JP3152387B2 - Quinazoline compound, monoclonal antibody specific to C9 base, and method for immunological measurement of pufferfish venom - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel quinazoline compound and a salt thereof, 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (so-called C9 base), which is one of the alkaline hydrolysis products of the phlegm venom tetrodotoxin. ) -Specific monoclonal antibodies and antibody fragments thereof, and 2-amino-6-hydroxymethyl-8-using said monoclonal antibodies or antibody fragments thereof.
The present invention relates to an immunological measurement method for hydroxyquinazoline and an immunological measurement method for a tetrodotoxin compound.

[0002]

2. Description of the Related Art Puffer fish, which has been a favorite fish since ancient times, contains tetrodotoxin (hereinafter, TTX) in its body.
) And many other TTX congeners, resulting in the loss of many lives. This situation has not changed even in recent years, and the incidence of food poisoning patients is still continuing. Recently, it has been revealed that TTX and / or TTX homologues exist in fish and snails other than puffer fish. Therefore, in order to ensure the safety of eating fish and shellfish such as puffer fish, it is necessary to first accurately measure the amount of TTX and / or TTX homologue present in the edible portion of puffer fish. It is. In particular, since puffer fish are often treated as fresh food, it is important that the measurement method be simple and quick. In addition, since TTX and / or TTX homologs contain lethal toxins, it is necessary to consider safety during measurement.

[0003] In general, the analysis of TTX requires the use of a sample of 0.1%.
A so-called mouse test method (official method) is used in which an extract extracted by heating with 1% acetic acid or methanol added with acetic acid is intraperitoneally administered to mice, and the toxic effect is calculated from the lethal time. The method has drawbacks in that it requires a lot of preparation time, a large measurement error, and is indistinguishable from other paralytic poisons, because it involves handling mice. Alternatively, as an alternative, TTX and / or TTX
Non-toxic 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (usually called C9 base, both names used in combination in the present specification) produced by alkali hydrolysis of the homologue emits fluorescence. Utilizing this, instrumental analysis such as liquid chromatography or gas chromatography is also performed. Although this method has no problem in terms of accuracy, purification of the sample is complicated and time-consuming,
In addition, there is a disadvantage that the measuring device and the equipment are expensive and large.

On the other hand, in recent years, a TT using an immunological measurement method which has been applied to the measurement of low molecular weight compounds as a simple and rapid analysis method without requiring expensive equipment has been recently used.
A method of measuring X has been attempted [Shugo Watanabe, Recent Advances in Fugu Poison Research, Published by Koseisho Koseikaku, 1988, pp. 21-31; and Endo Matumura and Sadako Fukiya, J.
ournal of AOAC International, vol. 75, No. 5, 883
−886 (1992)]. Although this method enables easy and rapid measurement of TTX in the course of distribution, it is difficult to measure the homologues present simultaneously with TTX at the same sensitivity with the same sensitivity.

[0005]

The present inventors have conducted intensive studies on a method for measuring various tetrodotoxin compounds in a lump and in a simple manner. As a result, they synthesized a novel compound which is a derivative of C9 base, and obtained the novel compound. Is used as a hapten, a monoclonal antibody specifically reacts with a C9 base generated by alkaline hydrolysis from a tetrodotoxin compound, and furthermore, the tetrodotoxin compound can be accurately measured by using the monoclonal antibody. I found it. The present invention is based on these findings.

[0006]

Accordingly, the present invention provides a compound of the general formula (I):

Embedded image [Wherein, R ¹ is a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms, and R ² represents —C
H ₂ -A- (C ＝ O) _m -B-COOR ³ , A is O, S, or NH, B is an alkylene group or a phenylene group having 1 to 10 carbon atoms, and R ³ is hydrogen An atom or a linear or branched alkyl group having 1 to 4 carbon atoms,
m is 0 or 1], or a salt thereof.

[0007] The present invention also relates to a monoclonal antibody and an antibody fragment thereof characterized by reacting specifically with 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (C9 base). The present invention also relates to a method for immunologically measuring 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (C9 base), characterized by using the above-mentioned monoclonal antibody or antibody fragment. In addition , the present invention provides (1) a step of alkali-hydrolyzing a test sample and then neutralizing the test sample, and (2) an immunological analysis of the 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (C9 base). Depending on the measurement method, 2-amino-
6-hydroxymethyl-8-hydroxyquinazoline (C
And (3) determining the total amount of the tetrodotoxin compound from the measured value obtained in the step (2). Further, the present invention
The quinazoline compound represented by the general formula (I) or a salt thereof
Is used as a hapten,
The present invention relates to a method for producing a nal antibody.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The C9 base, 2-amino-6-hydroxymethyl-8-hydroxyquinazoline, has the formula (II):

Embedded image A compound represented by the formula (III-a):

Embedded image Can be easily obtained by alkaline hydrolysis of tetrodotoxin or various related compounds thereof. The alkaline hydrolysis is carried out, for example, by Toshio
The method disclosed by Goto et al. [Bull. Chem. Soc.
Japan, vol. 35, 1045-1046 (1962)].

[0009] In the present specification, the term "tetrodotoxin compound" refers to a compound obtained by subjecting it to alkaline hydrolysis.
A compound capable of producing 9 bases, including tetrodotoxin (TTX) itself and its related compounds. The “tetrodotoxin compound” is, for example, a compound represented by the formula (III):

Embedded image Wherein, R ^o is a hydroxyl group, or R ^a is hydrogen atom or a hydroxyl group, or, R ^o and R ^a to form a -O- together, R ^b is a hydrogen atom or a hydroxyl group Yes, R
^c is a hydrogen atom, a hydroxyl group, or a hydroxymethyl group, and R ^d is a hydrogen atom, a hydroxyl group, an aldehyde group, a methyl group, a hydroxymethyl group, or —CH (OH) CH (N
H ₂₎ is COOH, and R ^e is a hydroxyl group or -O
OC (CH ₂ ) ₂ COOH].

There are various toxic compounds among the tetrodotoxin compounds. For example, tilitoxin, 11-oxotetrodotoxin, or 11-nortetrodotoxin-6,6-diol exhibiting activity (ie, toxicity) equivalent to tetrodotoxin; 11-nortetrodotoxin (S) exhibiting a fraction of the activity of tetrodotoxin -Ol, 11-nortetrodotoxin (R)
-Ol, 11-deoxytetrodotoxin, 4-epitetrodotoxin, or 6-epitetrodotoxin; or tetrodotoxin-8-O-hemisuccinate exhibiting activity about 100 times lower than that of tetrodotoxin-
4,9-anhydrotetrodotoxin and the like can be mentioned.

Table 1 shows the structures of tetrodotoxin and tetrodotoxin-related compound represented by the formula (III). In Table 1, tetrodotoxin is denoted by T0, and similarly, tilitoxin is denoted by T1, 11-oxotetrodotoxin is denoted by T2, and 11-nortetrodotoxin is denoted by T0.
6,6-diol at T3, 11-nortetrodotoxin (S) -ol at T4, 11-nortetrodotoxin (R) -ol at T5, 11-deoxytetrodotoxin at T6, and 4-epitetrodotoxin at T7. ,
6-epitetrodotoxin is indicated by T8 and tetrodotoxin-8-O-hemisuccinate-4,9-anhydrotetrodotoxin is indicated by T9. In Table 1, "*"
Indicates that to form a -O- together with R ^o. Although not shown in Table 1, in compounds T0 to T8, R
^o is hydroxyl, R ^o in compound T9 form a -O- together with R ^a.

[0012]

TABLE 1 Compound Name ^{R a R b R c R d} R e T0 H OH OH CH 2 OH OH T1 H OH OH CH (OH) CH OH (NH 2) COOH T2 H OH OH CHO OH T3 H OH OH OH OH T4 H OH OH H OH T5 H OH H OH OH T6 H OH OH CH 3 OH T7 OH H OH CH 2 OH OH T8 H OH CH 2 OH OH OH T9 * OH OH OH OOC (CH 2) 2 COOH

In the present specification, Me represents a methyl group or a methylene group. Thus, for example, -OMe means a methoxy group, -OMeOMe means a methoxymethoxy group, and -NHCOOMe means a (N-methoxycarbonyl) amino group. Ph represents a phenylene group, and ^t Bu represents a ^t -butoxy group.

The quinazoline compound of the present invention represented by the general formula (I) is a novel compound. In the general formula (I), examples of the linear or branched alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, and an n-
Examples include a propyl group, an i-propyl group, an n-butyl group, an i-butyl group, an s-butyl group, and a t-butyl group.

In the general formula (I), the number of carbon atoms is 1 to 1.
As the alkylene group of 0, a linear or branched alkylene group, for example, a methylene group, an ethylene group, a trimethylene group, a propylene group, a tetramethylene group, 1,2-butylene, 1,3-butylene, 3-butylene, pentamethylene group, 1,2-amylene group, 1,3-amylene group,
1,4-amylene group, 2,3-amylene group, 2,4-methylene group, hexamethylene group, 1,2-hexylene group,
1,3-hexylene group, 1,4-hexylene group, 1,5
-Hexylene group, 2,3-hexylene group, 2,4-hexylene group, 2,5-hexylene group or 3,4-hexylene group.

In the general formula (I), the phenylene group includes an o-phenylene group, an m-phenylene group, and a p-phenylene group.
Includes phenylene groups.

In the general formula (I), the group R ¹ is a hydrogen atom or a methyl group, and R ² is —CH ₂ —A—
(C = O) _m -B-COOR ³ , A is O, S, or NH, B is an alkylene group or phenylene group having 1 to 10 carbon atoms, and R ³ is a hydrogen atom or 1 carbon atom. ~ 4
A quinazoline compound represented by the formula (I) or a salt thereof, wherein m is 0 or 1;

When A is O in the group R ² in the general formula (I), the group R ¹ is preferably a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms (more preferably Methyl group), and the group R ² is —CH ₂ —O— (C
ＯO) — (CH ₂ ) _n —COOR ³ , wherein R ³ is a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms (more preferably a hydrogen atom); The quinazoline compound represented by the above formula (I) or a salt thereof, which is an integer of 10 (more preferably 2) is preferable, and a compound represented by the formula (I
V):

Embedded image (2-amino-8-methoxyquinazolin-6-yl) methyl succinate or a salt thereof is particularly preferred.

When A is S in the group R ² in the general formula (I), the group R ¹ is a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms (more Preferably a hydrogen atom), and the group R ² represents —CH ₂ —S
— (CH ₂ ) _n —COOR ³ , wherein R ³ is a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms (more preferably a hydrogen atom), and n is an integer of 1 to 10. A quinazoline compound represented by the formula (I) or a salt thereof, which is (more preferably 1), is preferable, and a compound represented by the formula (V):

Embedded image (2-Amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid represented by or a salt thereof is particularly preferred.

When A is NH in the group R ² in the general formula (I), the group R ¹ is a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms (more Preferably a hydrogen atom), and the group R ² represents —CH ₂ —
NH-Ph-COOR ³ , wherein R ³ is a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms (more preferably a hydrogen atom), and the phenylene group is a p-phenylene group, The quinazoline compound represented by the formula (I) or a salt thereof is preferable, and the compound represented by the formula (VI):

Embedded image 4- [N- (2-amino-8-hydroxyquinazolin-6-yl) methyl] aminobenzoic acid represented by or a salt thereof is particularly preferred.

The salt of the quinazoline compound represented by the formula (I) includes a salt with an inorganic acid or an organic acid, or a salt with an inorganic base or an organic base. Examples of acid addition salts include, for example, hydrochloride, sulfate, methanesulfonate, or p-toluenesulfonate, and further, a dicarboxylic acid such as oxalic acid, malonic acid, succinic acid, maleic acid, or fumaric acid. And salts with monocarboxylic acids such as acetic acid, propionic acid and butyric acid.
The inorganic base suitable for forming the salt of the quinazoline compound of the present invention is, for example, a hydroxide, carbonate, or bicarbonate of ammonia, sodium, lithium, calcium, magnesium, aluminum or the like. Examples of the salt with an organic base include mono-, di-, and tri-
Examples thereof include an alkylamine salt, a hydroxylamine salt, a guanidine salt, an N-methylglucosamine salt, and an amino acid salt.

The quinazoline compound of the present invention represented by the general formula (I) can be prepared by various methods. When A is S or NH in the general formula (I), for example, the general formula (VII):

Embedded image Wherein R ¹¹ is a hydroxyl protecting group, for example, a lower alkyl group or a lower alkoxy lower alkyl group; R ⁴ is an amino protecting group, for example, a lower alkyl carbonyl group or a lower alkoxy carbonyl group; atoms, for example, a bromine atom, a chlorine atom, or iodine atom with a compound represented by), the general formula ^{(VIII): R 31 OOC-} B- (C = O) m -D (VIII) ( wherein, B And m are as defined above, R ³¹ is a carboxy protecting group, for example, a lower alkyl group, and D is S
H or NH ₂ ), whereby the quinazoline compound of the present invention represented by the general formula (I) can be prepared.

That is, the compound represented by the general formula (VII) and the compound represented by the general formula (VIII) are
In a suitable solvent (eg, dimethyl sulfoxide) in the presence of a suitable base (eg, cesium carbonate or potassium carbonate) at 0 ° C. to 80 ° C. (eg, 55 ° C.) for 10 minutes to 12 hours, preferably 20 minutes. After reacting for 60 minutes to 60 minutes, general formula (IX):

Embedded image Wherein R ¹¹ , R ⁴ , R ³¹ , A, B, and m have the same meanings as described above,
The quinazoline compound of the present invention represented by the general formula (I) can be obtained by removing various protecting groups as necessary. If necessary, the quinazoline compound of the present invention represented by the formula (I) can be mutually converted into another quinazoline compound of the present invention represented by the formula (I).

When A is O in the general formula (I), for example, the general formula (X):

Embedded image (Wherein R ¹² is a hydrogen atom or a hydroxyl protecting group, for example, a lower alkyl group or a lower alkoxy lower alkyl group;
R ⁵ is a hydrogen atom or an amino protecting group, for example, a lower alkylcarbonyl group or a lower alkoxycarbonyl group), and a compound represented by the general formula (XI):

Embedded image (Wherein B and m have the same meaning as described above), whereby the quinazoline compound of the present invention represented by the general formula (I) can be prepared.

That is, the compound represented by the general formula (X) and the compound represented by the general formula (XI) are mixed in a suitable solvent (eg, pyridine) in the presence of a suitable base (eg, pyridine). The reaction is performed for 1 hour to 10 hours, preferably 2 hours to 4 hours under the following general formula (XI)
I):

Embedded image (Wherein R ¹² , R ⁵ , B and m have the same meanings as described above), followed by removal of various protecting groups as necessary to obtain the compound represented by the general formula (I) )
The quinazoline compound of the present invention represented by the following formula can be obtained. If necessary, the quinazoline compound of the present invention represented by the formula (I) can be mutually converted into another quinazoline compound of the present invention represented by the formula (I).

The monoclonal antibody of the present invention reacts specifically with C9 base. The preferred monoclonal antibody of the present invention specifically reacts with C9 base, does not react with the tetrodotoxin compound represented by the general formula (III) (for example, TTX), and does not react with tetrodonic acid. In addition, it reacts with the quinazoline compound represented by the general formula (I). However, when R ¹ is an alkyl group, the reactivity may differ depending on the type of the alkyl group.

A hybridoma producing the monoclonal antibody of the present invention can be prepared by using an appropriate compound as a hapten and screening using a C9 base. Separation of the hybridoma producing the monoclonal antibody of the present invention and preparation of the monoclonal antibody can be carried out by a conventional method, for example, as described in the Seikagaku Laboratory Course (edited by the Biochemical Society of Japan) or the Immunobiochemical Research Method (edited by the Japanese Biochemical Society). It can be done according to the method.

As used herein, the term “hapten” refers to a substance which has the ability to bind to an antibody but has no immunogenicity by itself and which expresses immunogenicity when bound to a carrier. means. Therefore, haptens generally have a functional group for binding to a carrier. The hapten that can be used in the preparation of the hybridoma that produces the monoclonal antibody of the present invention is not particularly limited as long as it is a compound that can produce an antibody that specifically reacts with C9 base when immunized with mammals or birds. For example, the quinazoline compound of the present invention represented by the general formula (I), preferably the succinic acid (2-amino-8-methoxyquinazoline-6) represented by the formula (IV)
-Yl) methyl, (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid represented by the above formula (V), or 4- [N-
(2-amino-8-hydroxyquinazolin-6-yl)
Methyl] aminobenzoic acid, particularly preferably of the above formula (V)
(2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid represented by the following formula:

The term "carrier" as used herein means a substance that binds (conjugates) to a hapten and imparts immunogenicity to the hapten. In the present invention, known carriers generally used conventionally can be used. Accordingly, as the carrier, for example, a biopolymer carrier, for example, having a molecular weight of about 10,000 or more, preferably about 40,000 to about 100
Ten thousand proteins (eg, mammalian serum albumin, immunoglobulin, ovalbumin, polylysine, or keyhole limpet hemocyanin), or polysaccharides (eg, dextran, amylose, or amylopectin), or cells (eg, mammalian erythrocytes or BCG bacteria).

The functional group for binding the hapten to the carrier is not particularly limited as long as it is a functional group capable of binding to each of the above-mentioned carriers, and is appropriately selected according to the carrier to be used. be able to. Examples of a functional group capable of binding to an amino group of a protein include a carboxyl group, an amino group, a hydroxyl group, and a thiol group. Examples of the functional group capable of binding to a thiol group of a protein include, for example, a thiol group or an amino group, and examples of the functional group capable of binding to a hydroxyl group of a polysaccharide include, for example,
Thiol or aldehyde groups can be mentioned.

The binding between the hapten and the carrier can be carried out using a conventionally known method. For example, when using bovine serum albumin as a biopolymer carrier, for example, a mixed acid anhydride method, an active ester method, a glutaraldehyde method, a m-maleimidobenzoyl-N-hydrosuccinoimide method, or a carbodiimide method. A conjugate of bovine serum albumin and a hapten can be prepared by a coupling method.

The conjugate can be used as an immunogen to immunize mammals or birds (for example, rabbits, mice, or chickens) by a conventional method. For example, an immunogen solution is emulsified and mixed with an equal amount of Freund's complete adjuvant or incomplete adjuvant, and is inoculated (primary immunization) into mammals or birds (eg, mice or rabbits).
The same operation is performed at intervals of 2 to 4 weeks, and immunization is performed several times.
Several days after the final immunization, the spleen is aseptically removed from a mammal or bird and crushed with a stainless steel mesh or the like to prepare spleen cells, which are used in the cell fusion step.

Myeloma cells (myeloma cells), which are the other parent cells used for cell fusion, are various known cell lines, for example, p3.NS-1 / Ag4.1 [Eur.
J. Immunol., 5, 511-517 (1975)], SP2 / 0-Ag
14 [Nature, 276, 269-270 (1978)], or P3-X
63-Ag8.653 [J. Immunol., 123, 1548-1550]
(1979)].

Cell fusion can be carried out by a conventional method, for example, by spleen cells and myeloma cells in a medium containing a known fusion promoter [eg, commercially available polyethylene glycol for cell fusion (molecular weight: 1500; manufactured by Boehringer Mannheim)]. The myeloma cells can be mixed with the spleen cells of the mouse at a ratio of about 1/10 to 1/5 according to a conventional method. After fusion, only hybridomas are grown using a hybridoma selection medium (eg, HAT medium), and the presence or absence of antibody production in the culture supernatant can be confirmed by, for example, reactivity with C9 base. The hybridoma producing the desired antibody can be usually cloned (monoclonal antibody-producing cell) using a known cell cloning method, for example, a limiting dilution method. The cloned hybridoma can be subcultured in a known medium, and can be easily stored in liquid nitrogen for a long period of time, for example.

The monoclonal antibody of the present invention can be easily prepared by culturing a hybridoma producing the desired monoclonal antibody. As a medium for culturing a hybridoma, in the case of in vitro, for example, any medium suitable for culturing under 5% carbon dioxide and 37 ° C. conditions can be used. (Hereinafter referred to as FBS) (hereinafter referred to as Dulbecco / 10% FBS medium)
It is preferable to use In the case of in vivo, culture is preferably performed in the peritoneal cavity of a suitable mammal, for example, a mouse.

The culture supernatant or the ascites of a suitable mammal to which the hybridoma has been administered intraperitoneally in vitro can be directly used as an antibody solution. Also, the target antibody can be separated and purified using these antibody solutions as starting materials. For the separation and purification of antibodies, methods generally used for separation and purification of proteins can be used. Examples of such methods include, for example,
There are methods such as ammonium sulfate salting out, ion exchange chromatography, molecular sieve column chromatography using molecular sieve gel, affinity chromatography using protein A or protein G binding polymer, or dialysis.

In the present invention, an antibody fragment containing an antigen-binding site for C9 base, such as Fab, Fa
b ', F (ab') ₂ , Fv and the like. These fragments can be obtained, for example, by digesting the monoclonal antibody of the present invention with a proteolytic enzyme in a conventional manner, and then separating and purifying the protein in a conventional manner.

Since the method for immunologically measuring C9 base according to the present invention is carried out using the above-mentioned monoclonal antibody or antibody fragment, C9 base can be accurately measured. The method for immunologically measuring C9 base according to the present invention comprises:
Except for using a monoclonal antibody or antibody fragment that specifically reacts with the C9 base, otherwise conventionally known immunoassays, such as enzyme immunoassay, latex agglutination, or radioimmunoassay Etc. can be applied as it is. In the enzyme immunoassay, for example, an antigen labeling method, which is usually called an antigen labeling method, uses a reaction in which a hapten labeled with an enzyme competes with a C9 base in a sample for the immobilized antibody or antibody fragment, and the like. Can be applied. Here, the immunological assay method for C9 base according to the present invention will be described below in the case of an antigen labeling method among enzyme immunoassay methods.

The method for immunologically measuring C9 base according to the present invention includes, for example, (1) a step of immobilizing an antibody or an antibody fragment; (2) a step of mixing a test sample and a labeled hapten solution; (3) A) a step of contacting and reacting the immobilized antibody with the mixed solution; (4) a step of separating a labeled hapten bound to the immobilized antibody and a labeled hapten not bound; The method comprises a step of measuring a signal derived from a label of a labeled hapten bound to one or the other, preferably an immobilized antibody, separated in the step (4).

The test sample which can be measured by the method for immunologically measuring C9 base according to the present invention is not particularly limited. For example, a test sample containing C9 base or C9 base can be used.
A test sample that may contain a base can be used. Further, as the test sample, a sample containing or possibly containing a tetrodotoxin compound is subjected to, for example, a known alkaline hydrolysis treatment [Toshio Goto et al., Bull.
l. Chem. Soc. Japan, vol. 35, 1045-1046 (1962)]
Alternatively, a processing solution obtained by performing the method can be used. In the method for immunologically measuring C9 bases according to the present invention, C9 bases can be confirmed or quantified using a hapten labeled with an enzyme label or another labeling substance. As the hapten, for example, a quinazoline compound represented by the formula (I) can be used. In the case of the enzyme immunoassay, a known labeling enzyme,
For example, peroxidase or alkaline phosphatase can be used. Instead of enzymes, for example, radioisotopes (eg, ³² P, ³⁵ S, or ³ H), vitamins (eg, biotin), fluorescent substances (eg, fluorescein isothiocyanate), or chemiluminescent substances (eg, Acridinium) can also be used.

In the method for immunologically measuring C9 base according to the present invention, after the reaction between the immobilized antibody and the labeled hapten is completed, the labeled hapten bound to the immobilized antibody and the immobilized antibody are Separate unbound labeled hapten. Separation can be performed, for example, by filtration, centrifugation, or washing with a buffer. After separation, a signal from any label, preferably a label bound to the immobilized antibody, is measured. When measuring a signal, it is preferable to change the reaction system to conditions favorable for signal measurement. For example, when the signal is fluorescence or light emission, the signal is detected under the condition that quenching does not occur. The amount of C9 base in the test sample can be determined from the above-mentioned signal measurement values, for example, by using a calibration curve prepared in advance. In the method for immunologically measuring C9 base according to the present invention, an appropriate control solution (for example, a solution of synthetic C9 base) can be used as a control.

The method for immunologically measuring a tetrodotoxin compound according to the present invention comprises the following steps: (1) a step of alkali-hydrolyzing a test sample and then neutralizing the same (hereinafter referred to as a decomposition step);
The step of measuring the amount of C9 bases in the treatment solution obtained in the step (1) (C9 base measurement step), and the step (2) obtained in the step (2), by the method for immunological measurement of the C9 bases described above. A step of determining the total amount of the tetrodotoxin compound from the measured values (hereinafter, referred to as a determining step). The test sample that can be measured by the method for immunologically measuring a tetrodotoxin compound according to the present invention is not particularly limited. A sample, for example, a seafood (for example, a puffer fish, a goby, an octopus, or a snail) or a processed seafood, or an extract thereof can be used.

In the degradation step (1) in the method for immunologically measuring a tetrodotoxin compound of the present invention, for example, 5%
After alkali hydrolysis by heating in potassium hydroxide at 90 ° C. to 100 ° C. for 30 minutes, the reaction solution is neutralized by adding an appropriate amount of acid to obtain a treatment solution. Here, neutralization means that the pH is adjusted to about 6.5 to 7.5. As the acid, an organic acid such as acetic acid, citric acid, or succinic acid, or an inorganic acid such as phosphoric acid, hydrochloric acid, or sulfuric acid can be used. When the tetrodotoxin compound is present in the test sample, the tetrodotoxin compound is decomposed into C9 base by alkaline hydrolysis. At this time, irrespective of the type of the tetrodotoxin compound, 1 mol of the C9 base is generated from 1 mol of the tetrodotoxin compound. To be generated.

In the step (2) of measuring the C9 base in the method for immunologically measuring a tetrodotoxin compound of the present invention, the same procedure as in the method for immunologically measuring a C9 base of the present invention is carried out, whereby the decomposition step is performed. The amount of C9 base in the treatment solution obtained in (1) can be measured.

As described in the decomposition step (1), the C9 base contained in the treatment solution is derived from an equimolar amount of the tetrodotoxin compound. Step (3)
Then, the amount of the tetrodotoxin compound initially contained in the test sample to be measured can be easily determined from the measurement value obtained in the C9 base measurement step (2). In the method for immunologically measuring a tetrodotoxin compound according to the present invention, a suitable control solution (for example, a synthetic C9 solution)
Can be used as a control.

[0046]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Preparation Example 1: Preparation of 3,5 -dimethylanisole 3,5-xylenol (50 g; 0.409 mol) was added to ethanol / dimethylformamide (6: 1) (500
ml), and potassium carbonate (63 g; 0.450 m
ol) aqueous solution at room temperature, and further methyl iodide (56 m
1; 0.899 mol), and the mixture was heated and refluxed overnight. Ethanol was distilled off, water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline and dried over magnesium sulfate. The title compound (40.5 g; yield = 73%) was obtained as a pale yellow liquid by distillation under reduced pressure (20 mmHg, 89 ° C.).

Preparation Example 2: Preparation of 5-methoxyisophthalic acid
Ltd. of potassium permanganate (188 g; 1.19 mol) was dissolved in water (1500 ml), at reflux, resulting in Preparation Example 1 3,5-dimethyl anisole (40.
5g; 0.298 mol) was added dropwise and refluxed for 4 hours. The reaction solution was filtered through celite, the pH of the filtrate was adjusted to 1 by adding concentrated hydrochloric acid under ice-cooling, and the precipitated crystals were collected by filtration and dried to give the title compound (31.0 g; yield = 54).
%) As white crystals.

Preparation Example 3: Dimethyl 5-methoxyisophthalate
Preparation of chill at −20 ° C. thionyl chloride (34.5 ml; 0.4
74 mol) was dropped into methanol (250 ml), and then 5-methoxyisophthalic acid (31.0 g; 0.158 mol) obtained in Preparation Example 2 was added. The mixture was heated to room temperature and stirred for 1.5 hours. The temperature was further raised and refluxed for 1.5 hours. The methanol was distilled off, ethyl acetate was added to dissolve the crystals, and a cooled saturated aqueous sodium hydrogen carbonate solution, water,
And saturated brine, and dried over magnesium sulfate. The solvent was distilled off, and the obtained crude product was recrystallized from ethyl acetate to give the title compound (18.6 g; yield =
54%) as white crystals.

Preparation Example 4: 5-methoxy-4-nitroiso
Preparation of dimethyl phthalate The dimethyl 5-methoxyisophthalate (5.53 g; 0.0247 mol) obtained in Preparation Example 3 was concentrated in concentrated sulfuric acid (4.
0 ml) and fuming nitric acid (1.03 ml; 0.02 ml).
47 mol) in concentrated sulfuric acid (10 ml) was added at -20 ° C, and the mixture was stirred for 30 minutes. Ice was added, the mixture was extracted with ethyl acetate, washed with a cold saturated aqueous sodium hydrogen carbonate solution, water, and saturated saline, and dried over magnesium sulfate. The solvent was distilled off to obtain a crude product. The title compound was obtained as white crystals by recrystallization from acetone. Melting point: 136 to 137 ° C (acetone) IR (CHCl ₃ ) cm ^-1 : 3100, 3050 (aromatic CH), 1740 (C = O), 1550 (N =
O), 1340 (N = O) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 3.9
3 (3H, s), 3.99 (6H, s), 7.93 (1
H, d, J = 1.2 Hz), 8.27 (1H, d, J =
1.2 Hz) MS m / z: 269 (M ⁺ 100%), 238 (M
⁺ -OMe 42.5%), 207 (M ⁺ -OMe-O
_{Me 45.3%) C 11 H 11} NO 7 Calculated by: C, 49.07; H, 4.12 ; N, 5.20 Found: C, 49.05; H, 4.20 ; N, 5.22

Preparation Example 5 4-Amino-5-methoxyiso
Preparation of dimethyl phthalate The crude dimethyl 5-methoxy-4-nitroisophthalate obtained in Preparation Example 4 above (5 g; 0.0186 mol)
Was dissolved in ethanol (80 ml), and platinum oxide (51.
9 mg; 0.229 × 10 ⁻³ mol) and subjected to catalytic reduction at 4 atm for 12 hours. The reaction solution was filtered through celite, the solvent of the filtrate was distilled off, and the obtained crude product was subjected to silica gel column chromatography (hexane: ethyl acetate = 5: 1).
To give the title compound [3.05 g; yield = 52% (from the product of Preparation Example 3)]. Melting point: 137 to 138 ° C (ethanol) IR (CHCl ₃ ) cm ^-1 : 3520, 3390 (N-
H), 3050, 3010 (aromatic CH), 1690
(C = O) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 3.8
8 (3H, s), 3.89 (3H, s), 3.92 (3
H, s), 7.45 (1H, d, J = 1.8 Hz),
8.26 (1H, d, J = 1.8 Hz) MS m / z: 239 (M ⁺ 100%), 224 (M
⁺ -NH ₂ 15.8%), 179 (M ⁺ -CO ₂ Me)
_{17.9%) C 11 H 13 NO} 5 Calculated by: C, 54.94; H, 5.46 ; N, 5.83 Found: C, 55.23; H, 5.48 ; N, 5 .86

Preparation Example 6: 2-amino-3,5-bis (H
Preparation of (Droxymethyl) anisole Dimethyl 4-amino-5-methoxyisophthalate obtained in Preparation Example 5 (9.28 g; 0.0388 mol)
Of tetrahydrofuran (60 ml) was added to lithium aluminum hydride (2.21 g; 0.0582 m
ol) in a solution of tetrahydrofuran (100 ml) at 0 ° C.
And stirred at room temperature for 1 hour. A 3N aqueous solution of sodium hydroxide (12 ml) and water (12 ml) were added to the reaction solution.
Was added under ice cooling, and the mixture was filtered through celite, and the solvent in the filtrate was distilled off. The obtained crude product was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 2) to give the title compound (4.53 g; yield = 63%). Melting point: 90-91 ° C (ethyl acetate) IR (CHCl ₃ ) cm ^-1 : 3610,3440,33
80 (N-H, O- H), 2990,2940 ( aromatic ^{C-H) 1 H-NMR} (200MHz, CDCl 3) δ: 3.8
7 (3H, s), 4.55 (2H, s), 4.66 (2
H, s), 6.71 (1H, d, J = 1.8 Hz),
6.81 (1H, d, J = 1.8 Hz) MS m / z: 183 (M ⁺ 100%), 164 (M
⁺ -OH-2H 17.3%), 136 (M ⁺ -OMe
-NH ₂ 22.1%) Calculated by _{C 9 H 13 NO 3: C} , 59.00; H, 7.15; N, 7.65 Found: C, 58.93; H, 7.07 ; N, 7.50

Preparation 7: 1,2-dihydro-6-hydro
Xymethyl-8-methoxy-4H-3,1-benzoxy
Obtained in Preparation Preparation Example 6 of spoon-2-one 2-amino-3,5-bis (hydroxymethyl) anisole (413.5mg; 2.2
Potassium carbonate (472.4 mg; 3.42 mmol) was added to a solution of 6 mmol) in methanol (10 ml) at 0 ° C., and methyl chloroformate (0.21 ml; 2.71 mm) was added.
ol) at room temperature and stirred for 2.5 hours. Further, methyl chloroformate (0.11 ml; 1.36 mmol) was added, and the mixture was stirred for 30 minutes. Methanol was distilled off, water was added, and the mixture was extracted with ethyl acetate / 2-butanol (3: 1). The organic layer was washed with a saturated saline solution and dried over sodium sulfate. The crude product obtained by evaporating the solvent was recrystallized from ethyl acetate to give the title compound (314.9 m
g; yield = 67%) as white crystals. Melting point: 178-179 ° C (ethyl acetate) MS m / z: 209 (M ⁺ 91.1%), 164
(M ⁺ -C (= O) -OH 28.5%), 136
^{(M + -NH-C (=} O) -O-CH 2 25.6%) Calculated by _{C 10 H 11 NO 4: C} , 57.41; H, 5.30; N, 6.70 Found : C, 57.30; H, 5.26; N, 6.43

Preparation Example 8: 1,2-Dihydro-8-methoxy
C-6-methoxymethoxymethyl-4H-3,1-ben
Preparation of duoxazin-2-one 1,2-dihydro-6-hydroxymethyl-8-methoxy-4H-3,1-benzoxazin-2-one (314.9 mg; 1.51 mmol) obtained in Preparation Example 7 above
To a solution of 1) in tetrahydrofuran (3 ml) / dimethylformamide (3 ml), diisopropylethylamine (0.42 ml; 2.41 mmol) was added at 0 ° C, and chloromethyl methyl ether (0.15 ml; 1.97 m) was added.
mol), and the mixture was stirred at room temperature overnight. A saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline and dried over sodium sulfate. The solvent was removed, and the obtained crude product was purified by silica gel column chromatography (hexane: ethyl acetate = 2: 1) to give the title compound (334 mg; yield = 88).
%). Melting point: 96-97 ° C (ethyl acetate) IR (CHCl ₃ ) cm ^-1 : 3425 (N-H), 29
50 (aromatic CH), 1725 (C = O), 1045
(COC) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 3.4
2 (3H, s), 3.90 (3H, s), 4.53 (2
H, s), 4.70 (2H, s), 5.29 (2H,
s), 6.72 (1H, s), 6.85 (1H, s),
7.43 (1H, s) MS m / z: 253 (M + 100%), 209 (M + -CH 3 OCH 2 7.4%), 192
(M ⁺ -MeOMe-CH ₃ -H 26.2%) , 14
8 (M ⁺ -MeOMe-CH ₃ -CO ₂ 27.4%) Calculated with C ₁₂ H ₁₅ NO ₅ : C, 56.91; H, 5.97; N, 5.53 Found: C, 56 .71; H, 5.92; N, 5.41

Preparation 9: 2-amino-3-hydroxymeth
Preparation of tyl-5-methoxymethoxymethylanisole 1,2-dihydro-8-methoxy-6-methoxymethoxymethyl-4H-3,1-benzoxazin-2-one (234.6 mg) obtained in Preparation Example 8 above ; 27
mmol) in methanol (2 ml) / water (1 ml).
(1.854 mmol) at 0 ° C. and stirred at room temperature for 2 hours. Methanol was distilled off, saturated saline was added, extracted with ethyl acetate, and dried over sodium sulfate. The solvent was distilled off, and the obtained crude product was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give the title compound (162.2 mg; yield = 77).
%). Melting point: 45-46 ° C (ethyl acetate) IR (CHCl ₃ ) cm ^-1 : 3600, 3460, 34
00 (OH, NH), 3000, 2950, 290
0 (aromatic C-H), 1045 (C -O-C) 1 H-NMR (200MHz, CDCl 3) δ: 3.3
9 (3H, s), 3.85 (3H, s), 4.46 (2
H, s), 4.61 (2H, s), 4.65 (2H,
s), 6.70 (1H, s), 6.78 (1H, s) MS m / z: 227 (M ⁺ 82.7%), 165
(M ⁺ -OMeOMe-H 100%), 148 (M ⁺
-OMeOMe-CH ₃ -2H 47.7%) Calculated by _{C 11 H 17 NO 4: C} , 58.13; H, 7.54; N, 6.16 Found: C, 58.12; H, 7.47; N, 6.27

Preparation 10: 3-hydroxymethyl-2-
(N-methoxycarbonyl) amino-5-methoxymethoate
Carboxymethyl obtained in anisole Preparation Preparation Example 9 2-Amino-3-hydroxymethyl-5-methoxymethoxy-methyl anisole (41.
6 mg; 0.183 mmol) of dichloromethane (2 m
l) To the solution was added sodium bicarbonate (18.5 mg;
220 mmol) at 0 ° C., and methyl chloroformate (0.0142 ml; 0.183 mmol) was added, followed by stirring at room temperature for 30 minutes. Further, methyl chloroformate (0.
0142 ml; 0.183 mmol) at 0 ° C. and stirred at room temperature for 30 minutes. The solvent was distilled off, water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated saline solution and dried over sodium sulfate. The solvent was distilled off to obtain a crude product. The obtained crude product was purified, and showed the following physicochemical properties. Melting point: 93-94 ° C (ethyl acetate) IR (CHCl ₃ ) cm ^-1 : 3425 (O-H), 29
60 (aromatic CH), 1725 (C = O), 1050
(COC) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 3.4
1 (3H, s), 3.77 (3H, s), 3.85 (3
H, s), 4.54 (2H, s), 4.57 (2H,
s), 4.71 (2H, s), 6.50 (1H, s),
6.88 (1H, d, J = 1.6 Hz), 7.08 (1
H, d, J = 1.6 Hz) MS m / z: 285 (M ⁺ 19.7%), 223
^{_{(M + -OCH 3 -OCH 3 60.6}} %), 149
(M ⁺ -CH ₂ OMeOMeOMe-CH ₂ OH 10
0%) Calculated by _{C 12 H 19 NO 6: C} , 54.73; H, 6.71; N, 4.91 Found: C, 54.63; H, 6.63 ; N, 4.83

Preparation Example 11: 3-methoxy-2- (N-meth
Toxicarbonyl) amino-5-methoxymethoxymethyl
Preparation of Rubenzaldehyde Oxalyl chloride (0.0199 ml; 0.223
mmol) in dichloromethane (1 ml) was added to dimethyl sulfoxide (0.0324 ml; 0.458 mmol).
l) in dichloromethane (1 ml) was added at −65 ° C., and the mixture was stirred for 2 minutes, and then crude 3-hydroxymethyl-2- (N-methoxycarbonyl) amino-5-methoxymethoxy obtained in Preparation Example 10 above A solution of methylanisole (59.2 mg; 0.208 mmol) in dichloromethane (3 ml) was added and stirred for 15 minutes. Furthermore, triethylamine (0.145 ml; 1.04 mmol)
After stirring for 5 minutes, the temperature was raised, and the mixture was stirred at room temperature for 20 minutes. Ice water was added, extracted with dichloromethane, the organic layer was washed with saturated saline, dried over sodium sulfate, and the solvent was distilled off to obtain a crude product. The obtained crude product was purified, and showed the following physicochemical properties. Melting point: 81-82 ° C (ether) IR (CHCl ₃ ) cm ^-1 : 3420 (N-H), 29
40 (aromatic CH), 1740 (ester C = O),
1700 (aldehyde C = O), 1055 (CO-
C) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 3.4
2 (3H, s), 3.79 (3H, s), 3.92 (3
H, s), 4.60 (2H, s), 4.72 (2H,
s), 7.15 (2H, d, J = 1.8 Hz and
1H, br s), 7.44 (1H, d, J = 1.8H)
z), 10.03 (1H, s) MS m / z: 283 (M ⁺ 100%), 222 (M
^{_{+ -OCH 2 OCH 3 30.7%)}} , 194 (M + -
_{NHC (= O) OCH 3 -CH} 3 59.8%) Calculated by _{C 13 H 17 NO 6: C} , 55.12; H, 6.05; N, 4.95 Found: C, 54.94 H, 6.08; N, 4.97

Preparation Example 12: 2-amino-3-methoxy-
Preparation of 5-methoxymethoxymethylbenzaldehyde The crude 3-methoxy-2- obtained in Preparation Example 11 above.
(N-methoxycarbonyl) amino-5-methoxymethoxymethylbenzaldehyde (49.5 mg; 0.17
5 mmol) in methanol (2 ml) / water (1 ml).
l; 0.264 mmol) at room temperature and the temperature is raised to 10
Reflux for minutes. The methanol was distilled off, an aqueous ammonium chloride solution was added, and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated saline solution and dried over sodium sulfate. The solvent was distilled off, and the obtained crude product was purified by silica gel column chromatography (hexane: ethyl sulfate = 4: 1) to give the title compound (31.2 mg; yield = 76%)
(From the product of Preparation 9)] as a yellow oil. IR (CHCl ₃ ) cm ⁻¹ : 3510, 3470 (N−
H), 2950 (aromatic CH), 1675 (C = O) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 3.4
2 (3H, s), 3.90 (3H, s), 4.52 (2
H, s), 4.70 (2H, s), 6.43 (2H,
s), 6.90 (1H, d, J = 1.6 Hz), 7.1
1 (1H, d, J = 1.6 Hz), 9.88 (1H,
s) MS m / z: 225 (M ⁺ 100%), 164 (M
⁺ -OMeOMe 100%), 149 (M ⁺ -OMe
_{OMe-CH 3 10.0%),} 136 (M + -OMe
_{OMe-CHO 18.6%) C 11} H 15 NO 4 Calculated by: C, 58.65; H, 6.71 ; N, 6.22 Found: C, 58.35; H, 6.59 ; N, 6.02

Preparation Example 13 2-amino-8-methoxy-
Preparation of 6-methoxymethoxymethylquinazoline To 2-ethoxyethanol (10 ml) was added sodium hydride (144 mg; 3.6 mmol) at 0 ° C., and guanidine hydrochloride (368.4 mg; 3.86 mmol) was added.
After l) was added and the mixture was stirred at room temperature for 30 minutes, the reaction solution was filtered through celite. The obtained filtrate was combined with the 2-amino-3-methoxy-5-methoxymethoxymethylbenzaldehyde obtained in Preparation Example 12 (575.6 mg; 2.5
6 mmol), and the solvent was distilled off.
Heated at 0 ° C. for 1 hour. The obtained crude product was purified by silica gel column chromatography (ethyl acetate),
The title compound (287 mg; yield = 45%) was obtained as yellow crystals. Melting point: 119-120 ° C (ethyl acetate) IR (CHCl ₃ ) cm ^-1 : 3481, 3304 (N-
H), 2949 (aromatic CH), 1423, 1573
(C = C, C = N) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 3.4
5 (3H, s), 4.04 (3H, s), 4.67 (2
H, s), 4.75 (2H, s), 5.53 (2H,
s), 7.09 (1H, s), 7.28 (1H, s),
8.98 (1H, s) MS m / z: 249 (M ⁺ 100%), 188 (M
⁺ -OMeOMe 25.0%), 187 (M ⁺ -OM
eOMe-H 71.6%) Calculated by _{C 12 H 15 N 3 O 3} : C, 57.82; H, 6.07; N, 16.86 Found: C, 57.75; H, 6 . 10; N, 16.68

Preparation Example 14 2- (Nt-butylcarbo)
Nyl) amino-8-methoxy-6-methoxymethoxy
Preparation of tilquinazoline 2-amino-8-methoxy- obtained in Preparation Example 13 above
6-methoxymethoxymethylquinazoline (1.75 g;
7.03 mmol) of 1,4-dioxane (20 ml)
To the solution was added triethylamine (2.3 ml; 16.2 mm
ol) and pivaloyl chloride (2.2 ml; 17.
6 mmol) at 100 ° C. and stirred at 80 ° C. for 1 hour. After completion of the reaction, ethyl acetate was added, and the mixture was washed with a saturated aqueous solution of sodium hydrogencarbonate, water, and saturated saline, and dried over magnesium sulfate. The solvent was distilled off, and the obtained crude product was purified by silica gel column chromatography (ethyl acetate) to give the title compound (2.11 g; 6.3).
(4 mmol; yield = 90%) as yellow crystals. Melting point: 133-134 ° C (ethyl acetate / ether) IR (CHCl ₃ ) cm ^-1 : 3446 (N-H), 29
60 (aromatic CH), 1704 (C = O), 158
1, 1450 (C = C, C = N), 1049 (C-O-
C) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 1.3
7 (9H, s), 3.45 (3H, s), 4.08 (3
H, s), 4.73 (2H, s), 4.77 (2H,
s), 7.20 (1H, s), 7.44 (1H, s),
8.37 (1H, s), 9.31 (1H, s) MS m / z: 333 (M ⁺ 90.7%), 332
(M ⁺ -H 100%), 271 (M ⁺ -OMeOMe
-H 84.5%) Calculated by _{C 17 H 23 N 3 O 4} : C, 61.24; H, 6.95; N, 12.61 Found: C, 60.72; H, 6.91 N, 12.68

Preparation Example 15: 6-bromomethyl-2- (N
-T-butylcarbonyl) amino-8-hydroxyquina
Preparation of zoline 2- (Nt-butylcarbonyl) amino-8-methoxy-6-methoxymethoxymethylquinazoline obtained in Preparation Example 14 above (18.7 mg; 0.056 mmol)
To a dichloromethane (2 ml) solution was added boron tribromide (0.021 ml; 0.225 mmol) at 0 ° C., and the mixture was stirred at room temperature for 2.5 hours. The solvent is distilled off and the pH is 5
And add saturated aqueous sodium bicarbonate to pH
The mixture was added to 4 to 5 and extracted with ethyl acetate. The organic layer was washed with a saturated saline solution and dried over sodium sulfate. The solvent was distilled off to obtain a crude product. The obtained crude product was purified, and showed the following physicochemical properties. ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 1.3
9 (9H, s), 4.59 (2H, s), 7.38 (1
H, s), 7.39 (1H, s), 8.46 (1H,
s), 9.19 (1H, s) MS m / z: 339 [M ⁺ (Br = 81) 31.7].
%], 337 [M ⁺ (Br = 79) 21.2%], 2
58 (M ⁺ -Br 41.8%), 173 (M ⁺ -Br
-C (= O) ^-t Bu 52.8%)

Preparation Example 16: 6-bromomethyl-2- (N
-T-butylcarbonyl) amino-8-methoxymethoxy
Preparation of shiquinazoline 6-bromomethyl-2- (N
To a solution of -t-butylcarbonyl) amino-8-hydroxyquinazoline (126 mg; 0.373 mmol) in tetrahydrofuran (5 ml) was added diisopropylethylamine (0.65 ml; 3.73 mmol) at 0 ° C, and chloromethyl methyl ether (0%) was added. .34 ml;
47 mmol) and stirred at room temperature for 40 minutes. A saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over magnesium sulfate. The crude product obtained by evaporating the solvent was subjected to silica gel column chromatography (hexane: ethyl acetate =
1: 1) to give the title compound (82.4 mg;
0.216 mmol; yield = 58%). Melting point: 107 ° C. (ether / ethyl acetate) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 1.3
7 (9H, s), 3.59 (3H, s), 4.70 (2
H, s), 5.51 (2H, s), 7.54 (1H,
s), 7.57 (1H, s), 8.35 (1H, s),
9.30 (1H, s) MS m / z: 382 (M ⁺⁺ 1 8.4%), 381
[M ⁺ (Br = 79) 8.8%], 321 (M ⁺ -O
MeOMe 100%), 302 (M ⁺ -Br 13
%)

Preparation Example 17: [2- (Nt-butyl carb)
Bonyl) amino-8-methoxymethoxyquinazoline-6
Preparation of Ethyl-Methylthioacetate Preparation Example 16 was added to a solution of cesium carbonate (21 mg; 0.0653 mmol) in dimethyl sulfoxide (1 ml).
A solution of 6-bromomethyl-2- (Nt-butylcarbonyl) amino-8-methoxymethoxyquinazoline (20.8 mg; 0.0544 mmol) obtained in 1 above in dimethyl sulfoxide (1 ml) and ethyl mercaptoacetate (7 μl; 06553 mmol) with 55
C. and stirred for 30 minutes. The solvent was distilled off, ethyl acetate was added, and the mixture was filtered through celite. The solvent of the filtrate was distilled off, and the obtained crude product was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give the title compound (15.0 mg; yield = 65%). Obtained. Melting point: 88-89 ° C (ether / hexane) IR (CHCl ₃ ) cm ^-1 : 3450 (N-H), 30
00 (aromatic CH), 1730 (C = O) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 1.2
8 (3H, t, J = 7 Hz), 1.36 (9H, s),
3.06 (2H, s), 3.57 (3H, s), 3.9
5 (2H, s), 4.17 (2H, q, J = 7 Hz),
5.50 (2H, s), 7.45 (1H, d, J = 1.
8Hz), 7.59 (1H, d, J = 1.8Hz),
8.34 (1H, s), 9.28 (1H, s) MS m / z: 421 (M ⁺ 18.7%), 409
(M ⁺ -CH ₃ 100%), 243 (M ⁺ -CH _{2-
SCH 2 C (= O) OEt} 51.1%) C 20 H 27 N 3 O 5 by S Calculated: C, 57.00; H, 6.46 ; N, 9.97; S, 7.59 Found Values: C, 56.71; H, 6.42; N, 9.85; S, 7.62.

Example 1: (2-amino-8-hydroxy
Preparation of quinazolin-6-yl) methylthioacetic acid [2- (Nt-butylcarbonyl) amino-8-methoxymethoxyquinazoline-6 obtained in Preparation Example 17 above.
-Yl] ethyl methylthioacetate (239 mg; 0.56
8 mmol) in methanol (6 ml).
73 ml) and 1N potassium hydroxide aqueous solution (2.27)
ml; 2.27 mmol) and stirred at room temperature for 3 hours. Under ice cooling, 3N hydrochloric acid (6 ml) was added, and the mixture was heated under reflux for 2 hours. After completion of the reaction, the solvent was distilled off, and the obtained crude yellow crystals were recrystallized from isopropanol to obtain crystals of an acid salt. This was recrystallized from pH 5 buffer to give the title compound,
That is, the compound of the present invention represented by the above formula (V) (70 mg; yield = 47%) was obtained as yellow crystals. Melting point: 126 to 127 ° C. (pH 5 buffer) IR (CHCl ₃ ) cm ⁻¹ : 3350 (N—H, O—
H), 3150 (carboxylic acid OH), 3050, 17
13 (C = O), 1674 (C = N) ¹ H-NMR (500 MHz, DMSO: CD ₃ OD =
10: 1) δ: 3.11 (2H, s), 3.82 (2
H, s), 7.05 (1H, s), 7.14 (1H,
s), 8.99 (1H, s) MS m / z: 265 (M ⁺ 38.6%), 174.
(M ⁺ -SCH ₂ CO ₂ H 100%)

Example 2: 4- [N- (2-amino-8-
Hydroxyquinazolin-6-yl) methyl] aminobenzo
Preparation of perfumed acid (1) 4- [N- [2- (Nt-butylcarbonyl)]
Amino-8-methoxymethoxyquinazolin-6-yl]
Preparation of ethyl [methyl] aminobenzoate 6-bromomethyl-2- (N
To a solution of -t-butylcarbonyl) amino-8-methoxymethoxyquinazoline (39.3 mg; 0.103 mmol) in dimethyl sulfoxide (1000 ml), potassium carbonate (28.5 mg: 0.206 mmol) and p-
Ethyl aminobenzoate (34 mg; 0.206 mmol)
l) was added and the mixture was stirred at room temperature for 30 minutes. Evaporate the solvent,
Ethyl acetate was added, and the mixture was filtered through Celite. The solvent of the filtrate was distilled off, and the obtained crude product was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 2) to give 4- [N- [2- (N-t-butyl). Carbonyl) amino-8-methoxymethoxyquinazoline-6
Yl] methyl] ethyl aminobenzoate (31.1 mg;
0.0690 mmol; yield = 67%). Melting point: 140-145 ° C (ether / ethyl acetate) IR (CHCl ₃ ) cm ^-1 : 3400 (N-H), 30
00 (aromatic CH), 1710 (C = O), 1013
(COC), 1642, 1592, 1540, 15
00 (C = C, C = N) ¹ H-NMR (200 MHz, CDCl ₃ ) δ: 1.3
5 (3H, t, J = 7.2 Hz), 1.36 (9H,
s), 3.57 (3H, s), 4.30 (2H, q, J
= 7.2 Hz), 5.21 (2H, d, J = 4.8H)
z), 4.96 (1H, t, J = 4.8 Hz), 5.4
7 (2H, s), 6.61 (2H, d, J = 8.8H)
z), 7.41 (1H, d, J = 1.6 Hz), 7.5
0 (1H, d, J = 1.6 Hz), 7.86 (2H,
d, J = 8.8 Hz), 8.37 (1H, s), 9.1
7 (1H, s) MS m / z: 466 (M ⁺ 27.4%), 465.
(M ⁺ -H 83.7%), 452 (M ⁺ -CH ₃ 2
8.1%), 451 (M ⁺ -CH ₃ -H 100%),
^{406 (M + -OMeOMe 25.5%)} C 25 H 30 N 4 O 5 in accordance Calculated: C, 64.36; H, 6.48 ; N, 12.01 Found: C, 64.06; H , 6.54; N, 11.84.

(2) Preparation of 4- [N- (2-amino-8-hydroxyquinazolin-6-yl) methyl] aminobenzoic acid 4- [N- [2- (N -T-butylcarbonyl) amino-8-methoxymethoxyquinazolin-6-yl] methyl] ethylaminobenzoate (46.
6 mg; 0.0998 mmol) in a mixture of tetrahydrofuran / water and a 1 N aqueous potassium hydroxide solution (1.20 mmol).
0 mmol) at 0 ° C. and refluxed for 1 hour. After completion of the reaction, the solvent was distilled off, 3N hydrochloric acid (5 ml) was added at 0 ° C., and the mixture was stirred at room temperature for 2 hours. The solvent was removed, and the obtained crude yellow crystals were recrystallized from isopropanol to obtain hydrochloride crystals. This was recrystallized from a pH 5 buffer to give 4-
[N- (2-amino-8-hydroxyquinazoline-6-
Yl) methyl] aminobenzoic acid, ie, of the formula (V
The compound of the present invention represented by I) (21 mg; yield = 6)
8%) as dark green crystals. IR (CHCl ₃ ) cm ⁻¹ : 3419, 3047, 3336 (N—H, O—H), 2
972 (aromatic CH), 1674 (C = O), 160
5 (C = C) ¹ H-NMR (500 MHz, DMSO: CD ₃ OD =
10: 1) δ: 4.34 (2H, s), 6.63 (2
H, d, J = 10 Hz), 7.07 (1H, s), 7.
21 (1H, s), 7.65 (2H, d, J = 10H
z), 9.01 (1H, s) MS m / z: 310 (M ⁺ 4.6%), 266 (M
⁺ -CO ₂ H 29.5%), 174 (M ⁺ -NH-P
h-CO ₂ H 92.0%), 137 (M ⁺ -CH ₂ N
_{H-Ph-CO 2 H,} -OH, -NH 2 100%)

Example 3 Succinic acid (2-amino-8-methyl
Preparation of Toxiquinazolin-6-yl) methyl 2-amino-8-methoxy- obtained in Preparation Example 13 above
6-methoxymethoxymethylquinazoline (205 mg;
0.823 mmol) in methanol (1 ml).
3N hydrochloric acid (2 ml) was added and reacted at room temperature for 24 hours. The reaction solution was distilled off under reduced pressure, adjusted to pH 7.0 with a saturated aqueous solution of sodium hydrogen carbonate, extracted with an ethyl acetate / 2-butanol solution (3: 1), and dried. The solvent was distilled off, and the obtained yellow crystals were recrystallized from acetone to give 2-amino-6-hydroxymethyl-8-methoxyquinazoline (143 mg; yield = 85%). The obtained 2-amino-6-hydroxymethyl-8-methoxyquinazoline (101 mg; 0.494 mmol) in pyridine (3 m
l) Add succinic anhydride (49 mg; 0.49 mmol) to the solution
l) was added at room temperature and stirred for 3 hours. After completion of the reaction, pyridine was distilled off under reduced pressure, and the obtained residue was recrystallized from dimethylsulfoxide / methanol to give the title compound, that is, the compound of the present invention represented by the above formula (IV) (105
mg; yield = 69%). Melting point: 210-215 ° C (DMSO / methanol) ¹ H-NMR (DMSO-d6): 2.56 (4H,
m), 3.89 (3H, s), 5.15 (2H, s),
6.93 (2H, brs), 7.10 (1H, s),
7.32 (1H, s), 9.03 (1H, s) ^13C -NMR (DMSO-d6): 28.9 (x
2), 55.4, 65.5, 112.0, 118.0,
119.5, 129.5, 143.8, 152.6, 1
60.7, 161.9, 172.1, 173.5 IR (Kbr): 3361, 3313 (OH, NH),
3184,2941,1726 (C = O), 1655,
1630, 1599, 1227, 1171 MS: 305 (M +, 47), 276 (14), 205
(100), 197 (69), 177 (58)

Example 4 Preparation of Monoclonal Antibody (1) Binding of (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic Acid to Carrier Protein by Active Ester Method (2) -Amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid 3.5 μmo
1 is dimethyl sulfoxide (hereinafter referred to as DMSO)
Dissolved in 50 μl. To this solution was added DMSO (10 μl).
1) dissolved in N-hydroxysuccinimide (4 μm
ol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (4 μmol) dissolved in DMSO (20 μl) was further added. After reacting at room temperature for 90 minutes, a borate buffer (85 mM boric acid, 60 mM NaCl, pH 8.0) was added to the reaction solution.
Bovine serum albumin (hereinafter BSA) dissolved in 500 μl
Referred to as) or Kiho Lumpur limpet hemocyanin (hereinafter referred to as KLH) 10 mg was further added and reacted again at room temperature for 90 minutes. After completion of the reaction, a Dulbecco's phosphate buffer [0.2 mg / ml-KCl, 0.2 mg / m
_{l-KH 2 PO 4, 8mg} / ml-NaCl, 1.15
mg / ml-Na ₂ HPO ₄ ; hereinafter, referred to as PBS (−)], and a conjugate of (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid and KLH, and (2 -Amino-8-hydroxyquinazoline-
Conjugates of 6-yl) methylthioacetic acid and BSA were each prepared.

(2) Immunization of mice with a conjugate of (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid and KLH Balb / c mice were immunized with (2-amino-8-hydroxy Quinazolin-6-yl) methylthioacetic acid and KL
This was carried out by dissolving 100 μg of the conjugate with H in 50 μl of PBS (−), emulsifying and mixing with an equal amount of complete Freund's adjuvant, and then intraperitoneally administering the mixture to mice. One month later, 1/4 of the initial dose was boosted,
A final immunization was performed three months later.

(3) Preparation of Monoclonal Antibody Cell fusion was performed using the spleen of a mouse three days after the final immunization. First, the excised spleen was washed three times with Dulbecco's medium, and then the spleen cells were taken out into the medium. The cell suspension was washed three times with Dulbecco's medium, mixed with mouse myeloma cells P3-X63-Ag8.653 at a cell number ratio of 5: 1 (spleen cells: myeloma cells), and centrifuged ( (1200 rpm; 5 minutes) to obtain a cell fraction. To this, 1 ml of a 50% polyethylene glycol (molecular weight 1500) solution was slowly added to fuse the cells. Cell fusion was stopped by gradually adding 10 ml of Dulbecco's medium and further adding 1 ml of FBS.
The fused cells were suspended in HAT medium further supplemented with 100 mM hypoxanthine, 0.4 mM aminopterin and 16 mM thymidine in Dulbecco's medium supplemented with 10% FBS, and placed in a 96-well polystyrene plate at 2 × 10 ^5. The cells were dispensed to give cells / well and cultured at 37 ° C. in the presence of 5% carbon dioxide for 10 to 14 days. After the culture, the reactivity of the antibody in the well was screened using the conjugate of (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid and BSA as an antigen for analysis.

(4) Screening of hybridomas producing antibodies that react with the conjugate of (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid and BSA The reactivity of the antibodies was determined by the ELISA method as follows. Was measured. First, a conjugate of (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid and BSA was dissolved in PBS (−) to a concentration of 1 μg / ml, and 50 μl was added to a 96-well microtiter plate.
/ Well, and then allowed to stand at 4 ° C. overnight to solidify. Next, a borate buffer containing 1% BSA (hereinafter referred to as a blocking buffer) 25
Replaced with 0 μl / well and blocked for 1 hour at room temperature. After washing, the culture supernatant 1 of the hybridoma was added thereto.
After adding 00 μl / well, the reaction was carried out at room temperature for 1 hour. After washing three times with a borate buffer, 100 μl / well of a peroxidase-conjugated anti-mouse IgG antibody (secondary antibody; manufactured by Capel) diluted 1000-fold with a borate buffer supplemented with 0.3% BSA was added. The reaction was performed for 1 hour. After washing three times with a borate buffer, a substrate solution of peroxidase [0.1 M sodium acetate buffer (pH 5.5),
100 μg / ml-TMBZ, 0.006% H ₂ O ₂ ]
And stop the reaction with 1N sulfuric acid.
Was measured for absorbance. About 4000 wells were screened and (2-amino-8-hydroxyquinazoline-6-
Yl) responsive to binding of the methylthio acid and BSA U
The E le found 32 wells.

(5) Screening for Antibody Reacting with C9 Base For the 32 wells found in the above (4), first, the culture supernatant in the wells was serially diluted using the ELISA method shown in the above (4). React with each other to obtain a maximum absorbance of 50
The dilution that gives a% absorbance was determined. Next, the reactivity between the C9 base and the antibody was qualitatively examined by the indirect competitive inhibition ELISA method. (2-Amino-
After immobilizing the conjugate of 8-hydroxyquinazolin-6-yl) methylthioacetic acid and BSA, it was blocked. 50 parts of C9 base serially diluted with borate buffer was added thereto.
μl / well was dispensed, and immediately, 50 μl / well of the culture supernatant adjusted so that the final concentration was the dilution ratio obtained above was added and mixed, and a competitive reaction was performed at room temperature for 1 hour.
After washing with a borate buffer three times, a secondary antibody is added again in the same manner as in the above (4), washing is performed, a substrate solution of peroxidase is added to develop a color, and the reaction is stopped with 1N sulfuric acid. Was measured for absorbance. The cells in the wells that had reacted with the C9 base were cloned by limiting dilution to obtain monoclonal antibody-producing cells. As a result, six types of the monoclonal antibody-producing cells of the present invention were established. Table 2 shows the subclasses of the monoclonal antibodies produced by each hybridoma. Hereinafter, the name of each cell shown in Table 2 is also used as the name of the monoclonal antibody produced from the cell.

[0072]

TABLE 2 Hybridoma monoclonal antibody subclass C9 9-2 IgG1 C9 11-1 IgG1 C9 13-3 IgG3 C9 21-7 IgG1 C9 47-42 IgG1 C9 104-1 IgG1

Example 5: Indirect competitive inhibition ELISA
Measurement of C9 bases The indirect competitive inhibition E shown in Example 4 (5) was performed for each of the six monoclonal antibodies shown in Example 4 (5).
C9 base was quantitatively measured by the LISA method. The results are shown in FIG. As is clear from FIG. 1, all the monoclonal antibodies reacted with the C9 base. In particular, the monoclonal antibody C922-7 (-△-in FIG. 1) exhibited the highest reactivity with C9 base, and was able to measure C9 base in a measurement range of about 5 to 100 ng / ml.

Example 6: By direct competitive inhibition ELISA
That C9 base measurements (1) hybridoma C9 21-7 which produced monoclonal antibody showed the highest reactivity against purified C9 bases of monoclonal antibodies, and a relatively high monoclonal antibody reactive to C9 base From the culture supernatant of the produced hybridoma C9104-1, monoclonal antibodies were purified as follows. First, ammonium sulfate was added to the culture supernatant to 50% saturation, followed by stirring at 4 ° C. for 1 hour. The resulting precipitate was solubilized by adding distilled water, dialyzed with 5 mM phosphate buffer (pH 7.0), adsorbed on a Mono S column (manufactured by Bio-Rad), and the monoclonal antibody was purified by a pH gradient.

(2) Preparation of peroxidase-linked (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid (2-amino-8-hydroxyquinazolin-6-yl)
3.5 μmol of methylthioacetic acid was added to DMSO (50 μl)
Was dissolved. To this solution, N-hydroxysuccinimide (4 μmol) dissolved in DMSO (10 μl) was added, and then 1-molecule dissolved in DMSO (20 μl).
Ethyl-3- (3-dimethylaminopropyl) carbodiimide (4 μmol) was added and reacted at room temperature for 1.5 hours. To this, 40 μl of 1M sodium bicarbonate was added, and peroxidase (5 mg) dissolved in 250 μl of borate buffer was further added and mixed, and the mixture was further reacted at room temperature for 3 hours. After the reaction, unreacted substances were removed using Sephadex G25 (manufactured by Pharmacia) to obtain a peroxidase-linked (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid solution.

(3) C by direct competitive inhibition ELISA
Measurement of 9 bases The direct competitive inhibition ELISA was performed as follows. First, each of purified monoclonal antibody C921-7 or monoclonal antibody C9104-1 was 5 μg / ml.
, And added to a 96- well microtiter plate so as to have a concentration of 50 µl / well , and then left at 4 ° C overnight to solidify the solid phase. . Next, the buffer was replaced with 300 μl / well of blocking buffer and blocking was performed at room temperature for 1 hour. On the other hand, the C9 base diluted stepwise with a borate buffer and the (2)
Was mixed with the peroxidase-bound (2-amino-8-hydroxyquinazolin-6-yl) methylthioacetic acid solution prepared in 1), and the mixture was dispensed at 100 μl / well, followed by reaction at room temperature for 1 hour. After washing three times with a borate buffer, the color was developed with a substrate solution of peroxidase, the reaction was stopped, and the absorbance at 450 nm was measured. The result is shown in FIG. In FIG. 2, the results of the reactivity between the C9 base and the monoclonal antibody C922-7 are indicated by "open squares"
The results of the reactivity of 9 bases with the monoclonal antibody C9104-1 are indicated by "open diamonds", respectively. As is clear from FIG. 2, in the direct competitive inhibition ELISA, the monoclonal antibody C9104-1 reacts slightly with high sensitivity to the C9 base, and about 5 to 1 is obtained using either antibody.
C9 base could be measured in the measurement range of 00 ng / ml. When the reactivity with TTX was examined in place of the C9 base, neither antibody reacted at a concentration of 80 μg / ml at all. Monoclonal antibody C9 104
The measurement results of -1 and TTX are shown by "white circles" in FIG. Therefore, the monoclonal antibodies C9104-1 and C910, which are the monoclonal antibodies of the present invention.
21-7 did not react with TTX and was found to be specific for the C9 base.

[0077]

By using the compound of the present invention as a hapten, it is possible to prepare the monoclonal antibody of the present invention which specifically reacts with the C9 base which is an alkaline hydrolysis product of the tetrodotoxin compound. The immunological measurement method of the present invention using the monoclonal antibody can easily and quickly measure C9 base, and thus can collectively measure tetrodotoxin compounds. Among these related compounds, compounds having no lethal activity by the official method (mouse test method), compounds that cannot be measured by the conventional method using an instrumental analyzer such as HPLC, and TTX itself are measured. The immunoassays used in this method include compounds that cannot be measured due to weak or no cross-reactivity, and are particularly effective for measuring tetrodotoxin compounds and screening for organisms that produce them. Also, the method of the present invention,
Since no equipment other than a spectrophotometer is required, it is possible to measure tetrodotoxin compounds without a large facility in the distribution process, etc.Furthermore, the tetrodotoxin compounds are detoxified by alkali hydrolysis at the first stage of measurement. Therefore, the safety of the measurer can be ensured. Further, since the sample is detoxified, the sample after measurement can be flowed into the sewage by adjusting the pH. As described above, it is considered that the use of the method of the present invention for the measurement of a tetrodotoxin compound can contribute to the research of pufferfish venom and the prevention of food poisoning due to the tetrodotoxin compound.

[Brief description of the drawings]

FIG. 1 is a graph showing the reactivity between C9 base and the monoclonal antibody of the present invention in an indirect competitive inhibition ELISA.

FIG. 2 is a graph showing the reactivity of C9 base or tetrodotoxin with the monoclonal antibody of the present invention in a direct competitive inhibition ELISA.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 33/577 C12N 15/00 C (72) Inventor Keiji Takemoto 2--11-15 Minami Hibarigaoka, Takarazuka-shi, Hyogo 411 (56 References: Journal of the Fisheries Society of Japan; Vol. 59, No. 7, pp. 1177-1182 (1993) Archives of Toxicology; vol. 44 (No. 4) p 291-297 (1980) Journal of AOAC International; vol. 25 (No. 2) p225-228 (1987) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 239/84 C12N 5/16 C12N 15/06 C12P 21/08 G01N 33/53 G01N 33 / 577 CA (STN) REGISTRY (STN)

Claims (7)

(57) [Claims] 1. A compound of the general formula (I): [Wherein, R ¹ is a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms, and R ² represents —C
H ₂ -A- (C ＝ O) _m -B-COOR ³ , A is O, S, or NH, B is an alkylene group or a phenylene group having 1 to 10 carbon atoms, and R ³ is hydrogen An atom or a linear or branched alkyl group having 1 to 4 carbon atoms,
m is 0 or 1], or a salt thereof. 2. 2-amino-6-hydroxymethyl-8
A monoclonal antibody, which specifically reacts with hydroxyquinazoline. 3. A fragment of the monoclonal antibody according to claim 2, which comprises an antigen-binding site that specifically reacts with 2-amino-6-hydroxymethyl-8-hydroxyquinazoline. Fragment. 4. A hybridoma which produces the monoclonal antibody according to claim 2. 5. The monoclonal antibody according to claim 2,
Alternatively, the antibody fragment according to claim 3 is used, wherein 2-amino-6-hydroxymethyl-8- is used.
An immunoassay for hydroxyquinazoline. 6. The method according to claim 5, wherein (1) the test sample is subjected to alkali hydrolysis and then neutralized, and (2) the immunological assay method according to claim 5, wherein Measuring the amount of 2-amino-6-hydroxymethyl-8-hydroxyquinazoline, and (3) determining the total amount of the tetrodotoxin compound from the measured value obtained in the step (2). An immunoassay for a tetrodotoxin compound. 7. The compound of claim 1, or a salt thereof,
3. Use according to claim 2, characterized in that it is used as a putten.
A method for producing a monoclonal antibody.