WO2003006507A1 - Method of preparing antinarcotic shellfish toxin antibody, novel antibody, elisa kit with the use of the antibody and labelled toxin sample prepared by the method - Google Patents

Method of preparing antinarcotic shellfish toxin antibody, novel antibody, elisa kit with the use of the antibody and labelled toxin sample prepared by the method Download PDF

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WO2003006507A1
WO2003006507A1 PCT/JP2002/005899 JP0205899W WO03006507A1 WO 2003006507 A1 WO2003006507 A1 WO 2003006507A1 JP 0205899 W JP0205899 W JP 0205899W WO 03006507 A1 WO03006507 A1 WO 03006507A1
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antibody
group
sulfhydryl
saxitoxin
toxin
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PCT/JP2002/005899
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French (fr)
Japanese (ja)
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Masaaki Kodama
Shigeru Sato
Kunihiro Shinagawa
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Japan Science And Technology Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to a method for producing an antibody against paralytic shellfish toxin and the antibody. Furthermore, the present invention relates to an ELISA kit using the above antibody and a labeled toxin preparation for a method for measuring paralytic shellfish toxin binding obtained by the above method. Background art
  • Paralytic shellfish poisoning poisoning presents symptoms characterized by nerve paralysis, and the causative toxins have been found to be goniotoxins and saxitoxins [Takeshi Amoto, paralytic shellfish toxins, food hygiene inspections] Guidelines (edited by RIKEN, supervised by the Ministry of Health and Welfare, Ministry of Health and Welfare), pp. 300-305, Japan Food Hygiene Association, 1999. These causative toxins are collectively referred to as paralytic shellfish poisons, and at present 20 or more gonitotoxins and saxitoxins are known as shellfish toxins found in shellfish such as mussels and anopheles.
  • This shellfish poison is a paralytic toxin that has the property of blocking sodium channels.It is contained in several types of phytoplankton belonging to the dinoflagellates, and shellfish that feed on it accumulates poison and poisons it. . Eating this shellfish causes food poisoning with high mortality, which is a major social problem in fisheries and food hygiene.
  • paralyzed shellfish poison 0.5-1.0 mg Oral ingestion causes symptoms such as sensory paralysis, nausea and diarrhea.
  • the lethal dose in humans is 1-3 mg.
  • Contaminated shellfish contaminated areas due to paralytic shellfish poisoning are expanding worldwide, and in these contaminated areas the toxicity of shellfish shipped to prevent food poisoning due to poisoned shellfish. Are tested by the mouse test method or the HPLG method. However, these measurement methods have many problems in terms of accuracy, cost and labor required for measurement, and development of a simple method for measuring poisons is desired.
  • the assay for lethal activity is as follows: injection of the mouse test solution intraperitoneally, from the moment of injection to the last gasping when the mouse dies with typical paralytic shellfish poisoning symptoms.
  • the time (lethal time) is recorded in seconds, and the amount of toxin is estimated from a dose-matching time curve made with standard poisons.
  • High-performance liquid chromatography uses a sample containing paralytic shellfish toxin. This is a method in which paralytic shellfish toxin is separated by high performance liquid chromatography, an oxidizing agent is added to the eluate, and the eluate is allowed to react in alkali, and the fluorescence of the resulting fluorescent substance is measured. Is a method that promotes Na + influx into a system in which the activity of Na-KATPase, an enzyme that excretes Na + out of the cell, is inhibited by ⁇ ⁇ abin in a neuroblast culture system.
  • a toxin that blocks the influx of Na + and infers its amount from the activity of the poison that blocks the swelling and death of cells caused by veratridine When a sample containing a poison is added, the cells are rounded and thin.
  • This method utilizes the fact that spore death is suppressed in proportion to the amount of poison.
  • the immunological method uses an antibody against paralytic shellfish toxin to directly measure the amount of antibody that binds to the toxin, for example, by an enzyme immunoassay (for example, ELISA), etc. This is a method for quantifying paralytic shellfish poison.
  • FS Ghu et al. reported that parasitism shellfish poisons were detected by ELISA using the anti-saxitoxin antibody nosaxitoxin-horseradish peroxidase conjugate or the anti-neosaxitoxin antibody Z neosaxitoxin-one horseradish peroxidase conjugate. It discloses Atsushi's method of measuring total amounts. Antibodies were prepared according to FS Ghu and X. Fan, J. Assoc. Off. Anal. Chem., 1985, 68: 13-16 and FS Chu, J. AOAC Int. 1992, 75: 341-345. The procedure is the same as that of Johnson et al.
  • a polyclonal antibody is obtained by subcutaneously injecting conjugates obtained by reacting leishimpet (Limpet) hemocyanin with saxitoxin or neosaxitoxin in a weakly acidic aqueous solution containing formalin.
  • the antigens used for the production of these antibodies are those in which a protein is bound to two guanidium groups present in the molecule of saxitoxin via a formalin [Johnson et al., Chu; And Fan, supra; V. Renz and G. Terplan, Arch, Lebensmitter I hyg., 1988, 39, 25-56; A. Cembe IIa, etc., "Specificity and cross-react ivity of an adsorption-inhibition. enzyme- I inked immunoassay for the detection of paralytic shellfish tox ins. In Toxic Marine Phytop I ankton "(E. Granel i et al.), Elsevier Science Pub Iisers, New York, pp.
  • the lethal activity measurement method and the neuroblast cell method have problems in animal and cell management, detection sensitivity, accuracy, specificity, etc., and the high-performance liquid chromatography method is easy to operate. Although it has high accuracy, it has the drawbacks of requiring expensive equipment, different sensitivity depending on the type of narcotic shellfish poison, and lack of supply of a purified external standard.
  • the immunological method has a problem in that the reactivity of the antibody differs depending on the type of saxitoxin derivative. Therefore, there has been a demand for the development of a simple and accurate method for narcotic shellfish poison that can be measured with a constant sensitivity regardless of the type of shellfish poison.
  • the inventors of the present invention used the narcotic shellfish toxin daltaotin (PSP-GSH) complex (Japanese Patent Application No. 10-91797) as a hapten and converted it into a protein molecule.
  • PSP-GSH narcotic shellfish toxin daltaotin
  • a polyclonal antibody reacting paralytic shellfish toxin with respect to all components was obtained and disclosed (JP-A-2000-344799).
  • the binding amount of hapten to protein molecules is small, the anti-titer of the obtained antiserum is low, and the binding method is required to introduce the poison into molecules such as proteins. Improvement was deemed necessary.
  • a first object of the present invention is to provide a method for producing an antibody against paralytic shellfish toxin using a binding method with the above-mentioned drawbacks for introducing a poison into another molecule such as a protein.
  • a second object of the present invention is to provide an antibody prepared using the improved binding method according to the present invention.
  • a third object of the present invention is to provide a novel ELISA kit using the antibody of the present invention.
  • a fourth object of the present invention is to provide a method for producing a labeled poison sample for use in a simple measurement method for paralytic shellfish poison, which is prepared using the improved binding method of the present invention, and a labeled poison standard prepared by this method. In the provision of goods. Disclosure of the invention
  • the present inventors prepared a toxic derivative having a free sulfhydryl group using a dithiol compound having a plurality (two) of sulfhydryl groups, and converted this derivative into a sulfhydryl group-directed functional group.
  • a poison and a protein are separately modified, and an antigen is obtained by a reaction between the two.
  • the antigen according to the present invention has a large amount of hapten per protein molecule, the inventors have found that an antiserum having a high antibody titer can be obtained and the antibody can be obtained in high yield.
  • the kit for ELISA poison determination can be used. Can be provided.
  • a poison can be efficiently bound to a carrier such as a resin or a fluorescent substance.
  • a carrier such as a resin or a fluorescent substance.
  • the toxicity is slightly reduced.However, since the active site of the toxin of the antigen according to the present invention is exposed, it is possible to prepare a labeled toxin with the toxicity remaining. .
  • FIG. 1 shows the structural formula and specific toxicity of paralytic shellfish poison (PSP).
  • PSP paralytic shellfish poison
  • FIG. 2 shows a method for producing an antigen (BSA-EDT-PSP conjugate) in the present invention.
  • FIG. 3 is a graph showing the change in the antibody titer of a rabbit that has been immunized with the BSA-E ding-STX conjugate of the present invention.
  • FIG. 4 is a graph showing the amount of PSP absorbed by antiserum.
  • FIG. 5 is a graph showing the reaction of each PSP component to an anti-STX antibody on an STX solid phase plate.
  • FIG. 6 is a graph showing the reaction of components other than PSP to an anti-STX antibody on an STX solid phase plate.
  • FIG. 7 shows the structure of a non-isotope-labeled PSP derivative according to the present invention.
  • FIG. 8 is a graph showing separation of STX (PAEM-EDT-STX) with a fluorescent label on Bio-Gel P-2 column chromatography.
  • FIG. 9 is a graph showing the specific toxicity of each PSP component, PSP-thiol complex and labeled PSP derivative.
  • sulfhydryl is added at position 11 of saxitoxin.
  • a dithiol compound having a plurality of (two) groups is bonded to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group, while a protein in which a sulfhydryl group-directing functional group is introduced into an amino group.
  • a method for producing an antibody against paralytic shellfish poison is provided.
  • the above-mentioned dithiol compound having a sulfhydryl group is preferably selected from the group consisting of 1,2-ethanedithiol, dithiothreitol, 2,3-dimenolecapto1-1-pronone dithioerythritol sulfonate.
  • the compound having a sulfhydryl group-directing functional group is preferably selected from the group consisting of maleimide, thiophthalimide, and an active halogen compound.
  • a dithiol compound having a plurality (two) of sulfhydryl groups at the 11-position of saxitoxin is bonded to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group.
  • a labeled compound such as agarose or biotin into which a thiol-directing functional group has been introduced, a fluorescent substance, a gold colloid, or the like, is reacted with this derivative, and the labeled toxin preparation for use in the measurement of simple binding of anesthetic narcotic toxin.
  • a labeled poison sample using the method for producing a labeled poison sample is non-isotope biotin-labeled and fluorescent-labeled samples, and their production methods are shown in FIG.
  • any functional group having a free sulfhydryl group directivity in particular, maleimide, thiophthalimide, active halogen and the like can be quantitatively converted into a thiol group under mild conditions in a neutral aqueous solution.
  • the narcotic shellfish toxin can be easily obtained in a much higher yield and easily than the binding method using the PSP-GSH complex or other amide condensation. It can even bind proteins and labeled compounds.
  • the saxitoxin-EDT complex is a mixture of goniotoxin (gonitoxin 2, 3 or a combination thereof) and EDT having a dithiol group. When heated (approximately 70 ° C), the goniotoxin is converted to goniotoxin via the EDT molecule. It can be obtained by a substitution reaction with the oso 3 — group at the 11-position of toxins.
  • this complex is a so-called hapten antigen, ie a substance that binds to the antibody but has no ability to elicit the antibody.
  • hapten antigen can be used as an immunogen by covalently binding a protein to a large molecule, but the protein of the present invention also has a similar function. is there.
  • An example of such a protein is albumin limpet hemocyanin (KLH).
  • the present invention also provides, in the second embodiment, any of the paralytic shellfish toxin components saxitoxin, goniotoxin 2,3, neosaxitoxin, goniotoxin 1,4, goniotoxin 5, and C toxin 1,2 It provides an antibody against paralytic shellfish that exhibits a relative reactivity of 50% or more (reactivity with saxitoxin is 100%).
  • Antibodies of the present invention include paralytic shellfish poisons such as IN-H derivatives (saxitoxin, goniotoxin 2,3), IN-0H derivatives (neosaxitoxin, goniotoxin 1,4), Sulfate derivative (gonio toxin 5, and all of the C toxins 1, 2) can be recognized almost in the same way.
  • expressions such as goniotoxin 2 and 3J mean a mixture of goniotoxin 2 and goniotoxin 3.
  • the antigen used in the production of the antibodies of the present invention is saxitoxin-EDT-protein complex.
  • This complex can be produced as follows.
  • a raw material for the saxitoxin portion goniotoxin 2, goniotoxin 3 or a mixture of any ratio thereof can be used ( these raw materials are based on 0.1N hydrochloric acid of shellfish such as mussels, hamadari and scallop).
  • the heated extract is treated with activated carbon, gel filtration chromatography (using Bio-GelP-2 (Bio-Rad)), ion-exchange chromatography (Bio-Rex70 (Bio-Rad)), etc. And can be purified.
  • the SH group of EDT changes the carbon atom at position 11 of the goniotoxin due to its electron donating property. and nucleophilic attack, resulting 0s0 3 - group is disengaged, the carbon atom of the 1 position 1 - S- (EDT) is attached, this Yotsute Sakishi butoxy Hmm EDT complex is produced.
  • the reaction takes place by heating the reactants under neutral conditions. Generally ⁇ 6 ⁇ 5 ⁇ 7 ⁇ 5, preferably suitable buffer [rho Ita7.0 ⁇ 7.5, for example phosphate Anmoniumu buffer, the reaction may be carried out using acetic acid Anmoniumu buffer solution.
  • the reaction temperature is generally room temperature. From about 100 ° G, preferably from 50 ° G to 80 ° G, more preferably about 70 ° C.
  • the desired product is purified by preparative high-performance liquid chromatography, gel filtration chromatography, ion exchange chromatography, adsorption chromatography, reverse phase distribution chromatography, etc. It can be performed alone or in combination and purified.
  • the product can be identified by ordinary measurement methods such as infrared spectroscopy, mass spectrometry, R-method, elemental analysis, and amino acid analysis.
  • Compounds with low resolution belong to hapten, and have no antigenicity per se, but a complex that covalently binds to antigenic protein as a carrier is a specific antibody against hapten.
  • Antigen It is thought that when a hapten-protein complex is injected by immunization, the hapten portion is recognized as an antigenic determinant, and an anti-hapten antibody is produced. In this case, it is necessary to prepare a hapten antigen by a selective binding method that does not easily form an extra antigenic determinant. Recognition of the hapten partial structure is weak near the binding site between the hapten and the protein serving as a carrier, and it is easy to recognize a distant partial structure.
  • the saxitoxin-e ding complex Japanese Patent Application No. 10-917907 having a structure in which an EDT group is bonded to the 11-position of saxitoxin previously discovered by the present inventors is regarded as an immunizing antigen. Not only is it preferred, but it also has the advantage of producing paralytic shellfish toxins that can detect almost all types of paralytic shellfish toxins with high reactivity.
  • albumin, hemocyanin and the like can be used, but not limited thereto, and any protein capable of inducing the antibody of the present invention can be used.
  • the EDT portion of the complex is used to combine the carrier protein (with a reactive functional group introduced) with the maleimide method ( ⁇ ⁇ Kita gawa et al., Biosh em. 92: 585-590, 1982) or by the azolevodiimide method (D. Exley, FEBS Lett., 91: 162-165, 1978), etc.
  • a complex can be formed.
  • saxitoxin-EDT-bovine serum albumin complex For example, in the examples described below, an example of producing a saxitoxin-EDT-bovine serum albumin complex will be described.
  • a saxitoxin-EDT conjugate is first produced, and albumin is converted via its free amino group.
  • the carrier protein is bound to saxitoxin-EDT by mercaptosuccinylation and reaction of the two.
  • the obtained complex can be purified by appropriately combining conventional protein purification methods. Such methods include, for example, salting out, solvent precipitation, gel filtration chromatography, ion exchange chromatography, HPLC, affinity chromatography, electrophoresis, chromatofocusing, limiting External filtration and the like are included.
  • a neosaxitoxin-EDT complex is obtained.
  • a neosaxitoxin-EDT-protein complex can be obtained by reacting the complex with a carrier protein in the same manner as described above, and the resulting product is saxitoxin-EDT-protein.
  • the protein complex it can be used to produce antibodies against paralytic shellfish toxins (see below).
  • An antibody to the saxitoxin-EDT-protein complex of the present invention can be prepared by immunizing an animal such as a mouse, a rat, a rabbit, a goat, or the like.
  • the immunogen solution is prepared using Freund's complete adjuvant, incomplete adjuvant, livia adjuvant system (RIBII MUNOCH EM RES EARCH IN NC), muramirirepeptide (Calzyme), hydroxylamine aluminum,
  • the mixture is emulsified and mixed with an adjuvant such as lipopolysaccharide, injected into animals subcutaneously, intradermally, or intramuscularly. The same procedure is repeated at intervals of 2 to 4 weeks, and booster immunization is performed several times.
  • the amount of antigen varies depending on the type of animal to be challenged with the antigen, and is, for example, 10 ig to several mg for a heron, and several jug to several 10 mg for a mouse.
  • the antiserum against paralytic shellfish toxin obtained as described above can be further purified to an IgG antibody by, for example, treating it with ammonium sulfate fractionation and then ion-exchange chromatography.
  • antibodies can be purified by affinity chromatography using a gel in which saxitoxin is chemically bound to a dextran gel or agarose gel. For production and purification of antibodies, see, for example, Introduction to Academic Studies ”(1 982).
  • an increase in antibody titer is observed from about one month after immunization, and in the case of antisera against saxitoxin, about 0.5 months after the antiserum binds 0.5 nmol of saxitoxin per ml of antiserum A titer is obtained.
  • the antiserum had a slightly lower reaction with neosaxitoxin, but the sera 5 months after immunization showed little difference in reactivity with both shellfish toxins.
  • Fig. 2 shows the reactivity of sera 4 and 5 months after immunization with various components of paralytic shellfish toxin.
  • the antibody of the present invention immunologically reacts with almost all components of paralytic shellfish toxin, and particularly, saxitoxin, goniotoxin 2, 3, neosaxotoxin, and goniotoxin, which are paralytic shellfish toxin components.
  • the saxitoxin-EDT.-protein complex of the present invention As an antigen, it is also possible to prepare a monoclonal antibody by an ordinary method. Specifically, after immunizing a rodent such as a mouse rat with the complex, the spleen is aseptically removed, spleen cells are prepared, fused with myeloma cells, and the hybridoma is transformed into a HAT medium. Or the like, and subcultured in an animal cell culture medium, or transplanted and cultured in the abdominal vagina of the rodent, and the monoclonal antibody can be collected from ascites.
  • Antibodies obtained as described above can be used to measure shellfish toxin in specimens of foods etc. suspected of containing paralytic shellfish toxin. ⁇ Measurement should be performed using a conventional antigen-antibody reaction.
  • a solid phase method or a homogeneous method, a competitive method or a non-competitive method, a sandwich method, or the like can be used.
  • enzymes such as alkaline phosphatase, 1 25 teeth 82 radioactive isotopes such ⁇ Fluorescent substances such as FITG and chemiluminescent substances such as acridium can be used.
  • Et al of the antibodies of the present invention can scan Trang resin (e.g. S e phad ex TM acids) activated with cyanogen bromide or the like, Agarosu resin (e.g. B i o-Ge M acids), a resin etc. Isolate and remove problematic shell poison when combined Column carrier for use.
  • Trang resin e.g. S e phad ex TM acids
  • Agarosu resin e.g. B i o-Ge M acids
  • GTX gonyautox in
  • saxi toxin group saxi toxin group
  • G-toxin group PSP components Fig. 1
  • GTX1 and GTX4, GTX2 and GTX3, and C1 and G2 are each an equilibrium mixture with a molar ratio of about 3: 1 (abbreviated as GTX1 + 4, GTX2 + 3, and C1 + 2, respectively).
  • GTX1 +4 at 50 / mo I was dissolved in 50 mL of 50m EDT-1 Om sodium phosphate buffer (pH 7.4) containing 20% THF and allowed to stand at room temperature for 2 days.
  • EDT was removed by extracting with ethyl ether three times, and ether remaining in the aqueous phase was distilled off under reduced pressure at 30% to obtain a reaction mixture containing EDT-neoSTX.
  • This was added to a Bio-Gel P-2 column (1.5 x 15 cm), the column was washed with 200 mL of water, and the adsorbed components were eluted with 0.1 M acetic acid.
  • the EDT-neoSTX complex was isolated while quantifying the GTX1, 4, neoSTX and EDT-neoSTX concentrations in each fraction by HPLG fluorescence.
  • EDT-STX was prepared and separated by the same treatment using 50 imol of GTX2 + 3 as a starting material.
  • the BSA-STX conjugate was diluted with PBS to a concentration of 500 ⁇ g / mL. Emulsion mixed with an equal volume of adjuvant was injected intradermally into two egrets (STX-BSA-1, STX-BSA-2) once or twice a month at 1 mL doses. BSA-neoSTX conjugate Similarly, I was immunized with 233 Great Egret (neoSTX-BSA-1 and neoSTX-BSA-2). The antibody titer of the serum obtained by collecting blood from the rabbit during the immunization was determined according to the following procedure.
  • the plate was washed three times with PBST, and 100 L of HRP-labeled secondary antibody diluted 1000-fold with PBS (-) was added. After shaking at 37 ° G for 1 hour, the plate was washed four times with PBST, and a chromogenic substrate solution (0PD-H202) was added at 100 / L. After shaking at 37 ° C for 30 minutes, the reaction was stopped by adding 100 L of 0.5N sulfuric acid, and the absorbance (0D) at 492 nm was measured using a microplate reader.
  • the filtrate obtained using an ultracentrifugal filtration kit (Ultrafree-MC, NL 5000, MiI Ipore) was analyzed by the HPLG fluorescence method.
  • the plate was washed three times with PBST, and 100 L of HRP-labeled secondary antibody diluted 1000-fold with PBS (-) was added. After shaking at 37 ° C for 1 hour, the plate was washed four times with PBST, and 100 L of a chromogenic substrate solution was added. After shaking at 37 ° G for 30 minutes, the reaction was stopped by adding 100 iL of 0.5N sulfuric acid, and the absorbance (0D) at 492 nm was measured using a microplate reader. The test was performed in triplicate.
  • PSP in the test solution was quantified by the fluorescence method of HPLC (0shima, 1995).
  • the amount of conjugated PSP in the solution, ie, the thiol-PSP complex such as EDT-STX, and the amount of PSP bound to the protein can be determined by dividing a part of the solution with 0.1 M sodium phosphate containing
  • the STX group obtained by diluting with buffer buffer (PH7.4), boiling for 5 minutes, and analyzing by HPLC fluorescence method, was compared with the sum of the GTX group and STX group before mercaptoethanol treatment. Calculated.
  • a BSA-EDT-STX conjugate and a BSA-EDT-neoST.X conjugate prepared by the same procedure were prepared according to the procedure shown in FIG. Previously, we created an antigen by introducing a glutathione-PSP complex into BSA, but this time, a new EDT-mediated conjugation method allows a larger number of PSP molecules to be antigen-transfected. (Table 1). BSA concentration in antigen solution and amount of PSP bound to BSA Protein in antigen solution PSP: BSA
  • BSA-EDT-STX (new antigen) 1497 5.4 2.3
  • BSA-GS-STX 684 1.0
  • BSA-GS-neoSTX 788 0.1>
  • FIG. 3 shows the change in the antibody titer of Egret (STX-EDDING-BSA-1 and STX-EDT-STX-2) immunized with the BSA-EDT-STX conjugate.
  • a significant increase in the antibody titer was observed in both of the two egrets three months after the start of immunization, and the antibody titer after four months continued to increase and decrease.
  • the antibody titers of the rabbits immunized with the NeoSTX-EDT-STX conjugate changed in a similar manner, but the increase in the antibody titer was lower than that immunized with the BSA-EDT-STX conjugate. All of the magpies were bled 7 months after the start of immunization, and the serum obtained was used for the following tests.
  • FIG. 4 shows the amount of each PSP component absorbed by the serum obtained from BSA-EDT-STX-1 and the serum obtained from BSA-neoSTX-1.
  • the serum of unimmunized egrets contained less than 0.1 nmo I of each PSP component per mL, which was below the detection limit.
  • 1 mL of BSA-STX-1 serum absorbed 1 to 3 nmol of PSP.
  • the same method can be used to detect any of the components with 10 to 100 times the sensitivity of the re-mouth test.
  • the antibody did not react at all with biological components partially similar in structure to PSP, such as adenosine (Ade) having a purine skeleton, force phain (Gaf), and arginine (Arg) having a guanidyl group (No. 6). Figure).
  • adenosine (Ade) having a purine skeleton
  • force phain Gaf
  • Arg arginine
  • crossing was observed at a high concentration of the pufferfish tetrodotoxin (TTX), which has the same pharmacological effect on the BSA-STX-1 antibody as PSP.
  • TTX pufferfish tetrodotoxin
  • the polyclonal antibody prepared using BSA-EDT-STX as an antigen according to the present invention had a slightly lower affinity for neoSTX, but showed almost the same affinity for other paralytic shellfish poison components.
  • the ELISA prepared as a test using this antibody was able to detect toxins with a sensitivity 10 to 100 times higher than that in the mouse test, although the values differed slightly depending on the toxic components.

Abstract

A method of preparing an antibody against a paralytic shell fish toxin which comprises bonding an arbitrary dithiol compound having a plural number of sulfhydryl groups to the 11-position of saxitoxin to give a saxitoxin sulfhydryl derivative having free sulfhydryl groups, separately preparing a protein having an arbitrary sulfhydryl-targeting functional group introduced into an amino group, then reacting the saxitoxin sulfhydryl derivative with the modified protein, immunizing a nonhuman animal with the antigen obtained by the reaction, and then obtaining an antiserum recognizing the paralytic shellfish toxin from the animal; an ELISA kit with the use of the above antibody; and a labeled toxin sample prepared by the above method. Thus, an antinarcotic shellfish toxin can be conveniently and accurately assayed at a constant sensitivity regardless of the shellfish toxin type.

Description

明細書 抗麻酔性貝毒抗体の製法、新規抗体、該抗体を用いる EL I SA測定キッ 卜、 該製法による系標識毒標品 技術分野  Description Production method of anti-narcotic shellfish toxin antibody, new antibody, ELISA kit using the antibody, system-labeled venom sample by the production method
本発明は、 麻痺性貝毒に対する抗体の製法およびその抗体に関する。 さらに本発明は、 上記抗体を用いる EL I SA 測定キッ トおよび上記製法に よリ得られる麻痺性貝毒結合測定法用標識毒標品に関する。 背景技術  The present invention relates to a method for producing an antibody against paralytic shellfish toxin and the antibody. Furthermore, the present invention relates to an ELISA kit using the above antibody and a labeled toxin preparation for a method for measuring paralytic shellfish toxin binding obtained by the above method. Background art
麻痺性貝毒中毒は神経麻痺を主徴とする症状を呈し、 その原因毒はゴ ニォ トキシン類およびサキシ トキシン類であることが判明している [安 元健、 麻痺性貝毒、 食品衛生検査指針 （理化学編、 厚生省生活衛生局監 修） 、 第 300— 305頁、 日本食品衛生協会、 1 991 年]。 これら原因毒は麻 痺性貝毒と総称され、 現在、 ィガイ、 ハマダリ等の貝類に見出される貝 毒と してゴニォ トキシン類、サキシ トキシン類で 20種以上が知られてい る。  Paralytic shellfish poisoning poisoning presents symptoms characterized by nerve paralysis, and the causative toxins have been found to be goniotoxins and saxitoxins [Takeshi Amoto, paralytic shellfish toxins, food hygiene inspections] Guidelines (edited by RIKEN, supervised by the Ministry of Health and Welfare, Ministry of Health and Welfare), pp. 300-305, Japan Food Hygiene Association, 1999. These causative toxins are collectively referred to as paralytic shellfish poisons, and at present 20 or more gonitotoxins and saxitoxins are known as shellfish toxins found in shellfish such as mussels and anopheles.
この貝毒はナ ト リウムチャネルをブロックする性質を有する麻痺性毒 であり、 渦鞭毛藻に属する植物プランク トンの数種類に含まれ、 これを 餌と して食べた貝が毒をため込み毒化する。 この貝を食すると死亡率の 高い食中毒を引き起こすため、 水産および食品衛生上大きな社会問題に なっている。 因みに、 麻痺性貝毒 0. 5〜 1 . 0 m g経口摂取しただけで感覚 麻痺、 悪心、 下痢などの症状がでる。 ヒ トにおける致死量は 1 〜 3 m g である。 麻痺性貝毒による貝類の汚染地域は世界的に拡大傾向にあり、 これらの汚染地域では毒化貝による食中毒防止のため出荷する貝の毒性 をマウス試験法あるいは HPLG法によ り検査している。 しかしこれらの測 定法は精度、 測定にかかる費用や労力の点で多く の問題があり、 毒の簡 易測定法の開発が望まれている。 This shellfish poison is a paralytic toxin that has the property of blocking sodium channels.It is contained in several types of phytoplankton belonging to the dinoflagellates, and shellfish that feed on it accumulates poison and poisons it. . Eating this shellfish causes food poisoning with high mortality, which is a major social problem in fisheries and food hygiene. By the way, paralyzed shellfish poison 0.5-1.0 mg Oral ingestion causes symptoms such as sensory paralysis, nausea and diarrhea. The lethal dose in humans is 1-3 mg. Contaminated shellfish contaminated areas due to paralytic shellfish poisoning are expanding worldwide, and in these contaminated areas the toxicity of shellfish shipped to prevent food poisoning due to poisoned shellfish. Are tested by the mouse test method or the HPLG method. However, these measurement methods have many problems in terms of accuracy, cost and labor required for measurement, and development of a simple method for measuring poisons is desired.
このため麻痺性貝毒を定量する方法が種々開発されている。 たとえば 公定法であるマウスを用いた致死活性測定法 [安元健、前掲； F. Jel lett 等 、 Toxicon, 30 (10) ： 1143-1156 (1992) ； W. Horwitz, Paralytic she I I f i sh po i son. In Official Methods of Analysis of the Association of Official Analytical Chemists" (Assoc. Official Anaに chem. , Washington D. C. ) pp. 881-882 (1990)]、 高速液体ク ロマ トグラフィー法 [ Y. Osh ima 等、 Mycotox i ns and Phycotoxins ' 88 (S. Nator i 等編）， pp.319-326, Elsevier, Amsterdam (1989) ] ；特開平 9 - 133669号公報] 、 神経芽細胞を用し、る方法 [K. Kogure等， Toxicon, 26: 191 - 197 (1989) ]、 抗体を用しゝる方法 [F. S. Ghu等， J. Agri. Food Chem. , 44： 4043-4047 (1996)] などが知られている。 致死活性による測定法は、 マウス試験原 液を腹腔内に注射し、 注射の終了 した瞬間からマウスが典型的な麻痺性 貝毒による症状を示して死亡する際の最後のあえぎまでの時間 （致死時 間） を秒単位で記録し、 標準毒で作製した用量一致時間曲線から毒量を 推定する方法である。 高速液体ク ロマ トグラフィー法は、 麻痺性貝毒を 含む試料を高速液体ク ロマ 卜グラフィ一にかけて麻痺性貝毒を分離し、 溶出液に酸化剤を加えてアルカ リ 中で反応させた後、 生じた蛍光性物質 の蛍光を測定する方法である。 神経芽細胞を用いる方法は、 神経芽細胞 の培養系において、 N a +を細胞外に排出する酵素 N a - K A T P a s e の活性をゥアバイ ンで阻害した系に N a +流入を促進するベラ ト リ ジ ンと N a +の流入を阻止する毒を持抗させ、 ベラ ト リ ジンによ り起こる 細胞の膨潤死を阻止する毒の活性からその量を推定するもので、 このァ ッセィ系に麻痺性貝毒を含む試料を添加したときには細胞の丸ま り と細 胞死が毒量に比例して抑制されることを利用した方法である。 さらに、 免疫学的方法は、 麻痺性貝毒に対する抗体を用いて、 毒に結合する抗体 量を、 例えば酵素免疫測定法 （例えば ELISA) 等の方法で直接測定し、 そ の抗体量から試料中の麻痺性貝毒を定量する方法である。 For this reason, various methods for quantifying paralytic shellfish poison have been developed. For example, the official method for assaying lethal activity using mice [Takeshi Yasumoto, supra; F. Jel lett et al., Toxicon, 30 (10): 1143-1156 (1992); W. Horwitz, Paralytic she II fi sh po i son. In Official Methods of Analysis of the Association of Official Analytical Chemists "(Assoc. Official Ana, chem., Washington DC) pp. 881-882 (1990)], high-performance liquid chromatography [Y. Osh ima Mycotox ins and Phycotoxins '88 (S. Nator i et al.), Pp. 319-326, Elsevier, Amsterdam (1989)]; Japanese Patent Application Laid-Open No. 9-133669], a method using neuroblasts [K. Kogure et al., Toxicon, 26: 191-197 (1989)], and methods using antibodies [FS Ghu et al., J. Agri. Food Chem., 44: 4043-4047 (1996)]. The assay for lethal activity is as follows: injection of the mouse test solution intraperitoneally, from the moment of injection to the last gasping when the mouse dies with typical paralytic shellfish poisoning symptoms. The time (lethal time) is recorded in seconds, and the amount of toxin is estimated from a dose-matching time curve made with standard poisons.High-performance liquid chromatography uses a sample containing paralytic shellfish toxin. This is a method in which paralytic shellfish toxin is separated by high performance liquid chromatography, an oxidizing agent is added to the eluate, and the eluate is allowed to react in alkali, and the fluorescence of the resulting fluorescent substance is measured. Is a method that promotes Na + influx into a system in which the activity of Na-KATPase, an enzyme that excretes Na + out of the cell, is inhibited by バ イ abin in a neuroblast culture system. And a toxin that blocks the influx of Na + and infers its amount from the activity of the poison that blocks the swelling and death of cells caused by veratridine. When a sample containing a poison is added, the cells are rounded and thin. This method utilizes the fact that spore death is suppressed in proportion to the amount of poison. Furthermore, the immunological method uses an antibody against paralytic shellfish toxin to directly measure the amount of antibody that binds to the toxin, for example, by an enzyme immunoassay (for example, ELISA), etc. This is a method for quantifying paralytic shellfish poison.
特に、 F. S. Ghu等 （前掲） は抗サキシ トキシン抗体ノサキシ トキシン —西洋ヮサビペルォキシダーゼ結合体または抗ネオサキシ トキシン抗体 Zネオサキシ トキシン一西洋ヮサビペルォキシダーゼ結合体を用いる ELISA によって麻痺性貝毒の全量を測定するアツセィ方法を開示してい る。 抗体の調製は、 F. S. Ghu と X. Fan, J. Assoc. Off. Anal. Chem, 1985, 68: 13 - 16および F. S. Chu, J. AOAC Int. 1992, 75 : 341 - 345に従って調 製されてし、る力《、 その手順は、 Johnson等 (Proc. Soc. Exp. Biol . Med. , 1964， 117, 425) の方法に準じ、 牛血清アルブミン、 po I y I ys i ne も しく は keyho le I i mpet (カサガイ） のへモシァニンをホルマリンを含む弱酸 性水溶液中でサキシ トキシンまたはネオサキシ トキシンと反応させて得 た結合体を、 ゥサギに皮下注射してポリクローナル抗体を得ることによ つている。  In particular, FS Ghu et al. (Supra) reported that parasitism shellfish poisons were detected by ELISA using the anti-saxitoxin antibody nosaxitoxin-horseradish peroxidase conjugate or the anti-neosaxitoxin antibody Z neosaxitoxin-one horseradish peroxidase conjugate. It discloses Atsushi's method of measuring total amounts. Antibodies were prepared according to FS Ghu and X. Fan, J. Assoc. Off. Anal. Chem., 1985, 68: 13-16 and FS Chu, J. AOAC Int. 1992, 75: 341-345. The procedure is the same as that of Johnson et al. (Proc. Soc. Exp. Biol. Med., 1964, 117, 425), using bovine serum albumin, poIyIsine, or keyho. A polyclonal antibody is obtained by subcutaneously injecting conjugates obtained by reacting leishimpet (Limpet) hemocyanin with saxitoxin or neosaxitoxin in a weakly acidic aqueous solution containing formalin.
具体的には、 これらの抗体の作製に用いられた抗原は、 サキシ トキシ ンの分子内に存在する 2つのグァニジゥム基にホルマリ ンを介してタン パク質を結合したもの [Johnson 等， 上記 ； Chu と Fan, 上記 ； V. Renz と G. Terplan, Arch, Lebensm i tte I hyg. , 1988, 39， 25-56； A. Cembe I I a 等， "Specificity and cross-react i v i ty of an adsorption-inhibition enzyme- I inked immunoassay for the detection of paralytic shel lfish t ox ins. In Toxic Marine Phytop I ankton" (E. Granel i 等編) ， Elsevier Sci ence Pub I i shers, New York, pp.339 - 344， 1989] 、 サキシ 卜キシンの 1 2位に存在する 2つの 0H基のうちの 1 つをはずし、 残った 0H基にタ ンパク質を結合したもの [ R. Carlson等、 Deve I opment of i mmunoassays for paralytic she I If ish poisoning. A radio immunoassay for sax i tox i n. In Seafood Toxins" , ( E. P. Rage I is編) ， ACS Symposium Series 262, American Chemical Society, Washington, D. C.， pp.181-192"] に大另 lj できる。 前者の抗原は、 タンパク質の結合部位が 2つのグァニジゥム基 のいずれかあるいは両方かが明確でなく、 作製された抗体の反応性は発 表グループにより異なるが、 いずれのグループの抗体もネオサキシ トキ シンとの反応性は良いもので 2 0 %程度 （但し、 サキシ トキシンとの反 応性を 1 0 0 %とする。 ） であり、 サキシ トキシンとの同様 IN-H誘導体 であるゴニォ トキシン 2 , 3およぴデカルバモイルザキシ トキシンとの 反応性も 5 0 %程度と低い。 また、 力ルバモイルー N—スルフェー ト誘 導体である C トキシンとは全く結合しない。 一方、 後者の抗体は、 ネオ サキシ トキシンとの反応性が 1 %以下と報告され、 他の毒成分との反応 性は調べられてない。 さらに、 同様の抗原を用いたモノクローナル抗体 の作製も試みられているが、 これらは反応性、 感度とも低い （R. Hack 等， Food and Agricultural immunology, 1990, 2, 47-48) 。 Specifically, the antigens used for the production of these antibodies are those in which a protein is bound to two guanidium groups present in the molecule of saxitoxin via a formalin [Johnson et al., Chu; And Fan, supra; V. Renz and G. Terplan, Arch, Lebensmitter I hyg., 1988, 39, 25-56; A. Cembe IIa, etc., "Specificity and cross-react ivity of an adsorption-inhibition. enzyme- I inked immunoassay for the detection of paralytic shellfish tox ins. In Toxic Marine Phytop I ankton "(E. Granel i et al.), Elsevier Science Pub Iisers, New York, pp. 339-344, 1989 ], With one of the two 0H groups present at the 12-position of saxitoxin removed and a protein bound to the remaining 0H group [R. Carlson et al., Deve I opment of immunoassays for paralytic she I If ish poisoning.A radio immunoassay for sax i tox in.In Seafood Toxins ", (Ep Rage I is), ACS Symposium Series 262, American Chemical Society, Washington, DC, pp. 181-192" You can do lj. In the former antigen, it is not clear whether the binding site of the protein is one or both of the two guanidium groups, and the reactivity of the produced antibodies differs depending on the published group, but the antibodies of both groups are different from those of neosaxitoxin. Has a good reactivity of about 20% (however, the reactivity with saxitoxin is 100%), and goniotoxins 2, 3 and IN-H derivatives similar to saxitoxin.反 応 Reactivity with decarbamoylzax toxin is low, about 50%. Also, it does not bind to C-toxin, which is a carbamoyl-N-sulfate derivative. On the other hand, the latter antibody is reported to be less than 1% reactive with neosaxitoxin, and its reactivity with other toxic components has not been determined. In addition, production of monoclonal antibodies using similar antigens has been attempted, but their reactivity and sensitivity are low (R. Hack et al., Food and Agricultural immunology, 1990, 2, 47-48).
致死活性測定法および神経芽細胞法においては、 動物や細胞の管理、 検出感度、 精度、 特異性などの点で問題がある し、 高速液体ク ロマ トグ ラフィ一法においては、 操作は簡単で、 精度も高いが、 高価な機器を必 要と し、 麻酔性貝毒の種類によって感度が異なること、 精製された外部 標準の供給がないことなどの欠点を有している。 また、 免疫学的方法に おいては、 抗体の反応性がサキシ トキシン誘導体の種類によって異なる などの点で問題がある。 したがって、 麻酔性貝毒の簡便で精度高く、 し かも貝毒の種類によらず感度一定に測定できる方法の開発が望まれてき た。  The lethal activity measurement method and the neuroblast cell method have problems in animal and cell management, detection sensitivity, accuracy, specificity, etc., and the high-performance liquid chromatography method is easy to operate. Although it has high accuracy, it has the drawbacks of requiring expensive equipment, different sensitivity depending on the type of narcotic shellfish poison, and lack of supply of a purified external standard. In addition, the immunological method has a problem in that the reactivity of the antibody differs depending on the type of saxitoxin derivative. Therefore, there has been a demand for the development of a simple and accurate method for narcotic shellfish poison that can be measured with a constant sensitivity regardless of the type of shellfish poison.
さきに、 本願発明者等は、 麻酔性貝毒一ダルタォチン （PSP-GSH) 複合 体 （特願平 10-91797号公報） をハプテンと し、 これをタンパク質分子に 導入した抗原を調製し、 ゥサギに投与したところ、 麻痺性貝毒を全前成 分に対して反応するポリク ロ一ナル抗体を得てこれを開示した （特開 2000- 344799号公報） 。 しかし、 この GSH との複合体を用いる方法ではタ ンパク質分子に対するハプテンの結合量が少なく、 得られた抗血清の抗 体価も低く、 毒をタンパク質等分子に導入するためには結合法の改良が 必要であると考えられた。 Earlier, the inventors of the present invention used the narcotic shellfish toxin daltaotin (PSP-GSH) complex (Japanese Patent Application No. 10-91797) as a hapten and converted it into a protein molecule. When the introduced antigen was prepared and administered to rabbits, a polyclonal antibody reacting paralytic shellfish toxin with respect to all components was obtained and disclosed (JP-A-2000-344799). However, in this method using a complex with GSH, the binding amount of hapten to protein molecules is small, the anti-titer of the obtained antiserum is low, and the binding method is required to introduce the poison into molecules such as proteins. Improvement was deemed necessary.
本発明の第 1 目的は、 毒をタンパク質等他の分子に導入するための、 上記欠点の改良された結合法を用いる、 麻痺性貝毒に対する抗体の製法 の提供にある。 また本発明の第 2 目的は、 本発明による改良結合法を用 いて調製した抗体の提供にある。 さらに本発明の第 3 目的は、 本発明に おける抗体を用いる新規 EL I SA キッ トの提供にある。 また本発明の第 4 目的は、 本発明における改良結合法を用いて調製される、 麻痺性貝毒簡 易測定法に使用するための標識毒標品の製法およびこの製法により調製 した標識毒標品の提供にある。 発明の開示  A first object of the present invention is to provide a method for producing an antibody against paralytic shellfish toxin using a binding method with the above-mentioned drawbacks for introducing a poison into another molecule such as a protein. A second object of the present invention is to provide an antibody prepared using the improved binding method according to the present invention. A third object of the present invention is to provide a novel ELISA kit using the antibody of the present invention. Further, a fourth object of the present invention is to provide a method for producing a labeled poison sample for use in a simple measurement method for paralytic shellfish poison, which is prepared using the improved binding method of the present invention, and a labeled poison standard prepared by this method. In the provision of goods. Disclosure of the invention
この度、 発明者等は、 スルフヒ ドリル基を複数 （ 2つ） 持つジチォ一 ル化合物を用いて遊離スルフ ヒ ドリル基を持つ毒誘導体を調製し、 この 誘導体をスルフ ヒ ドリル基指向性官能基をァミノ基に導入したタンパク 質と反応させると、 毒を効率的にタンパク質に導入でき、 より多くのハ プテン分子を導入した抗原の作成が可能であることを見いだした。 本発 明における上記製法は、 毒とタンパク質とを別個に修飾し、 両者の反応 により抗原を得ることに特徴がある。 本発明による抗原は、 タンパク質 分子当りのハプテン量が大きいので、高い抗体価を持つ抗血清が得られ、 抗体が高収率で得られることが発明者等により見いだされた。 本発明に おける改良結合法により調製した抗体を用いると、 毒の EL I SA 測定キッ 卜の提供が可能になる。 Recently, the present inventors prepared a toxic derivative having a free sulfhydryl group using a dithiol compound having a plurality (two) of sulfhydryl groups, and converted this derivative into a sulfhydryl group-directed functional group. By reacting with the protein introduced into the group, it was found that the poison could be efficiently introduced into the protein, and that an antigen with more hapten molecules introduced could be created. The production method of the present invention is characterized in that a poison and a protein are separately modified, and an antigen is obtained by a reaction between the two. Since the antigen according to the present invention has a large amount of hapten per protein molecule, the inventors have found that an antiserum having a high antibody titer can be obtained and the antibody can be obtained in high yield. By using the antibody prepared by the improved binding method of the present invention, the kit for ELISA poison determination can be used. Can be provided.
また本発明における抗体の製法によれば、 毒を樹脂または蛍光物質等 の担体と効率的に結合させ得ることも発明者等は見いだした。 蛍光物質 等マーカーを結合させた場合、 毒性は若干低減するが、 本発明による抗 原は毒の活性部位が露出されているので、 毒性を残したままでの標識毒 標品の調製が可能になる。 図面の簡単な説明  The inventors have also found that, according to the method for producing an antibody of the present invention, a poison can be efficiently bound to a carrier such as a resin or a fluorescent substance. When a marker such as a fluorescent substance is bound, the toxicity is slightly reduced.However, since the active site of the toxin of the antigen according to the present invention is exposed, it is possible to prepare a labeled toxin with the toxicity remaining. . BRIEF DESCRIPTION OF THE FIGURES
第 1 図は、 麻痺性貝毒 （PSP ) の構造式と比毒性を示す。  FIG. 1 shows the structural formula and specific toxicity of paralytic shellfish poison (PSP).
第 2図は、 本発明における抗原（BSA- EDT-PS P結合体） の製法を示す。 第 3図は、 本発明における BSA-E叮- STX 結合体を免疫したゥサギの抗 体価の推移を示したグラフである。  FIG. 2 shows a method for producing an antigen (BSA-EDT-PSP conjugate) in the present invention. FIG. 3 is a graph showing the change in the antibody titer of a rabbit that has been immunized with the BSA-E ding-STX conjugate of the present invention.
第 4図は、 抗血清による PSPの吸収量を示したグラフである。  FIG. 4 is a graph showing the amount of PSP absorbed by antiserum.
第 5図は、 各 PS P成分の STX固相プレー ト上での抗 STX抗体に対する 反応を示したグラフである。  FIG. 5 is a graph showing the reaction of each PSP component to an anti-STX antibody on an STX solid phase plate.
第 6図は、 PS P以外の成分の STX固相プレー ト上での抗 STX抗体に対す る反応を示したグラフである。  FIG. 6 is a graph showing the reaction of components other than PSP to an anti-STX antibody on an STX solid phase plate.
第 7図は、 本発明における非アイソ トープ系標識付き PSP 誘導体の構 造を示す。  FIG. 7 shows the structure of a non-isotope-labeled PSP derivative according to the present invention.
第 8図は、 蛍光標識付き STX ( PAEM-EDT-STX ) の B i o- Ge l P-2カラムク ロマ トグラフィ一上での分離を示したグラフである。  FIG. 8 is a graph showing separation of STX (PAEM-EDT-STX) with a fluorescent label on Bio-Gel P-2 column chromatography.
第 9図は、 各 PSP成分、 PSP—チオール複合体および標識付き PS P誘導 体の比毒性を示したグラフである。 発明を実施するための最良の形態  FIG. 9 is a graph showing the specific toxicity of each PSP component, PSP-thiol complex and labeled PSP derivative. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の第 1 の態様では、 サキシ トキシンの 1 1 位にスルフヒ ドリル 基を複数 （ 2つ） 持つジチオール化合物を結合させて遊離スルフ ヒ ドリ ル基を持つサキシ トキシンスルフ ヒ ドリル誘導体を調製し、 他方、 スル フ ヒ ドリル基指向性官能基をァミノ基に導入したタ ンパク質を調製し、 次いで上記サキシ トキシンスルフ ヒ ドリル誘導体と上記修飾タ ンパク質 とを反応させて得られる抗原を非ヒ ト動物に免疫し、 該動物から麻痺性 貝毒を認識し得る抗血清を得ることを含む、 麻痺性貝毒に対する抗体の 製法が提供される。 In the first embodiment of the present invention, sulfhydryl is added at position 11 of saxitoxin. A dithiol compound having a plurality of (two) groups is bonded to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group, while a protein in which a sulfhydryl group-directing functional group is introduced into an amino group. And then immunizing a non-human animal with an antigen obtained by reacting the saxitoxin sulfhydryl derivative with the modified protein to obtain an antiserum capable of recognizing paralytic shellfish toxin from the animal. A method for producing an antibody against paralytic shellfish poison is provided.
スルフヒ ドリル基を有する上記ジチオール化合物は、 1 ， 2—エタ ンジ チオール、 ジチオス レイ ト一ル、 2， 3—ジメノレカプ ト一 1 ープロ ノ ンス ルホン酸ジチォエリス リ トールからなる群から選択するのが好ま しい。 スルフ ヒ ドリル基指向性官能基を持つ化合物は、 マ レイ ミ ド、 チォフタ ルイ ミ ド、 活性ハロゲン化合物からなる群から選択するのが好ましい。  The above-mentioned dithiol compound having a sulfhydryl group is preferably selected from the group consisting of 1,2-ethanedithiol, dithiothreitol, 2,3-dimenolecapto1-1-pronone dithioerythritol sulfonate. . The compound having a sulfhydryl group-directing functional group is preferably selected from the group consisting of maleimide, thiophthalimide, and an active halogen compound.
また本発明の第 2の態様では、 第 1 態様における製法によ り得られた 抗体が提供される。  In a second aspect of the present invention, there is provided an antibody obtained by the production method of the first aspect.
また本発明の第 3の態様では、 第 1 態様において得られた抗体を用い る新規 EL I SAキッ 卜が提供される。  In a third aspect of the present invention, there is provided a novel ELISA kit using the antibody obtained in the first aspect.
また本発明の第 4の態様では、 サキシ トキシンの 1 1 位にスルフ ヒ ド リル基を複数 （ 2つ） 持つジチオール化合物を結合させて遊離スルフ ヒ ドリル基を持つサキシ トキシンスルフ ヒ ドリル誘導体を調製し、 次いで チオール指向性官能基を導入したァガロースまたはビォチン、 蛍光物質、 金コ ロイ ドなどの標識化合物をこの誘導体に反応させることからなる、 麻酔性貝毒簡易結合測定に用いるための標識毒標品が提供される。  In a fourth embodiment of the present invention, a dithiol compound having a plurality (two) of sulfhydryl groups at the 11-position of saxitoxin is bonded to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group. Then, a labeled compound such as agarose or biotin into which a thiol-directing functional group has been introduced, a fluorescent substance, a gold colloid, or the like, is reacted with this derivative, and the labeled toxin preparation for use in the measurement of simple binding of anesthetic narcotic toxin. Is provided.
さ らに本発明は第 5の態様においては、 上記標識毒標品の製法を用い た標識毒標品が提供される。 本発明における毒標品は非アイ ソ トープ系 のビォチン標識系および蛍光標識系標品であり、 これらの製法を第 7図 に示す。 本発明における上記製法は、 遊離スルフ ヒ ドリル基指向性の任意の官 能基、 特にマレイ ミ ド、 チォフタルイ ミ ド、 活性ハロゲン等が中性水溶 液中、 温和な条件下にチオール基と定量的に反応することを応用したも のであり、 PSP-GSH複合体も しくはその他のアミ ド縮合を用いる結合法に 比べ、 本発明によれば遙かに高収率かつ容易に麻酔性貝毒をタンパク質 や標識化合物とを結合さえることが可能である。 Further, according to a fifth aspect of the present invention, there is provided a labeled poison sample using the method for producing a labeled poison sample. The poison samples according to the present invention are non-isotope biotin-labeled and fluorescent-labeled samples, and their production methods are shown in FIG. In the production method of the present invention, any functional group having a free sulfhydryl group directivity, in particular, maleimide, thiophthalimide, active halogen and the like can be quantitatively converted into a thiol group under mild conditions in a neutral aqueous solution. According to the present invention, the narcotic shellfish toxin can be easily obtained in a much higher yield and easily than the binding method using the PSP-GSH complex or other amide condensation. It can even bind proteins and labeled compounds.
以下、 ジチオール化合物と してエタンジチオール （EDT ) を採用した場 合の本発明における抗体の製法を述べる。  Hereinafter, a method for producing an antibody in the present invention when ethanedithiol (EDT) is used as the dithiol compound will be described.
サキシ トキシン一 EDT複合体は、 ゴニォ トキシン （ゴニォ トキシン 2 , 3、 あるいはこれらの組合せ） とジチオール基を有する EDT とを混合 - 加熱 （約 7 0 °C ) すると、 EDTのィォゥ分子を介してゴニォ トキシン類の 1 1 位の o s o 3—基と置換反応することにより得ることができる。本発明 では、 この複合体はいわゆるハプテン抗原、 すなわち抗体とは結合する が抗体を誘発する能力のない物質である。 一般にこのようなハプテン抗 原にタンパク質を共有結合させて大分子とすることによって免疫原と し 得る場合もあることが知られているが、 本発明におけるタンパク質もま た同様の働きをもつものである。 そのようなタンパク質と して、 たとえ ばアルブミンゃカサガイへモシァニン（KLH)が例示できる。 The saxitoxin-EDT complex is a mixture of goniotoxin (gonitoxin 2, 3 or a combination thereof) and EDT having a dithiol group. When heated (approximately 70 ° C), the goniotoxin is converted to goniotoxin via the EDT molecule. It can be obtained by a substitution reaction with the oso 3 — group at the 11-position of toxins. In the present invention, this complex is a so-called hapten antigen, ie a substance that binds to the antibody but has no ability to elicit the antibody. It is generally known that in some cases, such a hapten antigen can be used as an immunogen by covalently binding a protein to a large molecule, but the protein of the present invention also has a similar function. is there. An example of such a protein is albumin limpet hemocyanin (KLH).
本発明はまた、 第 2の態様において、 麻痺性貝毒成分であるサキシ ト キシン、 ゴニォ トキシン 2 , 3、 ネオサキシ トキシン、 ゴニォ トキシン 1 ， 4、 ゴニォ トキシン 5、 および C トキシン 1 ， 2のいずれとも 5 0 %以上 の相対反応性 （但し、 サキシ トキシンとの反応性を 1 0 0 %とする。 ） を示す、 麻痺性貝に対する抗体を提供する。  In the second aspect, the present invention also provides, in the second embodiment, any of the paralytic shellfish toxin components saxitoxin, goniotoxin 2,3, neosaxitoxin, goniotoxin 1,4, goniotoxin 5, and C toxin 1,2 It provides an antibody against paralytic shellfish that exhibits a relative reactivity of 50% or more (reactivity with saxitoxin is 100%).
本発明の抗体は、 麻痺性貝毒である I N- H誘導体 （サキシ トキシン、 ゴ ニォ トキシン 2 , 3 ) 、 I N- 0H誘導体 （ネオサキシ トキシン、 ゴニォ トキ シン 1 , 4 ) 、 力ルバモイルー N—スルフェー ト誘導体 （ゴニォ トキシン 5、 および C トキシン 1 , 2 ) のすベてをほぼ同様に認識することができ る。 ここで Γゴニォ トキシン 2 , 3 J などの表現はゴニォ トキシン 2 とゴ ニォ トキシン 3 との混合物を意味する。 麻痺性貝毒の名称および構造に ついては、 たとえば、 安元健、 麻痺性貝毒、 食品衛生検査指針 （理化学 編、 厚生省生活衛生局監修） 、 第 300〜305頁、 日本食品衛生協会、 1901 年 ； Intergovernmental Oceanograph i c Commission ( 10C) Manual and Guides No.33 (1995) , UNESCO' s Workshops, France, pp.81 - 94などに記 載されている。 本発明の実施態様により、 本発明の抗体は上記方法によ つて得られたものである。 Antibodies of the present invention include paralytic shellfish poisons such as IN-H derivatives (saxitoxin, goniotoxin 2,3), IN-0H derivatives (neosaxitoxin, goniotoxin 1,4), Sulfate derivative (gonio toxin 5, and all of the C toxins 1, 2) can be recognized almost in the same way. Here, expressions such as goniotoxin 2 and 3J mean a mixture of goniotoxin 2 and goniotoxin 3. For the name and structure of paralytic shellfish poison, see, for example, Ken Yasumoto, Paralytic Shellfish Poison, Food Sanitation Inspection Guidelines (edited by RIKEN, supervised by Ministry of Health and Welfare, Ministry of Health and Welfare), pp. 300-305, Japan Food Sanitation Association, 1901 Intergovernmental Oceanographic Commission (10C) Manual and Guides No.33 (1995), UNESCO's Workshops, France, pp.81-94. According to an embodiment of the present invention, the antibody of the present invention has been obtained by the above method.
本発明の抗体の製造において使用される抗原は、 サキシ トキシン一 EDT —タンパク質複合体である。 この複合体は以下のようにして製造するこ とができる。 サキシ トキシン部分の原料と してゴニォ トキシン 2、 ゴニ ォ トキシン 3またはそれらの任意割合の混合物を使用することができる ( これらの原料成分は、 ィガイ、 ハマダリ、 ホタテガイ等の貝類の 0.1 規 定塩酸による加熱抽出物から活性炭処理、 ゲル濾過クロマ トグラフィー (たとえば Bio-GelP-2 (バイオラッ ド社） 使用） 、 イオン交換クロマ 卜グ ラフィー （たとえば Bio-Rex70 (バイオラッ ド社） 使用） などの処理を経 て精製することができる。 The antigen used in the production of the antibodies of the present invention is saxitoxin-EDT-protein complex. This complex can be produced as follows. As a raw material for the saxitoxin portion, goniotoxin 2, goniotoxin 3 or a mixture of any ratio thereof can be used ( these raw materials are based on 0.1N hydrochloric acid of shellfish such as mussels, hamadari and scallop). The heated extract is treated with activated carbon, gel filtration chromatography (using Bio-GelP-2 (Bio-Rad)), ion-exchange chromatography (Bio-Rex70 (Bio-Rad)), etc. And can be purified.
上記のようにして得られてゴニォ トキシンに EDT (還元型が好ましい。） を加熱下に反応させると、 EDTの SH基がその電子共与性のためにゴニォ トキシンの 1 1 位の炭素原子を求核攻撃し、 その結果 0S03—基が離脱し、 該 1 1 位の炭素原子に - S- (EDT) が結合し、 これによつてサキシ トキシ ンー EDT複合体が生成する。該反応は、 中性条件下で反応物質を加熱する ことによって起こる。 一般に ρΗ6· 5〜7· 5、 好ましく は ΡΗ7.0〜7.5の適切 な緩衝液、 たとえば燐酸アンモニゥム緩衝液、 酢酸アンモニゥム緩衝液 等を用いて反応を行うことができる。 また、 反応温度は、 一般に室温か ら約 1 00°G、 好ましく は 50°Gから 80°G、 よ り好ましく は約 70°Cである。 反応後、 目的の生成物を、 調製用高速液体ク ロマ トグラフィー、 ゲル濾 過ク ロマ トグラフィー、 イオン交換ク ロマ トグラフィー、 吸着ク ロマ ト グラフィー、 逆相配分ク ロマ トグラフィー等の手段を単独で又は組合わ せて実施し、 精製することができる。 生成物の同定は、 赤外分光法、 質 量分析法、 關 R法、 元素分析、 アミノ酸分析などの通常の測定法によって 行う ことができる。 When the goniotoxin obtained as described above is reacted with EDT (preferably in a reduced form) under heating, the SH group of EDT changes the carbon atom at position 11 of the goniotoxin due to its electron donating property. and nucleophilic attack, resulting 0s0 3 - group is disengaged, the carbon atom of the 1 position 1 - S- (EDT) is attached, this Yotsute Sakishi butoxy Hmm EDT complex is produced. The reaction takes place by heating the reactants under neutral conditions. Generally ρΗ6 · 5~7 · 5, preferably suitable buffer [rho Ita7.0～7.5, for example phosphate Anmoniumu buffer, the reaction may be carried out using acetic acid Anmoniumu buffer solution. The reaction temperature is generally room temperature. From about 100 ° G, preferably from 50 ° G to 80 ° G, more preferably about 70 ° C. After the reaction, the desired product is purified by preparative high-performance liquid chromatography, gel filtration chromatography, ion exchange chromatography, adsorption chromatography, reverse phase distribution chromatography, etc. It can be performed alone or in combination and purified. The product can be identified by ordinary measurement methods such as infrared spectroscopy, mass spectrometry, R-method, elemental analysis, and amino acid analysis.
サキシ トキシンのような低分離化合物はハプテンに属し、 それ自体は 抗原性をもたないが、 抗原性をもつタ ンパク質を担体と して、 これと共 有結合した複合体はハプテンに対する特異抗体を産生する抗原となる。 ハプテン一タ ンパク質複合体を免疫注射するとハプテン部分は抗原決定 基と して認識され、 抗ハプテン抗体が産生されると考えられている。 こ のとき、 余分な抗原決定基ができにく い選択的な結合方法でハプテン抗 原を作製する必要がある。 ハプテンの部分構造に対する認識は、 ハプテ ンと担体であるタ ンパク質の結合部位付近では弱く 、 よ り離れた部分構 造を認識しやすい。 したがって、 タ ンパク質と結合する際には、 ハプテ ン中の適切な結合位置を選択することが大切である。 本発明者等により 先に見出されたサキシ トキシンの 1 1 位に EDT 基が結合した構造をもつ サキシ トキシン一 E叮複合体 （特願平 1 0 - 91 797号） は、 免疫抗原と して 好ま しいだけでなく、 麻痺性貝毒のほぼ全ての種類を高い反応性で検出 可能とする麻痺性貝毒を産出 し得る利点をもつ。  Compounds with low resolution, such as saxitoxin, belong to hapten, and have no antigenicity per se, but a complex that covalently binds to antigenic protein as a carrier is a specific antibody against hapten. Antigen. It is thought that when a hapten-protein complex is injected by immunization, the hapten portion is recognized as an antigenic determinant, and an anti-hapten antibody is produced. In this case, it is necessary to prepare a hapten antigen by a selective binding method that does not easily form an extra antigenic determinant. Recognition of the hapten partial structure is weak near the binding site between the hapten and the protein serving as a carrier, and it is easy to recognize a distant partial structure. Therefore, when binding to proteins, it is important to select an appropriate binding site in the hapten. The saxitoxin-e ding complex (Japanese Patent Application No. 10-91797) having a structure in which an EDT group is bonded to the 11-position of saxitoxin previously discovered by the present inventors is regarded as an immunizing antigen. Not only is it preferred, but it also has the advantage of producing paralytic shellfish toxins that can detect almost all types of paralytic shellfish toxins with high reactivity.
担体となるタ ンパク質と してはアルブミン、 へモシァニンなどを使用 できるが、 これらに限定されず、 本発明の抗体を誘発可能とする任意の タ ンパク質を使用できる。サキシ トキシン一EDT複合体に担体タ ンパク質 を結合する際には、 該複合体の EDT 部分を利用して、 （反応性官能基を 導入した） 担体タ ンパク質とマ レイ ミ ド法 （Τ · K i t a gawa等， B i o sh em . 92 : 585-590 , 1 982 ) やカ ゾレボジイ ミ ド法 （D . Ex l ey 等， FEBS Lett . , 91 : 1 62-1 65， 1 978)などで結合させて、 サキシ トキシン一 GSH—タンパク質 複合体を生成することができる。 たとえば後述の実施例にはサキシ トキ シン一 EDT—牛血清アルブミ ン複合体の製造例を示すが、 ここでは先ずサ キシ トキシン一 EDT結合体を作り、一方アルブミ ンをその遊離アミノ基を 介してメルカプ トサクシ二ル化し、 両者を反応させることにより担体タ ンパク質をサキシ トキシン - EDTに結合させる。得られた複合体の精製は、 慣用のタンパク質精製法を適宜組合わせて実施できる。 そのような方法 には、 たとえば、 塩析、 溶媒沈殿、 ゲル濾過ク ロマ 卜グラフィー、 ィォ ン交換クロマ トグラフィー、 H PLC、ァフィ二ティ一ク ロマ トグラフィー、 電気泳動、 ク ロマ トフオーカシング、 限外濾過などが含まれる。 As a protein serving as a carrier, albumin, hemocyanin and the like can be used, but not limited thereto, and any protein capable of inducing the antibody of the present invention can be used. When binding the carrier protein to the saxitoxin-EDT complex, the EDT portion of the complex is used to combine the carrier protein (with a reactive functional group introduced) with the maleimide method (Τ · Kita gawa et al., Biosh em. 92: 585-590, 1982) or by the azolevodiimide method (D. Exley, FEBS Lett., 91: 162-165, 1978), etc. A complex can be formed. For example, in the examples described below, an example of producing a saxitoxin-EDT-bovine serum albumin complex will be described. Here, a saxitoxin-EDT conjugate is first produced, and albumin is converted via its free amino group. The carrier protein is bound to saxitoxin-EDT by mercaptosuccinylation and reaction of the two. The obtained complex can be purified by appropriately combining conventional protein purification methods. Such methods include, for example, salting out, solvent precipitation, gel filtration chromatography, ion exchange chromatography, HPLC, affinity chromatography, electrophoresis, chromatofocusing, limiting External filtration and the like are included.
ゴニ才 トキシン 2 , 3に換えて、 ゴニォ トキシン 1 、 ゴニォ トキシン 4 又はそれらの混合物を原料と して EDT と反応させる場合には、 ネオサキ シ トキシン- EDT複合体が得られる。 さ らにこの複合体を上記と同様に担 体タ ンパク質と反応させるこ とによってネオサキシ トキシン一 EDT—タ ンパク質複合体を得ることができ、 得られた生成物はサキシ トキシン一 EDT—タ ンパク質複合体と同様に麻痺性貝毒に対する抗体の産生に使用 するこ とができる （以下参照） 。  When the gonitotoxin 1, goniotoxin 4 or a mixture thereof is reacted with EDT instead of gonii toxins 2 and 3, a neosaxitoxin-EDT complex is obtained. Further, a neosaxitoxin-EDT-protein complex can be obtained by reacting the complex with a carrier protein in the same manner as described above, and the resulting product is saxitoxin-EDT-protein. Like the protein complex, it can be used to produce antibodies against paralytic shellfish toxins (see below).
本発明のサキシ トキシン一 EDT—タンパク質複合体を、マウス、ラッ 卜、 ゥサギ、 ャギ、 等の動物に免疫することによって該複合体に対する抗体 を作製することができる。 通常、 免疫原溶液を、 フロイン 卜の完全アジ ュバン ト、 不完全アジュバン ト、 リ ビアジュバン ト システム （ R I B I I MUNOCH EM RES EARCH I NC ) 、 ムラ ミリレペプチ ド （Ca l zyme社） 、 水酸ィ匕 アルミニウム、 リポポリサッカライ ドなどのアジュバン 卜と乳化混合し、 動物に皮下、 皮内、 または筋肉に注射した後、 2〜 4週間の間隔で同様 の操作を行い数回追加免疫し、 放血後抗血清 （ポリ ク ローナル抗体） を 得ることができる。 定期的に放血血清をサキシ トキシンとの反応性につ いて測定し、高力価が達成されるまで追加免疫を実施する。抗原の量は、 抗原刺激を受ける動物の種類によって異なるが、 たとえばゥサギの場合 1 0 i g〜数 mg、 マウスの場合数 ju g〜数 1 0mgである。 An antibody to the saxitoxin-EDT-protein complex of the present invention can be prepared by immunizing an animal such as a mouse, a rat, a rabbit, a goat, or the like. Usually, the immunogen solution is prepared using Freund's complete adjuvant, incomplete adjuvant, livia adjuvant system (RIBII MUNOCH EM RES EARCH IN NC), muramirirepeptide (Calzyme), hydroxylamine aluminum, The mixture is emulsified and mixed with an adjuvant such as lipopolysaccharide, injected into animals subcutaneously, intradermally, or intramuscularly.The same procedure is repeated at intervals of 2 to 4 weeks, and booster immunization is performed several times. (Polyclonal antibody) Obtainable. Periodically, the exsanguinated serum is measured for reactivity with saxitoxin and boosters are performed until a high titer is achieved. The amount of antigen varies depending on the type of animal to be challenged with the antigen, and is, for example, 10 ig to several mg for a heron, and several jug to several 10 mg for a mouse.
上記のようにして得られた麻痺性貝毒に対する抗血清はさらに、 たと えば硫安分画、 ついでイオン交換クロマ トグラフィ一で処理することに よ り I gG 抗体に精製することができる。 あるいはサキシ トキシンをデキ ス トランゲルまたはァガロースゲルに化学的に結合させたゲルを用いる ァフィ二テイクロマ トグラフィ一によって抗体を精製することができる, 抗体の作製および精製については、 たとえば生物化学実験法 1 5 「免疫 学入門」 （1 982)学会出版センターに詳細に記載されており参照可能であ る。  The antiserum against paralytic shellfish toxin obtained as described above can be further purified to an IgG antibody by, for example, treating it with ammonium sulfate fractionation and then ion-exchange chromatography. Alternatively, antibodies can be purified by affinity chromatography using a gel in which saxitoxin is chemically bound to a dextran gel or agarose gel. For production and purification of antibodies, see, for example, Introduction to Academic Studies ”(1 982).
本発明の方法では、 免疫後 1 ヶ月程度から抗体価の上昇が見られ、 サ キシ トキシンに対する抗血清の場合約 4 ヶ月後に抗血清 1 m I あた り 0. 5nmo l のサキシ トキシンを結合する力価が得られる。本抗血清はネオサ キシ トキシンとの反応は幾分低かったが、 免疫 5ヶ月後の血清では両者 の貝毒との反応性はほとんど差がなかった。 また、 第 2図に免疫 4、 5 ヶ月目の血清の麻痺性貝毒の各種成分との反応性を示すが、 4ヶ月目の 血清はネオサキシ トキシンおよびゴニォ トキシン 1 ， 4との反応性が幾 分劣るが、 5ヶ月目の血清は各成分との反応に大きな差が認められなか つた。 このように、 本発明の抗体は麻痺性貝毒のほぼすベての成分と免 疫学的に反応し、 特に麻痺性貝毒成分であるサキシ トキシン、 ゴニオ ト キシン 2 , 3、 ネオサキシ トキシン、 ゴニォ トキシン 1 , 4、 ゴニォ トキ シン 5、 および C トキシン （ C 1 、 C 2 ) のいずれとも 50 %以上、 好ま しくは 60 %以上の相対反応性（但し、サキシ トキシンとの反応性を 1 00 % とする。 ） を示す。 これに対して従来公知の抗体の多くは、 麻痺性貝毒 の全成分に共通のグァニジゥム基にタ ンパク質を結合させたものを抗原 と しているため、 一部の麻痺性貝毒成分に強い特異性を示す反面、 ほと んど結合しない成分も存在する。 貝毒の検出の信頼度を上げるためには、 抗体はほとんどすべての貝毒成分と同程度に反応するのが望ましい。 In the method of the present invention, an increase in antibody titer is observed from about one month after immunization, and in the case of antisera against saxitoxin, about 0.5 months after the antiserum binds 0.5 nmol of saxitoxin per ml of antiserum A titer is obtained. The antiserum had a slightly lower reaction with neosaxitoxin, but the sera 5 months after immunization showed little difference in reactivity with both shellfish toxins. Fig. 2 shows the reactivity of sera 4 and 5 months after immunization with various components of paralytic shellfish toxin. The sera 4 months after immunization showed some reactivity with neosaxitoxin and goniotoxin 1, 4. Although inferior, the serum at 5 months showed no significant difference in the reaction with each component. As described above, the antibody of the present invention immunologically reacts with almost all components of paralytic shellfish toxin, and particularly, saxitoxin, goniotoxin 2, 3, neosaxotoxin, and goniotoxin, which are paralytic shellfish toxin components. Relative reactivity of 50% or more, and preferably 60% or more, to any of toxins 1, 4, goniotoxin 5, and C toxin (C1, C2) (however, the reactivity with saxitoxin is 100%). ) Is shown. In contrast, many of the conventionally known antibodies are paralytic shellfish poison The protein is bound to a guanidium group that is common to all of the components, and the antigen is used as the antigen.Therefore, although it shows strong specificity for some paralytic shellfish poison components, there are also components that hardly bind I do. To increase the reliability of shellfish poison detection, it is desirable that the antibody reacts to the same extent as almost all shellfish poison components.
本発明のサキシ トキシン一 EDT.—タ ンパク質複合体を抗原と して用い ることによって定法によ りモノク ローナル抗体を作製することも可能で ある。 具体的には、 マウスゃラッ 卜などのげつ歯類に該複合体を免疫後 に脾臓を無菌的に取り出 し、 脾臓細胞を調製した後、 ミエローマ細胞と 融合し、 ハイプリ ドーマを HAT 培地等の選別用培地で選別し、 動物細胞 培養用培地中で継代培養するか、 または該げっ歯類の腹膣内に移植培養 し腹水からモノ ク ローナル抗体を採取することができる。 モノ ク ローナ ゾレ抗体の作製 Iこっしゝて ίま、 M i I st e i n と Kho l e r, N atu re256： 495 ( 1 976) ； 属性化学実験講座、 免疫生化学研究法 （日本生化学会編） 等に記載され る方法を使用できる。  By using the saxitoxin-EDT.-protein complex of the present invention as an antigen, it is also possible to prepare a monoclonal antibody by an ordinary method. Specifically, after immunizing a rodent such as a mouse rat with the complex, the spleen is aseptically removed, spleen cells are prepared, fused with myeloma cells, and the hybridoma is transformed into a HAT medium. Or the like, and subcultured in an animal cell culture medium, or transplanted and cultured in the abdominal vagina of the rodent, and the monoclonal antibody can be collected from ascites. Preparation of Monoclonal Zole Antibody I Kosipatema, MiIstein and Kholer, Nature 256: 495 (1 976); Laboratory of Attribute Chemistry, Immunobiochemical Research Method (edited by The Biochemical Society of Japan) ) Etc. can be used.
上記のようにして得られた抗体は、 麻痺性貝毒を含む疑いのある食品 等の検体中の貝毒の測定に使用することができる ό 測定は慣用の抗原抗 体反応を用いて行う ことができ、 固相法も し く は均質法、 競合法も し く は非競合法、 サン ドイ ッチ法などの方法を使用することができる。 たと えば、 サン ドイ ッチ法を用いる場合には、 過剰量の標識化第二抗体を用 いるが、 標識と してペルォキシダーゼ、 アルカ リ フォスファターゼ等の 酵素、 1 25し 82Ρ等の放射性同位体、 F I TG等の蛍光物質、 ァク リ ジニゥム などの化学発光物質を用いるこ とができる。 標識の種類に依存して、 EL I SAなどの酵素抗体法、 ラジオィムノアツセィ、 蛍光抗体法の使用が可 能である。 さ らに、 本発明の抗体は、 臭化シアン等で活性化されたデキ ス トラン樹脂 （たとえば S e phad exTM類） 、 ァガロース樹脂 （たとえば B i o- Ge M類） 、 等の樹脂に結合するときには問題の貝毒を分離除去する ためのァフィ二ティーカラム担体とすることができる。 Antibodies obtained as described above can be used to measure shellfish toxin in specimens of foods etc. suspected of containing paralytic shellfish toxin.ό Measurement should be performed using a conventional antigen-antibody reaction. A solid phase method or a homogeneous method, a competitive method or a non-competitive method, a sandwich method, or the like can be used. For example, when using a San Doi pitch method, although use of excess amount of a labeled second antibody, and a labeled Peruokishidaze, enzymes such as alkaline phosphatase, 1 25 teeth 82 radioactive isotopes such Ρ Fluorescent substances such as FITG and chemiluminescent substances such as acridium can be used. Depending on the type of label, it is possible to use an enzyme antibody method such as ELISA, radioimmunoassay, or a fluorescent antibody method. Et al of the antibodies of the present invention can scan Trang resin (e.g. S e phad ex TM acids) activated with cyanogen bromide or the like, Agarosu resin (e.g. B i o-Ge M acids), a resin etc. Isolate and remove problematic shell poison when combined Column carrier for use.
ジチオール化合物と してエタ ンジチオール（EDT)以外の化合物を採用 した場合の実施形態も、 上記とほぼ同 じである。  An embodiment in which a compound other than ethanedithiol (EDT) is used as the dithiol compound is almost the same as the above.
本発明に係わる他の関連文献は次のようである ：  Other relevant documents relating to the present invention are as follows:
Ho I I i ngworth T. Weke I I MM (1990) Paralytic shel lfish poison. In: Official Methods of Ana lysis, 15th edition, K. Hel lrich ed. , Association of Official Analytical Chemists, Arl ington, Virginia, pp.881-882. Ho II inngworth T. Weke II MM (1990) Paralytic shel lfish poison.In: Official Methods of Analysis, 15th edition, K. Hel lrich ed., Association of Official Analytical Chemists, Arlington, Virginia, pp.881- 882.
Oshima Y (1995) : Post - column der i vat i zat i on HPLC methods for paralytic she I If ish poisons. In: Manual on Harmful Marine Algae, 10C Manuals and Guides No.33, Ha I I egraef f GM, Anderson DM, Cembe I I a AD eds. , Intergovernmental Oceanograph i c Commission of UNESCO, Paris, pp.81-94.  Oshima Y (1995): Post-column der i vat i zat i on HPLC methods for paralytic she I If ish poisons.In: Manual on Harmful Marine Algae, 10C Manuals and Guides No.33, Ha II egraef f GM, Anderson DM , Cembe II a AD eds., Intergovernmental Oceanographic Commission of UNESCO, Paris, pp.81-94.
Sato S Saka i R, Kodama (2000) Identification of th i oether intermediates in the reductive transformation of gonyautox i ns into sax i tox i ns by th i os. Bi oorg. Med. Chem. Lett. 10:1787-1789.  Sato S Saka i R, Kodama (2000) Identification of th i oether intermediates in the reductive transformation of gonyautox ins into sax i tox ins by th i os. Bioorg. Med. Chem. Lett. 10: 1787-1789.
Sommer H. Meyer KF (1937) Paralytic shel lfish poisoning. Arch. Pathol . 24 560-568. 以下の実施例によって本発明をさ らに具体的に説明するが、 本発明は これらの実施例によって限定されるものではない。 本発明の記載に関連する略号は次のようである ： 試料および試薬類 ァシュハン 卜 Complete Adjuvant Freund' s Wako Sommer H. Meyer KF (1937) Paralytic shellfish poisoning. Arch. Pathol. 24 560-568. The present invention will be described more specifically by the following examples, but the present invention is limited by these examples. Not something. Abbreviations relevant to the description of the invention are as follows: Samples and Reagents Ashhunt Complete Adjuvant Freund's Wako
Bio-Ge I P-2 ゲル爐過樹月旨、 mesh size: fine B io-Rad  Bio-Ge I P-2 gel furnace, mesh size: fine B io-Rad
B i ot i n-PE (AC5)  B i ot i n-PE (AC5)
ヒ才ナンフヘル試薬 Dojindo  Dojindo
ma I e I m I ae N - [2 -（2 - Pyr idyl ami no) e thy门 ma I e I m I ae N-[2-(2-Pyr idyl ami no) e thy 门
PAE Wako  PAE Wako
ma I e i m i de  ma I e i m i de
BSA 牛血清アルブミ ン Wako、 IRA grade D SO ジメチルスルフォキシ ド Wako、 特級 BSA Bovine serum albumin Wako, IRA grade DSO Dimethyl sulfoxide Wako, Special grade
EDT 1, 2-ェタ ンジチオール Wako, 特級  EDT 1, 2-ethanedithiol Wako, special grade
EDTA エチレンジァミ ン 4酢酸 Wako、 特級  EDTA Ethylenediamine tetraacetic acid Wako, Special grade
一マレイミ ド酷酸 N—ヒ ドロキシコハク  One Maleimid Severe Acid N-Hydroxysuccinic
GMBS Do j i ndo  GMBS Do j i ndo
酸イミ ドエステル 反応プレー ト EL I SA p I ate 96 - we I I I wak i  Acid imidoester reaction plate EL I SA p Iate 96-we I I wak i
0 - phenylenediamine - 2HG1  0-phenylenediamine-2HG1
OPD Wako  OPD Wako
(tab I et)  (tab I et)
HRP 標識抗ゥサギーャギ、 使用時  HRP-labeled anti-saggy goat, when used
酵素標識二次抗体 Dako  Enzyme-labeled secondary antibody Dako
1000倍希釈  1000-fold dilution
マイクロプレートリーダー MPR A4 Toso  Microplate reader MPR A4 Toso
Mg, Ca free リン酸緩衝生理食塩水  Mg, Ca free phosphate buffered saline
PBS (-）  PBS (-)
(pH7.4)  (pH7.4)
0.5%BSA 0.1%Tween60 を含む  Contains 0.5% BSA 0.1% Tween60
PBSB PBS (一）  PBSB PBS (one)
PBST 0.1 %Tween20を含む PBS (-）  PBST 0.1% Tween20 in PBS (-)
Sephadex G - 50 ケル爐過樹月旨、 mesh size: f ine Pharmacia  Sephadex G-50 Kel Furnace, mesh size: fine Pharmacia
THF テ トラ ヒ ドロフラン Wako、 特級  THF Tetra Hydrofuran Wako, Special grade
ニュージーラン ドホワイ 卜、 雄、  New Zealand White, male,
ゥサギ tt; a ¾tl 1 Κσ  ゥ egret tt; a ¾tl 1 Κσ
実施例 1 Example 1
麻痺性貝毒（PSP)精製毒および EDT-PSP複合体の調製  Preparation of purified paralytic shellfish poison (PSP) poison and EDT-PSP complex
大船渡湾産毒化ホタテガイから常法によ り、 gonyautox i n (GTX)群、 sax i toxin群および G - toxin群 PSP成分 （第 1 図） を単離した。 精製した 成分のうち GTX1 と GTX4、 GTX2 と GTX3 および C1 と G2はそれぞれモル比 約 3 ： 1 の平衡混合物 （それぞれ GTX1+4、 GTX2+3、 C1+2 と略記） と して 使用した。 50 / mo I の GTX1 +4 を 20%の THF を含む 50m EDT-1 Om リ ン酸 ナ ト リ ウム緩衝液 （pH7.4)50mLに溶解し、 室温で 2 日間静置した。 ジェ チルエーテルで 3回抽出して EDT を除去し、 水相に残存するエーテルを 30%で減圧下留去して、 EDT-neoSTX を含む反応混合液を得た。 これを Bio-Gel P- 2のカラム （1.5 x15cm) に添加し、 水 200mLでカラムを洗浄 後、 0.1 M酢酸で吸着成分を溶出 した。 1 OmLずつ分取し、各画分中の GTX1， 4、 neoSTX な らびに EDT-neoSTX 濃度を HPLG 蛍光法で定量しつつ、 EDT - neoSTX複合体を単離した。 50 i mol の GTX2+3 を出発物質と して同様 に処理し、 EDT - STXを調製し、 分離した。 The gonyautox in (GTX) group, saxi toxin group and G-toxin group PSP components (Fig. 1) were isolated from the Ofunato Bay poisoned scallop by a conventional method. Of the purified components, GTX1 and GTX4, GTX2 and GTX3, and C1 and G2 are each an equilibrium mixture with a molar ratio of about 3: 1 (abbreviated as GTX1 + 4, GTX2 + 3, and C1 + 2, respectively). used. GTX1 +4 at 50 / mo I was dissolved in 50 mL of 50m EDT-1 Om sodium phosphate buffer (pH 7.4) containing 20% THF and allowed to stand at room temperature for 2 days. EDT was removed by extracting with ethyl ether three times, and ether remaining in the aqueous phase was distilled off under reduced pressure at 30% to obtain a reaction mixture containing EDT-neoSTX. This was added to a Bio-Gel P-2 column (1.5 x 15 cm), the column was washed with 200 mL of water, and the adsorbed components were eluted with 0.1 M acetic acid. The EDT-neoSTX complex was isolated while quantifying the GTX1, 4, neoSTX and EDT-neoSTX concentrations in each fraction by HPLG fluorescence. EDT-STX was prepared and separated by the same treatment using 50 imol of GTX2 + 3 as a starting material.
実施例 2  Example 2
PSPに対する特異抗体作成用抗原の作成  Preparation of antigen for preparing specific antibody against PSP
BSA20mg を 2mLの PBS (—） Iこ 容角军し、これ Iこ 5mgの 6MBS100 μしの DMS0 に溶解したものを混合して室温で 1 時間静置した。 これを 1mMの EDTA を 含む 0.1M リ ン酸ナ ト リウム緩衝液 （pH6.0) で充填した Sephadex G- 25 のカラム（1.5 X 20cm)に添加し、 同溶液で溶出 した。 得た高分子画分を合 ―し、 炭酸ナ ト リ ゥム溶液で pH を 7.4に調整した後、 遠心限外濾過キッ 卜 （UFV4BGG25, 隱し 10, 000， Mi l l ipore) で 4mL に濃縮した。 この 2mL に 5 mo I の EDT-neoSTX を溶解し、 5 °Gで 2 日間静置した。 次に同混合 物を PBS (-)に対して 4回透析し、内液を neoSTX - BSA結合体溶液と して回 収した。 STX - BSA結合体も同様の手順で調製した。 抗原溶液中の BSA濃度 は 280nmの吸収によ り、 結合 PSP濃度は HPLG蛍光法によ り算出 した。 実施例 3  BSA (20 mg) was dissolved in 2 mL of PBS (-) I, and 5 mg of this solution dissolved in 100 μL of 6 MBS in DMS0 was mixed and allowed to stand at room temperature for 1 hour. This was added to a Sephadex G-25 column (1.5 X 20 cm) packed with 0.1 M sodium phosphate buffer (pH 6.0) containing 1 mM EDTA, and eluted with the same solution. The obtained polymer fractions were combined, adjusted to pH 7.4 with a sodium carbonate solution, and concentrated to 4 mL with a centrifugal ultrafiltration kit (UFV4BGG25, concealed 10,000, Millipore). did. 5 mL of EDT-neoSTX was dissolved in 2 mL of the solution, and allowed to stand at 5 ° G for 2 days. Next, the same mixture was dialyzed four times against PBS (-), and the internal solution was collected as a neoSTX-BSA conjugate solution. The STX-BSA conjugate was prepared in a similar procedure. The BSA concentration in the antigen solution was calculated by absorption at 280 nm, and the bound PSP concentration was calculated by HPLG fluorescence method. Example 3
ゥサギへの免疫および抗体価の測定  免疫 Measurement of immunity and antibody titer to herons
BSA-STX結合体を 500〃 g/mLの濃度に PBSで希釈した。等量のアジュ バン トと混合したェマルジヨ ン 2ヶ月に 1 、 2回の頻度で 1mL ずつ、 2 羽のゥサギ（STX - BSA - 1、 STX-BSA - 2)に皮内注射した。 BSA - neoSTX結合体 も同様 Iこ 2 33のゥサギ （neoSTX— BSA— 1、 neoSTX—BSA— 2) Iこ免疫した。 免 疫中ゥサギよ り随時採血して得た血清の抗体価を下記の手順で求めた。 The BSA-STX conjugate was diluted with PBS to a concentration of 500 μg / mL. Emulsion mixed with an equal volume of adjuvant was injected intradermally into two egrets (STX-BSA-1, STX-BSA-2) once or twice a month at 1 mL doses. BSA-neoSTX conjugate Similarly, I was immunized with 233 Great Egret (neoSTX-BSA-1 and neoSTX-BSA-2). The antibody titer of the serum obtained by collecting blood from the rabbit during the immunization was determined according to the following procedure.
96we I I の反応プレー 卜の各 we I I に、 PBS (-）に対して 50 Mの濃度に調製 した STX溶液を 50 Lずつ分注し 37°Cで 2時間振とう した。 プレー トを PBST で 2回洗浄後、 0.3%ゼラチンを含む PBS(- ) 300 L を添加して 37°C で 1 時間振とう した。 PBSTで 2回洗浄後、 PBSBに対して、 100倍希釈し た抗血清を、 50 L ずつ分注し、 PBST で 3回洗浄した。 37°Gで 1 時間振 とう後、 PBSTで 3回洗浄し、 PBS (-）に対して、 1000倍希釈した HRP標識 二次抗体を、 100 Lずつ添加した。 37°Gで 1 時間振とう後、 PBSTで 4回 洗浄し、 発色基質溶液 （0PD-H202) を 100 / L添加した。 37°Cで 3 0分振 とう した後、 0.5N硫酸 100 Lを添加して反応を停止し、 マイクロプレー トリーダーで 492nmの吸収（0D) を測定した。 To each weII of the 96weII reaction plate, 50 L of an STX solution prepared at a concentration of 50 M with respect to PBS (-) was dispensed, and the mixture was shaken at 37 ° C for 2 hours. After washing the plate twice with PBST, 300 L of PBS (-) containing 0.3% gelatin was added, and the plate was shaken at 37 ° C for 1 hour. After washing twice with PBST, antiserum diluted 100-fold with PBSB was dispensed in 50 L aliquots, and washed three times with PBST. After shaking at 37 ° G for 1 hour, the plate was washed three times with PBST, and 100 L of HRP-labeled secondary antibody diluted 1000-fold with PBS (-) was added. After shaking at 37 ° G for 1 hour, the plate was washed four times with PBST, and a chromogenic substrate solution (0PD-H202) was added at 100 / L. After shaking at 37 ° C for 30 minutes, the reaction was stopped by adding 100 L of 0.5N sulfuric acid, and the absorbance (0D) at 492 nm was measured using a microplate reader.
実施例 4  Example 4
抗血清による PSPの吸収試験  PSP absorption test with antiserum
STX BSA-1 および neoSTX- BSA- 1 から得た血清および無免疫のゥサギ から得た血清 （正常血清） それぞれ 50 Lに 1 Mの PSP各成分 （ G1 +2、 GTX1+4、 GTX2+3、 GTX5、 STX、 neoSTX) の標準溶液を等量混合し、 5°Gで 15 時間静置した。 限外遠心濾過キッ 卜 （ Ultrafree - MC， N L 5000， Mi I I ipore) で得た濾液を HPLG蛍光法で分析した。  Serum from STX BSA-1 and neoSTX- BSA-1 and serum from non-immunized egret (normal serum) 1 M of PSP in 50 L each (G1 +2, GTX1 + 4, GTX2 + 3, An equal amount of a standard solution of GTX5, STX, and neoSTX) was mixed and allowed to stand at 5 ° G for 15 hours. The filtrate obtained using an ultracentrifugal filtration kit (Ultrafree-MC, NL 5000, MiI Ipore) was analyzed by the HPLG fluorescence method.
実施例 5  Example 5
ポリクローナル抗体を用いる PSPの EL I SA分析法の開発  Development of ELISA method for PSP using polyclonal antibody
96we I I の反応プレー 卜の各 we I I に、 PBS (-）に対して 50 Mの濃度に調 製した STX溶液を 50jU Lずつ分注し、 37°Gで 2時間振とう した。 プレー トを PBSTで 2回洗浄後、 0.3%ゼラチンを含む PBS (-) 300 Lを添加して 37°Cで 1 時間振とう した。 PBSTで 2回洗浄後、 PSP各成分の PBSすなわ ち G1+2、 GTX1+4, GTX2+3、 GTX5、 GTX6、 neoSTX, dcSTX、 STX を PBS (-） で 0、 0.1、 1、 10、 100、 1000、 10000 nM に希釈した溶液をそれぞれ 50 し 添加した。 次いで PBSB で 200 倍に希釈した抗血清 （BSA- STX-1) を 50 L ずつ添加して 37°Cで 1 時間振と う した。 同一プレ一 ト上に PSP と 抗血清溶液を添加しない we I I を用意してゼロブランク （Bo)と した。 37°C で 1 時間振と う後、 PBSTで 3回洗浄し、 PBS (-）に対して、 1000倍希釈し た HRP標識二次抗体を、 100 Lずつ添加した。 37°Cで 1 時間振と う後、 PBSTで 4回洗浄し、 発色基質溶液を 100 L添加した。 37°Gで 3 0分振と う した後、 0.5N硫酸 100 i L を添加して反応を停止し、 マイク 口プレー ト リーダーで 492nmの吸収（0D)を測定した。 試験は 3連で行った。 An STX solution prepared at a concentration of 50 M with respect to PBS (-) was dispensed in 50 jUL portions into each weII of the 96weII reaction plate, and shaken at 37 ° G for 2 hours. After washing the plate twice with PBST, 300 L of PBS (-) containing 0.3% gelatin was added, and the plate was shaken at 37 ° C for 1 hour. After washing twice with PBST, the PBS of each component of PSP, that is, G1 + 2, GTX1 + 4, GTX2 + 3, GTX5, GTX6, neoSTX, dcSTX, and STX in PBS (-) Then, 50 solutions each diluted to 0, 0.1, 1, 10, 100, 1000, and 10,000 nM were added to each solution. Then, 50 L of antiserum (BSA-STX-1) diluted 200-fold with PBSB was added and shaken at 37 ° C for 1 hour. On the same plate, weII without PSP and antiserum solution was prepared and set as zero blank (Bo). After shaking at 37 ° C for 1 hour, the plate was washed three times with PBST, and 100 L of HRP-labeled secondary antibody diluted 1000-fold with PBS (-) was added. After shaking at 37 ° C for 1 hour, the plate was washed four times with PBST, and 100 L of a chromogenic substrate solution was added. After shaking at 37 ° G for 30 minutes, the reaction was stopped by adding 100 iL of 0.5N sulfuric acid, and the absorbance (0D) at 492 nm was measured using a microplate reader. The test was performed in triplicate.
実施例 6  Example 6
ビォチン標識 STXの作成  Preparation of biotin-labeled STX
凍結乾燥状態の精製 EDT-STX複合体（15 i mol)を、 1.4mLの 10mM リ ン酸 緩 衝 液 （ pH7, 4 ) に 溶 解 し 、 こ れ に 6mg ( 10 fl mo I ) の Biot i n-PE (AC5) 2-ma 16!1^(^を 600〃 1_の DMSOに溶解したものを混合した c 5°Cで一晚静置後、 0.05M酢酸で 5mL に定容し、 3連の SepPak C18 p I us カー ト リ ッジを 0.05M酢酸 15mLで洗浄後、 0.05M酢酸一メタノール（ 1： 1 v/v) 10mLで吸着成分を溶出 した。 含水酸性メタノール溶出液に含まれる ビォチン標識 STXの濃度を HPLC蛍光法で定量するとともに、 同画分の毒 性をマウス試験法で調べた。 Lyophilized purified EDT-STX complex (15 imol) was dissolved in 1.4 mL of 10 mM phosphoric acid buffer (pH 7, 4), to which 6 mg (10 fl moI) of Biot i n-PE (AC5) 2 -ma 16! 1 ^ ( one ^ at 600〃 1_ of DMSO mixed c 5 ° C a solution obtained by dissolving the晚静after standing, and constant volume of 5mL in 0.05M acetic acid, The triplicate SepPak C18 pIus cartridge was washed with 15 mL of 0.05 M acetic acid, and the adsorbed components were eluted with 10 mL of 0.05 M acetic acid-methanol (1: 1 v / v). The concentration of biotin-labeled STX was quantified by HPLC fluorescence, and the toxicity of the same fraction was examined by a mouse test method.
実施例 7  Example 7
蛍光標識 STXの作成  Preparation of fluorescently labeled STX
6mgの PAEM-2塩酸塩 ( w253.69) を精製 EDT - STX複合体 (23/ mo I ) と ともに 2.3mLの 50mM リ ン酸緩衝液中、 室温で 2 日間静置した。 反応混合 物を Bio-GEL P-2のカラム （1.5 x 20cm) に添加し、 200mLでカラムを洗 浄後 0.1M 酢酸、 次いで 0.5M酢酸で吸着成分を溶出した。 希酢酸溶出液 は 10mLずつ分取し、各画分を HPLG蛍光法で調べ PEAE- EDT-STX結合体（蛍 光標識 STX) の溶出をモニターした。 単離した PAEM-EDT - STX結合体の毒 性をマウス試験法で調べた。 6 mg of PAEM-2 hydrochloride (w253.69) together with the purified EDT-STX complex (23 / moI) was allowed to stand in 2.3 mL of 50 mM phosphate buffer at room temperature for 2 days. The reaction mixture was added to a Bio-GEL P-2 column (1.5 x 20 cm), the column was washed with 200 mL, and the adsorbed components were eluted with 0.1 M acetic acid and then with 0.5 M acetic acid. The dilute acetic acid eluate was collected in 10 mL aliquots, and each fraction was examined by the HPLG fluorescence method. The PEAE-EDT-STX conjugate The elution of the photolabeled STX) was monitored. The toxicity of the isolated PAEM-EDT-STX conjugate was determined by a mouse assay.
実施例 8  Example 8
PSPならびに PSP—ナオール複合体の定量  Determination of PSP and PSP-naol complex
供試溶液中の PSPは HPLCの蛍光法（0shima， 1995)で定量した。 溶液中 の結合型 PSPすなわち EDT- STX等のチオール一 PSP複合体およびタ ンパク に結合した PSP等の量は、 溶液の一部を、 10%メルカプ トエタノールを含 む 0.1M リ ン酸ナ ト リウム緩衝液 （PH7.4) で希釈し、 5分間煮沸した後 HPLC蛍光法で分析して得られた STX群と、 メルカプ トエタノール処理前 の GTX群と STX群の和を比較することによ り算出した。  PSP in the test solution was quantified by the fluorescence method of HPLC (0shima, 1995). The amount of conjugated PSP in the solution, ie, the thiol-PSP complex such as EDT-STX, and the amount of PSP bound to the protein can be determined by dividing a part of the solution with 0.1 M sodium phosphate containing The STX group obtained by diluting with buffer buffer (PH7.4), boiling for 5 minutes, and analyzing by HPLC fluorescence method, was compared with the sum of the GTX group and STX group before mercaptoethanol treatment. Calculated.
実施例 9  Example 9
マウス試験法  Mouse test method
供試液のマウス毒性を A.0. A. G.法 (Hoi I ingworth and Wekel I, 1990) に従って求めた。 ddY 系雄マウス （体重 2 0 ± 1 g) を使用 し、 1 試料に つき 5尾のマウスを用い致死時間の中間値から PSP の用量致死時間曲線 (Sommer and Meyer, 1937) に従って投与した毒値 (mouse unit = MU) を算出 した。 1 MU は 15分で 1 匹のマウスを殺す毒量に相当する。  The mouse toxicity of the test solution was determined according to the A.0.A.G. method (Hoi Iingworth and Wekel I, 1990). Using ddY male mice (body weight 20 ± 1 g), using 5 mice per sample, administering the toxin value according to the PSP dose lethal time curve (Sommer and Meyer, 1937) from the median lethal time (mouse unit = MU) was calculated. One MU is equivalent to the toxic dose that kills one mouse in 15 minutes.
実施例 1 0  Example 10
PSP—タ ンパク結合体 （抗原） の作成  Preparation of PSP-protein conjugate (antigen)
第 2図に示す手順で BSA- EDT- STX 結合体および同様の手順で調製した BSA-EDT - neoST.X結合体を調製した。 以前我々はグルタチオン- PSP複合体 を BSA に導入して抗原を作成しているが、 今回新たに作成した EDT を介 する結合法によ り、 よ り多数の PSP 分子がタ ンパクを導入した抗原の作 成に成功した （表 1 ) 。 抗原溶液中の BSA濃度および BSAに結合した PSP量 抗原溶液のタ ンパク PSP:BSA A BSA-EDT-STX conjugate and a BSA-EDT-neoST.X conjugate prepared by the same procedure were prepared according to the procedure shown in FIG. Previously, we created an antigen by introducing a glutathione-PSP complex into BSA, but this time, a new EDT-mediated conjugation method allows a larger number of PSP molecules to be antigen-transfected. (Table 1). BSA concentration in antigen solution and amount of PSP bound to BSA Protein in antigen solution PSP: BSA
抗原  Antigen
濃度 ( g/mL) モル比 (%)Concentration ( g / mL) Molar ratio (%)
BSA- EDT- STX (新規抗原） 1497 5.4 2.3 BSA-EDT-neoSTX 607 8.9 4. 1 BSA - GS - STX 684 1.0 0.4 BSA-GS-neoSTX 788 0. 1> 実施例 1 1 BSA-EDT-STX (new antigen) 1497 5.4 2.3 BSA-EDT-neoSTX 607 8.9 4.1 BSA-GS-STX 684 1.0 0.4 BSA-GS-neoSTX 788 0.1> Example 1 1
ゥサギ血清の抗体価の推移  抗体 Changes in antibody titer of heron serum
第 3図に BSA-EDT- STX結合体を免疫したゥサギ（ STX - E叮- BSA - 1 および STX-EDT-STX-2) の抗体価の推移を示す。 2羽のゥサギとも免疫開始後 3 ヶ月目よリ抗体価の有意な上昇が認められ、 4ヶ月目以降の抗体価は増 減を繰り返しながら推移した。 NeoSTX - EDT-STX結合体を免疫したゥサギ の抗体価もほぼ同様に推移したが、 BSA- EDT-STX結合体を免疫した場合に 比べて抗体価の上昇は低かった。 いずれのゥサギも免疫開始後 7 ヶ月を 過ぎた時点で全採血し、 得た血清を以下の試験に用いた。  FIG. 3 shows the change in the antibody titer of Egret (STX-EDDING-BSA-1 and STX-EDT-STX-2) immunized with the BSA-EDT-STX conjugate. A significant increase in the antibody titer was observed in both of the two egrets three months after the start of immunization, and the antibody titer after four months continued to increase and decrease. The antibody titers of the rabbits immunized with the NeoSTX-EDT-STX conjugate changed in a similar manner, but the increase in the antibody titer was lower than that immunized with the BSA-EDT-STX conjugate. All of the magpies were bled 7 months after the start of immunization, and the serum obtained was used for the following tests.
実施例 1 2  Example 1 2
抗血清による PSP成分の吸収  Absorption of PSP components by antiserum
第 4図に BSA— EDT— STX— 1カヽら得た血清および BSA— neoSTX— 1カゝら得た血 清による各 PSP 成分の吸収量を示した。 無免疫のゥサギの血清 （正常血 清） は 1 mL当たりいずれの PSP成分も 0. 1 nmo I 未満と検出限界未満であ つた。 これに対して BSA-STX-1 血清 1mLは 1 ~3nmol の PSP を吸収した。  FIG. 4 shows the amount of each PSP component absorbed by the serum obtained from BSA-EDT-STX-1 and the serum obtained from BSA-neoSTX-1. The serum of unimmunized egrets (normal serum) contained less than 0.1 nmo I of each PSP component per mL, which was below the detection limit. In contrast, 1 mL of BSA-STX-1 serum absorbed 1 to 3 nmol of PSP.
BSA-neoSTX-1 血清による PSPの吸収はこれに比べ低かった。 Absorption of PSP by BSA-neoSTX-1 serum was lower.
実施例 1 3  Example 13
STX固相化プレー ト上での各 PSP成分の競合 PSP 成分の顕著な吸収が認められた BSA- STX- 1 血清を用いて、 PSP の EL ISAによる定量法の開発を試みた。 種々検討の結果、 STXの中性リ ン酸 緩衝液を添加して固相化した反応プレー 卜を用いる i nd i rect two step 法によ り定量的に PSP 成分を分析することが可能となった。 第 5図に示 すよう Iこ IG50値を与える濃度 Iま STX、 GTX2+3で 4〜 7nM、 neoSTX、 dcS丁 X、 GTX5、 GTX1+4で 30〜 100nM、 GTX6で約 200nMであった。 このように PSP の成分によ り抗体に対する反応性は若干異なるものの、 いずれの成分に 対しても同法によリマウス試験法の 10〜 100 倍の高感度での検出が可能 である。抗体は、プリン骨格を持つアデノシン（Ade)や力フェイン（Gaf)、 およびグァニジル基を持つアルギニン （Arg) など、 PSP と部分的に構造 の類似する生体成分には全く反応しなかった （第 6図） 。 しかし興味あ るこ とに BSA - STX - 1 抗体に対して PSP と同じ薬理作用を持つフグ毒テ 卜 ロ ドトキシン （TTX) は高濃度で交差が認められた。 Competition of each PSP component on STX immobilized plate An attempt was made to develop a method for quantitative determination of PSP by ELISA using BSA-STX-1 serum, which showed significant absorption of PSP components. As a result of various studies, it became possible to quantitatively analyze PSP components by the indirect two-step method using a reaction plate immobilized by adding a neutral phosphate buffer solution of STX. Was. As shown in FIG. 5, the concentration giving the IG50 value was 4 to 7 nM for STX and GTX2 + 3, 30 to 100 nM for neoSTX, dcS-X, GTX5 and GTX1 + 4, and about 200 nM for GTX6. As described above, although the reactivity with the antibody differs slightly depending on the PSP components, the same method can be used to detect any of the components with 10 to 100 times the sensitivity of the re-mouth test. The antibody did not react at all with biological components partially similar in structure to PSP, such as adenosine (Ade) having a purine skeleton, force phain (Gaf), and arginine (Arg) having a guanidyl group (No. 6). Figure). Interestingly, however, crossing was observed at a high concentration of the pufferfish tetrodotoxin (TTX), which has the same pharmacological effect on the BSA-STX-1 antibody as PSP.
実施例 1 4  Example 14
標識付き PSP誘導体の調製および比毒性  Preparation and specific toxicity of labeled PSP derivatives
マレイ ミ ド基とスルフヒ ドリル基は中性水溶液中穏和な条件下で反応 し結合体を形成する。 マレイ ミ ド基を持つ種々のラベル化合物が市販さ れているが、 本試験ではこれらの中からピオチンラベル試薬と水溶性の 蛍光ラベル試薬を選び、 これらを導入して標識付き STX 誘導体を作成し た （第 7図） 。 これら 2種の誘導体に加え、 スルフヒ ドリル基のマスキ ング剤である NEM を同様の手順で導入した NEM-EDT - STX誘導体を調製し た。 これら標識付き誘導体は Bio- Gel P - 2 等のカラムクロマ 卜グラフィ —により他の PSP 成分から分離することが出来た （第 8図） 。 精製して 得た誘導体を腹膣内投与したマウスは PSP を投与した場合と同じ症状を 示して死亡した。 グルタチオン一 PSP複合体 （Sato et aに， 2000) およ び EDT - STX誘導体は STXの 100分の 1 未満の比毒性しか示さなかったの に対し、 EDT- STXを出発物質とするこれら標識付き PSP誘導体は、 STXの 3 0分の 1 ないし 7分の 1 と、 N—suけ ocarbamoyl 基を持つ天然成分 (C1 ~C4, GTX5) のそれに匹敵する比毒性を示した （第 9図） 。 産業上の利用可能性 Maleimide and sulfhydryl groups react under mild conditions in neutral aqueous solutions to form conjugates. A variety of labeled compounds having a maleimide group are commercially available.In this test, a biotin-labeled reagent and a water-soluble fluorescent labeling reagent were selected, and these were introduced to produce labeled STX derivatives. (Figure 7). In addition to these two derivatives, NEM-EDT-STX derivatives were prepared in which NEM, a masking agent for sulfhydryl groups, was introduced in the same procedure. These labeled derivatives could be separated from other PSP components by column chromatography such as Bio-Gel P-2 (Fig. 8). Mice given the purified derivative intraperitoneally showed the same symptoms as those given PSP and died. Glutathione-PSP complex (Sato et a, 2000) and EDT-STX derivatives showed less than 100 times less specific toxicity than STX On the other hand, these labeled PSP derivatives starting from EDT-STX are 1/3 to 1/7 of STX, and natural components (C1 to C4, GTX5) having N-su-carbamoyl group. It showed comparable specific toxicity (Fig. 9). Industrial applicability
本発明による BSA- EDT - STX を抗原と して調製したポリクロ一ナル抗体 は、 neoSTXに対する親和性は若干低かったが、 他の麻痺性貝毒成分には ほぼ同様の親和性を示した。本抗体を用いて試験的に作成した ELISAは、 毒成分によって値は若干異なるものの、 マウス試験の 1 0 〜 1 0 0倍の 感度で毒を検出することができた。  The polyclonal antibody prepared using BSA-EDT-STX as an antigen according to the present invention had a slightly lower affinity for neoSTX, but showed almost the same affinity for other paralytic shellfish poison components. The ELISA prepared as a test using this antibody was able to detect toxins with a sensitivity 10 to 100 times higher than that in the mouse test, although the values differed slightly depending on the toxic components.

Claims

請 求 の 範 囲 The scope of the claims
1 . サキシ トキシンの 1 1 位にスルフヒ ドリル基を複数持つジチオール 化合物を結合させて遊離スルフヒ ドリル基を持つサキシ トキシンスルフ ヒ ドリル誘導体を調製し、 他方、 スルフヒ ドリル基指向性の官能基をァ ミノ基に導入したタ ンパク質を調製し、 次いで上記サキシ トキシンスル フ ヒ ドリル誘導体と上記調製タ ンパク質とを反応させて得られる抗原を 非ヒ ト動物に免疫し、 該動物から麻痺性貝毒を認識する抗血清を得るこ とを含む、 麻痺性貝毒に対する抗体の製法。 1. A saxitoxin sulfhydryl derivative having a free sulfhydryl group is prepared by bonding a dithiol compound having a plurality of sulfhydryl groups at the 1-position of saxitoxin, while a sulfhydryl group-directing functional group is substituted with an amino group. A non-human animal is immunized with an antigen obtained by reacting the saxitoxin sulfhydryl derivative with the prepared protein, and recognizes paralytic shellfish toxin from the animal. A method for producing an antibody against paralytic shellfish toxin, comprising obtaining an antiserum that is resistant to paralysis.
2 . スルフヒ ドリル基を有するジチオール化合物が、 1 ， 2—エタ ンジチ オール、 ジチオス レィ トール、 ジチォエリスリ トール、 2， 3 —ジメルカ プ トー 1 一プロパンスルホン酸からなる群から選択される、 請求の範囲 第 1 項記載の抗体の製法。 2. The dithiol compound having a sulfhydryl group is selected from the group consisting of 1,2-ethanedithiol, dithiothreitol, dithioerythritol, 2,3-dimercapto-1 monopropanesulfonic acid. The method for producing the antibody according to claim 1.
3 . スルフ ヒ ドリル基指向性の官能基が、 マ レイ ミ ド、 チオフタルイ ミ ド、 活性ハロゲン基からなる群から選択される、 請求の範囲第 1 項記載 の抗体の製法。 3. The method for producing an antibody according to claim 1, wherein the functional group having a sulfhydryl group directing property is selected from the group consisting of maleimide, thiophthalimide, and an active halogen group.
4 . 請求の範囲第 1 項記載の方法によ り得られる抗体。 4. An antibody obtained by the method according to claim 1.
5 . 請求の範囲第 1 項記載の製法によ り得られる抗体を用いる EL I SA 測 定キッ 卜。 5. An ELISA measurement kit using an antibody obtained by the production method according to claim 1.
6 . サキシ トキシンの 1 1 位にスルフ ヒ ドリル基を複数持つジチオール 化合物を結合させて遊離スルフ七 ドリル基を持つサキシ トキシンスルフ ヒ ドリル誘導体を調製し、 他方、 スルフ ヒ ドリル基指向性の官能基を導 入した標識化合物を調製し、 次いで上記サキシ トキシンスルフ ヒ ドリル 誘導体と上記修飾標識化合物とを反応させて得られる、 麻痺性貝毒簡易 結合測定法に使用するための標識毒標品の製法。 6. A dithiol compound having multiple sulfhydryl groups at the 11-position of saxitoxin is bound to form a saxitoxin sulfone having a free sulfhydryl group. A paralyptic compound obtained by preparing a hydryl derivative and, on the other hand, preparing a labeled compound into which a sulfhydryl group-directed functional group is introduced, and then reacting the saxitoxin sulfhydryl derivative with the modified labeled compound. A method for producing labeled poison samples for use in the simple binding assay for shellfish poison.
7 . スルフ ヒ ドリル基を有するジチオール化合物が、 1， 2—エタ ンジチ オール、 ジチオスレィ トール、 2 , 3—ジメルカプ ト一 1 一プロパンスル ホン酸からなる群から選択される、 請求の範囲第 6項記載の標識毒標品 の製法。 7. The dithiol compound having a sulfhydryl group is selected from the group consisting of 1,2-ethanedithiol, dithiothreitol, and 2,3-dimercapto1-1-propanesulfonic acid. The manufacturing method of the labeled poison standard product described.
8 . スルフ ヒ ドリル基指向性の官能基が、 マ レイ ミ ド、 チオフタルイ ミ ド、 活性ハロゲン基からなる群から選択される、 請求の範囲第 6項記載 の標識毒標品の製法。 8. The method for producing a labeled poison sample according to claim 6, wherein the sulfhydryl group-directing functional group is selected from the group consisting of maleimide, thiophthalimid, and an active halogen group.
9 . 標識化合物が、 ァガロース、 ビォチン、 蛍光物質および金コ ロイ ド からなる群から選択される、 請求の範囲第 6項記載の標識毒標品の製法。 9. The method according to claim 6, wherein the labeled compound is selected from the group consisting of agarose, biotin, a fluorescent substance, and gold colloid.
1 0 . 請求の範囲第 6項記載の製法によ り得られる標識毒標品。 10. A labeled poison sample obtained by the production method according to claim 6.
PCT/JP2002/005899 2001-07-04 2002-06-13 Method of preparing antinarcotic shellfish toxin antibody, novel antibody, elisa kit with the use of the antibody and labelled toxin sample prepared by the method WO2003006507A1 (en)

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US8173601B2 (en) 2007-02-09 2012-05-08 Acceleron Pharma, Inc. Activin-ActRIIa antagonists and uses for treating multiple myeloma
US10513525B2 (en) 2009-05-07 2019-12-24 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for studying, imaging, and treating pain
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CN102459273A (en) * 2009-05-07 2012-05-16 里兰斯坦福初级大学理事会 Methods and compositions for studying, imaging, and treating pain
US9174999B2 (en) 2009-05-07 2015-11-03 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for studying, imaging, and treating pain
CN107011344A (en) * 2009-05-07 2017-08-04 里兰斯坦福初级大学理事会 Method and composition for studying, being imaged and treating pain
US10106549B2 (en) 2014-04-09 2018-10-23 Siteone Therapeutics, Inc. 10′,11′-modified saxitoxins useful for the treatment of pain
US10112953B2 (en) 2015-09-30 2018-10-30 Siteone Therapeutics, Inc. 11,13-modified saxitoxins for the treatment of pain
US10920256B2 (en) 2016-02-12 2021-02-16 Vestlandets Innovasjonsseiskap AS Process
US11566271B2 (en) 2016-02-12 2023-01-31 Vestlandets Innovasjonsseiskap AS Processes to make neosaxitoxin and analogues thereof
US11236097B2 (en) 2017-03-29 2022-02-01 Siteone Therapeutics, Inc. 11,13-modified saxitoxins for the treatment of pain
US11279706B2 (en) 2017-03-29 2022-03-22 Siteone Therapeutics, Inc. 11,13-modified saxitoxins for the treatment of pain
US11834456B2 (en) 2017-03-29 2023-12-05 Siteone Therapeutics, Inc. 11,13-modified saxitoxins for the treatment of pain

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