JP2003012699A - Method for manufacturing anti-paralytic shellfish poison antibody, new antibody, elisa measuring kit using the antibody, and system-labeling poison standard sample prepared by the manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the determination of a paralytic shellfish poison simple, having high accuracy, independent of the kind of shellfish toxin and having a constant sensitivity. SOLUTION: A method for preparing an antibody against a paralytic shellfish poison comprises the following steps: an arbitrary dithiol compound having two or more sulfhydryl groups is bound to the 11th position of saxitoxin to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group; on the other hand, a protein into whose amino group an arbitrary functional group having sulfhydryl group directivity has been introduced, is prepared; the saxitoxin sulfhydryl derivative is made to react with the modified protein to prepare an antigen; and an animal other than human is immunize with the obtained antigen, and an antiserum recognizing the paralytic shellfish poison is obtained from the animal. An ELISA assaying kit using the antibody. A standard sample of labeling poison prepared by the preparation method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an antibody against paralytic shellfish toxin and the antibody. Further, the present invention relates to an ELISA assay kit using the above antibody and a labeled poison preparation for the paralytic shellfish toxin binding assay obtained by the above production method.

[0002]

2. Description of the Related Art Paralytic shellfish poisoning has a symptom mainly showing nerve palsy, and its causative poisons have been found to be goniotoxins and saxitoxins [Ken Yasumoto, paralytic shellfish poison, Food Hygiene Inspection Guidelines (Science and Chemistry, Ministry of Health and Welfare Bureau supervision), pages 300-305, Japan Food Hygiene Association, 1991
Year]. These causative toxins are collectively called paralytic shellfish poisons, and at present, more than 20 species of goniotoxins and saxitoxins are known as shellfish poisons found in shellfish such as mussels and clams.

This shellfish poison is a paralytic poison that has the property of blocking sodium channels, and it is contained in several types of phytoplankton that belong to dinoflagellates, and the shellfish that eats with it accumulates the poison and poisons it. Eating this shellfish causes food poisoning with a high mortality rate, which is a major social problem in fisheries and food hygiene. By the way, paralytic shellfish poison 0.5
Symptoms such as numbness, nausea, and diarrhea occur only by ingesting ~ 1.0 mg orally. The lethal dose in humans is 1-3 mg. Areas where shellfish are contaminated with paralytic shellfish poisons are expanding worldwide. In these areas, the toxicity of shellfish shipped to control food poisoning by poisoned shellfish is tested by mouse test method or HPLC.
Inspected by law. However, these measurement methods
There are many problems in terms of cost and labor for measurement, and development of a simple method for measuring poison is desired.

Therefore, various methods for quantifying paralytic shellfish poison have been developed. For example, an official method for measuring lethal activity using mice [Ken Yasumoto, supra; J. F. Jellett et al., To
xicon, 30 (10): 1143-1156 (1992); W. Horwitz, Paralyt
ic shellfish poison. In "Official Methods of Analy
sisi of the Association of Official Analytical Che
mists "(Assoc. Official Anal. chem., Washingtion D.
C.) pp. 881-882 (1990)], high performance liquid chromatography method [Y. Oshima et al., Mycotoxins and Phycotoxins '88.
(Edited by S. Natori et al.), Pp.319-326, Elsevier, Amsterdam
(1989)); JP-A-9-133669], a method using neuroblast cells [K. Kogure et al., Toxicon, 26: 191-197 (1989)],
Method using antibody [FS Chu et al., J. Agri. Food Che
m., 44: 4043-4047 (1996)] and the like are known. The lethal activity is measured by injecting a mouse test stock solution into the abdominal cavity, and the time from the end of the injection to the last gasping when the mouse dies with typical paralytic shellfish poisoning (lethal time). ) Is recorded in seconds, and the amount of poison is estimated from the dose-matching time curve prepared with the standard poison. The high performance liquid chromatography method applies high performance liquid chromatography to a sample containing paralytic shellfish to separate the paralytic shellfish poison, and after adding an oxidizing agent to the eluate and reacting it in alkali, This is a method of measuring fluorescence. The method using neuroblasts is the enzyme Na-KATPas that excretes Na + out of the cell in a neuroblast culture system.
The system in which the activity of e was inhibited by ouabain was tested with veratridine which promotes Na + influx and a poison which blocks the influx of Na +, and its amount is estimated from the activity of the toxin that blocks swelling death of cells caused by veratridine. , This method utilizes the fact that when a sample containing paralytic shellfish poison is added to this assay system, cell rounding and cell death are suppressed in proportion to the amount of poison. Further, an immunological method is to use an antibody against paralytic shellfish toxin to directly measure the amount of the antibody that binds to the toxin by a method such as enzyme-linked immunosorbent assay (eg, ELISA), and measure the amount of the antibody in the sample. This is a method for quantifying paralytic shellfish poison.

In particular, FS Chu et al. (Supra) use an anti-saxitoxin antibody / saxitoxin-horseradish peroxidase conjugate or an anti-neo-saxitoxin antibody / neosaxitoxin-horseradish peroxidase conjugate.
An assay method for measuring the total amount of paralytic shellfish toxin by ELISA is disclosed. Antibody preparation is done by FS Chu and X. Fan,
J. Assoc. Off. Anal. Chem, 1985, 68: 13-16 and F.
Although prepared according to S. Chu, J. AGACInt. 1992, 75: 341-345, the procedure is described by Johnson et al. (Proc. Soc. Exp.
Biol. Med., 1964, 117, 425), and a conjugate obtained by reacting bovine serum albumin, polylysine or hemocyanin of keyhole limpet (limpet) with saxitoxin or neosaxitoxin in a weakly acidic aqueous solution containing formalin. Is subcutaneously injected into a rabbit to obtain a polyclonal antibody.

[0006] Specifically, the antigen used for the production of these antibodies is one in which a protein is bound to two guanidinium groups present in the molecule of saxitoxin via formalin [Johnson et al., Supra; Chu and Fan. , Above; V. Renz
And G. Terplan, Arch, Lebensmittelhyg., 1988, 39, 25.
-56; A. Cembella et al., "Specificity and cross-reactivi.
ty ofan adsorption-inhibition onzyme-linked immuno
assay for the detection of paralytic shellfish toxi
ns. In Toxic Marine Phytoplankton "(edited by E. Graneli et al.), Elsevier Science Publishers, New York, pp.33
9-344,1989], one of two OH groups at the 12-position of saxitoxin was removed, and the remaining OH group was bound to a protein [R. Carlson et al., "Development of immu
noassays for paralytic shellfish poisoning.A radi
o immunoassay for sexitoxin. InSwafood Toxins ",
(EP Ragelis Edition), ACS Symposium Serics 262, Ame
ricanChemical Society, Washington, DC, pp.181-19
2 "]. In the former antigen, the binding site of the protein is unclear in one or both of the two guanidinium groups. The antibody also has good reactivity with neosaxitoxin at about 20% (however, the reactivity with saxitoxin is 100%), and the same IN-H derivative, goniotoxin 2, 3 and detox, as with saxitoxin. The reactivity with carbamoylsaxitoxin is as low as about 50%, and it does not bind to the carbamoyl-N-sulfate derivative C toxin at all, whereas the latter antibody has a reactivity with neosaxitoxin of 1 or less.
% Or less, reactivity with other toxic components has not been investigated. Furthermore, attempts have been made to produce monoclonal antibodies using similar antigens, but these have low reactivity and low sensitivity (R. Hack et al., Food and Agriculturel immunology,
1990, 2, 47-48).

[0007]

Problems to be Solved by the Invention The lethal activity assay method and the neuroblast method have problems in terms of animal and cell management, detection sensitivity, accuracy, specificity, etc. However, it has the drawbacks that it is easy to operate and highly accurate, but it requires expensive equipment, that the sensitivity varies depending on the type of anesthetic shellfish poison, and that no purified external standard is supplied. Further, the immunological method has a problem in that the reactivity of the antibody varies depending on the type of the saxitoxin derivative. Therefore, it has been desired to develop a method that is simple and highly accurate for anesthetic shellfish poisoning, and that can measure with constant sensitivity regardless of the type of shellfish poison.

The present inventors used the anesthetic shellfish-glutaotine (PSP-GSH) complex (Japanese Patent Application No. 10-91797) as a hapten and prepared an antigen by introducing this into a protein molecule. When administered to rabbits, a polyclonal antibody that reacts with paralytic shellfish poison to all the anterior components was obtained and disclosed (JP-A-2000-344799). However, in the method using the complex with GSH, the amount of hapten bound to the protein molecule is small, the antibody titer of the obtained antiserum is low, and improvement of the binding method is necessary to introduce the toxin into the protein molecule. Was thought to be.

The first object of the present invention is to provide a method for producing an antibody against paralytic shellfish venom using the improved binding method for introducing the venom into other molecules such as proteins. A second object of the present invention is to provide an antibody prepared by 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 a method for producing a labeled poison preparation for use in a simple method for measuring paralytic shellfish poison prepared by using the improved binding method of the present invention, and a labeled poison preparation prepared by this production method. Is provided.

The present inventors have prepared a poisonous derivative having a free sulfhydryl group using a dithiol compound having a plurality (two) of sulfhydryl groups, and introduced this derivative into the amino group of a sulfhydryl group-directed functional group. It was found that the poison can be efficiently introduced into the protein by reacting with the protein, and it is possible to prepare an antigen having more hapten molecules introduced therein. The above-mentioned production method of the present invention is characterized in that the poison and the protein are modified separately and the antigen is obtained by the reaction of both. The antigen according to the invention is
It has been found by the present inventors that an antiserum having a high antibody titer can be obtained and the antibody can be obtained in a high yield because of the large amount of hapten per protein molecule. Use of the antibody prepared by the improved binding method of the present invention makes it possible to provide a venom ELISA assay kit.

According to the method for producing an antibody of the present invention,
The inventors have also found that the 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, but since the active site of the antigen of the present invention is exposed, the labeled venom preparation can be prepared with the toxicity remaining.

Therefore, in the first aspect of the present invention, a plurality of sulfhydryl groups (2
One) to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group by binding with a dithiol compound, while preparing a protein having a sulfhydryl group-directed functional group introduced into an amino group, and then the saxitoxin sulfhydryl derivative and the modified protein described above. There is provided a method for producing an antibody against paralytic shellfish poisoning, which comprises immunizing a non-human animal with an antigen obtained by reacting the parasite and obtaining antiserum capable of recognizing the paralytic shellfish poison.

The dithiol compound having a sulfhydryl group is preferably selected from the group consisting of 1,2-ethanedithiol, dithiothreitol and dithioerythritol 2,3-dimercapto-1-propanesulfonic acid. The compound having a sulfhydryl group-directed functional group is
It is preferably selected from the group consisting of maleimide, thiophthalimide and active halogen compounds.

In a second aspect of the present invention, there is provided the antibody obtained by the production method of the first aspect.

Further, in a third aspect of the present invention, a novel ELISA kit using the antibody obtained in the first aspect is provided.

Further, in the fourth aspect of the present invention, a dithiol compound having a plurality of sulfhydryl groups (two) at the 11-position of saxitoxin is bound to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group, and then a thiol-directed functional group is prepared. There is provided a labeled venom preparation for use in a simple binding assay of an anesthetic shellfish poison, which comprises reacting a labeled compound such as agarose or biotin having a group, a fluorescent substance, and gold colloid with this derivative.

Further, in a fifth aspect of the present invention, there is provided a labeled poison preparation using the method for producing the labeled poison preparation described above.
The toxic preparations in the present invention are non-isotopic biotin-labeled and fluorescent-labeled preparations.
Shown in.

In the above-mentioned production method of the present invention, any functional group directed to a free sulfhydryl group, particularly maleimide, thiophthalimide, active halogen, etc., reacts quantitatively with a thiol group in a neutral aqueous solution under mild conditions. According to the present invention, the anesthetic shellfish can be bound to a protein or a labeled compound with much higher yield and more easily than the binding method using the PSP-GSH complex or other amide condensation. It is possible to bear.

The method for producing the antibody of the present invention when ethanedithiol (EDT) is used as the dithiol compound will be described below.

The saxitoxin-EDT complex is a mixture of goniotoxin (goniotoxin 2, 3 or a combination thereof) and EDT having a dithiol group and heating (about 7).
At 0 ° C.), it can be obtained by substitution reaction with the 11-position OSO ₃ ⁻ group of the goniotoxin via the sulfur molecule of EDT. In the present invention, this complex is a so-called hapten antigen, that is, a substance that binds to the antibody but has no ability to induce the antibody. It is generally known that a protein may be covalently bound to such a hapten antigen to form a large molecule, which may be used as an immunogen.
The protein of the present invention also has a similar function. Examples of such proteins include albumin and limpet hemocyanin (KLH).

The present invention also provides, in a second aspect, the paralytic shellfish toxin components saxitoxin, goniotoxin 2,
3, neosaxitoxin, goniotoxin 1,4, goniotoxin 5, and C toxin 1,2 are both 5
Provided is an antibody against paralytic shellfish, which exhibits a relative reactivity of 0% or more (however, the reactivity with saxitoxin is 100%).

The antibody of the present invention is an IN-H derivative (saxitoxin, goniotoxin 2, 3) which is a paralytic shellfish poison, IN-O.
H derivatives (neo-saxitoxin, goniotoxin 1,
4), all of the carbamoyl-N-phosphate derivatives (goniotoxin 5, and C toxin 1,2) can be recognized almost in the same way. Here, expressions such as “goniotoxin 2, 3” mean a mixture of goniotoxin 2 and goniotoxin 3. For the name and structure of paralytic shellfish poison, see, for example, Takeshi Yasumoto, Paralytic shellfish poison, Food Hygiene Inspection Guideline (Science and Chemistry, Ministry of Health and Welfare Bureau supervision).
Pages 0-305, Japan Food Hygiene Association, 1901; Intergovernmen
tal Oceanographic Commision (IOC) Manual and Guides
No.33 (1995), UNESCO's Workshops, France, pp.81-94
Etc. According to an embodiment of the present invention, the antibody of the present invention is obtained by the above method.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The antigen used in the production of the antibody of the present invention is a saxitoxin-EDT-protein complex. This composite can be manufactured as follows. As a raw material for the saxitoxin portion, goniotoxin 2, goniotoxin 3, or a mixture thereof in any ratio can be used. These raw material components are treated with activated carbon from a heated extract of shellfish such as mussels, clams and scallops with 0.1N hydrochloric acid, gel filtration chromatography (for example, using Bio-Gel P-2 (Bio-Rad)), ion exchange chromatography ( For example Bio-Re
x70 (Bio-Rad) can be used for purification.

When the goniotoxin obtained as described above is reacted with EDT (reduced form is preferable) under heating, the SH group of EDT is the electron-donating property, so that the 11th carbon of the goniotoxin is present. atoms and nucleophilic attack, the result OSO ₃ ^-
The group is released, and -S- (EDT) is bonded to the 11th carbon atom, thereby forming a saxitoxin-EDT complex. The reaction occurs by heating the reactants under neutral conditions. Generally pH 6.5-7.5, preferably ph7.0-
The reaction can be carried out using a suitable buffer of 7.5, such as ammonium phosphate buffer, ammonium acetate buffer and the like. The reaction temperature is generally from room temperature to about 100.
℃, preferably 50 to 80 ℃, more preferably about 70 ℃. After the reaction, the desired product is purified by preparative high performance liquid chromatography, gel filtration chromatography, ion exchange chromatography, adsorption chromatography, reverse phase partition chromatography, etc., alone or in combination. be able to. The product can be identified by a usual measurement method such as infrared spectroscopy, mass spectrometry, NMR method, elemental analysis and amino acid analysis.

Low-isolation compounds such as saxitoxin belong to haptens and have no antigenicity by themselves, but a protein having antigenicity is used as a carrier, and a complex covalently bound to the protein produces a specific antibody against the hapten. It becomes an antigen. It is considered 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. At this time, it is necessary to produce the hapten antigen by a selective binding method in which an extra antigenic determinant is difficult to form. The recognition of the partial structure of the hapten is weak near the binding site between the hapten and the carrier protein, and it is easy to recognize the partial structure farther away. Therefore, when binding to a protein, it is important to select an appropriate binding position in the hapten. The saxitoxin-EDT complex having a structure in which an EDT group is bound to the 11-position of saxitoxin previously discovered by the present inventors (Japanese Patent Application No.
0-91797) is not only preferable as an immune antigen,
It has the advantage of being able to produce paralytic shellfish poison, which enables highly reactive detection of almost all types of paralytic shellfish poison.

Albumin, hemocyanin and the like can be used as the carrier protein, but the protein is not limited to these, and any protein capable of inducing the antibody of the present invention can be used. When binding a carrier protein to the saxitoxin-EDT complex, the carrier protein (introducing a reactive functional group) and the maleimide method (T. Kitagawa et al., J. Bioshem. 92: 585-590,19
82) and the carbodiimide method (D. Exley et al., FEBS Lett., 91:
162-165, 1978) and the like to bind saxitoxin-GS
H-protein complexes can be generated. For example, in the examples described below, an example of producing a saxitoxin-EDT-bovine serum albumin complex is shown.Here, first, a saxitoxin-EDT conjugate is prepared, while albumin is mercaptosuccinylated via its free amino group, and both are combined. By reacting the carrier protein with saxitoxin-E
Connect to DT. Purification of the obtained complex can be carried out 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, ultrafiltration, etc. are included.

When goniotoxin 1, goniotoxin 4 or a mixture thereof is used as a raw material instead of goniotoxins 2 and 3 and reacted with EDT, neosaxitoxin-
An EDT complex is obtained. Further, by reacting this complex with a carrier protein in the same manner as above, a neosaxitoxin-EDT-protein complex can be obtained,
The resulting product can be used for the production of antibodies against paralytic shellfish poison as well as the saxitoxin-EDT-protein complex (see below).

By immunizing animals such as mouse, rat, rabbit, goat, etc. with the saxitoxin-EDT-protein complex of the present invention, an antibody against the complex can be prepared. Usually, an immunogen solution is emulsified and mixed with an adjuvant such as Freund's complete adjuvant, incomplete adjuvant, RIBI IMMUNOCHEM RESEARCH INC, muramyl peptide (Calzyme), aluminum hydroxide, lipopolysaccharide, and the like. After subcutaneous injection, intradermal injection, or intramuscular injection, the same operation is performed at intervals of 2 to 4 weeks, and booster immunization is performed several times to obtain antiserum (polyclonal antibody) after exsanguination. Bleeding sera are routinely measured for reactivity with saxitoxin and booster immunizations are performed until high titers are achieved. The amount of the antigen varies depending on the type of animal that receives the antigen stimulation, but is 10 μg to several mg in the case of rabbit and several μg to several 10 mg in the case of mouse.

The antiserum against paralytic shellfish poison obtained as described above can be further purified into an IgG antibody by treating with, for example, ammonium sulfate fractionation and then ion exchange chromatography. Alternatively, the antibody can be purified by affinity chromatography using a gel in which saxitoxin is chemically coupled to dextran gel or agarose gel. The production and purification of the antibody are described in detail in, for example, Biochemistry Experimental Method 15 "Introduction to Immunology" (1982) Academic Society Publishing Center, and can be referred to.

In the method of the present invention, an increase in antibody titer is observed from about 1 month after immunization, and in the case of antisera against saxitoxin, titers of 0.5 nmol of saxitoxin per ml of antiserum are obtained after about 4 months. This antiserum had a somewhat low reaction with neosaxitoxin, but there was almost no difference in the reactivity with both shellfish poisons in the serum 5 months after immunization. In addition, FIG. 2 shows the reactivity of sera at the 4th and 5th month of immunization with various components of paralytic shellfish toxin, but the sera at the 4th month of the immunity shows some reactivity with neosaxitoxin and goniotoxin 1,4. Although inferior, the serum of the 5th month did not show a large difference in the reaction with each component. Thus, the antibody of the present invention immunologically reacts with almost all components of paralytic shellfish poison,
Particularly paralytic shellfish toxin components, saxitoxin, goniotoxin 2, 3, neosaxitoxin, goniotoxin 1,
4, goniotoxin 5, and C toxin (C1, C
Both of 2) show relative reactivity of 50% or more, preferably 60% or more (however, the reactivity with saxitoxin is 100%). On the other hand, many of the conventionally known antibodies are
Since the antigen is a protein bound to a guanidinium group, which is common to all the components of paralytic shellfish poison, it has strong specificity to some of the paralytic shellfish poison components, but it also has a component that hardly binds. To increase the reliability of shellfish poison detection, it is desirable that the antibody reacts with almost all shellfish poison components.

It is also possible to prepare a monoclonal antibody by a standard method by using the saxitoxin-EDT-protein complex of the present invention as an antigen. Specifically, after immunizing a rodent such as a mouse or a rat with the complex, the spleen is aseptically removed, and spleen cells are prepared,
The hybridoma is fused with myeloma cells, hybridomas are selected with a selection medium such as HAT medium and subcultured in an animal cell culture medium, or transplanted into the abdominal vagina of the rodent, and cultured ascites to obtain a monoclonal antibody. Can be collected. For the production of monoclonal antibodies, see Milstein and Kholer,
Nature 256: 495 (1976); methods described in attribute chemistry experiment course, immunobiochemistry research method (edited by the Japanese Biochemical Society), etc. can be used.

The antibody obtained as described above can be used for the measurement of shellfish poison in samples such as foods suspected of containing paralytic shellfish poison. The measurement can be performed by using a conventional antigen-antibody reaction, and solid phase method or homogeneous method, competitive method or non-competitive method, sandwich method and the like can be used. For example, when the sandwich method is used, an excess amount of labeled secondary antibody is used, but as a label, an enzyme such as peroxidase or alkaline phosphatase, ¹²⁵ I, ⁸²
A radioactive isotope such as P, a fluorescent substance such as FITC, and a chemiluminescent substance such as acridinium can be used. Depending on the type of label, enzyme antibody method such as ELISA, radioimmunoassay, or fluorescent antibody method can be used. Furthermore, the antibody of the present invention separates the shellfish toxin in question when bound to a resin such as dextran resin (eg, Sephadex ^™ ) or agarose resin (eg, Bio-Gel ^™ ) activated with cyanogen bromide or the like. It can be used as an affinity column carrier for removal.

Ethanedithiol as dithiol compound
The embodiment when a compound other than (EDT) is adopted is almost the same as the above.

Other relevant documents relating to the present invention are as follows: Hollingworth T. Wekell MM (1990): Paralytic shellfis.
h posison. In: OfficialMethods of Analysis, 15th ed
ition, K. Hellrich ed., Association of Official An
alytical Chemists, Arlington, Virginia, pp.881-88
2. Oshima Y (1995): Post-column derivatization HPLC me
thods for paralytic shellfish poisons. In: Manual o
n Harmful Marine Algae, 10C Manuals and Guides No.
33, Hallegraeff GM, Anderson DM, Cembella AD eds.,
Intergovernmental Oceanographic Commission of UNE
SCO, Paris, pp.81-94. Sato S, Sakai R, Kodama M (2000): Identification of
thioether intermediates in the reductive transfor
mation of gonyautoxins into saxitosins by thios. B
ioorg. Med. Chem. Lett. 10: 1787-1789. Sommer H. Meyer KF (1937): Paralytic shellfish pois
oning. Arch. Pathol. 24: 560-568.

[0035]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Abbreviations relevant to the description of the present invention are as follows: Samples and reagents Adjuvant: Complele Adjuvant Freund's Wako Bio-Gel P-2 gel filtration resin, mesh size: fine Bio-rad Biotin PE (AC5) ₂ Maleimide biotin labeling reagent Dojindo PAEM N- [2- (2-Pyridyiamino) ethyl] maleimide Wako BSA: Bovine serum albumin Wako, IRA grande DMSO: Dimethyl sulfoxide Wako, special grade EDT: 1,2-ethanedithiol Wako, special grade EDTA : Ethylenediamine tetraacetic acid Wako, special grade GMBS: γ-maleimidobutyric acid N-human hydroxysuccinic acid imid ester Dojindo reaction plate: ELISA plate 96-well Iwaki OPD: o-phenylenediamine-2HC1 (tablet) Wako Enzyme-labeled secondary antibody: HRP-labeled anti-antibody Usagi-Yagi, 1000 times diluted when used Dako Microplate reader: MPR A4 Toso PBS (-): Mg, Ca free Phosphate buffered saline (pH7.4) PBSB: PBS containing 0.5% BSA and 0.1% Tween60 ( -) PBST: 0.1% Tween20 Contains PBS (-) Sephadex G-50 gel filtration resin, mesh size: fine Pharmacia THF: Tetrahydrofuran Wako, special grade Rabbit: New Zealand white, male, weight about 1Kg

Example 1 Purification of paralytic shellfish poison (PSP) and preparation of EDT-PSP complex From Ofunato Bay poisoned scallop, gonyautoxi
n (GTX) group, saxitoxin group and C-toxin group PSP components (Fig. 1) were isolated. GTX1 and GTX4, GTX2 among the purified components
And GTX3 and C1 and C2 were used as an equilibrium mixture (abbreviated as GTX1 + 4, GTX2 + 3 and C1 + 2, respectively) in a molar ratio of about 3: 1. 50 mM EDT-10 with 50 μmol GTX1 + 4 in 20% THF
It was dissolved in 50 mL of mM sodium phosphate buffer (pH 7.4) and left standing at room temperature for 2 days. ED extracted with diethyl ether 3 times
T was removed, and the ether remaining in the aqueous phase was distilled off under reduced pressure at 30% to obtain a reaction mixture containing EDT-neoSTX. This is Bio-
It was added to a column (1.5 x 15 cm) of Gel p-2, the column was washed with 200 mL of water, and the adsorbed component was eluted with 0.1 M acetic acid. 10 mL aliquots were collected, and EDT-neoSTX complexes were isolated while quantifying the GTX1,4, neoSTX and EDT-neoSTX concentrations in each fraction by HPLC fluorescence method. Similarly treated with 50 μmol GTX2 + 3 as starting material,
DT-STX was prepared and separated.

Example 2 Preparation of antigen for preparing specific antibody against PSP 20 mg of BSA was dissolved in 2 mL of PBS (-), and 5 mg of GMBS 100 μ was added to this.
What was melt | dissolved in DMSO of L was mixed, and it left still at room temperature for 1 hour. This is a Sephadex G-25 column (1.5x20 cm) packed with 0.1 mM sodium phosphate buffer (pH 6.0) containing 1 mM EDTA.
And eluted with the same solution. The obtained polymer fractions were combined and the pH was adjusted to 7.4 with a carbonic acid-removing solution, and then the solution was centrifuged with an ultrafiltration kit (UFV4BCG25, NMWL 10,000, Millipore).
It was concentrated to 4 mL. 5 μmol of EDT-neoSTX was dissolved in this 2 mL and left standing at 5 ° C. for 2 days. Next, the mixture was dialyzed against PBS (-) four times, and the inner solution was recovered as a neoSTX-BSA conjugate solution. The STX-BSA conjugate was also prepared by the same procedure. The BSA concentration in the antigen solution was 280 nm and the bound PSP concentration was H.
It was calculated by the PLC fluorescence method.

Example 3 Immunization of Rabbit and Measurement of Antibody Titer The BSA-STX conjugate was diluted with PBS to a concentration of 500 μg / mL. Emulsion combined with an equal amount of adjuvant, once every two months,
2 rabbits (STX-BSA-1, STX-B)
SA-2) was injected intradermally. The BSA-neoSTX conjugate was similarly immunized in two rabbits (neoSTX-BSA-2). The antibody titer of the serum obtained by collecting blood from the immunized rabbit at any time was determined by the following procedure. For each well of the 96-well reaction plate, for PBS (-)
Dispense 50 μL of STX solution prepared to a concentration of 50 μM at 37 ° C
I shook it for 2 hours. After washing the plate twice with PBST,
300 μL each of PBS (-) containing 3% gelatin was dispensed and washed 3 times with PBST. After shaking for 1 hour at 37 ℃, wash 3 times with PBST,
100 μL of HRP-labeled secondary antibody diluted 1000 to PBS (-)
Each was added. After shaking at 37 ° C. for 1 hour, the plate was washed 4 times with PBST, and 100 μL of a coloring substrate solution (OPD-H202) was added. 37 ° C
After shaking for 30 minutes at room temperature, add 100 μL of 0.5N sulfuric acid to stop the reaction, and absorb at 492 nm (O 2
D) was measured.

Example 4 Absorption test of PSP by antiserum 1 per 50 μL of serum obtained from STX BSA-1 and neoSTX-BSA-1 and serum obtained from non-immunized rabbit (normal serum), respectively
μM PSP components (C1 + 2, GTX1 + 4, GTX2 + 3, GTX5, STX, n
EoSTX) standard solutions were mixed in equal amounts and left at 5 ° C for 15 hours. Ultracentrifugal filtration kit (Ultrafree-MC, NMWL 5000, M
The filtrate obtained by illipore) was analyzed by HPLC fluorescence method.

Example 5 Development of ELISA method for PSP using polyclonal antibody 50 μl for PBS (-) was added to each well of a 96-well reaction plate.
Dispense 50 μL each of STX solution adjusted to the concentration of M at 37 ℃
Shake for time. 0.3% after washing the plate twice with PBST
300 μL of PBS (−) containing gelatin was added and shaken at 37 ° C. for 1 hour. After washing twice with PBST, PBS of each component of PSP
C1 + 2, GTX1 + 4, GTX2 + 3, GTX5, GTX6, neoSTX, dcSTX, S
50 μL of a solution prepared by diluting TX with PBS (−) to 0.1, 1, 10, 100, 1000, and 10000 nM was added. Next, add 50 μL of antiserum (BSA-STX-1) diluted 200 times with PBSB and incubate at 37 ℃.
I shook it for 1 hour. A zero blank (Bo) was prepared by preparing a well containing neither PSP nor antiserum solution on the same plate. After shaking for 1 hour at 37 ℃, wash 3 times with PBST, and then use PBS (-)
100 μL of HRP-labeled secondary antibody diluted 1000 times was added to each. After shaking for 1 hour at 37 ℃, wash 4 times with PBST,
100 μL of chromogenic substrate solution was added. After shaking at 37 ° C. for 30 minutes, 100 μL of 0.5N sulfuric acid was added to stop the reaction, and the absorption (OD) at 492 nm was measured with a microplate reader. The test was performed in triplicate.

Example 6 Preparation of biotin-labeled STX 1.4 mL of freeze-dried purified EDT-STX complex (15 μmol) was added.
Dissolved in 10 mM phosphate buffer (pH 7.4) of 6 mg (10
μmol of Biolin-PE (AC5) 2-maleimide dissolved in 600 μL of DMSO was mixed. After standing overnight at 5 ° C, wash with 5 mL of 0.05M acetic acid and then 0.05 mL of 0.05M acetic acid-methanol (1: 1 v / v).
The adsorbed components were eluted at. The concentration of biotin-labeled STX contained in the hydrous acidic methanol eluate was quantified by HPLC fluorescence method and examined by the toxicity mouse test method of the same fraction.

Example 7 Preparation of Fluorescently Labeled STX 6 mg of PAEM-2 hydrochloride (Mw253.69) was allowed to stand with purified EDT-STX complex (23 μmol) in 2.3 mL of 50 mM phosphate buffer for 2 days at room temperature. did. The reaction mixture was added to a column of Bio-GEL P-2 (1.5 x 20 cm), the column was washed with 200 mL, and then the adsorbed component was eluted with 0.1 acetic acid and then 0.5 M acetic acid. Collect 10 mL of dilute acetic acid eluate and examine each fraction by HPLC fluorescence method PEAE-E
Elution of DT-STX conjugate (fluorescently labeled STX) was monitored.
The toxicity of isolated PAEM-EDT-STX conjugates was examined by the mouse test method.

Example 8 Quantification of PSP and PSP-Naol Complex PSP in the test solution was quantified by HPLC fluorescence method (Oshima, 1995). The amount of the bound PSP in the solution, that is, the thiol-PSP complex such as EDT-STX and the PSP bound to the protein, is 0.1 M sodium phosphate buffer (pH 7.4) containing a part of the solution containing 10% mercaptoethanol. ) And boil for 5 minutes, then H
It was calculated by comparing the sum of the STX group obtained by analysis by the PLC fluorescence method and the GTX group before the mercaptoethanol treatment.

Example 9 Mouse Test Method The mouse toxicity of the test solution was evaluated by the AOAC method (Hollingworth and W
ekell, 1990). ddy male mouse (weight ±
1 g), 5 mice per sample were used, and the median lethal time to the PSP dose lethal time curve (Sommer and
The administered poison value according to Mcyer, 1937 (mouse unil = M
U) was calculated. The IMU corresponds to the toxic dose that kills one mouse in 15 minutes.

Example 10 Preparation of PSP-protein conjugate (antigen) A BSA-EDT-STX conjugate and a BSA-EDT-neoSTX conjugate prepared by the same procedure as shown in FIG. 2 were prepared. Previously, we introduced the glutathione-PSP complex into BSA to create an antigen, but the newly created EDT-mediated binding method succeeded in creating an antigen in which a larger number of PSP molecules introduced a protein. (Table 1).

[Table 1]

Example 11 Transition of antibody titer of rabbit serum FIG. 3 shows a rabbit immunized with BSA-EDT-STX conjugate (STX-EDT-B.
SA-1 and STX-EDT-STX-2) antibody titer changes are shown. Two
A significant increase in the antibody titer was observed from the 3rd month after the start of immunization in both rabbits, and the antibody titer from the 4th month onward continued to increase and decrease. The antibody titers of rabbits immunized with NeoSTX-EDT-STX conjugates were almost the same, but the increase in antibody titers was lower than that when immunized with BSA-EDT-STX conjugates.
All rabbits were exsanguinated 7 months after the start of immunization, and the obtained serum was used in the following test.

Example 12 Absorption of PSP component by antiserum FIG. 4 shows serum obtained from BSA-EDT-STX-1 and BSA-neoSTX-1.
The amount of absorption of each PSP component by the serum obtained from was shown. Non-immunized rabbit serum (normal serum) contains either PSP per mL
The components were also less than 0.1 nmol, which was below the detection limit. In contrast, 1 mL of BSA-STX-1 serum absorbed 13 nmol PSP. BSA
-The absorption of PSP by neoSTX-1 serum was lower than that.

Example 13 Competition of each PSP component on STX-immobilized plate Using BSA-STX-1 serum in which remarkable absorption of PSP component was observed, an attempt was made to develop a quantification method of PSP by ELISA. . As a result of the main investigation, it became possible to quantitatively analyze PSP components by the indirect two step method using a reaction plate immobilized with STX neutral phosphate buffer. As shown in Fig. 5, the concentration that gives the IC50 value is 4 7n for STX and GTX2 + 3.
30 100nM for M, neoSTX, dcSTX, GTX5, GTX1 + 4, approx. For GTX6
It was 200 nM. As described above, although the reactivity of the antibody to the antibody varies slightly depending on the component of PSP, it is possible to detect any component at a sensitivity as high as 10 100 times that of the mouse test method by the same method. The antibody did not react at all with biological components partially similar in structure to PSP, such as adenosine (Ade) having a purine skeleton, caffeine (Caf), and arginine (Arg) having a guanidyl group (Fig. 6). ). Interestingly, however, the puffer fish venom tetrodotoxin (TTX), which has the same pharmacological action as PSP against the BSA-STX-1 antibody, was found to be crossed at a high concentration.

Example 14 Preparation and Specific Toxicity of Labeled PSP Derivative Maleimide group and sulfhydryl group react in a neutral aqueous solution under mild conditions to form a conjugate. Various labeling compounds having a maleimide group are commercially available. In this test, a biotin labeling reagent and a water-soluble fluorescent labeling reagent were selected from these and introduced to prepare a labeled STX derivative (Fig. 7). ). In addition to these two kinds of derivatives, NEM-EDT-STX derivatives were prepared by introducing NEM, which is a sulfhydryl group masking agent, by 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 treated with the purified derivative intravaginally died with the same symptoms as when PSP was administered. Glutathione-
Whereas the PSP complex (Sato et al., 2000) and EDT-STX derivatives showed less than 100 times less specific toxicity than STX, these labeled PSP derivatives starting with EDT-STX 1 / 30th to 1 / 7th of STX and N-sulfocarbam
The specific toxicity was comparable to that of the natural component having oy1 groups (C14, GTX5, 6) (Fig. 9).

[0051]

The polyclonal antibody prepared using BSA-EDT-STX according to the present invention as an antigen had a slightly low affinity for neoSTX, but showed a similar affinity to other paralytic shellfish toxins. ELISA prepared experimentally using this antibody
Was able to detect the venom with a sensitivity 10 to 100 times higher than that in the mouse test, although the value varied slightly depending on the toxic component.

[Brief description of drawings]

FIG. 1 is a diagram showing the structural formula and nontoxicity of paralytic shellfish poison (PSP).

FIG. 2 is a diagram showing a method for producing an antigen (BSA-EDT-PSP conjugate) in the present invention.

FIG. 3 is a graph showing changes in the antibody titer of rabbits immunized with the BSA-EDT-STX conjugate of the present invention.

FIG. 4 is a diagram showing the amount of PSP absorbed by antisera.

FIG. 5 is a diagram showing the reaction of each PSP component with an anti-STX antibody on an STX solid-phase plate.

FIG. 6: Anti-STX of components other than PSP on STX solid-phase plate
It is the figure which showed the reaction with respect to an antibody.

FIG. 7: PSP with non-isotopic label in the present invention
It is the figure which showed the structure of the derivative.

FIG. 8: Bio-Gel P of fluorescently labeled STX (PAEM-EDT-STX)
FIG. 2 is a diagram showing separation on -2 column chromatography.

FIG. 9 is a diagram showing the specific toxicity of each PSP component, PSP-thiol complex, and labeled PSP derivative.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] June 24, 2002 (2002.6.2)
4)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Method of producing anti-narcotic shellfish antibody, novel antibody,
ELISA measurement kit using the antibody, system-labeled poison preparation by the production method

[Claims]

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an antibody against paralytic shellfish toxin and the antibody. Further, the present invention relates to an ELISA assay kit using the above antibody and a labeled poison preparation for the paralytic shellfish toxin binding assay obtained by the above production method.

[0002]

2. Description of the Related Art Paralytic shellfish poisoning has a symptom mainly showing nerve palsy, and its causative poisons have been found to be goniotoxins and saxitoxins [Ken Yasumoto, paralytic shellfish poison, Food Hygiene Inspection Guidelines (Science and Chemistry, Ministry of Health and Welfare Bureau supervision), pages 300-305, Japan Food Hygiene Association, 1991
Year]. These causative toxins are collectively called paralytic shellfish poisons, and at present, more than 20 species of goniotoxins and saxitoxins are known as shellfish poisons found in shellfish such as mussels and clams.

This shellfish poison is a paralytic poison that has the property of blocking sodium channels, and it is contained in several types of phytoplankton that belong to dinoflagellates, and the shellfish that eats with it accumulates the poison and poisons it. Eating this shellfish causes food poisoning with a high mortality rate, which is a major social problem in fisheries and food hygiene. By the way, paralytic shellfish poison 0.5
Symptoms such as numbness, nausea, and diarrhea occur only by ingesting ~ 1.0 mg orally. The lethal dose in humans is 1-3 mg. Areas where shellfish are contaminated with paralytic shellfish poisons are expanding worldwide. In these areas, the toxicity of shellfish shipped to control food poisoning by poisoned shellfish is tested by mouse test method or HPLC.
Inspected by law. However, these measurement methods
There are many problems in terms of cost and labor for measurement, and development of a simple method for measuring poison is desired.

Therefore, various methods for quantifying paralytic shellfish poison have been developed. For example, the official method of measuring lethal activity using mice [Ken Yasumoto, supra; JF Jellett et al., Toxico
n, 30 (10): 1143-1156 (1992); W. Horwitz, Paralytic
shellfish poison. In "Official Methods of Analysi
s of the Association of Official Analytical Chemis
ts "(Assoc. Official Anal. chem., Washington DC)
pp. 881-882 (1990)], high performance liquid chromatography method [Y. Oshima et al., Mycotoxins and Phycotoxins'88 (S. N.
atori et al.), pp.319-326, Elsevier, Amsterdam (198
9)]; JP-A-9-133669], methods using neuroblasts [K. Kogure et al., Toxicon, 26: 191-197 (1989)], methods using antibodies [FS Chu et al., J. Agri. Food Chem., 4
4: 4043-4047 (1996)] and the like are known. The lethal activity is measured by injecting a mouse test stock solution into the abdominal cavity, and the time from the end of the injection to the last gasping when the mouse dies with typical paralytic shellfish poisoning (lethal time). ) Is recorded in seconds, and the amount of poison is estimated from the dose-matching time curve prepared with the standard poison. The high performance liquid chromatography method applies high performance liquid chromatography to a sample containing paralytic shellfish to isolate the paralytic shellfish toxin.
This is a method in which an oxidizing agent is added to the eluate and the mixture is reacted in an alkali, and then the fluorescence of the fluorescent substance produced is measured. The method using neuroblasts is the method of using Na in a neuroblast culture system.
Toxin that inhibits swelling death of cells caused by veratridine is caused by veratridine that promotes Na + influx and a toxin that blocks influx of Na + in a system in which the activity of the enzyme Na-KATPase that excretes + is extracellularly inhibited by ouabain. The amount is estimated from the activity of Escherichia coli, and it is a method that utilizes the fact that when a sample containing paralytic shellfish poison is added to this assay system, cell rounding and cell death are suppressed in proportion to the amount of poison. Further, an immunological method is to use an antibody against paralytic shellfish toxin to directly measure the amount of the antibody that binds to the toxin by a method such as enzyme-linked immunosorbent assay (eg, ELISA), and measure the amount of the antibody in the sample. This is a method for quantifying paralytic shellfish poison.

In particular, FS Chu et al. (Supra) uses an anti-saxitoxin antibody / saxitoxin-horseradish peroxidase conjugate or an anti-neo-saxitoxin antibody / neosaxitoxin-horseradish peroxidase conjugate.
An assay method for measuring the total amount of paralytic shellfish toxin by ELISA is disclosed. Antibody preparation is done by FS Chu and X. Fan,
J. Assoc. Off. Anal. Chem, 1985, 68: 13-16 and F.
It is prepared according to S. Chu, J. AOAC Int. 1992, 75: 341-345, the procedure of which is described by Johnson et al. (Proc. Soc. Exp.
Biol. Med., 1964, 117, 425), and a conjugate obtained by reacting bovine serum albumin, polylysine or hemocyanin of keyhole limpet (limpet) with saxitoxin or neosaxitoxin in a weakly acidic aqueous solution containing formalin. Is subcutaneously injected into a rabbit to obtain a polyclonal antibody.

Specifically, the antigens used for the production of these antibodies are those in which a protein is bound to two guanidinium groups present in the molecule of saxitoxin via formalin [Johnson et al., Supra; Chu and Fan. , Above; V. Renz
And G. Terplan, Arch, Lebensmittelhyg., 1988, 39, 25.
-56; A. Cembella et al., "Specificity and cross-reactivi
ty of an adsorption-inhibition enzyme-linked immun
oassay for the detection of paralytic shellfish to
xins. In Toxic Marine Phytoplankton "(E. Graneli et al.), Elsevier Science Publishers, New York, pp.33
9-344,1989], one of two OH groups at the 12-position of saxitoxin was removed, and a protein was bound to the remaining OH group [R. Carlson et al., "Development of immu"
noassaysfor paralytic shellfish poisoning. A radio
immunoassay for saxitoxin. In Seafood Toxins ",
(EP Ragelis Edition), ACS Symposium Series 262, Ame
rican Chemical Society, Washington, DC, pp.181-1
92 "]. In the former antigen, the binding site of the protein is not clear for either or both of the two guanidinium groups, and the reactivity of the produced antibody differs depending on the presentation group. The antibody also has good reactivity with neosaxitoxin and is about 20% (however, the reactivity with saxitoxin is 100%).
Similar to saxitoxin, reactivity with goniotoxin 2, 3 and decarbamoylsaxitoxin, which are IN-H derivatives, is as low as about 50%. Further, it does not bind to C toxin which is a carbamoyl-N-sulfate derivative at all.
On the other hand, the latter antibody was reported to have a reactivity with neosaxitoxin of 1% or less, and its reactivity with other toxic components has not been examined. Furthermore, attempts have been made to produce monoclonal antibodies using similar antigens, but these have low reactivity and low sensitivity (R. Hack et al., Food and Agricultural immunolog.
y, 1990,2, 47-48).

[0007]

Problems to be Solved by the Invention The lethal activity assay method and the neuroblast method have problems in terms of animal and cell management, detection sensitivity, accuracy, specificity, etc. However, it has the drawbacks that it is easy to operate and highly accurate, but it requires expensive equipment, that the sensitivity varies depending on the type of anesthetic shellfish poison, and that no purified external standard is supplied. Further, the immunological method has a problem in that the reactivity of the antibody varies depending on the type of the saxitoxin derivative. Therefore, it has been desired to develop a method that is simple and highly accurate for anesthetic shellfish poisoning, and that can measure with constant sensitivity regardless of the type of shellfish poison.

The present inventors used the anesthetic shellfish-glutaotine (PSP-GSH) complex (Japanese Patent Application No. 10-91797) as a hapten and prepared an antigen by introducing this into a protein molecule. When administered to rabbits, a polyclonal antibody that reacts with paralytic shellfish to all pre-ingredients was obtained and disclosed (Japanese Patent Laid-Open No. 2000-344799). However, in the method using the complex with GSH, the amount of hapten bound to the protein molecule is small, the antibody titer of the obtained antiserum is low, and improvement of the binding method is necessary to introduce the toxin into the protein molecule. Was thought to be.

The first object of the present invention is to provide a method for producing an antibody against paralytic shellfish venom using the improved binding method for introducing the venom into other molecules such as proteins. A second object of the present invention is to provide an antibody prepared by 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 a method for producing a labeled poison preparation for use in a simple method for measuring paralytic shellfish poison prepared by using the improved binding method of the present invention, and a labeled poison preparation prepared by this production method. Is provided.

The present inventors have prepared a poisonous derivative having a free sulfhydryl group using a dithiol compound having a plurality (two) of sulfhydryl groups, and introduced this derivative into the amino group of a sulfhydryl group-directed functional group. It was found that the poison can be efficiently introduced into the protein by reacting with the protein, and it is possible to prepare an antigen having more hapten molecules introduced therein. The above-mentioned production method of the present invention is characterized in that the poison and the protein are modified separately and the antigen is obtained by the reaction of both. The antigen according to the invention is
It has been found by the present inventors that an antiserum having a high antibody titer can be obtained and the antibody can be obtained in a high yield because of the large amount of hapten per protein molecule. Use of the antibody prepared by the improved binding method of the present invention makes it possible to provide a venom ELISA assay kit.

According to the method for producing an antibody of the present invention,
The inventors have also found that the 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, but since the active site of the antigen of the present invention is exposed, the labeled venom preparation can be prepared with the toxicity remaining.

Therefore, in the first aspect of the present invention, a plurality of sulfhydryl groups (2
One) to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group by binding with a dithiol compound, while preparing a protein having a sulfhydryl group-directed functional group introduced into an amino group, and then the saxitoxin sulfhydryl derivative and the modified protein described above. There is provided a method for producing an antibody against paralytic shellfish poisoning, which comprises immunizing a non-human animal with an antigen obtained by reacting the parasite and obtaining antiserum capable of recognizing the paralytic shellfish poison.

The dithiol compound having a sulfhydryl group is preferably selected from the group consisting of 1,2-ethanedithiol, dithiothreitol and dithioerythritol 2,3-dimercapto-1-propanesulfonic acid. The compound having a sulfhydryl group-directed functional group is
It is preferably selected from the group consisting of maleimide, thiophthalimide and active halogen compounds.

In a second aspect of the present invention, there is provided the antibody obtained by the production method of the first aspect.

Further, in a third aspect of the present invention, a novel ELISA kit using the antibody obtained in the first aspect is provided.

Further, in the fourth aspect of the present invention, a dithiol compound having a plurality of sulfhydryl groups (two) at the 11-position of saxitoxin is bound to prepare a saxitoxin sulfhydryl derivative having a free sulfhydryl group, and then a thiol-directed functional group is prepared. There is provided a labeled venom preparation for use in a simple binding assay of an anesthetic shellfish poison, which comprises reacting a labeled compound such as agarose or biotin having a group, a fluorescent substance, and gold colloid with this derivative.

Further, in a fifth aspect of the present invention, there is provided a labeled poison preparation using the method for producing the labeled poison preparation described above.
The toxic preparations in the present invention are non-isotopic biotin-labeled and fluorescent-labeled preparations.
Shown in.

In the above-mentioned production method of the present invention, any functional group directed to a free sulfhydryl group, particularly maleimide, thiophthalimide, active halogen, etc., reacts quantitatively with a thiol group in a neutral aqueous solution under mild conditions. According to the present invention, the anesthetic shellfish can be bound to a protein or a labeled compound with much higher yield and more easily than the binding method using the PSP-GSH complex or other amide condensation. It is possible to bear.

The method for producing the antibody of the present invention when ethanedithiol (EDT) is used as the dithiol compound will be described below.

The saxitoxin-EDT complex is a mixture of goniotoxin (goniotoxin 2, 3 or a combination thereof) and EDT having a dithiol group and heating (about 7).
At 0 ° C.), it can be obtained by substitution reaction with the OSO3-group at position 11 of goniotoxins via the sulfur molecule of EDT. In the present invention, this complex is a so-called hapten antigen, that is, a substance that binds to the antibody but has no ability to induce the antibody. It is generally known that a protein can be used as an immunogen by covalently binding a protein to such a hapten antigen to form a large molecule, but the protein of the present invention also has a similar function. Examples of such proteins include albumin and limpet hemocyanin (KLH).

The present invention also provides, in a second aspect, the paralytic shellfish toxin components saxitoxin, goniotoxin 2,
3, neosaxitoxin, goniotoxin 1,4, goniotoxin 5, and C toxin 1,2 are both 5
Provided is an antibody against paralytic shellfish, which exhibits a relative reactivity of 0% or more (however, the reactivity with saxitoxin is 100%).

The antibodies of the present invention are IN-H derivatives (saxitoxin, goniotoxin 2, 3), which are paralytic shellfish poisons, and IN-OH.
Derivatives (neosaxitoxin, goniotoxin 1,
4), all of the carbamoyl-N-sulfate derivatives (goniotoxin 5, and C toxin 1,2) can be recognized almost in the same way. Here, expressions such as "goniotoxin 2, 3" mean a mixture of goniotoxin 2 and goniotoxin 3. For the name and structure of paralytic shellfish poison, see, for example, Takeshi Yasumoto, Paralytic shellfish poison, Food Hygiene Inspection Guideline (Science and Chemistry, Ministry of Health and Welfare Bureau supervision).
Pages 0-305, Japan Food Hygiene Association, 1901; Intergovernmen
tal Oceanographic Commission (IOC) Manualand Guide
s No.33 (1995), UNESCO's Workshops, France, pp.81-9
4, etc. According to an embodiment of the present invention, the antibody of the present invention is obtained by the above method.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The antigen used in the production of the antibody of the present invention is a saxitoxin-EDT-protein complex. This composite can be manufactured as follows. As a raw material for the saxitoxin portion, goniotoxin 2, goniotoxin 3, or a mixture thereof in any ratio can be used. These raw material components are mussels, clams, scallops, and the like, activated carbon treatment from a heated extract of 0.1N hydrochloric acid of shellfish, gel filtration chromatography (for example, using Bio-Gel P-2 (Bio-Rad)),
It can be purified through a treatment such as ion exchange chromatography (for example, using Bio-Rex70 (Bio-Rad)).

When the goniotoxin obtained as described above is reacted with EDT (reduced form is preferable) under heating, the SH group of EDT is the electron-donating property, so that the 11th carbon of the goniotoxin is present. Nucleophilic attack on the atom, resulting in the elimination of the OSO3- group, and -S- (EDT) is bound to the 11th carbon atom,
This produces a saxitoxin-EDT complex.
The reaction occurs by heating the reactants under neutral conditions. Generally, the reaction can be carried out using an appropriate buffer solution having a pH of 6.5 to 7.5, preferably pH 7.0 to 7.5, such as ammonium phosphate buffer, ammonium acetate buffer and the like. The reaction temperature is generally room temperature to about 100 ° C, preferably 50 ° C to 80 ° C, 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 partition chromatography, etc., alone or in combination. be able to. The product can be identified by a usual measurement method such as infrared spectroscopy, mass spectrometry, NMR method, elemental analysis and amino acid analysis.

Low-isolation compounds such as saxitoxin belong to haptens and have no antigenicity by themselves, but a protein having antigenicity is used as a carrier, and a complex covalently bound to the protein produces a specific antibody against the hapten. It becomes an antigen. It is considered 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. At this time, it is necessary to produce the hapten antigen by a selective binding method in which an extra antigenic determinant is difficult to form. The recognition of the partial structure of the hapten is weak near the binding site between the hapten and the carrier protein, and it is easy to recognize the partial structure farther away. Therefore, when binding to a protein, it is important to select an appropriate binding position in the hapten. The saxitoxin-EDT complex having a structure in which an EDT group is bound to the 11-position of saxitoxin previously discovered by the present inventors (Japanese Patent Application No.
0-91797) is not only preferable as an immune antigen,
It has the advantage of being able to produce paralytic shellfish poison, which enables highly reactive detection of almost all types of paralytic shellfish poison.

Albumin, hemocyanin and the like can be used as the carrier protein, but the protein is not limited to these, and any protein capable of inducing the antibody of the present invention can be used. When binding a carrier protein to the saxitoxin-EDT complex, the carrier protein (introducing a reactive functional group) and the maleimide method (T. Kitagawa et al., J. Bioshem. 92: 585-590,19
82) and the carbodiimide method (D. Exley et al., FEBS Lett., 91:
162-165, 1978) and the like to bind saxitoxin-GS
H-protein complexes can be generated. For example, in the examples described below, an example of producing a saxitoxin-EDT-bovine serum albumin complex is shown.Here, first, a saxitoxin-EDT conjugate is prepared, while albumin is mercaptosuccinylated via its free amino group, and both are combined. By reacting the carrier protein with saxitoxin-E
Connect to DT. Purification of the obtained complex can be carried out by appropriately combining conventional protein purification methods. Such methods include, for example, salting out, solvent precipitation, gel filtration chromatography, ion exchange chromatography, HPLC,
It includes affinity chromatography, electrophoresis, chromatofocusing, ultrafiltration and the like.

When goniotoxin 1, goniotoxin 4 or a mixture thereof is used as a raw material instead of goniotoxins 2 and 3 and reacted with EDT, neosaxitoxin-
An EDT complex is obtained. Further, by reacting this complex with a carrier protein in the same manner as above, a neosaxitoxin-EDT-protein complex can be obtained,
The resulting product can be used for the production of antibodies against paralytic shellfish poison as well as the saxitoxin-EDT-protein complex (see below).

By immunizing animals such as mouse, rat, rabbit, goat, etc. with the saxitoxin-EDT-protein complex of the present invention, an antibody against the complex can be prepared. Usually, an immunogen solution is emulsified and mixed with an adjuvant such as Freund's complete adjuvant, incomplete adjuvant, Libi adjuvant system (RIBI IMMUNOCHEM RESEARCH INC), muramyl peptide (Calzyme), aluminum hydroxide, lipopolysaccharide, and the like. After subcutaneous injection, intradermal injection, or intramuscular injection, the same operation is performed at intervals of 2 to 4 weeks, and booster immunization is performed several times to obtain antiserum (polyclonal antibody) after exsanguination. Regularly measure exsanguinated serum for reactivity with saxitoxin,
Boosts are given until high titers are achieved. The amount of antigen varies depending on the type of animal receiving antigen stimulation, but for example, in the case of rabbits 10 μg to several mg, in the case of mice several μg
It is a few 10 mg.

The antiserum against paralytic shellfish poison obtained as described above can be further purified into an IgG antibody by treating with, for example, ammonium sulfate fractionation and then ion exchange chromatography. Alternatively, the antibody can be purified by affinity chromatography using a gel in which saxitoxin is chemically coupled to dextran gel or agarose gel. The production and purification of the antibody are described in detail in, for example, Biochemistry Experimental Method 15 "Introduction to Immunology" (1982) Academic Society Publishing Center, and can be referred to.

In the method of the present invention, an increase in antibody titer is observed from about 1 month after immunization, and in the case of antisera against saxitoxin, titers of 0.5 nmol of saxitoxin per ml of antiserum are obtained after about 4 months. This antiserum had a somewhat low reaction with neosaxitoxin, but there was almost no difference in the reactivity with both shellfish poisons in the serum 5 months after immunization. In addition, FIG. 2 shows the reactivity of sera at the 4th and 5th month of immunization with various components of paralytic shellfish toxin, but the sera at the 4th month of the immunity shows some reactivity with neosaxitoxin and goniotoxin 1,4. Although inferior, the serum of the 5th month did not show a large difference in the reaction with each component. Thus, the antibody of the present invention immunologically reacts with almost all components of paralytic shellfish poison,
Particularly paralytic shellfish toxin components, saxitoxin, goniotoxin 2, 3, neosaxitoxin, goniotoxin 1,
4, goniotoxin 5, and C toxin (C1, C
Both of 2) show relative reactivity of 50% or more, preferably 60% or more (however, the reactivity with saxitoxin is 100%). On the other hand, many of the conventionally known antibodies are
Since the antigen is a protein bound to a guanidinium group, which is common to all the components of paralytic shellfish poison, it has strong specificity to some of the paralytic shellfish poison components, but it also has a component that hardly binds. To increase the reliability of shellfish poison detection, it is desirable that the antibody reacts with almost all shellfish poison components.

It is also possible to prepare a monoclonal antibody by a standard method by using the saxitoxin-EDT-protein complex of the present invention as an antigen. Specifically, after immunizing a rodent such as a mouse or a rat with the complex, the spleen is aseptically removed, and spleen cells are prepared,
Fused with myeloma cells, the hybridomas are selected with a selection medium such as HAT medium and subcultured in a medium for animal cell culture, or transplanted into the abdominal vagina of the rodent, and cultured for monoclonal antibody from ascites. Can be collected. For the production of monoclonal antibodies, see Milstein and Kholer,
Nature256: 495 (1976); Methods described in Attribute Chemistry Laboratory, Immunobiochemistry Research Method (edited by the Japanese Biochemical Society), etc. can be used.

The antibody obtained as described above can be used for the measurement of shellfish poison in samples such as foods suspected of containing paralytic shellfish poison. The measurement can be performed using a conventional antigen-antibody reaction, and a solid phase method or a homogeneous method, a competitive method or a non-competitive method, a sandwich method and the like can be used. For example, when using the sandwich method, an excess amount of labeled second antibody is used, but as a label, peroxidase, an enzyme such as alkaline phosphatase, or the like,
Radioisotopes such as 125I and 82P, fluorescent substances such as FITC, and chemiluminescent substances such as acridinium can be used.
Depending on the type of label, enzyme antibody method such as ELISA, radioimmunoassay, or fluorescent antibody method can be used. Furthermore, the antibody of the present invention separates and removes the shellfish toxin when it binds to a resin such as dextran resin (for example, Sephadex ™ s) activated with cyanogen bromide or the like, agarose resin (for example, Bio-Gel ™ s), or the like. Can be used as an affinity column carrier.

Ethanedithiol as dithiol compound
The embodiment when a compound other than (EDT) is adopted is almost the same as the above.

Other relevant documents relating to the present invention are as follows: Hollingworth T. Wekell MM (1990): Paralytic shellfi
sh poison. In: Official Methods of Analysis, 15th
edition, K. Hellrich ed., Association of Official
Analytical Chemists, Arlington, Virginia, pp.881-8
82. Oshima Y (1995): Post-column derivatization HPLC m
ethods for paralytic shellfish poisons. In: Manual
on Harmful Marine Algae, 10C Manuals and Guides N
o.33, Hallegraeff GM, Anderson DM, Cembella AD ed
s., Intergovernmental Oceanographic Commission of
UNESCO, Paris, pp.81-94. Sato S, Sakai R, Kodama M (2000): Identification o
f thioether intermediates in the reductive transfo
rmation of gonyautoxins into saxitoxins by thios.
Bioorg. Med. Chem. Lett. 10: 1787-1789. Sommer H. Meyer KF (1937): Paralytic shellfish poi
soning. Arch. Pathol. 24: 560-568.

[0035]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Abbreviations relevant to the description of the present invention are as follows: Samples and Reagents Adjuvant: Complete Adjuvant Freund's Wako Bio-Gel P-2 gel filtration resin, mesh size: fine Bio-Rad Biotin-PE (AC5 ) 2-Maleimide biotin labeling reagent Dojindo PAEM N- [2- (2-Pyridyiamino) ethyl] maleimide Wako BSA: Bovine serum albumin Wako, IRA grade DMSO: Dimethyl sulfoxide Wako, special grade EDT: 1,2-ethanedithiol Wako , Special grade EDTA: Ethylenediaminetetraacetic acid Wako, Special grade GMBS: γ-Maleimid-butyric acid N-human oxysuccinic acid imid ester Dojindo reaction plate: ELISA plate 96-well Iwaki OPD: o-phenylenediamine-2HC1 (tablet) Wako Enzyme-labeled secondary antibody: HRP labeled anti-rabbit, diluted 1000 times when used Dako Microplate reader: MPR A4 Toso PBS (-): Mg, Ca free Phosphate buffered saline (pH7.4) PBSB: 0.5% BSA and 0.1% Tween60 Including PBS (-) PBST: 0.1% Tween20 No PBS (-) Sephadex G-50 gel filtration resin, mesh size: fine Pharmacia THF: Tetrahydrofuran Wako, special grade rabbit: New Zealand White, male, weighing approximately 1Kg

Example 1 Purification of paralytic shellfish toxin (PSP) and preparation of EDT-PSP complex From the Ofunato Bay poisoned scallop, gonyautoxi was prepared by a conventional method.
n (GTX) group, saxitoxin group and C-toxin group PSP components (Fig. 1) were isolated. GTX1 and GTX4, GTX2 among the purified components
And GTX3 and C1 and C2 were used as an equilibrium mixture (abbreviated as GTX1 + 4, GTX2 + 3 and C1 + 2, respectively) in a molar ratio of about 3: 1. 50 mM EDT-10 with 50 μmol GTX1 + 4 in 20% THF
It was dissolved in 50 mL of mM sodium phosphate buffer (pH 7.4) and left standing at room temperature for 2 days. ED extracted with diethyl ether 3 times
T was removed, and the ether remaining in the aqueous phase was distilled off under reduced pressure at 30% to obtain a reaction mixture containing EDT-neoSTX. This is Bio-
Gel P-2 was added to the column (1.5x15 cm), the column was washed with 200 mL of water, and the adsorbed component was eluted with 0.1 M acetic acid. The EDT-neoSTX complex was isolated while quantifying GTX1,4, neoSTX and EDT-neoSTX concentrations in each fraction by HPLC fluorescence method. Similarly treated with 50 μmol GTX2 + 3 as starting material,
DT-STX was prepared and separated.

Example 2 Preparation of antigen for preparing specific antibody against PSP 20 mg of BSA was dissolved in 2 mL of PBS (-), and 5 mg of GMBS 100 μ was added to this.
What was melt | dissolved in DMSO of L was mixed, and it left still at room temperature for 1 hour. This is a Sephadex G-25 column (1.5x20 cm) packed with 0.1 M sodium phosphate buffer (pH 6.0) containing 1 mM EDTA.
And eluted with the same solution. After combining the obtained polymer fractions and adjusting the pH to 7.4 with sodium carbonate solution, centrifugal ultrafiltration kit (UFV4BCG25, NMWL 10,000, Millipore)
It was concentrated to 4 mL. 5 μmol of EDT-neoSTX was dissolved in this 2 mL and left standing at 5 ° C. for 2 days. Next, the mixture was dialyzed against PBS (-) four times, and the inner solution was recovered as a neoSTX-BSA conjugate solution. The STX-BSA conjugate was also prepared by the same procedure. The BSA concentration in the antigen solution was 280 nm and the bound PSP concentration was H.
It was calculated by the PLC fluorescence method.

Example 3 Immunization of Rabbit and Measurement of Antibody Titer The BSA-STX conjugate was diluted with PBS to a concentration of 500 μg / mL. Two rabbits (STX-BSA-1, S) each containing 1 mL of emulsion mixed with an equal amount of adjuvant once every two months
TX-BSA-2) was injected intradermally. Similarly, the BSA-neoSTX conjugate was immunized in two rabbits (neoSTX-BSA-1, neoSTX-BSA-2). The antibody titer of the serum obtained by collecting blood from the immunized rabbit at any time was determined by the following procedure. Each well of a 96-well reaction plate
In addition, 50% STX solution prepared in PBS (-) at a concentration of 50 μM
Each μL was dispensed and shaken at 37 ° C for 2 hours. PBS plate
After washing twice with T, 300 μL of PBS (−) containing 0.3% gelatin was added and shaken at 37 ° C. for 1 hour. After washing twice with PBST, PB
An antiserum diluted 100 times to SB was dispensed in 50 μL aliquots and washed 3 times with PBST. After shaking for 1 hour at 37 ℃, PBST
The cells were washed 3 times with PBS, and 100 µL of HRP-labeled secondary antibody diluted 1000-fold with PBS (-) was added. After shaking at 37 ℃ for 1 hour, wash with PBST four times, and use the chromogenic substrate solution (OPD-H202).
100 μL was added. After shaking for 30 minutes at 37 ℃, 0.5N sulfuric acid
The reaction was stopped by adding 100 μL, and the absorbance (OD) at 492 nm was measured with a microplate reader.

Example 4 Absorption test of PSP by antiserum 1 per 50 μL of serum obtained from STX BSA-1 and neoSTX-BSA-1 and serum obtained from non-immunized rabbit (normal serum), respectively
μM PSP components (C1 + 2, GTX1 + 4, GTX2 + 3, GTX5, STX, n
EoSTX) standard solutions were mixed in equal amounts and left at 5 ° C for 15 hours. Ultracentrifuge filtration kit (Ultrafree-MC, NMWL 5000,
The filtrate obtained by Millipore) was analyzed by HPLC fluorescence method.

Example 5 Development of ELISA method for PSP using polyclonal antibody 50 μl for PBS (-) was added to each well of a 96-well reaction plate.
50 μL of the STX solution adjusted to the concentration of M was dispensed and shaken at 37 ° C. for 2 hours. Wash the plate twice with PBST and then 0.3
PBS (-) containing 300% of gelatin was added and shaken at 37 ° C for 1 hour. After washing twice with PBST, PBS of each component of PSP, namely C1 + 2, GTX1 + 4, GTX2 + 3, GTX5, GTX6, neoSTX, dcST
X, STX with PBS (-) 0, 0.1, 1, 10, 100, 1000, 10000 nM
50 μL of each diluted solution was added. Then PBSB
50 μL of antiserum (BSA-STX-1) diluted 200-fold with 1 was added and shaken at 37 ° C. for 1 hour. Prepare a well without addition of PSP and antiserum solution on the same plate and prepare a zero blank (B
o) After shaking for 1 hour at 37 ℃, wash 3 times with PBST,
HRP-labeled secondary antibody diluted 1000-fold with PBS (-)
00 μL was added to each. After shaking for 1 hour at 37 ℃, use PBST for 4
After washing twice, 100 μL of the coloring substrate solution was added. 30 at 37 ° C
After shaking by shaking, 100 μL of 0.5 N sulfuric acid was added to stop the reaction, and the absorption (OD) at 492 nm was measured with a microplate reader. The test was performed in triplicate.

Example 6 Preparation of biotin-labeled STX 1.4 mL of lyophilized purified EDT-STX complex (15 μmol) was added.
Dissolved in 10 mM phosphate buffer (pH 7.4), and add 6 mg (10
μmol of Biotin-PE (AC5) 2-maleimide dissolved in 600 μL of DMSO was mixed. After standing overnight at 5 ℃, adjust the volume to 5mL with 0.05M acetic acid, and use 3 SepPak C18 plus cartridges.
After washing with 15 mL of 0.05M acetic acid, 0.05M acetic acid-methanol (1: 1
The adsorbed components were eluted with v / v) 10 mL. The concentration of biotin-labeled STX contained in the hydrous acidic methanol eluate was quantified by HPLC fluorescence method, and the toxicity of the same fraction was examined by mouse test method.

Example 7 Preparation of Fluorescently Labeled STX 6 mg of PAEM-2 hydrochloride (Mw253.69) was allowed to stand with purified EDT-STX complex (23 μmol) in 2.3 mL of 50 mM phosphate buffer for 2 days at room temperature. did. The reaction mixture was applied to the Bio-GEL P-2 column.
(1.5 x 20 cm), the column was washed with 200 mL, and the adsorbed component was eluted with 0.1 M acetic acid and then 0.5 M acetic acid. Collect 10 mL of dilute acetic acid eluate and examine each fraction by HPLC fluorescence method PEAE-E
Elution of DT-STX conjugate (fluorescently labeled STX) was monitored.
The toxicity of the isolated PAEM-EDT-STX conjugate was examined by the mouse test method.

Example 8 Quantification of PSP and PSP-Naol complex PSP in the test solution was quantified by HPLC fluorescence method (Oshima, 1995). The amount of the bound PSP in the solution, that is, the thiol-PSP complex such as EDT-STX and the PSP bound to the protein, is 0.1 M sodium phosphate buffer (pH 7. It was calculated by comparing the STX group obtained by diluting with 4), boiling for 5 minutes and then analyzing by HPLC fluorescence method, and the sum of GTX group and STX group before mercaptoethanol treatment.

Example 9 Mouse Test Method The mouse toxicity of the test solution was evaluated by the AOAC method (Hollingworth and W
ekell, 1990). ddY male mouse (body weight 2
0 ± 1 g) and 5 mice per sample were used, and the median lethal time to the dose lethal time curve of PSP (Sommer
and Meyer, 1937).
= MU) was calculated. One MU corresponds to the amount of poison that kills one mouse in 15 minutes.

Example 10 Preparation of PSP-protein conjugate (antigen) A BSA-EDT-STX conjugate and a BSA-EDT-neoSTX conjugate prepared by the same procedure as shown in FIG. 2 were prepared. Previously, we introduced the glutathione-PSP complex into BSA to create an antigen, but the newly created EDT-mediated binding method succeeded in creating an antigen in which a larger number of PSP molecules introduced a protein. (Table 1).

[Table 1]

Example 11 Transition of antibody titer of rabbit serum FIG. 3 shows rabbits immunized with BSA-EDT-STX conjugate (STX-EDT-B.
SA-1 and STX-EDT-STX-2) antibody titer changes are shown. Two
A significant increase in the antibody titer was observed from the 3rd month after the start of immunization in both rabbits, and the antibody titer from the 4th month onward continued to increase and decrease. The antibody titers of rabbits immunized with NeoSTX-EDT-STX conjugates changed in a similar manner, but the increase in antibody titers was lower than that when immunized with BSA-EDT-STX conjugates.
All rabbits were exsanguinated 7 months after the start of immunization, and the obtained serum was used in the following test.

Example 12 Absorption of PSP component by antiserum FIG. 4 shows serum obtained from BSA-EDT-STX-1 and BSA-neoSTX-1.
The amount of absorption of each PSP component by the serum obtained from was shown. Non-immunized rabbit serum (normal serum) contains either PSP per mL
The components were also less than 0.1 nmol, which was below the detection limit. In contrast, 1 mL of BSA-STX-1 serum absorbed 1-3 nmol of PSP. BS
Absorption of PSP by A-neoSTX-1 serum was lower than that.

Example 13 Competition of each PSP component on STX-immobilized plate Using BSA-STX-1 serum in which remarkable absorption of PSP component was observed, an attempt was made to develop a quantification method of PSP by ELISA. . 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 with STX neutral phosphate buffer. As shown in Fig. 5, the concentration giving the IC50 value was 4 to 7 nM for STX and GTX2 + 3.
neoSTX, dcSTX, GTX5, GTX1 + 4 30-100 nM, GTX6 about 20
It was 0 nM. As described above, although the reactivity to the antibody is slightly different depending on the components of PSP, any component can be detected with the sensitivity 10 to 100 times higher than that of the mouse test method by the same method. The antibody is an adenosine (A
de), caffeine (Caf), and arginine (Arg) having a guanidyl group, did not react at all with biological components partially similar in structure to PSP (Fig. 6). Interestingly, however, the puffer fish venom tetrodotoxin (TTX), which has the same pharmacological action as PSP against the BSA-STX-1 antibody, was found to be crossed at high concentrations.

Example 14 Preparation and Specific Toxicity of Labeled PSP Derivative Maleimide group and sulfhydryl group react in a neutral aqueous solution under mild conditions to form a conjugate. Various labeling compounds having a maleimide group are commercially available. In this test, a biotin labeling reagent and a water-soluble fluorescent labeling reagent were selected from these and introduced to prepare a labeled STX derivative (Fig. 7). ). In addition to these two kinds of derivatives, NEM-EDT-STX derivatives were prepared by introducing NEM, which is a sulfhydryl group masking agent, by 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 treated with the purified derivative intravaginally died with the same symptoms as when PSP was administered. Glutathione-PSP
The complex (Sato et al., 2000) and EDT-STX derivative are ST
It showed less than 100 times less specific toxicity than X, while E
These labeled PSP derivatives starting from DT-STX are S
It showed a specific toxicity comparable to that of natural components (C1-4, GTX5) having N-sulfocarbamoy1 group and 1/30 to 1/7 of TX (Fig. 9).

[0051]

EFFECT OF THE INVENTION The polyclonal antibody prepared by using BSA-EDT-STX as an antigen according to the present invention had a slightly low affinity for neoSTX, but showed almost the same affinity for other paralytic shellfish toxin components. ELIS prepared experimentally using this antibody
Although the value of A was slightly different depending on the poison component, the poison could be detected with sensitivity 10 to 100 times that of the mouse test.

[Brief description of drawings]

FIG. 1 is a diagram showing the structural formula and specific toxicity of paralytic shellfish poison (PSP).

FIG. 2 is a diagram showing a method for producing an antigen (BSA-EDT-PSP conjugate) in the present invention.

FIG. 3 is a graph showing changes in the antibody titer of rabbits immunized with the BSA-EDT-STX conjugate of the present invention.

FIG. 4 is a diagram showing the amount of PSP absorbed by antisera.

FIG. 5 is a diagram showing the reaction of each PSP component with an anti-STX antibody on an STX solid-phase plate.

FIG. 6: Anti-STX of components other than PSP on STX solid-phase plate
It is the figure which showed the reaction with respect to an antibody.

FIG. 7: PSP with non-isotopic label in the present invention
It is the figure which showed the structure of the derivative.

FIG. 8: Bio-Gel P of fluorescently labeled STX (PAEM-EDT-STX)
FIG. 2 is a diagram showing separation on -2 column chromatography.

FIG. 9 is a diagram showing the specific toxicity of each PSP component, PSP-thiol complex, and labeled PSP derivative.

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Claims (10)

[Claims] 1. A saxitoxin sulfhydryl derivative having a free sulfhydryl group is prepared by binding an arbitrary dithiol compound having a plurality of sulfhydryl groups at position 11 of saxitoxin, while a sulfhydryl group-directed functional group is introduced into an amino group. Preparing a protein, and then immunizing a non-human animal with an antigen obtained by reacting the saxitoxin sulfhydryl derivative with the modified protein to obtain antiserum recognizing paralytic shellfish poison from the animal, paralytic How to make antibodies against shellfish poison. 2. The dithiol compound having a sulfhydryl group is 1,2-ethanedithiol, dithiothreitol, dithioerythritol, 2,3-dimercapto-1.
-The method for producing an antibody according to claim 1, which is selected from the group consisting of propanesulfonic acid. 3. The method for producing the antibody according to claim 1, wherein the optional functional group having a sulfhydryl group-directed group is selected from the group consisting of maleimide, thiophthalimide, and an active halogen group. 4. An antibody obtained by the method according to claim 1. 5. An ELISA measurement kit using the antibody obtained by the method according to claim 1. 6. A labeled compound in which a saxitoxin sulfhydryl derivative having a free sulfhydryl group is prepared by binding an arbitrary dithiol compound having a plurality of sulfhydryl groups at the 11-position of saxitoxin, while introducing a sulfhydryl group-directed arbitrary functional group. Of the saxitoxin sulfhydryl derivative and the modified labeled compound described above, and a method of producing a labeled venom preparation for use in a simple binding assay method for paralytic shellfish poison. 7. The label according to claim 6, wherein the dithiol compound having a sulfhydryl group is selected from the group consisting of 1,2-ethanedithiol, dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid. How to make a poison standard. 8. The method for producing a labeled poison sample according to claim 6, wherein the optional functional group having a sulfhydryl group-directed group is selected from the group consisting of maleimide, thiophthalimide, and an active halogen group. 9. The labeling compound is agarose, biotin,
Selected from the group consisting of fluorescent substances and colloidal gold,
The method for producing the labeled poison sample according to claim 6. 10. A labeled poison preparation obtained by the method according to claim 6.