JP4680607B2 - Monoclonal antibody that recognizes ciguatoxins, and ciguatoxins detection kit using the same - Google Patents

Description

  The present invention relates to ciguatoxins, particularly a monoclonal antibody capable of recognizing ciguatoxin 51-OH-CTX3C or CTX1B having a hydroxyl group in the M ring portion, a hybridoma producing the monoclonal antibody, and a ciguatoxins detection kit using the monoclonal antibody About.

  The present invention also relates to a compound used for obtaining the above-mentioned monoclonal antibody, and a conjugate compound obtained by binding the compound to a carrier protein.

  Food poisoning ciguatera occurs frequently in wide coral reefs such as Polynesia, Hawaii, Okinawa, and the Caribbean, and more than 20,000 poisoning patients occur annually. Ciguatera toxin is produced by phytoplankton and is contained in fish and shellfish through the food chain. Therefore, once it occurs, a large number of edible fish are extensively poisoned and become a serious social problem. Therefore, rapid detection of ciguatera toxin is important for the prevention of food poisoning ciguatera.

  Research on the detection of ciguatoxin (CTX), the main causal toxin of food poisoning ciguatera, using immunological techniques began around 1977 with the development of radioimmunoassay. Immunological research requires antibodies that recognize ciguatoxins, but only a small amount of ciguatoxins is collected from nature (for example, 850 cigarette toxins from 4t poisonous moray cucumber are only 0.35 mg). ) Since it is difficult to produce by culturing, it is also difficult to produce antibodies.

The University of Hawaii Hokama et al. Prepared a conjugate in which ciguatoxin (1 μg), the main body of ciguatera toxin, was linked to human serum albumin by the carbodiimide method, and this was used as an antigen to immunize mice to prepare monoclonal antibodies (non-patented) Reference 1).
However, although this antibody binds to ciguatoxin, it exhibits strong cross-activity with okadaic acid, a marine polyether toxin having a structure similar to ciguatoxins, and the difference in affinity is only about 5 times (Non-patent Documents). 2).
In addition, it has been found that other marine polyether toxins such as brevetoxin, mitotoxin, and palytoxin also show cross-activity, but no detailed data has been published (see Non-Patent Document 3).
Reagents and kits (Cigua Check (trademark)) for detecting fish contaminated with ciguatoxins by immunological techniques using the antibody of Hokama et al. Have been developed.

  By the way, ciguatoxins are not a single compound but a mixture of various toxins and have the following general formula:

Are mainly known (compounds 1 to 4).

The inventors previously prepared three monoclonal antibodies using a protein conjugate that chemically synthesized the left ABC ring part of ciguatoxin and used this as a synthetic hapten. Only weak affinity was shown (see Non-Patent Document 4).
In addition, other groups have attempted immunization using a conjugate of a synthetic hapten (JKLM ring), but no monoclonal antibody has been obtained (see Non-Patent Document 5).

  Under such circumstances, the present inventors designed and synthesized a synthetic hapten containing the IJKLM ring part, which is a partial structure at the right end of ciguatoxin CTX3C (compound 1 in the above formula), and A hybridoma 3D11 having the accession number FERM PB-8293 was prepared by a method including a step of immunizing a mouse with a protein conjugate, and the monoclonal antibody 3D11 having high specificity for ciguatoxins was successfully produced using the hybridoma ( Patent Document 1).

  The binding dissociation constant (Kd) of monoclonal antibody 3D11 for ciguatoxin CTX3C was 122 nM. In addition, cross-activity between marine polyether toxin similar to ciguatoxin and monoclonal antibody 3D11 was confirmed to be cross-reactive with red tide venom brevetoxins, but the cross-activity was compared with binding to ciguatoxin. And it was very weak less than 1/350.

Furthermore, the present inventors commissioned a method comprising a step of designing and synthesizing a compound containing ABCDE ring part, which is a partial structure of the left end of ciguatoxin CTX3C, as a hapten, and immunizing a mouse with a protein conjugate of this synthetic hapten. A hybridoma 10C9 having the number FERM BP- 8292 was prepared, and a monoclonal antibody 10C9 highly specific to ciguatoxins was successfully prepared using the hybridoma (see Patent Document 2).

  In addition, the present inventors made a kit for detecting ciguatoxin CTX3C by a sandwich method with improved detection characteristics by combining the above two monoclonal antibodies (see Patent Document 3 and Non-Patent Document 6).

However, it is known that ciguatoxin, which is a major toxin of ciguatera food poisoning, has four types of analogs mainly having different substitution patterns of the A ring and the M ring as shown in the above formula (see above). Compounds 1 to 4), which all have different strengths as toxins, can act as toxins for ciguatera food poisoning. Therefore, in order to widely prevent ciguatera food poisoning, it is also necessary to detect ciguatoxins other than the above ciguatoxin CTX3C.
JP 2003-55400 A JP 2003-267978 A Japanese Patent Laid-Open No. 2003-267979 Toxicon Volume 15, (1977), 317 Journal of Clinical Laboratory Analysis Vol. 6, (1992), p. 54 Journal of AOAC International Volume 81, (1998), 727 Synthesis (1999), page 1431 Toxicon Volume 38 (2002), page 669 Journal of American Chemical Society Vol. 125, (2003), p. 7608

  An object of the present invention is to provide a means capable of detecting ciguatoxins, particularly cigartoxin 51-OH-CTX3C and CTX1B having a hydroxyl group in the M ring portion.

  In order to solve the above problems, the present inventors have found that the above problems can be solved by first synthesizing a compound having the structure of the HIJKLM ring part of ciguatoxins and using this as a hapten.

Therefore, the present invention is a monoclonal antibody that specifically recognizes ciguatoxins having a hydroxyl group at the M ring.
In addition, the present invention was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as the hybridoma 8H4 deposited under the accession number FERM P-20347 producing the above monoclonal antibody (accession number FERM P-20347). Date of entrustment: December 22, 2004. In this specification, this hybridoma is designated as the same name as antibody 8H4).
In addition, the present invention is produced by the monoclonal antibody described above and hybridoma 10C9 (deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on March 5, 2002) deposited under the deposit number FERM BP- 8292. A kit for detecting ciguatoxins by the sandwich method, comprising the monoclonal antibody to be prepared.

で表される化合物である。 Furthermore, the present invention provides the following formula (II):

It is a compound represented by these.

(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でもある。 The present invention also provides the following formula (I), wherein the compound of the above formula (II) is bound to a carrier protein:

(Where n represents an integer)
It is also a protein conjugate represented by.

  By using the present invention, it is possible to construct a system capable of detecting ciguatoxins that could not be detected by immunological techniques until now, particularly ciguatoxin 51-OH-CTX3C and CTX1B having a hydroxyl group in the M ring. It is possible to effectively prevent food poisoning ciguatera.

  The monoclonal antibody of the present invention is a monoclonal antibody that specifically recognizes ciguatoxins having a hydroxyl group in the M ring portion. Examples of ciguatoxins having a hydroxyl group at the M ring include ciguatoxin 51-OH-CTX3C (compound 2 above) and ciguatoxin CTX1B (compound 4 above).

  The monoclonal antibody of the present invention has a binding dissociation constant (Kd) for ciguatoxin 51-OH-CTX3C of about 30 to 100 nM and a Kd for ciguatoxin CTX1B of about 1 to 10 μM.

  The above monoclonal antibody can be obtained by immunizing an animal with the protein conjugate of the above formula (I). As a method for immunizing an animal, a known method can be used. Moreover, as an animal, the animal used in a normal immunological method can be used, for example, a goat, a rabbit, a mouse etc. are mentioned, A mouse is used preferably.

  The monoclonal antibody of the present invention is considered to recognize and bind to the HIJKLM ring part of ciguatoxins. Using another monoclonal antibody that recognizes a site different from the site of ciguatoxins recognized by this monoclonal antibody in combination with the monoclonal antibody of the present invention, a kit for detecting ciguatoxins with high sensitivity by the sandwich method is obtained. be able to. Here, the sandwich method refers to a method in which two or more antibodies are used, one antibody is immobilized, an antigen is bound to the antibody, and another antibody is bound to the antigen.

  Therefore, the kit for detecting ciguatoxins of the present invention comprises the monoclonal antibody of the present invention and other monoclonal antibodies that recognize a site different from the site of ciguatoxins recognized by the monoclonal antibody.

As the other monoclonal antibody, a monoclonal antibody produced by hybridoma 10C9 deposited under the deposit number FERM BP- 8292 can be preferably used. This monoclonal antibody was produced by the present inventors as a monoclonal antibody that can specifically bind to ciguatoxins using the ABCDE ring part of ciguatoxins as a synthetic hapten (see JP-A-2003-267978).

In the detection kit for ciguatoxins of the present invention, either the monoclonal antibody of the present invention or the other monoclonal antibody is preferably labeled. The label may be any label that is usually used in immunological techniques.For example, an enzyme label (for example, peroxidase, alkaline phosphatase, β-galactosidase, etc.), a fluorescent label (for example, fluorescein isothiocyanate (FITC), allophycocyanin (APC) ), Phycoerythrin (PE), carbocyanine, etc.), radiolabels (eg tritium ( ³ H), ¹²⁵ I, ¹³¹ I, etc.) and the like. In the present invention, it is more preferable to use an enzyme label.
As a method for labeling a monoclonal antibody, a known method can be used, and a commercially available kit for labeling antibodies can also be used.

  The compound of the above formula (I) can be obtained from the compound of the above formula (II). As a method for obtaining a compound of the formula (I), for example, a compound of the formula (II) and an activator are reacted, and the following formula (V):

(式中、-O-Lは活性化剤に由来する脱離基を表す)
で表される中間体を得て、これをキャリアータンパク質と反応させる方法を挙げることができる。

(Wherein -OL represents a leaving group derived from the activator)
An intermediate represented by the following can be obtained and reacted with a carrier protein.

  The activator may be any compound that is usually used in preparing protein conjugates. For example, N, N′-dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride And carbodiimides such as 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide meth-p-toluenesulfonate.

  By substituting -O-L with a carrier protein in the intermediate of the above formula (V), the protein conjugate of the above formula (I) can be obtained. The compound of formula (I) can be used as a protein conjugate having a moiety of formula (II) as a synthetic hapten.

  The carrier protein may be any one that is usually used as a carrier to be bound to a hapten in an immunological technique, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg albumin (OVA), Examples thereof include fibrinogen, and BSA, KLH, and OVA are preferable.

  As a method of reacting the intermediate of the above formula (V) with the carrier protein, a known method can be used. For example, a method of reacting the intermediate of the above formula (V) in a carrier protein solution can be mentioned.

The compound of the above formula (II) is a novel compound, which is also one aspect of the present invention.
The compound of formula (II) can be obtained by the following method 1.
The compound of formula (II) has the following formula (III):

And a compound of formula (IV):

The diastereomeric mixture which consists of a compound represented by these may be sufficient.
This diastereomeric mixture can be resolved by high performance liquid chromatography (HPLC) using normal phase, reverse phase columns. For example, a silica gel column or an octadecyl silica gel column can be used.

Hereinafter, the present invention will be described in more detail by describing embodiments of the compound, protein conjugate, monoclonal antibody, hybridoma, and ciguatoxin detection kit of the present invention. However, the present invention is limited to the following embodiments. It is not a thing.
A. Preparation of Compound of Formula (II) The compound of formula (II) described above was synthesized as a diastereomeric mixture of formula (III) and formula (IV) by Method 1 below.

Method 1

  Where TBAF represents tetra-n-butylammonium fluoride, THF represents tetrahydrofuran, MeOH represents methanol, TBPS represents (tert-butyldiphenylsilyl), TIPS represents (triisopropylsilyl), NAP Represents (naphthylmethyl).

In Method 1 above, Compound 5 was obtained by the method described in M. Inoue et al. J. Org. Chem. 2004, 69, 2797-2804.
TBAF (116 μL, 1.0 M THF solution, 116 μmol) was added to a THF solution (0.5 mL) of compound 5 (14.3 mg, 11.6 μmol) at room temperature. The reaction solution was stirred at room temperature for 5 hours, and then the solvent was distilled off. The residue was purified by ODS (octadecylsilyl) column (ODS Merck RP18, eluent: methanol: H ₂ O = 20: 1 to 100% methanol) to obtain compound 6 (4.5 mg, yield: 93%).

¹ H NMR (500 MHz, CDCl ₃ ) δ 7.84-7.77 (8H, m, NAP), 7.52-7.43 (6H, m, NAP), 4.82 (1H, d, J = 12.0 Hz, NAP), 4.75 (1H , d, J = 12.0 Hz, NAP), 4.62 (1H, d, J = 12.5 Hz, NAP), 4.58 (1H, d, J = 12.5 Hz, NAP), 4.27 (1H, m, H51), 3.98 ( 1H, dd, J = 9.5, 2.5 Hz, H52), 3.86 (1H, ddd, J = 11.5, 9.5, 4.5 Hz, H41), 3.84 (1H, dd, J = 9.5, 5.0 Hz, H52), 3.77 ( 1H, dd, J = 12.0, 4.5 Hz, H31), 3.62 (1H, d, J = 9.0 Hz, H45), 3.55 (1H, d, J = 10.5 Hz, H29), 3.47 (1H, d, J = 10.5 Hz, H29), 3.44 (1H, d, J = 3.0 Hz, H44), 3.41 (1H, dd, J = 9.0, 9.0 Hz, H46), 3.35 (1H, ddd, J = 12.0, 12.0, 3.0 Hz , H34), 3.12 (1H, ddd, J = 12.0, 12.0, 5.0 Hz, H33), 3.10 (1H, ddd, J = 11.5, 11.5, 4.5 Hz, H39), 2.99 (1H, ddd, J = 11.5, 11.5, 3.0 Hz, H38), 2.85 (1H, dd, J = 9.5, 4.5 Hz, H42), 2.28 (1H, ddd, J = 11.5, 4.5, 4.5 Hz, H40), 2.19 (1H, m, H43) , 2.16 (1H, m, H50), 2.15 (1H, m, H50), 2.13 (1H, m, H32), 1.87 (1H, m, H36), 1.84 (1H, m, H37), 1.75 (1H, m, H35), 1.67 (1H, ddd, J = 12.0, 12.0, 12.0 Hz, H32), 1.61 (1H, m, H37) , 1.55 (1H, m, H47), 1.50 (1H, m, H48), 1.44 (1H, m, H35), 1.42 (1H, ddd, J = 11.5, 11.5, 11.5 Hz, H40), 1.13 (3H, s, H53), 1.11 (3H, d, J = 8.0 Hz, Me55), 1.06 (3H, d, J = 6.0 Hz, Me56), 1.05 (3H, d, J = 7.0 Hz, Me54), 1.03 (3H , d, J = 6.5 Hz, Me57)

To a CH ₂ Cl ₂ solution of compound 6 (5.2 mg, 6.21 mmol), (MeO) ₂ CH ₂ CO ₂ Me (46 mg, 310 mmol) and a catalytic amount of p-toluenesulfonic acid monohydrate (p -TsOH · H ₂ O) was added. The reaction solution was stirred at room temperature for 8 hours, and 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 a silica gel column (eluent: hexane: ethyl acetate = 10: 1 to 3: 1) to obtain Compound 7 and Compound 8 as a 2: 1 mixture (3.9 mg, yield). (Rate: 68%).

To a methanol solution of a mixture of compound 7 and compound 8 (1.5 mg, 1.63 μmol), Pd (OH) ₂ / C (1 mg) was added and stirred for 36 hours in a hydrogen atmosphere. After filtration through celite, the solvent was distilled off. Hexane was added to the residue and extracted twice with methanol. The combined methanol phase was concentrated to obtain a mixture of compound 9 and compound 10 (1.1 mg, yield: 100%).

¹ H NMR (500 MHz, CDCl ₃ ) δ 5.00 (0.5H, t, J = 5.0, 5.0 Hz), 5.00 (0.5H, t, J = 5.0, 5.0 Hz), 4.50 (1H, m), 3.88 ( 1H, dd, J = 10.0, 4.5 Hz), 3.81-3.62 (6H, m), 3.70 (3H, s), 3.48 (2H, m), 3.38-3.23 (4H, m), 3.15 (1H, m) , 2.90 (1H, m), 2.86 (1H, dd, J = 9.5, 5.0 Hz), 2.71 (2H m), 2.27-1.95 (5H, m), 1.86-1.39 (9H, m), 1.24 (3H, s), 1.14 (3H, d, J = 7.5 Hz), 1.06 (3H, d, J = 6.0 Hz), 1.05 (3H, d, J = 7.5 Hz), 1.01 (3H, d, J = 6.5 Hz)
ESI-TOFMS: Calculated for C ₃₃ H ₅₂ O ₁₂ Na (M + Na ⁺ ): 663.401, measured value: 663.336.

To a mixture of compound 9 and compound 10 (3.8 mg, 5.9 μmol) in t-butanol / H ₂ O (volume ratio 4: 1, 750 mL), LiOH (5.0 mg, 0.12 mmol) was added at room temperature, Stir for 1 hour. To the reaction solution, KHSO ₄ was added until pH 5 and then extracted with ethyl acetate (5 mL). After drying the organic phase with MgSO ₄ , the solvent was distilled off to obtain a diastereomeric mixture of the compounds of formula (III) and formula (IV).

  To a solution of the above diastereomeric mixture of formula (III) and formula (IV) in dimethylformamide (DMF) (0.3 mL) at room temperature is added N-hydroxysuccinimide (6.8 mg, 59 μmol) and 1- (3-dimethylaminopropyl). ) -3-Ethylcarbodiimide hydrochloride (EDC, 5.7 mg, 30 μmol) was added and stirred for 2 hours. To this solution, N-hydroxysuccinimide (6.8 mg, 59 μmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, 5.7 mg, 30 μmol) were added and stirred for 11 hours. Ethyl (2 mL) was added. The organic phase was washed with water and the aqueous phase was extracted with ethyl acetate (5 mL). The combined organic phases were concentrated to synthesize a diastereomeric mixture represented by the above formula (Compound 11 and Compound 12) and dissolved in DMF (200 μL).

B. Production of protein conjugate Production of keyhole limpet hemocyanin (KLH) conjugate: Compound obtained by the above method in a solution of KLH (6.6 mg) in PBS (phosphate buffered saline; 1.0 mL) A DMF solution (100 μL) of a mixture of 11 and Compound 12 was added and stirred at room temperature for 5 minutes. After standing at room temperature for 1 day, it was dialyzed against PBS (1 L) at 4 ° C. After 6 hours, PBS (1 L) was changed, and after 12 hours, the dialysis tube was transferred to an Eppendorf tube and stored at -78 ° C.

Production of bovine serum albumin (BSA) conjugate: A DMF solution (100 μL) of a mixture of compound 11 and compound 12 obtained by the above method was added to a solution of BSA (6.6 mg) in PBS (1.0 mL) at room temperature. Stir for 5 minutes. After standing at room temperature for 1 day, it was dialyzed against PBS (1 L) at 4 ° C. After 6 hours, PBS (1 L) was changed, and after 12 hours, the dialysis tube was transferred to an Eppendorf tube and stored at -78 ° C.

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 71300 (the molecular weight of BSA was about 66400). Since the molecular weight of the hapten part, which is a part derived from the compound of the above formula (II), is 608, it can be seen that an average of 8 haptens are linked to the BSA conjugate.

C. Immunization RIBI adjuvant (manufactured by RIBI Immunol. Res. Inst.) Was added to the KLH conjugate (100 μg) obtained as described above, and the mixture was stirred well to obtain an emulsion, which was then added to Balb / c mice (5 Were administered intraperitoneally 3 times every 2 weeks. One week after the third immunization, mouse serum was collected, and the antibody titer of the serum was determined by the following ELISA (enzyme-linked immunosorbent assay) method using the BSA conjugate.

ELISA method
Place 50 μL of the above BSA conjugate (2 μg / mL) in each well of a 96-well ELISA plate (COSTAR 3596), leave it at room temperature for 2 hours, and then leave it overnight at 4 ° C. to place the conjugate on the plate. Adsorbed. The plate was washed 3 times with PBS-Tween (PBS containing 5% Tween 20 [ICI Tween-20 (polyoxyethylene (20) sorbitan monolaurate) equivalent No. 167-11515]), and then MILLI-Q. Washing was performed once with water (purified water deionized using Direct-Q manufactured by Millipore using a plurality of stages of resins and filters) to remove the unadsorbed conjugate. Anti-serum (or hybridoma culture supernatant, purified antibody solution) was added to the wells, allowed to stand at room temperature for 1 hour, and then washed with PBS-Tween and MILLI-Q water three times each. 50 μL of enzyme-labeled secondary antibody (goat anti-mouse IgG-horseradish peroxidase (HRP)) (manufactured by BIO-RAD, 170-6516, diluted 1000-fold) was placed in each well, allowed to stand at room temperature for 1 hour, PBS- Each was sequentially washed 3 times with Tween and MILLI-Q water. Add 100 μL of the substrate solution (4.0 mg of 1,2-phenylenediamine, 10 μL of hydrogen peroxide, 10 mL of 0.1 M citrate buffer (pH 5.0)), and proceed with the color reaction for several minutes. The reaction was stopped at 50 μL). Absorbance at 490 nm was measured using a microplate absorbance measurement apparatus (BIO-RAD, model 680).

Measurement of antibody titer in serum A dilution series sample was prepared by serially diluting serum collected from mice from 400 times to 51200 times using PBS containing 10% fetal bovine serum (FBS). 50 μL of each diluted series sample was placed on an ELISA plate (COSTAR 3596) adsorbed with the BSA conjugate as described above, and allowed to stand at room temperature for 1 hour, and then the absorbance at 490 nm was measured in the same manner as described above. The results of plotting the logarithm of serum dilution and absorbance for 5 mice are shown in FIG.
FIG. 1 shows that antibodies in serum bind to the BSA conjugate in a serum concentration-dependent manner.

D. Removal of spleen from high antibody titer mice, cell fusion, production of monoclonal hybridoma
Three weeks after the third immunization, mice showing the highest antibody titer among the 5 mice were selected. In the abdominal cavity of this mouse, RIBI adjuvant (manufactured by RIBI Immunol. Res. Inst.) Was added to KLH conjugate (100 μg), and the mixture was stirred well to give an emulsion, and the spleen was removed 3 days later . After removing tissue or organ fragments adhering to the spleen with forceps, serum-free medium (RPMI 1640 Medium (GIBCO, 11875-185) Penicillin Streptomycin (GIBCO, 15140-122) 1000 mL 10 The sample was added to a petri dish containing 15 mL, and the 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, and pipetted well and filtered to make a total volume of 30 mL. Centrifugation was performed at 800 rpm for 5 minutes at room temperature, and the supernatant was removed and tapped.

  A tube in which myeloma cells P3X63-Ag8.653 (manufactured by Dainippon Pharmaceutical Co., Ltd.) was frozen was taken out from a liquid nitrogen cell storage container at -180 ° C, and thawed quickly in a 37 ° C constant temperature bath. After the tube was thoroughly disinfected 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, growth medium (serum-free medium with 100 mL of FBS, L-glutamine (200 mM, GIBCO, 25030-081) 10 mL, Blyclone (BioResearch Ireland BR-001) 20 mL) 10 mL was added, the cells were suspended and transferred to a 50 mL culture flask. The flask was loosened and placed in a carbon dioxide incubator. It was subcultured every 2-3 days to make two 250 mL flasks (90-100 mL).

Spleen cells removed from the mouse (2 × 10 ⁸ cells) and the above myeloma cells (5 × 10 ⁷ cells) are mixed, centrifuged (800 rpm, 5 minutes, room temperature), the supernatant is removed, I tapped it. 30 mL of ECF buffer [manitol 45.5 g, 10 mM magnesium chloride 10 mL, 10 mM calcium chloride 10 mL, 20 mM Tris-HCl (pH 7.2) 10 mL dissolved in distilled water to 1000 mL) In addition, centrifugation (800 rpm, 5 minutes, room temperature), supernatant removal, and tapping were repeated twice, and then 4.8 mL of ECF buffer was added. This was dispensed 1.2 mL into each well of a 6-well plate (SUMIRON, MS-80060), and the cells were fused using the SSH-10 cell fusion device (Shimadzu) under the following conditions: Distance between electrodes : 1.0 mm; AC frequency: 1 MHz; AC initial applied voltage: 40 V; AC initial applied 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; Number of applied pulses: 1; Pulse voltage change: + 0V; AC last applied time: 0 seconds; AC voltage decay rate: 0% Contact enhancement: off.

  The obtained hybridoma cells were mixed with (Mouse-Mouse hybridoma) in HAT medium (132 mL of serum-free medium, 40 mL of FCS, L-glutamine (200 mM, GIBCO 25030-081), Blyclone (BioResearch Ireland BR- 001) 10 mL, and HAT (hypoxanthine-aminopterin-thymidine, GIBCO 31062-011) 4 mL] were suspended in 100 mL and transferred to 10 96-well plates in 100 μL aliquots. A hybridoma containing a hybridoma 8H4 that produces an antibody that binds to the BSA conjugate of the compound of the above formula (I) and formula (II) as a hapten after culturing for about 2 weeks in a carbon dioxide culture apparatus is used as a BSA conjugate. And screened by ELISA. Positive wells were selected, and after cloning twice, monoclonal hybridomas producing antibodies that became ELISA positive again were sequentially cultured and grown to about 200 mL each. As a result, hybridomas producing three types of monoclonal antibodies (8H4, 8B9 and 7C9) were obtained.

E. Evaluation of monoclonal antibodies
Purification of antibody and determination of subclass The culture supernatants of the above three hybridomas were purified with an anti-mouse IgG + IgM affinity column (NGK Industries, Ltd.) (binding buffer: phosphate buffer pH 7.0, for elution) Buffer: 0.2 M Gly-HCl pH 2.5). The purified antibodies were analyzed by SDS-PAGE and confirmed to be 95% pure respectively. The subclass of each antibody was determined using a typing kit (PIERCE 37501). As a result, 8H4 was IgG ₁ κ, 8B9 was IgG ₁ κ, and 7C9 was IgG _2b κ.

Antibody affinity analysis (competitive inhibition experiment)
For the three antibodies purified as described above, the dissociation constant (Kd) between the antibody and the hapten was determined as follows. A solution of a competitive inhibitor (PBS solution, 30 μL each) was prepared on an ELISA plate by serial 2-fold dilution. To this was added the above antibody solution (30 μL) and left at room temperature for 2 hours. 50 μL of the antibody / inhibitor mixture was added to a 96-well ELISA plate (COSTAR 3596) adsorbed with BSA conjugate (0.625 μg / mL) and allowed to stand at room temperature for 15 minutes. After washing the plate with PBS-Tween and MILLI-Q water three times each, 50 μL of enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) (manufactured by BIO-RAD, 170-6516, diluted 1000-fold) Each well was allowed to stand at room temperature for 1 hour, and then washed with PBS-Tween and MILLI-Q water three times each. Add 100 μL of substrate solution (4.0 mg of 1,2-phenylenediamine, 10 μL of hydrogen peroxide solution, 10 mL of 0.1 M citrate buffer (pH 5.0)), and proceed with the color reaction for several minutes. The reaction was stopped at 50 μL). Using a microplate absorbance measurement apparatus (BIO-RAD, model 680), the absorbance at 490 nm was measured to obtain a titration curve. With reference to the method of Friguet et al. (Journal of Immunological Method, Vol. 77 (1985), p. 305), the Kd of the inhibitor was determined from the slope of the straight line obtained by preparing the Klotz plot.
The results when Compound 6 was used as a competitive inhibitor are shown in Table 1. From Table 1, it can be seen that all three monoclonal antibodies bind strongly to the hapten compound 6.

  Of these three monoclonal antibodies, 8H4, which had the strongest binding to compound 6, was subjected to the same inhibition experiment using ciguatoxin 51-OH-CTX3C (compound 2). It was found to show high affinity for OH-CTX3C (Kd = 75 nM). Monoclonal antibody 8H4 shows only weak affinity for ciguatoxin CTX3C (compound 1), which does not have a hydroxyl group in the M ring (Kd = 3.2 μM), and other polyether marine toxins (brevetoxins A and B, okadaic acid). And mitotoxin) showed little cross-reactivity.

F. Detection kit for ciguatoxins by sandwich method
Enzyme labeling of 8H4 antibody (synthesis of 8H4-HRP used in sandwich method)
HRP labeling of 8H4 was performed using Peroxidase Labeling Kit-NH ₂ (trade name, manufactured by Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions. That is, 100 μL of the washing buffer attached to the kit and monoclonal antibody 8H4 (0.2 mg) were placed in a Filtration Tube. After gently mixing by pipetting, the mixture was centrifuged (8000 × g, 10 minutes). 100 μL of wash buffer was added and centrifuged again (8000 × g, 10 minutes). A solution obtained by mixing and dissolving 10 μL of reaction buffer and NH ₂ -Reactive Peroxidase by pipetting was added onto the membrane of Filtration Tube in which the antibody 8H4 was concentrated. The antibody on the membrane and the solution were mixed by pipetting and then left at 37 ° C. for 2 hours. 100 μL of washing buffer was added and centrifuged (8000 × g, 10 minutes). 200 μL of storage buffer was added to dissolve the labeled antibody by pipetting. The solution was placed in a 500 μL tube and stored at 4 ° C.

A sandwich detection method for ciguatoxin
Place 50 μL / well of PBS solution (4.3 μg / mL) of monoclonal antibody 10C9 produced by hybridoma 10C9 under the accession number FERM BP- 8292 in an ELISA plate (COASTER 3590) at 4 ° C. overnight. I left it alone. The solution was discarded, PBS containing 1% skim milk was added (400 μL / well), and the mixture was allowed to stand at room temperature for 1 hour. The solution was discarded, and after washing 3 times with PBS-Tween, a diluted solution of ciguatoxin 51-OH-CTX3C (compound 2) was added (50 μL / well) and allowed to stand at room temperature for 1 hour. After discarding the solution and washing 3 times with PBS-Tween, the above HRP-labeled monoclonal antibody 8H4 solution (1 mg / mL, 50 μL) was added, and the mixture was allowed to stand at room temperature for 1 hour. Discard the solution, wash 3 times with PBS-Tween, add 100 μL of the substrate solution (4.0 mg of 1,2-phenylenediamine, 10 μL of hydrogen peroxide, 10 mL of 0.1 M citrate buffer (pH 5.0)), and add several The color reaction was allowed to proceed for 1 minute, and then the reaction was stopped with 2N sulfuric acid (50 μL). Absorbance at 490 nm was measured using a microplate absorbance measurement apparatus (BIO-RAD, model 680). The result is shown in figure 2.

  From the results shown in Fig. 2, it is possible to detect ciguatoxin 51-OH-CTX3C with high sensitivity by combining 10C9, an antibody produced using hapten with ABCDE ring part of ciguatoxins, and enzyme-labeled 8H4. I understand.

FIG. 1 is a graph showing antibody titers of sera obtained from mice immunized with a BSA conjugate according to the present invention. FIG. 2 is a graph showing the detection characteristics of ciguatoxin 51-OH-CTX3C using the detection kit according to the sandwich method of the present invention.

Claims (11)

Hybridoma 8H4 deposited under accession number FERM P-20347, which produces a monoclonal antibody that specifically recognizes ciguatoxins having a hydroxyl group at the M ring. The following formula (I):

(Where n represents an integer)
Obtained by immunizing mice with in represented by protein conjugate of claim 1 hybridoma 8H4. A monoclonal antibody that specifically recognizes ciguatoxins having a hydroxyl group at the M ring, produced by the hybridoma 8H4 according to claim 1 or 2. The monoclonal antibody according to claim 3 , wherein the ciguatoxin having a hydroxyl group at the M ring portion is ciguatoxin 51-OH-CTX3C. A kit for detecting ciguatoxins by the sandwich method, comprising the monoclonal antibody according to claim 3 or 4 and a monoclonal antibody produced by hybridoma 10C9 deposited under the accession number FERM BP- 8292. The kit for detecting ciguatoxins according to claim 5 , wherein the monoclonal antibody according to claim 3 or 4 or the monoclonal antibody produced by hybridoma 10C9 deposited under the deposit number FERM BP- 8292 is labeled. The ciguatoxin detection kit according to claim 6 , wherein the label is an enzyme label. The following formula (II):

A compound represented by The compound of formula (II) has the following formula (III):

And a compound of formula (IV):

The compound of Claim 8 which is a diastereomeric mixture of the compound represented by these. The compound of claim 8 or 9 is bound to a carrier protein and has the following formula (I):

(Where 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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