirring at room temperature for 30 minutes, 5.2 ml of benzyl chloride was added dropwise, and 15
Stir for hours. Add 200 ml of water to the reaction mixture to dilute it,
After extraction with 400 ml of ethyl acetate, purification by silica gel chromatography was performed, and 5.27 g of 2,
3,4-Tribenzyl-6-trityl-1,5-AG was obtained. This product is ¹ H-NMR using deuterated chloroform.
A total of 30 H of aromatic protons was observed at 7.0 to 7.8 ppm.

3.33 g of the above compound was added to 20 ml of 80%
It was dissolved in acetic acid and stirred at 65 ° C. for 2 hours. The reaction solution is concentrated under reduced pressure, purified by silica gel chromatography, and then recrystallized from ethyl acetate and n-hexane.
1.48 g of colorless needle crystals were obtained.

The compound had a melting point of 85 to 86 ° C. and a D of 1.8 ppm in ¹ H-NMR using deuterated chloroform.
1H worth of protons that disappear when ₂ O is added, 3.0-
Proton for 8 H at 4.2 ppm, 4.4 to 5.1 pp
A proton of 6 H was observed in m and a proton of 15 H was observed in 7.28 ppm. In addition, with IR using KBr, 33
00, 2980, 2875, 1500, 1505, 11
Absorption was observed at 00 cm ^-1 .

[Brief description of drawings]

FIG. 1: 1,5 by EIA with 1,5-AG antibody
-Shows the calibration curve of AG and glucose.

Claims (2)

[Claims] 1. A test liquid containing 1,5-anhydroglucitol, and an antibody having a specific reactivity with 1,5-anhydroglucitol and labeled 1,5-anhydroglucitol. To cause an immunoreaction, and then to separate the labeled 1,5-anhydroglucitol produced by the immune reaction from the antibody and the unbound labeled 1,5-anhydroglucitol. Then, by measuring the label of either one of the separated ones, 1,5-
A method for quantifying 1,5-anhydroglucitol, which comprises quantifying anhydroglucitol. 2. An antibody having specific reactivity with 1,5-anhydroglucitol.