WO2012046563A1 - Monoclonal antibody which recognizes ciguatoxins ctx1b and 54-deoxy-ctx1b, and ciguatoxin detection kit using same - Google Patents

Abstract

The present invention relates to: a monoclonal antibody which is capable of recognizing a ciguatoxin that has a dihydroxybutenyl group in the A-ring moiety; a hybridoma which is capable of producing the monoclonal antibody; and a ciguatoxin detection kit which uses the monoclonal antibody. The present invention also relates to: a compound which is used for the purpose of obtaining the monoclonal antibody; and a conjugate compound wherein the compound is bound to a carrier protein.

Description

MONOCLONAL ANTIBODY Recognizing Shigatoxins CTX1B and 54-Deoxy-CTX1B, and Shigatoxins Detection Kit Using the Same

The present invention relates to shigatoxins, particularly a monoclonal antibody capable of recognizing ciguatoxin CTX1B or 54-deoxy-CTX1B having a dihydroxybutenyl group in the A ring, a hybridoma producing the monoclonal antibody, and a shigatoxin using the monoclonal antibody It relates to a kind detection kit.
The present invention also relates to a compound used to obtain the above-mentioned monoclonal antibody, and a conjugated compound formed by binding the compound to a carrier protein.

Food poisoning Shigatella occurs frequently in large coral reef areas such as Polynesia, Hawaii, the Caribbean, and Okinawa, and more than 50,000 people develop poisoning annually. Ciguatera toxin is produced by phytoplankton and accumulated in fish and shellfish via the food chain. Therefore, once ciguatera toxin is generated, it becomes a serious social problem because a large number of food fish are toxic in a wide range. Therefore, rapid detection of ciguatera toxin is important for the prevention of food poisoning ciguatera.

Studies on detection of ciguatoxins (ciguatoxins, CTX), which are the major causative toxins of food poisoning ciguatera, began with the development of radioimmunoassay from around 1977 with the development of radioimmunoassay. In studies by immunological techniques, an antibody that recognizes shigatoxins is required. However, since only a very small amount of shigatoxins is collected from nature (for example, 850 mice, only 0.35 mg of ciguatoxin obtained from 4 tons of moray eel) is difficult to produce by culture, an antibody that recognizes shigatoxins is It is difficult to manufacture.

Koka et al. Of the University of Hawaii have reported that they produced a conjugate in which ciguatoxin (1 μg), which is the main body of shigatera toxin, was linked to human serum albumin by the carbodiimide method and used as an antigen to immunize mice to produce a monoclonal antibody. (See Non-Patent Document 1).
However, although this antibody binds to ciguatoxin, it has also shown a strong cross reaction with okadaic acid, a marine polyether toxin having a similar structure to ciguatoxins. That is, the difference in affinity between this antibody for shigatoxin and okadaic acid is very small (see Non-Patent Document 2).
In addition, this antibody has been known to cross-react with other marine polyether toxins such as brevetoxin, mitotoxin, palytoxin, etc. However, detailed data has not been published (see Non-Patent Document 3).
Using the antibody of Hokama et al., A reagent (Cigua Check (trademark)) for detecting fish contaminated with shigatoxins by an immunological method has been developed.

で表される４種(化合物１～４)が主に知られている。 By the way, ciguatoxins are not single compounds. That is, ciguatoxins are a mixture of various toxins and have the following formula (III):

Four types (compounds 1 to 4) represented by are mainly known.

The present inventors previously chemically synthesized the ABC ring portion (partial structure on the left side of Formula (III)) of CTX 1 B (compound 4 in Formula (III) above) of ciguatoxin, and used it as a synthetic hapten. Three monoclonal antibodies were prepared using protein conjugates. However, all of these antibodies showed very weak affinity for ciguatoxin (see Non-patent Document 4).
Other research groups have also attempted to immunize animals using a conjugate of a synthetic hapten JKLM ring (partial structure on the right in Formula (III)) to produce a polyclonal antibody that recognizes ciguatoxin. However, no monoclonal antibody has been obtained (see Non-patent Document 5).

Under such circumstances, the present inventors designed and synthesized a synthetic hapten including the IJ KLM ring portion (partial structure on the right side of formula (III)) of ciguatoxin CTX 3 C (compound 1 in formula (III) above) By the method including the step of immunizing a mouse with a protein conjugate of this synthetic hapten, hybridoma 3D11 of Accession No. FERM BP-8293 is produced, and monoclonal antibody 3D11 highly specific to shigatoxins is produced using this hybridoma. Particularly successful (see Patent Document 1).
The dissociation constant (K _d ) of this monoclonal antibody 3D11 for shigatoxin CTX3C was 122 nM. In addition, the cross-reactivities of the marine polyether toxin having a structure similar to that of ciguatoxin and the monoclonal antibody 3D11 were examined. As a result, cross-reactivity with red tide poison brevetoxins was confirmed, but the cross-reactivity was very weak, and was about 1/350 or less as compared with the reactivity with ciguatoxin.

Furthermore, the present inventors have designed and synthesized a compound containing the ABCDE ring part (the partial structure on the left side of Formula (III)) of ciguatoxin CTX3C as a hapten, and immunize a mouse with a protein conjugate of this synthetic hapten. The hybridoma 10C9 of Accession No. FERM BP-8292 was produced by the method including, and it succeeded in preparing a monoclonal antibody 10C9 highly specific to shigatoxins using the hybridoma (see Patent Document 2).
In addition, the present inventors combined the above two types of monoclonal antibodies (3D11 and 10C9) to produce a kit for detecting sigatoxin CTX3C by a sandwich method with further improved detection characteristics (Patent Document 3 and Non-patent Document 3) 6).

Furthermore, the present inventors have synthesized, for the first time, a compound having the structure of the HIJKLM ring of ciguatoxins, in order to produce an antibody for detecting ciguatoxin 51-OH-CTX3C and CTX1B having a hydroxy group in the M ring. This is used as a hapten to immunize an animal to prepare hybridoma 8H4 deposited under Accession No. FERM BP-11400, which is used to specifically bind to shigatoxins 51-OH-CTX3C and CTX1B with high specificity. We succeeded in preparing monoclonal antibody 8H4. Furthermore, the present inventors have prepared a kit for detecting ciguatoxin 51-OH-CTX3C by a sandwich method having improved detection characteristics by combining the above-mentioned monoclonal antibodies 8H4 and 10C9 (Patent Document 4 and Non-patent Document 7) ).

Japanese Patent Application Publication No. 2003-55400 Patent No. 3845796 specification Patent No. 3820519 specification JP, 2006-193485, A

Hokama Y. et al., Toxicon Vol. 15, (1977), page 317 Hokama Y. et al., Journal of Clinical Laboratory Analysis Vol. 6, (1992), page 54 Hokama Y. et al., Journal of AOAC International Vol. 81, (1998), page 727 Oguri H. et al., Synthesis (1999), page 1431 Pauillac S. et al., Toxicon Vol. 38, (2000), page 669 Oguri H. et al. Journal of American Chemical Society Vol. 125, (2003), 7608 Tsumuraya T. et al. Toxicon Vol. 48, (2006), page 287

As described above, in the case of shigatoxin, which is the main toxin of ciguatera food poisoning, there are four kinds of analogues that differ mainly in the substitution pattern of the A ring portion and the M ring portion (compounds 1 to 4 described above). All of these, although varying in strength as toxins, can act as toxins in ciguatera food poisoning. Therefore, in order to widely prevent ciguatera food poisoning, it is necessary to detect all of these ciguatoxins. In particular, ciguatoxin CTX1B is considered to be the most universally existing ciguatera causative toxin in the world, so its detection is important.

In view of the above-described circumstances, the present invention aims to provide means capable of detecting ciguatoxins, particularly ciguatoxins CTX1B and 54-deoxy-CTX1B having a dihydroxybutenyl group in the A ring portion.

Surprisingly, the present inventors specifically synthesize a compound having the structure of the ABCDE ring part of ciguatoxins having a dihydroxybutenyl group in the A ring part and use it as a hapten to make the compound specific. It has been found that it is possible to obtain a monoclonal antibody that recognizes
Thus, the present invention provides a monoclonal antibody that specifically recognizes ciguatoxins having a dihydroxybutenyl group in the A ring portion.
In addition, the present invention is the Accession No. FERM BP-11401 producing the above-mentioned monoclonal antibody on Sep. 15, 2010, National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (Postal Code 305-8566, Ibaraki, Japan) The hybridoma 3G8 (herein, this hybridoma is denoted as "3G8" which is the same name as an antibody) is provided which has been deposited at Tsukuba 1-chome, 1-chome, 1st-address, 1st-center, 6th.

Furthermore, the present invention relates to the monoclonal antibody described above and Accession No. FERM BP-11400 on Dec. 22, 2004 National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566, Tsukuba, Ibaraki Prefecture, Japan) City, 1-chome, 1-chome, 1st, 1st, 1st, 1st, 6th center) 6) The monoclonal antibody produced by hybridoma 8H4, or Accession No. FERM BP-8293 on March 5, 2002, National Institute of Advanced Industrial Science and Technology The present invention provides a sigatoxins detection kit comprising a monoclonal antibody produced by hybridoma 3D11 deposited at the Patent Organism Depositary Center (Postal Code 305-8566, Central 1st, 1-1, 1-chome Higashi, Tsukuba, Ibaraki, Japan).

で表される化合物を提供する。 Furthermore, the present invention relates to the following formula (II):

To provide a compound represented by

（式中、nは整数を表す）
で表されるタンパク質コンジュゲートを提供する。 Further, the present invention provides a compound of the following formula (I) in which a compound of the above formula (II) is bound to a carrier protein:

(Wherein, n represents an integer)
To provide a protein conjugate represented by

By using the present invention, a system for detecting ciguatoxins which could not be detected by immunological techniques so far, particularly ciguatoxins CTX1B and 54-deoxy-CTX1B having a dihydroxybutenyl group in the A ring portion is constructed. be able to. Therefore, according to the present invention, more precise detection of ciguatoxins is possible, and more effective prevention of food poisoning ciguatera is expected.

FIG. 1 is a graph showing the antibody titer of mouse serum obtained by immunizing mice with a keyhole limpet hemocyanin (KLH) conjugate, which was evaluated using the BSA conjugate according to the present invention. FIG. 2 is a graph showing the detection characteristics of ciguatoxin CTX1B of the ciguatoxin detection kit according to the present invention.

The monoclonal antibody of the present invention is a monoclonal antibody that specifically recognizes ciguatoxins having a dihydroxybutenyl group in the A ring part in the above general formula (III). Examples of ciguatoxins having a dihydroxybutenyl group in the A ring portion include ciguatoxin CTX1B (compound 4 in the above formula (III)), shigatoxin 54-deoxy-CTX1B (compound 3 in the above formula (III)) and the like .
The monoclonal antibodies of the present invention have K _d of about 1 to 100 nM against ciguatoxins CTX1B and 54-deoxy-CTX1B.

The monoclonal antibody of the present invention can be obtained by immunizing an animal with the protein conjugate of the above-mentioned formula (I). Known methods can be used to immunize animals. The animals to be immunized include, for example, goats, rabbits and mice, and among them, mice are preferably used.

The monoclonal antibody of the present invention is considered to recognize and bind to the ABCDE ring of sigatoxins having a dihydroxybutenyl group in the A ring.
The monoclonal antibody of the present invention alone can detect ciguatoxins in an antigen-antibody reaction known per se.
As the monoclonal antibody of the present invention, preferred is, for example, a monoclonal antibody produced by hybridoma 3G8 deposited on Sep. 15, 2010 as the Accession No. FERM BP-11401 at the Patent Microorganisms Depositary, National Institute of Advanced Industrial Science and Technology. It can be used.

Alternatively, Shiga toxins are detected with higher sensitivity by using another monoclonal antibody that recognizes another site different from the site of shigatoxins recognized by the monoclonal antibody of the present invention in combination with the monoclonal antibody of the present invention. It can also be done.
Therefore, a ciguatoxins detection kit comprising the monoclonal antibody of the present invention and another monoclonal antibody that recognizes a site other than the site of ciguatoxins recognized by the monoclonal antibody is also one of the present invention.

As other monoclonal antibodies, monoclonal antibody 8H4 produced by hybridoma 8H4 deposited on December 22, 2004 as a deposit number of FERM BP-11400 at National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology, or Accession Number As the FERM BP-8293, a monoclonal antibody 3D11 produced by hybridoma 3D11 deposited on March 5, 2002 at the National Institute of Advanced Industrial Science and Technology, Patent Microorganisms Depositary on March 5, 2002 can be preferably used.
The monoclonal antibody 8H4 is a monoclonal antibody which can be specifically bound to sigatoxins having a hydroxy group in the M ring by using the HIJKLM ring of ciguatoxins having a hydroxy group in the M ring as the synthetic hapten by the present inventors. It was prepared as an antibody (see JP-A-2006-193485). In addition, the monoclonal antibody 3D11 is specifically directed to shigatoxins having no hydroxy group in the M ring, by the present inventors using the IJKLM ring of the ciguatoxins having no hydroxy group in the M ring as a synthetic hapten. It was prepared as a monoclonal antibody capable of binding (see Japanese Patent Laid-Open No. 2003-55400).

The two types of antibodies that specifically recognize different sites of ciguatoxins as described above can be used in conventionally known immunological methods to detect ciguatoxins. Conventionally known immunological methods include a sandwich method of detecting a target antigen using two or more types of antibodies.

In the ciguatoxins detection kit of the present invention, it is desirable that either the monoclonal antibody or the other monoclonal antibody of the present invention be labeled. The label may be any label commonly used in immunological methods, such as enzyme label (eg, peroxidase, alkaline phosphatase, β-galactosidase etc.), fluorescent label (eg, fluorescein isothiocyanate (FITC), allophycocyanin (APC) , Phycoerythrin (PE), carbocyanine, etc., radioactive labels (eg, tritium ( ³ H), ¹²⁵ I, ¹³¹ I etc.), gold colloid labels, etc. In the present invention, enzyme labels are more preferred.
Labeling of a monoclonal antibody can be performed by a known method. Alternatively, a commercially available kit for antibody labeling can be used.

The compounds of formula (I) above can be obtained from the compounds of formula (II) above. Specifically, the compound of the formula (I) is prepared by reacting the carrier protein with 3,3'-dithiobis (sulfosuccinimide propionate) (DTSSP) and then tris (2-carboxyethyl) phosphine hydrochloride ( Method of reducing amino group of basic amino acid residue (lysine, histidine and arginine residue) of carrier protein to thiol group by reduction with a reducing agent such as TCEP) and reacting it with a compound of formula (II) It can be obtained by
Conventionally, as a method of conjugating a compound containing a ABCDE ring part of ciguatoxins having no dihydroxybutenyl group in the A ring part with a carrier protein, the amino group of the basic amino acid residue of the carrier protein is Methods have been known to react with N-hydroxysuccinimidyl ester derivatives of compounds containing (see Patent No. 3845796), but in the present invention, the amino group of the basic amino acid residue of the carrier protein is By converting into thiol group and making it react, a protein conjugate capable of causing an animal to produce a target antibody can be obtained.

As the above-mentioned carrier protein, any protein may be used as long as it can be used as a carrier to be bound to a hapten in an immunological method, and bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg albumin (OVA), Fibrinogen and the like can be mentioned, and among them, BSA, KLH and OVA are preferable.

The compounds of the above-mentioned formula (II) are novel compounds, which are also one of the present invention.
Compounds of formula (II) can be obtained by the method 1 described below.

Hereinafter, the present invention will be described in more detail by describing embodiments of the compound of the present invention, a protein conjugate, a monoclonal antibody, a hybridoma and a ciguatoxins detection kit, but the present invention is not limited to the following embodiments.
A. Preparation of the Compound of Formula (II) The compound of Formula (II) above was synthesized by the following method 1.
Method 1

In the above formula, Fmoc represents (9-fluorenylmethyloxycarbonyl), DCC represents dicyclohexylcarbodiimide, DMAP represents N, N-dimethyl-4-aminopyridine, and DDQ represents 2,3-dichloro- Represents 5,6-dicyano-p-benzoquinone, PPTS represents pyridinium p-toluenesulfonate, MeOH represents methanol, THF represents tetrahydrofuran, DMF represents N, N-dimethylformamide, and BMPS is 3- 3- N-succinimidyl maleimide is represented, NAP represents (naphthylmethyl).

In Method 1 described above, Compound 5 was obtained by the method described in M. Hirama et al., Org. Lett., Volume 6, page 751 (2004).
To a solution of Fmoc-glycine (32.1 mg, 108.1 μmol) in CH ₂ Cl ₂ (1.1 mL) was added DMAP (9.89 mg, 81.0 μmol) and DCC (22.3 mg, 108.1 μmol) at room temperature. The reaction solution was stirred at room temperature for 5 minutes, and then a solution of compound 5 (5.0 mg, 5.4 μmol) in CH ₂ Cl ₂ (1.0 mL) was added. The reaction solution was further stirred for 14 hours, and then phosphate buffer (pH = 7) was added. After extraction with ethyl acetate, the separated organic phase was dried over MgSO ₄ . After the solvent was distilled off, the residue was purified by silica gel column (CH ₂ Cl ₂ / EtOAc 1: 0 to 1: 9) to obtain compound 6 (6.5 mg, yield: 100%).

¹ H NMR (400 MHz, CDCl 3) δ 7.82-7.66 (15H, m), 7.61-7.59 (2H, m), 7.54-7.37 (8H, m), 7.34-7.28 (4H, m), 5.93 (1H , dd, J = 15.6, 5.2 Hz), 5. 84 (1 H, m), 5.8 2 (1 H, m), 5.81-5. 80 (2 H, m), 5. 54-5. 68 (2 H, m), 5. 66 (1 H, m), 5.48 (1H, m), 5.32 (1H, m), 5.30 (1H, m), 5.11-5.07 (2H, m), 5.03-4.98 (2H, m), 4.80 (1H, d, J = 12.4 Hz) , 4.68 (1H, br), 4.66 (1H, d, J = 12.8 Hz), 4.63 (1H, d, J = 12.8 Hz), 4.62 (1H, d, J = 12.4 Hz), 4.44 (2H, m) , 4.24 (1H, m), 4.16 (1H, m), 4.11 (1H, m), 4.08 (1H, m), 4.03 (2H, m), 3.86 (1H, m), 3.67 (1H, ddd, J = 8.4, 8.4, 2.8 Hz), 3.60 (H, dd, J = 10.4, 6.8 Hz), 3.57 (1 H, m), 3.55 (1 H, dd, J = 8.8, 8.8 Hz), 3.52 (1 H, dd, J = 10.4, 4.0 Hz), 3.35 (1 H, m), 3.31 (1 H, m), 3.15 (1 H, m), 3. 13 (H, m), 2.67 (H, m), 2. 40 (1 H, m), 2.36 (1 H, m), 2.31 (1 H, m), 2.19 (1 H, m), 1.61 (1 H, m).
ESI-FT-MS: Calculated for C ₇₇ H ₇₃ NO ₁₂ Na (M + Na ⁺ ): 1226.5025, found: 1226.5024.

DDQ (12.3 mg, 54.0 μmol) was added to a solution of compound 6 (6.5 mg, 5.4 μmol) in CH ₂ Cl ₂ / H ₂ O (volume ratio 2: 1, 5.4 mL) at room temperature, and stirred for 30 minutes . After adding a saturated aqueous solution of Na ₂ S ₂ O ₃ to the reaction solution, the reaction solution was diluted with ethyl acetate and then a saturated aqueous solution of NaHCO ₃ was added. After extraction with ethyl acetate, the separated organic phase was dried over MgSO ₄ . After evaporation of the solvent, the residue was purified by silica gel column (CHCl ₃ / MeOH 1: 0 to 100: 1) to give compound 7 (3.3 mg) and compound 8 (3.0 mg).
ESI-FT-MS: Compound 7: calculated for _{C 55 H 57 NO 12 Na (} M + Na +): 946.3773, Found: 946.3774.
Compound 8: calculated for _{C 55 H 55 NO 12 Na (} M + Na +): 944.3616, Found: 944.3621.

To a solution of compound 7 (3.3 mg, 3.6 μmol) in CH ₂ Cl ₂ (1.8 mL), DDQ (6.5 mg, 28.6 μmol) was added at room temperature and stirred for 1 hour. After adding a saturated aqueous solution of Na ₂ S ₂ O ₃ to the reaction solution, the reaction solution was diluted with ethyl acetate and then a saturated aqueous solution of NaHCO ₃ was added. After extraction with ethyl acetate, the separated organic phase was dried over MgSO ₄ . After evaporating the solvent, the residue was purified by silica gel column (CHCl ₃ / MeOH 1: 0 to 100: 1) to obtain compound 8 (1.0 mg).

PPTS (6.5 mg, 26.0 μmol) was added at room temperature to a solution of compound 8 (4.0 mg, 4.3 μmol) in THF / MeOH (volume ratio 1: 1, 2.2 mL), and after stirring for 6 hours, the reaction solution was saturated Aqueous NaHCO ₃ solution was added. After extraction with ethyl acetate, the separated organic phase was dried over MgSO ₄ . After the solvent was distilled off, the residue was purified by silica gel column (CHCl ₃ / MeOH 1: 0 to 30: 1) to give compound 9 (1.3 mg, yield: 31%).
ESI-FT-MS: calculated for C ₄₄ H ₄₉ NO ₁₂ Na (M + Na ⁺ ): 806.3147, found: 806.3152.

To a solution of compound 9 (1.3 mg, 1.7 μmol) in DMF (1.7 mL) was added piperidine (1.6 μL, 16.6 μmol) at room temperature and stirred for 1.5 hours. After the solvent was distilled off, piperidine was distilled off by azeotropic distillation with toluene to obtain a crudely purified product.

To a solution of the crude product in DMF (1.7 mL) was added BMPS (0.9 mg, 3.3 μmol) at room temperature and stirred for 2 hours. After the solvent was distilled off, the residue was purified by silica gel column (CHCl ₃ / MeOH 100: 1 to 20: 1) to obtain a compound of formula (II) (1.2 mg, yield: 99%).

¹ H NMR (400 MHz, C ₅ D ₅ N) δ 9.58 (1 H, dd, J = 5.8, 5.8 Hz), 6.77 (2 H, m), 6. 39 (1 H, m), 6. 34 (1 H, m), 5.95 (1H, m), 5. 91 (1 H, m), 5. 89 (1 H, m), 5. 78 (1 H, m), 5. 76 (1 H, m), 5.72 (1 H, ddd, J = 13.2, 2.4, 2.4 Hz), 5.59 (1H, ddd, J = 13.2, 2.4, 2.4 Hz), 5.53 (1 H, m), 5.15 (1 H, dd, J = 17.2, 2.0 Hz), 5.09 (1 H, dd, J = 10.4, 2.0 Hz) , 4.86 (1H, br), 4.69 (1H, m), 4.36 (2H, m), 4.21 (1H, m), 4.15 (1H, m), 4.10 (1 H, dd, J = 9.2, 8.8 Hz), 4.08 (2H, dd, J = 7.4, 7.4 Hz), 4.03 (1 H, m), 3. 97 (2 H, m), 3. 77 (1 H, m), 3. 76 (1 H, dd, J = 8.8, 8.8 Hz), 3.50 (1H, m), 3.49 (1H, m), 3.42 (1H, dd, J = 9.2, 9.2 Hz), 3.35 (H, ddd, J = 11.2, 9.2, 4.0 Hz), 2.89 (2H, dd, J = 7.4, 7.4 Hz), 2.72 (H, ddd, J = 16.0, 8.0, 4.0 Hz), 2.55 (1 H, m), 2.53 (1 H, ddd, J = 11.2, 4.0, 4.0 Hz), 2.47 (1 H, m), 2.25 (1 H, ddd, J = 14.8, 7.6, 7.6 Hz), 1. 84 (1 H, ddd, J = 11.2, 11.2, 11.2 Hz).
ESI-FT-MS: Calcd. C ₃₆ H ₄₄ N ₂ O ₁₃ Na calculated (M + Na ⁺ ): 735.2736, found: 735. 2739.

B. Production of protein conjugates

Preparation of Keyhole Limpet Hemocyanin (KLH) Conjugate: 3,3'-Dithiobis (sulfosuccinimidoylpropionate) at room temperature against a solution of KLH (10.0 mg) in PBS (phosphate buffered saline; 4 mL) ) (DTSSP) (13.7 mg, 45.0 μmol) was added. After standing at room temperature for 3 hours, it was dialyzed against PBS (1 L) at 4 ° C. The PBS (1 L) was exchanged twice every 2 hours, and after 12 hours, the solution was removed from the dialysis membrane to obtain the above compound 10.

To a solution of compound 10 in PBS (3.0 mL, 1.72 mg / mL) was added a solution of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (1.8 mg, 6.4 μmol) in PBS (100 μL) at room temperature. After standing for 2 hours at room temperature, a solution of TCEP (1.8 mg, 6.4 μmol) in PBS (100 μL) is added again, and after standing for another 2 hours at room temperature, a solution of TCEP (1.8 mg, 6.4 μmol) in PBS (100 μL) Was added again. After standing at room temperature for 12 hours, it was dialyzed against PBS (1 L) at 4 ° C. The PBS (1 L) was exchanged twice every 2 hours, and after 12 hours, the solution was removed from the dialysis membrane to obtain the above compound 11.

To a solution of compound 11 in PBS (1.2 mL, 1.72 mg / mL) was added a solution of the compound of formula (II) (0.60 mg, 0.84 μmol) obtained by the above method at room temperature in DMF (100 μL). After gently stirring for 24 hours at room temperature, it was dialyzed against PBS (1 L) at 4 ° C. The PBS (1 L) was replaced twice every two hours, and after 12 hours, the solution of KLH conjugate was removed from the dialysis membrane and transferred to an eppendorf tube for storage.

Preparation of Bovine Serum Albumin (BSA) Conjugate: To a solution of BSA (20.0 mg) in PBS (10 mL), DTSSP (13.7 mg, 45.0 μmol) was added at room temperature. After standing at room temperature for 3 hours, it was dialyzed against PBS (1 L) at 4 ° C. The PBS (1 L) was exchanged twice every two hours, and after 12 hours, the solution was removed from the dialysis membrane to obtain the above compound 12.
To a solution of compound 12 in PBS (9.0 mL, 1.70 mg / mL) was added a solution of TCEP (1.8 mg, 6.4 μmol) in PBS (100 μL) at room temperature. After standing at room temperature for 2 hours, a solution of TCEP (1.8 mg, 6.4 μmol) in PBS (100 μL) is added again, and after standing at room temperature for 2 hours, a solution of TCEP (1.8 mg, 6.4 μmol) in PBS (100 μL) Was added again. After standing at room temperature for 12 hours, it was dialyzed against PBS (1 L) at 4 ° C. The PBS (1 L) was replaced twice every 2 hours, and after 12 hours, the solution was removed from the dialysis membrane to obtain the above compound 13.

To a solution of compound 13 in PBS (1.1 mL, 1.82 mg / mL) was added a solution of the compound of the formula (II) (0.60 mg, 0.84 μmol) obtained by the above method at room temperature in DMF (100 μL). After gently stirring for 24 hours at room temperature, it was dialyzed against PBS (1 L) at 4 ° C. The PBS (1 L) was replaced twice every 2 hours, and after 12 hours, the solution of BSA conjugate was removed from the dialysis membrane and transferred to an eppendorf tube for storage.

Analysis of Hapten Number The BSA conjugate obtained as described above was analyzed by MALDI-TOF-MS. The average molecular weight of the BSA conjugate was about 77400 (the molecular weight of BSA is about 66400). Since the molecular weight of the hapten moiety which is a moiety derived from the compound of the above-mentioned formula (II) is 802, it is understood that an average of 14 haptens are linked to the BSA conjugate.

C. Immunization To the conjugate (300 μg) of the compound of formula (II) obtained as described above and KLH, AbISCO ^TM -100 (ISCONOVA, Inc., 150 μL) as an adjuvant and PBS are added to make a solution volume. It was 1.2 mL. The solution thus prepared was intraperitoneally administered three times every two weeks to Balb / c mice (5 animals: Kiwa Experimental Animal Research Institute, Inc.) as an antigen solution (200 μL / mouse). . One week after the third administration, mouse serum was collected, and the antibody titer of the serum was determined by the following ELISA (enzyme-linked immunosorbent assay) method using a conjugate of the compound of formula (II) described above and BSA as an antigen. It was determined.

ELISA method Add 50 μl of BSA conjugate (2 μg / mL) to each well of a 96-well ELISA plate (COSTAR 3590), leave it for 2 hours at room temperature, and then leave it overnight at 4 ° C. The gate was adsorbed. The plate was washed 3 times with PBS-Tween [0.05% Tween-20 (PBS containing polyoxyethylene (20) sorbitan monolaurate (manufactured by ICI, Tween-20 equivalent product 170-6531 manufactured by Tween-20 equivalent product)]. The non-adsorbed BSA conjugate was removed, antiserum (or hybridoma culture supernatant, purified antibody solution) was added to the wells, allowed to stand at room temperature for 1 hour, and washed 3 times with PBS-Tween 50 μL of enzyme-labeled enzyme-labeled A secondary antibody (goat anti-mouse IgG-horseradish peroxidase) (BIO-RAD, 170-6516, 1000-fold diluted solution in PBS-Tween) is added to each well and left at room temperature for 1 hour, PBS-Tween The solution was washed 3 times each with 100 μL of substrate solution [Composition of substrate solution: 4.0 mg of 1,2-phenylenediamine, 10 μL of hydrogen peroxide solution, 10 mL of 0.1 M citric acid buffer (pH 5.0)] and allowed to stand for several minutes. After the color reaction proceeded, the reaction was stopped with 2 N sulfuric acid (50 μL) microplate absorbance meter (BI The absorbance at 490 nm was measured using O-RAD (Model 680).

Measurement of Antibody Titers in Serum Dilution series samples were prepared by serially diluting sera from 300 times to 38400 times with PBS containing 10% FBS. 50 μL of each dilution series sample was placed on the ELISA plate (COSTAR 3590) on which the BSA conjugate was adsorbed as described above, and left at room temperature for 1 hour, and then the absorbance at 490 nm was measured by the above method (FIG. 1) reference). The results of plotting serum dilution and absorbance for 5 mice are shown in FIG. From FIG. 1, it can be seen that antibodies in serum bind to the BSA conjugate in a serum concentration-dependent manner.

Extraction of spleen from high antibody titer mice, cell fusion, preparation of monoclonal hybridoma The solution was added AbISCO ^TM -100 (ISCONOVA, 50 μL) and PBS to the KLH conjugate (100 μg) obtained as described above The volume was adjusted to 0.4 mL to prepare an antigen solution. Three weeks after the third immunization, the antigen solution prepared above was boosted (200 μL) into the abdominal cavity of the mouse showing the highest antibody titer among the five mice, and the spleen was removed three days later. After removing fragments of tissues and organs adhering to the spleen with tweezers, use serum-free medium [RPMI 1640 medium (GIBCO, 11875-085) in 1000 mL penicillin / streptomycin (GIBCO, 15140-122) 10 Medium to which mL was added] The medium was transferred to a 15 mL petri dish, and cells in the spleen were suspended with tweezers. The spleen cell suspension was filtered and transferred to a 50 mL centrifuge tube. Furthermore, 15 mL of serum-free medium was added, pipetting was carried out well, and filtration was performed to a total volume of 30 mL. Centrifuge at 800 rpm for 5 minutes at room temperature, remove the supernatant and tap.

A tube in which myeloma cells P3X63-Ag8.653 (Dainippon Pharmaceutical Co., Ltd.) were frozen was taken out from a liquid nitrogen cell storage container at -180 ° C, and thawed quickly in a thermostat at 37 ° C. After thoroughly disinfecting the tube with alcohol cotton, the cell suspension in the tube was transferred to 30 mL of serum-free medium. The supernatant was removed by centrifugation at 800 rpm for 5 minutes at room temperature. After tapping, 100 mL of growth medium (serum free from serum, 100 mL of FBS, 10 mL of L-glutamine (200 mM, GIBCO, 25030-081), 20 mL of Brei clone (BR-001 manufactured by BioResearch Ireland) was added. Medium) 10 mL was added, cells were suspended, and transferred to a 50 mL culture flask. The stopper of the flask was loosened, placed in a carbon dioxide incubator, and cultured. The cells were subcultured every 2 to 3 days to make two 250 mL flasks (90 to 100 mL).

Mix spleen cells (2 × 10 ⁸ ) taken from the mouse with the above myeloma cells (5 × 10 ⁷ ), centrifuge (800 rpm, 5 minutes, room temperature), remove the supernatant, and tap. did. Thereafter, ECF buffer [buffer containing 10 ml of 10 mM magnesium chloride, 10 mL of 10 mM magnesium chloride, 10 mL of 20 mM Tris-HCl (pH 7.2) dissolved in distilled water to make 1000 mL] 30 After adding mL, centrifuging (800 rpm, 5 minutes, room temperature), removing the supernatant and tapping twice, the operation was repeated with 4.8 mL of ECF buffer. This was divided into 4 wells of 1.2 mL each well of a 6-well plate (manufactured by SUMIRON, MS-80060), and cells were fused under the following conditions using an SSH-10 cell fusion device (manufactured by Shimadzu) [ Distance between electrodes: 1.0 mm; AC frequency: 1 MHz; AC initial applied voltage: 40 V; AC initial application time: 10 seconds (s); pulse width: 40 (microseconds (μs)); pulse voltage: 230 V; pulse Electric field strength: 2.30 kV / cm; AC secondary applied voltage: 1.0 V; pulse application interval: 0 seconds; applied pulse number: 1; pulse voltage change: +0 V; AC final application time: 0 second; AC voltage attenuation factor: 0%; contact strengthening: off].

The obtained hybridoma cells (Mouse-Mouse hybridoma) were added to HAT medium [serum free medium 144 mL with FCS 40 mL, L-glutamine (200 mM, 25030-081 manufactured by GIBCO) 2 mL, Brei clone (BioResearch Ireland BR) -001) Medium containing 10 mL and HAT (31062-011 manufactured by GIBCO) 4mL was suspended in 60 mL, and 100 μL / well was transferred to six 96-well plates. A hybridoma which produces an antibody which is cultured for about 2 weeks in a carbon dioxide gas culture apparatus and which binds a conjugate of a compound of formula (II) and BSA (that is, a compound of BSA in the protein in formula (I)) is The conjugate was screened by ELISA. Positive wells were selected, and after cloning twice, hybridomas producing monoclonal antibodies that became ELISA positive were successively cultured and grown to about 200 mL each. Thus, hybridoma 3G8 producing a monoclonal antibody at high antibody titer was obtained. This hybridoma was deposited at National Institute of Advanced Industrial Science and Technology Patent Organism Depositary on Sep. 15, 2010 under Accession No. FERM BP-11401 (hereinafter, this hybridoma is referred to as "3G8" which is the same name as antibody. indicate).

E. Evaluation of monoclonal antibody Purification of antibody and determination of subclass The culture supernatant of hybridoma 3G8 prepared as described above was purified using an anti-mouse IgG + IgM affinity column (manufactured by NGK Industries, Ltd.) (binding buffer: Phosphate buffer pH 7.0, elution buffer: 0.2 M Gly-HCl pH 2.5). The subclass of antibody was determined using mouse monoclonal antibody isotyping kit IsoStrip (manufactured by Roche Applied Science, 11493027001). As a result, the subclass of antibody 3G8 was IgG ₁ κ.

Affinity analysis of antibody (competition inhibition experiment)
Next, the dissociation constant (K _d ) with the hapten was determined for the purified antibody. A solution of competitive inhibitor (compound 14 shown below) (30 μL each of a 10% MeOH-containing PBS solution) diluted in 2-fold was prepared sequentially in a plate for ELISA. The antibody solution (30 μL) was added to this, and left at room temperature for 2 hours. 50 μL of a mixture of antibody and inhibitor was added to a 96-well ELISA plate (COSTAR 3590) to which a BSA conjugate (1.25 μg / mL) was adsorbed, and left at room temperature for 15 minutes. After washing the plate three times with PBS-Tween, 50 μL of enzyme-labeled secondary antibody (Goat anti-mouse IgG-Horseradish peroxidase) (BIO-RAD, 170-6516, diluted 1: 1000 in PBS-Tween) The wells were placed and allowed to stand at room temperature for 1 hour, and then washed 3 times with PBS-Tween. After adding 100 μL of substrate solution [Composition of substrate solution: 4.0 mg of 1,2-phenylenediamine, 10 μL of hydrogen peroxide solution, 10 mL of 0.1 M citric acid buffer (pH 5.0)] and letting the color reaction proceed for several minutes The reaction was quenched with 2 N sulfuric acid (50 μL). The absorbance at 490 nm was measured using a microplate absorbance measurement apparatus (manufactured by BIO-RAD, model 680) to obtain a titration curve. Referring to the method of Friguet et al. [Journal of Immunological Method, Vol. 77 (1985), p. 305], the K _d of the inhibitor was determined from the slope of the straight line obtained by preparing the Klotz plot, K _d = It became 1.5 nM. That is, it is understood that the monoclonal antibody 3G8 strongly binds to the hapten compound 14.

The same inhibition experiment was performed using shigatoxin CTX1B (compound 4), and it was revealed that the antibody 3G8 exhibits high affinity for CTX1B (K _d = 14.9 nM). In addition, antibody 3G8 showed no cross-reactivity with ciguatoxin CTX3C (compound 1) and other polyether marine toxins (brebetoxins A and B, okadaic acid).

F. Shiga toxins detection kit by sandwich method
Enzymatic labeling of 8H4 antibody (synthesis of 8H4-HRP for sandwich method)
Antibody 8H4 (a monoclonal antibody that recognizes shigatoxins having an OH group in the M ring portion, produced by hybridoma 8H4 deposited under Accession No. FERM BP-11400 and deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology the reference) 2006-193485 JP, HRP-Antibody All-in- One TM Conjugation kit (Solulink Co., Ltd.) used according to its instructions and HRP labeled. Using the 8H4 antibody (100 μg), approximately 150 μL of an 8H4-HRP solution was obtained. The solution was placed in a 500 μL tube and stored at 4 ° C.

Method for detecting sandwich of ciguatoxin CTX1B A PBS solution (10 μg / mL) of the monoclonal antibody 3G8 obtained as described above was put in an ELISA plate (3590) manufactured by COSTAR, 50 μL per well, and left overnight at 4 ° C. The solution was discarded and PBS containing 1% skimmed milk was added (400 μL / well) and allowed to stand at room temperature for 1 hour. The solution was discarded, and after washing three times with PBS-Tween, a diluted solution of ciguatoxin CTX1B (compound 4) was added (50 μL / well) and allowed to stand at room temperature for 1 hour. After discarding the solution and washing three times with PBS-Tween, the above-mentioned HRP-labeled monoclonal antibody 8H4 solution (20 μg / mL, 50 μL) was added and allowed to stand at room temperature for 1 hour. Discard the solution and wash 3 times with PBS-Tween, then 100 μL of substrate solution [Composition of substrate solution: 1,2-phenylenediamine 4.0 mg, hydrogen peroxide solution 10 μL, 0.1 M citric acid buffer (pH 5.0) 10 mL ] Was added, and the color reaction was allowed to proceed for several minutes, and then the reaction was stopped with 2N sulfuric acid (50 μL). The absorbance at 490 nm was measured using a microplate absorbance measurement device (manufactured by BIO-RAD, model 680). The measurement results are shown in FIG.

From the results in FIG. 2, it is possible to detect shigatoxin CTX1B at a detection sensitivity of 0.25 nM or more with high sensitivity by combining 3G8 which is an antibody prepared using a hapten having an ABCDE ring portion of shigatoxin CTX1B and enzyme-labeled antibody 8H4. I know what I can do.

This application relates to Japanese Patent Application No. 2010-226734 filed on October 6, 2010, and all of the claims, specification, drawings and abstract thereof are herein incorporated by reference. Be incorporated.

Claims (11)

A monoclonal antibody that specifically recognizes shigatoxins having a dihydroxybutenyl group in the A ring portion. The following formula (I):

(Wherein, n represents an integer)
The monoclonal antibody according to claim 1, which can be obtained by immunizing an animal with a protein conjugate represented by The monoclonal antibody according to claim 2, wherein the animal is a mouse. The monoclonal antibody according to any one of claims 1 to 3, wherein the ciguatoxins having a dihydroxybutenyl group in the A ring part are ciguatoxin CTX1B or 54-deoxy-CTX1B. The monoclonal antibody according to any one of claims 1 to 4 is produced on Sep. 15, 2010 under Accession No. FERM BP-11401 on Sep. 15, 2010 Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Postal Code 305-) 8566, hybridoma 3G8 deposited at Central 1st, 1st Street, 1st Street, 1st Street, Tsukuba, Ibaraki Prefecture, Japan. A monoclonal antibody according to any one of claims 1 to 4 and a Patent Organism Depositary at National Institute of Advanced Industrial Science and Technology (AIC) on Dec. 22, 2004 under Accession No. FERM BP-11400 (Postal Code 305-8566, The patent of the National Institute of Advanced Industrial Science and Technology (AIST) on March 5, 2002 as hybridoma 8H4 or Accession No. FERM BP-8293, which was deposited at Central 1st, 1-1, 1-chome, Tsukuba, Ibaraki, Japan A kit for detecting ciguatoxins, which comprises a monoclonal antibody produced by hybridoma 3D11 deposited at the Organism Depositary Center (Postal Code 305-8566, Central 1st, 1-1, 1-chome Higashi, Tsukuba, Ibaraki, Japan). The monoclonal antibody according to any one of claims 1 to 4 or Accession No. FERM BP-11400 on Dec. 22, 2004 Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566, A monoclonal antibody produced by hybridoma 8H4 deposited at I-Chome 1-chome, 1-chome, Tsukuba, Ibaraki, Japan, or an accession number FERM BP-8293 as an independent administrative corporation on March 5, 2002 (2002) The monoclonal antibody produced by hybridoma 3D11 deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary (Postal Code 305-8566, Central 1st, Central 1st, 1st Street, 1st Street, Tsukuba, Ibaraki, Japan) is labeled. The Shiga toxins detection kit as described in 6. The kit for detecting ciguatoxins according to claim 6 or 7, wherein the label is an enzyme label. The following formula (II):

A compound represented by The following formula (I), wherein the compound according to claim 9 is bound to a carrier protein

(Wherein, n represents an integer)
A protein conjugate represented by The protein conjugate according to claim 10, wherein the carrier protein is bovine serum albumin, keyhole limpet hemocyanin or egg albumin.

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