JPS6377870A - Determination method for 1,5-anhydroglucitol, antibody and 1,5-anhydroglucitol derivative - Google Patents

Abstract

NEW MATERIAL:A 1,5-anhydroglucitol derivative expressed by formula I [any one of X, Y and Z is expressed by the formula A-alkylene-B- (A is -NH2 or -COOH; B is -O- or -COO-; alkylene is 1-12C)]. EXAMPLE:1,5-Anhydro-6-0-glycidyl-D-glucitol. USE:A diagnostic marker for diabetes capable of determining 1,5-anhydroglucitol by adding an antibody having specific reactivity with the 1,5-anhydroglucitol and labeled 1,5-anhydroglucitol to a 1,5-anhydroglucitol-containing test solution to carry out immunization reaction, separating the unbound labeled 1,5- anhydroglucitol and measuring the label of either one thereof. PREPARATION:A compound expressed by formula II [either one of R1-R3 is expressed by the formula A-alkylene-B-; the other two and R4 are OBn (Bn is benzyl), etc.], as necessary, is subjected to hydrolysis or catalytic reduction to remove protecting groups and afford the aimed compound expressed by formula I.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is applicable to 1 and 5 which are expected as diagnostic markers for diabetes.
- Method for quantifying anhydroglucitol (rl, hereinafter referred to as 5-AGJ), antibodies used for the measurement, and 1.5-
It is related by AG derivative number.

[Conventional technology]

1.5-AG is a compound that exists in human cerebrospinal fluid and plasma and has been reported to decrease in plasma levels in certain diseases, particularly diabetes. This 1. ．． It is not known that it is possible to create antibodies that react with 5-AG, and conventionally, 1.5-AG has been measured mainly by gas chromatography.

[Problems to be solved by the invention]

However, conventional methods require advanced techniques for sample pretreatment and maintenance and management of analytical equipment, and simple 1.5-AG
A measurement method was requested.

[Means for solving problems]

As a result of intensive research into a simple method for measuring 1,5-AG, the present inventors have found that 1,5-AG, a low-molecular compound and serum component that is generally difficult to generate hapten antibodies for, has been found to be specific for 1,5-AG. We found that it is possible to create reactive antibodies,
The present invention was completed by applying this created antibody to the quantification of 1,5 AG. That is, in the present invention, +1) 1.5-anhydroglucitol is added to the test solution containing 1.5-anhydroglucitol. A. A reactive antibody;
An immunoreaction was performed by adding labeled 1,5-anhydroglucitol, and then the conjugate of labeled 1.5-anhydroglucitol and the antibody produced by the immunoreaction was combined with the unbound labeled 1,5-anhydroglucitol. After separating from 1,5-anhydroglucitol, is one of the separated substances labeled? A method for quantifying 1,5-anhydroglucitol (21), which comprises quantifying 1,5-anhydroglucitol in the test liquid by measuring (21), 5-anhydroglucitol and (3) general Niyama [wherein, any one of X, Y, and Z is Nihe-alkylene-B-, and the other two represent -OH. C；CteA &'! , , NH2 or G': , C0OH
B represents 10- or -COO-, and alkylene has 1 to 12 carbon atoms. This invention relates to a novel 1,5-anhydroglucitol derivative represented by

The test liquid used in the present invention is not particularly limited as long as it is desired to measure the concentration of 5-AG, for example, cerebrospinal fluid, plasma, serum, urine, and 1.5-AGd, which can be easily measured once. Examples include processing solutions obtained by processing these test solutions.

The antibodies used in the present invention that have specific reactivity 2 are those that have 1.
Compared to 5-AG.

The reaction is negligible or not at all. 1
．． In the immunoassay for 5-AG, glucose, which is present in large amounts in serum and has a similar structure to 1.5-AG, poses a particular problem. In the case of glucose, it is desirable to use an antibody whose reactivity to glucose is 10% or less, more preferably 1% or less, of 1.5-AG.

The labeled 1,5 AG used in the present invention includes:
An example thereof is 1,5-AG labeled by a labeling method such as that used in the usual --butene immunoassay. Now, a number of methods have been proposed as immunoassay methods using labeled body 2, depending on the type of labeling substance (tracer), and any of these methods may be used. - For example, radioimmunoassay in which the tracer is a radioactive substance (
In RIA), is it a constituent element of 1.5-AG itself?
Substitutions with radioactive isotopes are available. Also, 1,
Also included are 1,5-AG derivatives having a side chain 2 in which radioactive iodine is introduced into the hydroxyl group at the 3rd, 4th, or 6th position of 5-AG.

A specific example of introducing a tracer is one in which tritium (3H) or C is introduced at the first position of 1,5-AG.

In enzyme immunoassay (EIA) in which the tracer is an enzyme, selection of the enzyme to be labeled and the method of binding to 1,5-AG is a problem. Enzymes that can be used in the EIA of the present invention include those commonly used in EIA, such as horseradish peroxidase, alkaline phosphatase, and β-galactosidase. .For the method of bonding with 5-AG, the compound of general formula (1), which is a 1.5-AG derivative, is used, and the enzyme protein is bonded directly or indirectly via the amine group or carboxyl group of the functional group A of the side chain. The conditions required for these enzymes for EIA and the method of binding are described in detail in Houka et al. There is.

Using the compound of general formula (1), any compound may be used as long as it can be bonded to the functional group of the side chain via an amine group or a carboxyl group χ, has strong fluorescence intensity, and is stable.

In addition, in order to increase the labeling density of the fluorescent substance, an appropriate carrier may be used. A large number of fluorescent substances may be bonded to one molecule of the 1,5-AG derivative via the phosphor. Specific fluorescent substances include fluorescein isothiocyanate, rhodamine,
Danzyl chloride, coumarin, etc. can be used.

Luminescent immunoassay (LIA) where the tracer is a luminescent substance
In this case, it is possible to use a compound of the general formula (1) which is a 1.5-AG derivative, or a compound chemically bonded with an isoluminol compound or the like via an amino group or a carboxyl group of the functional group A of the side chain.

Furthermore, various methods that involve a binding reaction between avidin and biotin can also be used to bind 1,5-AG and a labeled compound in each of the above-mentioned immunoassay methods.

BF separation operations used in conventional methods are used as appropriate.

For example, when using what is called a "surface phase method" in which antibodies are immobilized on a solid phase carrier, on the surface of the solid phase carrier,
Physically, chemically or immunologically linked. 1.5-AG
1.5-
After a competitive immune reaction (competitive method) with AG (antigen) and labeled 1,5 AG, labeled L5-AG bound to the antibody can be obtained by washing the solid phase support.

On the other hand, unbound labeled 1,5-AG can be collected by immunoreactive liquid. As commonly used solid phase carriers, beads molded using various synthetic resins and microtiter are known, and 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 label substances χ with high sensitivity, and is not limited by the measurement principle and apparatus.

The antigen used is an antigen for animal sensitization, that is, an immunogen, which is necessary for creating an antibody that specifically reacts with 1,5-AGK, and is a 1,5-AG derivative represented by the general formula (Tl). The antigenic carrier substance Z is chemically bonded to the antigenic carrier substance Z.It can be selected from the antigenic cavities.Most antigenic proteins and polypeptides have a
,000, preferably 15,000 or more, more preferably 50,000 or more.

Generally, proteins collected from one animal species become antigens when introduced into the bloodstream of another species.

Particularly useful proteins include albumin, globulins, enzymes, hemocyanin, glutelin, and significant non-protein components2.
There are proteins (for example, S protein) that have the molecule fz
Particularly preferred are albumin and globulin between 30,000 and 200,000. Synthetic polypeptides may also be used. Other references showing technology related to conventional antigenic carrier materials include:

7: 1-24 (1975): Weinryb
and 5hroff, DrugMetab-Rev
, 10:271-283 (1975): Brought
on and Strong, Cl1n, Chem
-22: 726-732 (1976); and
Playfair, etal, Br-Med.

Bull, 30: 24-31 (1974) Antigenic determinant density, i.e. the average number of habuten moieties bound to a carrier, is theoretically limited only by the number of available binding sites on one selected carrier molecule. However, in common cases where the carrier is a natural protein such as albumin, on average 1 to about 50,
More usually from 2 to about 20.

Regarding the binding method, use the enzyme label 1 for EIA described above.
．． It can be carried out in exactly the same manner as the method for producing 5-AG.

The method for producing antibodies using antigens derived from 1,5-AG derivatives used in the present invention may be performed according to any conventional technique (for example, Parker
) of Radioimmunoa-戊3g5y of Biologically Acti
ve Compounds, Prentice-H
all (Englewood C1)ffs, Ne
(w Jersey USA-1976)) Usually, rabbits, goats, mice, and guinea pigs are used.
the immunogen complex, usually mixed with an adipant, is administered to a host animal such as a horse or a horse.
Inject at the various sites listed above.

Additionally, injections are made at regular or irregular intervals from the same or different sites, after which blood is drawn and antibody titers are determined at 1 to determine whether optimal titers have been reached. Blood is drawn from the host animal to obtain the appropriate volume of specific antiserum. If desired, undesirable substances such as non-specific antibodies can be removed in a purification step before determining the suitability of the antiserum for use in the actual analysis. good.

Antibodies (e.g., antibodies commonly referred to as monoclonal antibodies) can be produced using somatic hybridization (fusion) techniques.
l hybridization technology
s) can also be obtained. (Lymphoyt
e Hybridomas, ed-Melcher%
et al., Springer-Verlag (N
ew York 1978) Nature266:
495 (1977), 5bience208:
692 (1980) and Metbods in E.
nzymology73(PartB)'3-46(1
981)) The 1,5-AG derivative used in the present invention has side chain binding via the 10H group at the 3rd, 4th or 6th position of 1,5-AG represented by the general formula (1). This is a new compound introduced.

In formula C, any one of X, Y, and Z is 2A-alkylene-B-, and the other two represent -OH. here,
A represents -NH2 or -COOH and B represents 10- or -C
OO-, and the alkylene has 1 to 12 carbon atoms. ] In the general formula, examples of alkylene include those having about 1 to 12 carbon atoms, such as methylene, ethylene, propylene, and butylene. These may be branched and have 2 to 1 carbon atoms.
It is preferably about 0, more preferably ethylene,
Examples include propylene and butylene.

Examples of the compound of general formula (1) of the present invention include the compounds shown in Table 1 below.

These compounds of the present invention are synthesized as follows.

That is, general formula (2) [wherein, any one of R1, R2, R3 is the formula A-alkylene-B-, and the other two and R4 are -NH-
CBZ, -COOH, or -COOBn? B indicates -〇- or -〇〇〇-, and alkylene indicates Z having 1 to 12 carbon atoms.

CBZ is benzyloxycarbonyl, Bn is benzyl, Tr is triphenylmethyl, and THP is tetrahydropyranyl. The 1,5-AG derivative of general formula (1) can be obtained by subjecting the compound represented by formula (1) to acid hydrolysis or catalytic reduction to remove the protective group as required.

Acid hydrolysis is carried out at 20-100°C in the presence of acid in an aqueous medium.
1 or 25 to 60 hours for 1 to 5 hours.

As the acid, mineral acids such as hydrochloric acid and sulfuric acid, and lower fatty acids of 01 to C4 such as formic acid, acetic acid, and trifluoroacetic acid can be used, and preferred ones are hydrochloric acid and acetic acid. When using mineral acid, the amount of acid added is such that the concentration in the reaction solution is 0.01.
~5N, preferably about 0.1 to 2N, and if fatty acids are used, add them so that the concentration in the reaction solution is 50% or more, preferably about 60 to 80%. Yo().

In fused reduction, alcohols such as alcohol, ethanol, etc., ethers such as tetrahydrofuran and dioxane, water, dimethylformamide, acetic acid, etc. can be used singly or in combination as necessary in the presence of a catalyst, usually in a solvent. Preferred examples include methanol and ethanol.

As the catalyst, palladium on carbon, palladium black, palladium chloride, palladium hydroxide, etc. can be used, and the preferred one is
The signature is palladium black.

Palladium chloride, etc. The amount of the catalyst may be about 10 to 20% of the amount of the raw material compound.

Representative compounds of general formula plates used as raw materials are shown below.

1. The compound of general formula (■) is as follows (■).

It is synthesized via reaction route 7 of either (IV) or (V).

Bn OR3 The compound of general formula (T) of the present invention is isolated and purified from a reaction solution by a conventional method and obtained as a free acid or a free base. [Effects] Next, the effects of the present invention will be explained by experimental examples. do.

Experimental example Creating a calibration curve In a test tube, add 1.5 AG or glucose standard yo,
Samples serially diluted 0.1 in PBS containing 1% BSA
ff1l, 1,5-AG anti-blood bottle 0.1% BSA made by immunizing rabbits with a conjugate of compound 4 and BSA
0.1 ml of a solution diluted 1000 times with PBS containing 7, 0.1 ml of a solution diluted 1000 times with PBS containing conjugate of compound 2 and HRP, ), O,X% BSA'& 0.3% of the above One DASP bead blocked with PBS containing BSA was added, and immunoreaction was performed overnight at 4°C.

After the reaction, the beads were washed 4 times with PBS containing 0.1% BSA and then washed with 0.05M phosphate buffer 1 (pH 7) containing 0.05% p-hydroxyphenylpropionic acid and o.oot% hydrogen peroxide. , 0) (substrate solution) was transferred to a new test tube containing 1 ml, and an enzyme reaction was performed at 37° C. for 1 hour. This reaction solution contains 1.5% sodium azide (0.25 N hydroxide) IJ solution (stopper) 0.1 ml
After adding 3 and stopping the reaction, use a fluorescence measuring device (manufactured by Fujirebio Co., Ltd.,
Auto FP-1) 'i, excitation wavelength 313rI
rr1). The fluorescence intensity of the reaction solution was measured at a detection wavelength of 405 mm, and a calibration curve was prepared as shown in FIG.

As is clear from the calibration curve of 1.5-AG in Figure 1, the detection limit of 1.5-AG with this measurement method was 0.1 μg/ml, and the sensitivity was sufficient to be applicable to clinical sample measurement. .

Additionally, the cross-reactivity of the antibody with glucose is only 0.2
%Met.

Therefore, the quantitative method of the present invention can be used as a practical quantitative method for 1.5-AG.

The conjugate of Compound 2 and HRP used in the above experimental examples was prepared by the following method.

Compound 2. 6.7μ, N-hydroxysuccinimide, 7.3μ, and 8,4Ing of DCC were dissolved in 0.4 ml of nodioxane, and reacted at room temperature for 5 hours with stirring. The ureide produced by the active esterification reaction was removed by filtration, and the filtration residue Y was further washed with dioxane (0.05 ml) to obtain a dioxane solution of the active ester of Compound 2. Next, the above active ester solution was added to a solution in which HRP (manufactured by Toyobo, RZ=3.41) 25-2■ was dissolved in 4.2 ml of 01)M phosphate buffer (pH 7.5), and the solution was stirred at room temperature. The reaction was continued for 5 hours while stirring. After removing the precipitate in the reaction solution by centrifugation, 0.9% sodium chloride was added. OI
The desired conjugate was obtained by dialyzing against M phosphate buffer (pH 7.0) (hereinafter referred to as PBS).Then, the solution of this conjugate was filtered through an ultrafiltration membrane PMIO. 1°5-A
4.4 ml of HRP-labeled G (HRPa degree 6.1)
1) g/m1. A protein recovery rate of 100% and an HRP activity recovery rate of 100% were obtained.

Moreover, DASP beads were created by the following method.

Polystyrene beads for EIA (wooden blocks, 6.5171)
1) ' ) 300 pieces with 2% concentration score roll 900 (
(manufactured by Kao) at room temperature overnight. Next, it was washed with water using No. 1, which eliminates air bubbles, and after thoroughly draining the water, it was spread on a filter paper and air-dried. Affinity purified goat anti-rabbit IgGCH&L
) Antibody (manufactured by Jackson ImmunoResearch) 2■ was added to 0.
0.05M phosphate buffer containing 1% sodium azide (pH
7,5) The above washed beads were soaked in 100 ml of the solution and left at room temperature for 2 days to physically adsorb the antibody.

The second antibody-immobilized beads (hereinafter referred to as DA) prepared in this way
SP beads) are prepared using P containing 0.3% BSA before use.
Nonspecific adsorption was blocked by soaking in BS solution and incubating at 45° C. for 10 hours, and the sample was subjected to EIA.

Below, preparation of 1.5-AG derivative and preparation of antigen.

The present invention will be explained in detail using specific examples in the order of antibody production and 1.5-AG immunoassay.

Example 1. 1,5-anhydro-6-0-glycyl-
Production of D-glucitol (Compound 1 in Table 1) 1,5-AG derivative (ff
D 434■ and 1 g of benzyloxycarbonylglycine chloride 3971) were stirred at room temperature for 20 hours in the presence of dioxane solvent and pyridine. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography to give 266.
An oily substance was obtained. In this product, protons corresponding to 20H of aromatic hydrogen polished rice were observed at 7.3 ppm in IH-NMR using deuterated chloroform. When positive and negative ion F A B -MS was measured using a mixture of glycerin and DMSO as a matrix, 626 corresponding to (λi+H) and CM
624 corresponding to -H)- was observed as each reference peak.

Next, 250 mg of the above compound was subjected to catalytic reduction using a palladium black heat catalyst in a mixture of methanol, acetic acid, and water.
heavy water? According to 'H-NMR, 104 mg of the title compound was obtained, from which aromatic hydrogen was removed from benzyl ether and benzyloxycarbonyl groups.

Compound 1 contains glycerin and DMSO in the matrix.
When positive ion FAB-MS Y was measured using a mixture of the above, the reference peak ytm was measured at 222, which corresponds to CM+H). In addition, in IR using the thin film method, there is an absorption based on ester at 1750 cm-', 16
Amino absorption at 30 cm-', etc., 1510, 14
Absorption was observed at 20.1240.1)00.1050 cm.

Example 2. 1. Preparation of 5-anhydro-6-0-(3-carboxy-propionyl)-D-glucitol (Compound 2 in Table 1) Add 3.17 g of benzylsuccinic acid chloride to 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 1.
Stirred for 5 hours. Dilute the reaction solution with ice water and add ethyl acetate to
After extraction twice with 5 ml, the extract was washed with 1N hydrochloric acid, washed with water, and dried over sodium sulfate. The product was then purified by silica gel chromatography for 7 times to obtain 680 ml of an oily substance.

Catalytic reduction of 650 mg of the above compound was carried out in a mixture of methanol, acetic acid and water using palladium black as a catalyst to obtain 1 g of the title compound 4801).

The compound was determined by positive ion FAB-MS in the same manner as in Example 1 (reference rice noodles were added to 265, which corresponds to M+J, and negative ion F
1 080 cm corresponding to (M-H)- in AB-MS
Absorption was observed in each case.

Example 3. 1. Preparation of 5-anhydro-6-0-carboxymethyl-D-glucitol (Omoteko's Compound 3) 53 ml of sodium hydride was placed in a two-headed flask, and under a nitrogen atmosphere, 0ml of DMF was added, and while stirring, 1,5-AG derivative (III) obtained by the method of Reference Example 1
Add 430 ffl. After stirring for 30 minutes, chloroacetic acid 1
) and stirred at room temperature for 15 hours. 75 ml of water was added to the reaction solution, extracted twice with ethyl acetate, washed with saturated brine and then with water, and purified by silica gel chromatography to obtain 170 ml of an oily substance. This product has a standard peak of 493, which corresponds to CM+H nail, in positive ion FAB-MS, and negative ion FAB-M
A reference peak was observed at 491 corresponding to CM-H)- in S.

1 g of the above compound 1401) was subjected to catalytic reduction in a mixture of methanol, acetic acid, and water using palladium black as a catalyst to obtain the title compound 55rr@.

Compound 3 is a positive ion F A B -M S (M+H
)”, the reference peak is set at 223, which corresponds to the negative ion FAB
-MS observed a reference peak at 221, which is half of CM-H).

Also, in IH using the thin film method, 3350°2930.
1740.1250. Ll, 00,1060cm"-'
Absorption was observed in each.

Implementation f9U 4. 1.5-anhydro-6-0-(3
-carboxy-propyl)-D-glucitol (Compound 4 in Table 1) Place 1.80ff1g of sodium hydride in a two-headed flask, add 20ml of DMF under a nitrogen stream, and while stirring, proceed as shown in Reference Example 1. 1.3 g of 1,5-AG derivative (III) obtained by the method described above was added dropwise. After stirring for 30 minutes 5
- Bromu 1 - Pentene 671 ■ Processing, 15 at room temperature
Stir for hours. 75 ml of water was added to the reaction solution, extracted twice with 75 ml of ethyl diacetate, washed with saturated saline, then washed with water twice, and purified with silica gel chromatography to obtain 1.01 g of an oily substance. Obtained.

980 rng of the above compound are dissolved in 8 ml of acetone, and while stirring, 2.4 g of Na I 04 dissolved in 10 ml of water and B ml of acetone are added dropwise. 8 dissolved in 4 ml of water while cooling to 0°C.
After boiling 0q of KMn04 and stirring at room temperature for 1 hour, 600rng of NaIO4 was added and stirring was continued for 1 hour. Add 100ml of water Z to the reaction solution, and add 75ml of ethyl acetate.
After extracting twice in step 1, washing with saturated saline and then water,
Purification was performed using silica gel chromatography, and 660
An oily substance was obtained.

630 mg of the above compound was mixed with methanol and acetic acid.

Catalytic reduction was carried out in a water mixture using palladium black as a catalyst to obtain 1 g of the title compound 2901).

Compound 4 has a reference peak at 251 corresponding to (M+H)'' in positive ion FAB-MS, and a reference peak at 251 corresponding to (M+H)'' in positive ion FAB-MS.
In hiS, a reference peak was observed at 249, which is half of (MH).

In addition, in IR using the thin film method, 2930°1720.
Absorption was observed at 1420.1370 and 1260.108108O', respectively.

Example 5. 1. Production of 5-anhydro-4-0-(3-carboxy-propyl)-D-glucitol (compound 5 in Table 1) 169 ml of sodium hydride was placed in a two-headed flask, and 10 ml of DMF was added under a nitrogen stream. ; and while stirring, 1.24 g of compound (fv) was added dropwise. After stirring for 30 minutes, 630 μ of 5-7' rom-1-pentene was added.
The mixture was stirred at room temperature for 15 hours. Add 50 ml of cold water to the reaction solution
After adding tg and extracting twice with 75 ml of ethyl acetate,
Purification was performed using silica gel chromatography, and 850
mg of oil was obtained.

The above compound 820Q! Dissolve in 8 ml of acetone,
While stirring, 1.4 g of Na I 04 'a' dissolved in 10 ml of water and f3 ml of acetone are added dropwise. Next, while cooling to 0° C., 50 μm of KMn O4 dissolved in 4 ml of water was added, and after stirring at room temperature for 1 hour, 350 mg of Na I 04 was further added and stirring was continued for 1 hour. The reaction solution was acidified with hydrochloric acid, extracted with ethyl acetate, and then subjected to silica gel chromatography 7 times.
An oily substance of 0.00 cm was obtained.

Add 80% acetic acid to 1 g of the above compound 5601).

Stirring was continued at 65°C for 2 hours. After adding 50 ml of water to the reaction solution and performing ethyl acetate extraction, purification was performed using silica gel chromatography.

320 cm of oily substance was combined.

280mg of the above compound in a mixture of methanol, acetic acid and water, palladium black? Catalytic reduction was carried out using a catalyst to obtain 1 g of the title compound 1501).

Compound 5 is a positive ion F A B -M S (M+H
)”, the reference peak is set at 251, which corresponds to the negative ion FAB
-MS, a reference peak was observed at 249 corresponding to CM-H)-.

In addition, in IR using the thin film method, 2930°1720.
Absorption was observed at 1460 and 1380 cm"-', respectively.

Example 6. 1. Production of 5-anhydro-3-0-(3-carboxy-propyl)-D-glucitol (Compound 6 in Table 1) Place 200 ml of sodium hydride in a two-headed flask, and add 10 ml of DMF under a nitrogen stream. Add and while stirring,
Compound (V1720) is added dropwise. After stirring for 30 minutes, 5-
Brome-1-pentefu 44mg? In addition, at room temperature 15
Stir for 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 purified by silica gel chromatography to obtain 1 g of an oily substance.

1.3 g of the above repeatedly synthesized compound was dissolved in ethanol, and in the presence of a catalytic amount of 1)-) luenesulfonic acid,
The protective group was removed by stirring at 65° C. for 1 hour. Next, in the same manner as in Reference Example 2, the 2-, 4-, and 6-position hydroxyl groups were benzylated, and then in the same manner as in Example 4.5, Na
After I 04, K Mn 04 treatment, catalytic reduction using palladium thermal catalyst Z? and the title compound 90
I got ■.

Compound 6 has a reference peak at 249 corresponding to (MH)- in negative ion FAB-MS? Observed.

In addition, in IR using the thin film method, 2950°1720,
Absorption was observed at 1250 cm-'.

Example 7. 1. Production of 5-anhydro-6-0-(6-amino-hexanoyl)-D-glucitol (compound 7 in Table 1) Compound (III) 434■ shown in Reference Example 1 was dissolved in 10 ml of pyridine, At room temperature, benzyloxycarbonyl-ε-aminocaproic acid chloride 535Z is added dropwise. After stirring at room temperature for 15 hours, 751T of water was added to the reaction solution.
II'Q was added, extracted twice with ethyl acetate, and purified by silica gel chromatography to obtain 366 cm of oily substance.

The obtained compound was dissolved in a mixture of methanol, acetic acid, and water.
Reduction was carried out using palladium black as a catalyst to obtain the title compound 104.

For Compound 7, a reference peak was observed at 278 corresponding to (M+H)'' in positive ion FAB-MS.

Also, in IR using the thin film method, 1740°1460.
1) Absorption was observed at 00 cm''-'.

Example 8. 1. Preparation of 5-anhydro-6-0-(1)-amino-undecanoyl)-D-glucitol (Compound 8 in Table 1) )-amino-undecanoic acid chloride 637■ The title compound 4001 was reacted in exactly the same manner except that it was changed to 637■. Ig was obtained.

Compound 7 is a positive ion F A B -M S (M+
A reference peak was observed at 348, which corresponds to H)+.

In addition, in IR using the thin film method, 2925°1740
．． Absorption was observed at 1460 cm-'.

(2) Preparation of antigen Various methods are known for preparing hapten antigens as mentioned above, but here, as an example, bovine serum albumin (hereinafter abbreviated as BSA) is used as an antigenic carrier material. , 1.5-AG derivatives) are chemically bonded.

The chemical bonding method differs depending on the introduced side chain terminal group [A in general formula (1)] of the 1.5-AG derivative (1), but when A is an amino group, a water-soluble carbodiimide method is used to bond the carboxyl group. In the case of BSA directly by active ester method
combined with. However, the method of chemical bonding is not limited by this. Furthermore, the terminal group A and BSA may be bonded through a spacer of a low molecular weight compound.

Reference example 1. 1. Creation of 5-anhydro-6-0-glycidol (compound 1) and BSA conjugate Compound 1.161
ng and 23.9 mg of B5A in 1.6 ml of 1/1
5M') acid buffer (pH 5, 6).

To this, while stirring at room temperature, water-soluble carbodiimide hydrochloride 2081) 18χ was added in several portions to pH 5-6.
I managed to react while begging. Furthermore, after reacting overnight at room temperature, the reaction mixture was dialyzed against physiological saline to obtain the desired conjugate. Next, this was concentrated using a concentrator equipped with an ultrafiltration membrane PMIO (manufactured by Amicon), and the thickness was reduced to 0.2 μm.
The protein was sterilized by filtration (manufactured by Millibore) and used as an antigen for immunization.The protein recovery rate in all steps was 88%.

BSA conjugates of Compound 7 and Compound 8 were also created using a similar method.

Reference example 2. 1. Preparation of 5-anhydro-6-0-(3-carboxy-propyl)-D-glycidol (compound 4) and BSA conjugate 4,145.8 mg of compound, 134 μg of N-hydroxysuccinimide, and N,N- 180.3 ml of dicyclohexylcarbodiimide (DCC) was dissolved in 3.5 ml of dioxane and reacted at room temperature for 3 to 4 hours with stirring. The active ester produced by the reaction was removed by filtration, and the filtration residue was washed with 42 ml of dioxa to obtain a dioxane solution of the active ester of Compound 1. Next, BSA'Y 15■/m
0.1 M so that the concentration is 1! acid buffer tpH7
The active ester solution χ was added to 25.7 ml of the BSA solution R dissolved in step 5), and the mixture was reacted overnight at room temperature with stirring.

The precipitate in the reaction solution was removed by centrifugation and separation, and then dialyzed against physiological saline to obtain the desired conjugate. next,
A concentrator using ultrafiltration membrane PM10' and centrifuge for this solution of combined substances? The antigen was concentrated and 0.2 μm filter sterilized to prepare an antigen for immunization. The protein recovery rate through all steps was 98%.

In addition, by the same method, compounds 2.3.5 and 60B
An SA conjugate was also created.

(3) Creation of antibodies Using Reference Example 2, Reference Example 3, and BSA-conjugated antibodies of the general formula (1) shown in Table 1 prepared according to their methods as immunogens, antibodies were isolated from domestic rabbits by a known method. 1.5-AG antibody? Created.

Example 9 A BSA conjugate of compound No. 4 was diluted with physiological saline, and
■ The concentration was adjusted to 1/ml. This melt a 1.5
ml, add 1.5 ml of Freunds compl.
Add the adjuvant, mix well, and subcutaneously inject iml/feather into domestic rabbits (2 to 3 birds for each antigen) (administered 4 times at 1-week intervals, then 3 times at 2-week intervals). After sufficient sensitization, blood was collected two weeks later, and the blood was centrifuged at 3000 rpm for 15 minutes to obtain serum. After heat treatment at 60°C for 30 minutes, the serum was stored at -20°C, and the blood was collected for each immunogen. 1,5-AG antiserum compound. Does the presence of antibodies against 1.5-AG in this serum indicate BSA? After absorption into the fixed resin, it was confirmed by the don't-immuno binding anomaly method.

A 1.5-AG antiserum was obtained in the same manner as above using a BSA conjugate of compounds 1-3.5-8.

(41), 5-AG immunoassay As an immunoassay method, as mentioned above, RIA is used.

Although it can be measured by various methods such as EIA, FIA, and LIA, here we will show an example of application to EIA. This is an example of what is called a competitive law.

Example 10°glucose and 1.5-AG at 10 μg/ml each,
A model test solution containing 1 μg/ml was prepared.

This is shown in the experimental example "E of 1,5-AG by competition method".
When measured according to the method of IAJ and quantified using the calibration curve shown in Figure 1, the concentration of 1.5-AG was 1.1 g.
/ml.

Compounds (Jm), ■), (V), which are the raw materials for the compound of general formula (2) and are key to the synthesis of the compound of general formula (2), are, for example,
It was synthesized according to the reaction formula shown below. As a specific example, a synthesis example of compound (m) is shown below.

Reference example 3. 1.5-Anhydro-2,3,4-tri〇-benzy-D-glucitol (m) NoMl Preparation 1
．． 5-AG 3.28 g'f Dissolved in 20 ml of pyridine, and while stirring under water cooling, trityl chloride 6, 1.2
gχ was added dropwise. The mixture was stirred for 1 hour under water cooling and further stirred for 15 hours at room temperature. Dilute the reaction solution with 200 ml of water and add 150 ml of water.
Extract twice with ml of ethyl acetate? After that, it was purified by silica gel chromatography to obtain 7.44 g of 6-dolythylated 1.5-AGV. In addition to the 1.5-AG signal in 'H-NMR using deuterated chloroform, this product also contained 15H derived from aromatic hydrogen at 7.3 to 7.9 ppm.
minutes of protons were observed.

900 ml of sodium hydride was placed in a two-headed flask, 40 ml of DMF was added under a nitrogen stream, and 4.06 g of the above compound was added dropwise while stirring. After stirring at room temperature for 30 minutes,
．． Seven drops of 2 ml of benzyl chloride were added, and the mixture was stirred for 15 hours. Add 200 ml of water to the reaction solution to dilute it to 400 ml.
After 7 extractions with ethyl acetate, purification was performed using silica gel chromatography to obtain 5.27 g of 2.3.4-
) Dibenzyl-6-trityl-1,5-AG was obtained. This product was found to be 7.0 in 'H-NMR using deuterated chloroform 7.
A total of 30 H aromatic protons were observed at ~7 and 8 ppm.

3.33 g of the above compound was dissolved in 20 m+ of 80% acetic acid and stirred at 65°C for 2 hours.

Reaction liquid? The residue was concentrated under reduced pressure, purified by silica gel chromatography, and then recrystallized from ethyl acetate and n-hexane to obtain 1.48 g of colorless needle crystals χ.

The compound has a melting point of 85-86°C, and in 'HNMR using single-fold quapoform, protons for IR disappear when D20 is added to δ1.8, protons for 8H at δ3.0-4.2, and protons for 8H at δ4. .4 to 5.1, 6H proton, δ7
．． 15H protons were observed at 28. Also, KBr
IR using 3300.2980, 2875.15
Absorption appointments were made on 00.1505.1)00 respectively.

[Brief explanation of the drawing]

FIG. 1 shows 1, by EIA using 1.5-AG antibody.
5 shows calibration curves for 5-AG and glucose.

Claims (3)

[Claims] (1) In the test solution containing 1,5-anhydroglucitol,
An antibody that has specific reactivity to 1,5-anhydroglucitol and labeled 1,5-anhydroglucitol are added to cause an immunoreaction, and then the labeled 1,5-anhydroglucitol produced by the immunoreaction is After separating the bound product of hydroglucitol and the antibody from the unbound labeled 1,5-anhydroglucitol, the label of either one of the separated products is measured to determine whether it is in the test liquid. 1,5-anhydroglucitol characterized by quantifying 1,5-anhydroglucitol in
Method for quantifying anhydroglucitol (2) an antibody having specific reactivity to 1,5-anhydroglucitol; (3) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, one of X, Y, and Z is the formula A-alkylene-B-, and the other two are - Indicates OH. Here A is
-NH_2 or -COOH, B is -O- or -
COO- is shown, and the alkylene is one having 1 to 12 carbon atoms. ] A novel 1,5-anhydroglucitol derivative represented by: