JP2005245211A - Method for detecting trichothecene-based mycotoxin and method for determining trichothecene-based mycotoxin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply and accurately detecting a trichothecene-based mycotoxin contained in a specimen such as a food, a feed, etc., and a method for simply and accurately determining the content of a trichothecene-based mycotoxin contained in a specimen such as a food, a feed, etc. <P>SOLUTION: The method for detecting a trichothecene-based mycotoxin in a specimen comprises bringing a T cell into contact with a specimen to commit the T cell to apoptosis and assaying the induced apotosis. The method for determining the content of a trichothecene-based mycotoxin in a specimen comprises bringing a T cell into contact with a specimen to commit the T cell to apoptosis and assaying the induced apoptosis. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a detection method and detection reagent for trichothecene mycotoxins contained in samples such as foods and feeds, and a quantification method and reagent for determination of trichothecene mycotoxins contained in samples such as foods and feeds.

  In the United States in 1972, deoxynivalenol, one of the trichothecene-based mycotoxins, was isolated from corn, a diet that caused poisoning in dairy cows. Since then, natural pollution of agricultural products and livestock feed, mainly cereals, due to mycotoxins has been reported throughout the world. As toxicity of mycotoxin, it has been suggested that it may cause secondary diseases in humans and livestock by suppressing the immune function of the living body. Toxicity studies on mycotoxins, which are frequently contaminated, have been actively conducted mainly in Europe and the United States, but most studies have been conducted at the level of animal experiments.

Recently, Yoshizawa et al. Revealed that cell death (apoptosis) was caused in thymic T cells of mice administered with T-2 toxin, which is one of the trichothecene mycotoxins (see Non-Patent Document 1).
Studies on the immunotoxicity of mycotoxins have been conducted using experimental animals so far (see Non-Patent Documents 1, 2, and 3). However, considering the possibility that there is a difference in susceptibility to toxicity due to species differences, it is ultimately necessary to examine toxicity to humans. As an animal symptom related to immunotoxicity, atrophy of the thymus has been reported (see Non-Patent Document 2), and it is clear that apoptosis is caused by immature T cells with CD4-positive / CD8-positive surface markers. (See Non-Patent Document 1). However, using these animal experiment phenomena in immunotoxicity testing methods is time consuming and labor intensive. Further, since these experimental systems use mice, they cannot be said to be appropriate systems for evaluating toxic effects on humans. Until now, research using cultured human cells has been attempted (see Non-Patent Documents 4 to 6), but the elucidation of the mechanism of action is completely advanced by the absence of cell lines that are highly sensitive to mycotoxins. There wasn't.

  On the other hand, a conference co-sponsored by WHO / FAO was held in Geneva, Switzerland in March 2001, and an acceptable level of mycotoxin intake was determined. In Japan, the Ministry of Health, Labor and Welfare issued a notification in May 2003 that set the standard value of deoxynivalenol in wheat as 1100 ppb. Trichothecene-based mycotoxins are often observed to be complexly contaminated with substances having different toxic strengths such as nivalenol (hereinafter sometimes referred to as NIV) and T-2 toxin. In order to determine the sum of the immunotoxicity of a plurality of trichothecene mycotoxins contained in the above, a method for quantitatively evaluating the immunotoxicity of these trichothecene mycotoxins is required.

Under these circumstances, trichothecene mycotoxins contained in samples such as food and feed can be detected easily and accurately, and the amount of trichothecene mycotoxins in the samples can be measured easily and accurately. It was an urgent task to establish a method for simply and accurately evaluating the above.
Heretofore, as a method for detecting trichothecene mycotoxins, a method using an antibody against trichothecene mycotoxins has been reported (see Patent Document 1).
“Toxicology and Applied Pharmacology”, Academic Press, 1998, Vol. 148, p. 205-214 “Toxicology and Applied Pharmacology”, Academic Press, 1993, Vol. 121, p. 8-14 “Toxicologic Pathology”, Society of Toxicologic Pathologist, 1998, Vol. 26, p. 674-681 “Natural Toxins”, John Wiley and Sons, 1995, Volume 3, p. 129-137 “Toxicology and Applied Pharmacology”, Academic Press, 2000, 164, p. 149-160 “Natural Toxins”, John Wiley and Sons, 1996, Vol. 4, p. 234-241 International Publication No. 01/018196 Pamphlet

  An object of the present invention is to provide a method and a reagent for easily and accurately detecting trichothecene mycotoxins contained in a sample such as food or feed, and a trichothecene mycotoxin content contained in a sample such as food or feed easily and accurately. It is to provide a method and a reagent for quantitative determination.

The present invention relates to the following (1) to (15).
(1) A method for detecting a trichothecene mycotoxin in a specimen, wherein the apoptosis is induced in the cell by bringing the T cell into contact with the specimen, and the induced apoptosis is quantified.
(2) A method for quantifying the trichothecene mycotoxin content in a specimen, which comprises inducing apoptosis by contacting the T cell with the specimen and quantifying the amount of apoptosis induced.

(3) The amount of trichothecene mycotoxin selected from the group consisting of deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T-2), and vercarin A in the amount of trichothecene mycotoxin in the sample The method according to (2) above, wherein the content is converted as:
(4) The method according to any one of (1) to (3) above, wherein the measurement of apoptosis is a method of measuring the activity of caspase activated by induction of apoptosis.

(5) The method according to (4) above, wherein the caspase is caspase-3.
(6) The method according to any one of (1) to (5) above, wherein the T cell is a T cell derived from a leukemia patient.
(7) The method according to any one of (1) to (6) above, wherein the T cells are CD4 positive and CD8 positive T cells.

(8) The method according to any one of (1) to (7) above, wherein the T cell is an LMAT cell (FERM BP-08598).
(9) A reagent for detecting trichothecene-based mycotoxins in a specimen, comprising T cells.
(10) A reagent for quantifying trichothecene mycotoxins in a specimen, comprising a T cell and a trichothecene mycotoxin standard solution.

(11) The reagent according to (9) or (10), further comprising a caspase substrate.
(12) The above (9) to (9), wherein the trichothecene mycotoxin is any trichothecene mycotoxin selected from the group consisting of deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T-2), and vercarin A. (11) The reagent according to any one of (11).

(13) The reagent according to any one of (9) to (12), wherein the T cell is a T cell derived from a leukemia patient.
(14) The reagent according to any one of (9) to (13) above, wherein the T cells are CD4 positive and CD8 positive T cells.
(15) The reagent according to any one of (9) to (13), wherein the T cell is an LMAT cell (FERM BP-08598).

  According to the present invention, a method and a reagent for easily and accurately detecting trichothecene mycotoxins contained in a sample such as food or feed, and a trichothecene mycotoxin contained in a sample such as food or feed can be easily and accurately quantified. Methods and reagents for doing so are provided.

(T cell)
The T cell used in the present invention may be any T cell in which apoptosis is induced by contact with a trichothecene mycotoxin, and specifically includes a T cell derived from a leukemia patient. Examples of T cells include CD4 negative and CD8 negative T cells, CD4 positive T cells, CD8 positive T cells, CD4 positive and CD8 positive T cells, preferably CD4 positive and CD8 positive T cells, More preferred are T cells that are CD4 positive and CD8 positive and have CD2 positive, CD21 positive, CD34 positive, CD95 negative, and TCRαβ negative properties. Specific examples include LMAT cells established from peripheral lymphocyte fraction cells of leukemia patients, human leukemia T cell line Jurkat cells (ATCC No.TIB-152), Molt-4 cells (ATCC CRL-1582), etc. However, LMAT cells are particularly preferred. As of January 21, 2004, LMAT cells have been deposited as FERM BP-08598 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Chuo 1st, East 1-chome, Tsukuba City, Ibaraki, Japan).

The T cells used in the present invention may be T cells other than those derived from leukemia patients. The T cells have the properties of T cells and have the property of infinite proliferation. Examples thereof include cells. Specific examples include T cells derived from healthy individuals that have undergone genetic recombination so as to have an infinitely proliferating property.
(Trichothecene mycotoxins)
Trichothecene mycotoxins are a group of 50 or more mycotoxins having a common skeleton, and are classified into three groups according to structural features. Hereinafter, the three groups are referred to as groups A, B, and C, respectively.

  Examples of group A include T-2 toxin (hereinafter referred to as T-2), HT-2 toxin (hereinafter referred to as HT-2) and the like, and group B includes deoxynivalenol (hereinafter referred to as DON). ), Nivalenol (hereinafter referred to as NIV), fusarenone X, and the like, and group C includes loridin A, vercarin A, and the like. It is said that C> A> B as the toxicity of the trichothecene mycotoxin.

(Sample)
The specimen used in the present invention is not particularly limited as long as it is a specimen that may contain trichothecene mycotoxins. However, crops that may be attached to molds that produce trichothecene mycotoxins, Examples include processed products and environmental factors that affect the growth of the crop.

  Examples of crops include any crops such as rice, wheat, beans, corn, and potatoes. The crop may be cultivated in a field such as a field, or may be present on the market, and its location is not limited. The processed product of the crop includes any food and drink processed using the above crop as a raw material. Samples used in the present invention also include so-called feeds, which are foodstuffs for livestock, and meat, milk, eggs, or processed products of livestock such as pigs, cows, and chickens that consume feed. Environmental factors that affect crop growth include various elements that make up the crop environment. Specific examples include inorganic factors such as moisture and soil, microorganisms that adhere to the inorganic factors, and organic residues such as spores of microorganisms floating in the air and plant stumps.

  The specimen described above can be used as a measurement sample as it is, but a sample obtained by subdividing the specimen can also be used as a measurement sample. Furthermore, trichothecene mycotoxins can be extracted from a specimen or a subdivided specimen using a solvent, and the resulting extract can be used as a measurement sample. A sample obtained by diluting the extract with a solvent or a concentrated solution can be used as a measurement sample.

As a method for subdividing the specimen, any method may be used as long as it does not involve decomposition or disappearance of trichothecene-based mycotoxins. For example, a method of grinding using a mortar and pestle, ultrasonic wave, mill, hammer, etc. And a method of grinding using a blade and a method of finely shearing using a blade.
Examples of a method for extracting trichothecene-based mycotoxins from a sample subdivided by the above method or a sample in an intact form using a solvent include a method in which a sample or a subdivided sample is directly immersed in a solvent. Further, at the time of extraction, the extraction efficiency can be increased by stirring operation, ultrasonic treatment or the like.

  The solvent for extracting the trichothecene-based mycotoxins may be any solvent that can dissolve the trichothecene-based mycotoxins. Examples thereof include water, organic solvents, water-containing organic solvents, and the like. Or a water-containing organic solvent is preferred. Examples of the water-soluble organic solvent include methanol, ethanol, propanol, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide, acetone, dioxane, dimethoxyethane and the like. The amount of the solvent for extracting the trichothecene mycotoxin is, for example, a ratio of the amount of the solvent to the sum of the volume of the extract and the amount of the solvent (= total amount of the extracted solution) is 25% or more, preferably 50% or more, 75 % Or more is more preferable. The water content in the water-containing organic solvent is, for example, 0.5 to 50%, but preferably 5 to 20%.

  Extracts containing trichothecene mycotoxins include not only a solution (extract) obtained by extracting trichothecene mycotoxins from a sample with a solvent, but also a diluted solution obtained by diluting the extract with the solvent, or the extract. And concentrates obtained in this manner. The concentrate of the extract can be obtained, for example, by distilling off the solvent in the extract under normal pressure or reduced pressure. These extracts, diluted solutions and concentrates can be used for measurement as they are, but those in a state where insolubles and the like are removed by passing through a column can also be used for measurement. Examples of the column that can be used for removing insolubles include a florisil / anhydrous sodium sulfate multilayer column, a silica gel / anhydrous sodium sulfate multilayer column, and the like.

(Caspase)
The caspase used in the present invention is not particularly limited as long as it is a caspase that is induced by apoptosis and whose activity is measured, and examples thereof include caspase 1 to caspase 14, and caspase 3 is preferable.
(Method for detecting trichothecene mycotoxins)
Examples of the method for detecting trichothecene mycotoxins include a method comprising the following steps.
[1] A step of contacting a specimen with a T cell to induce apoptosis in the T cell; and
[2] A step of detecting induced apoptosis.

There is no restriction | limiting in particular as long as it is an aspect which enables the said process as a specific aspect of each process.
The step [1] can be performed in a medium in which T cells are maintained. Any medium that maintains T cells can be used in either a serum medium or a serum-free medium.
The temperature at which the specimen and T cells are brought into contact is not particularly limited as long as apoptosis of the cell is induced, but is, for example, 0 to 50 ° C, preferably 10 to 45 ° C, and preferably 25 to 40 More preferably.

The time for contacting the specimen and the T cell is not particularly limited as long as apoptosis of the T cell is caused. For example, it is 0.5 to 36 hours, preferably 1 to 24 hours, and 5 to 12 Time is even more preferred.
In addition, the contact between the specimen and the T cell can be performed under normal aerobic conditions, but can also be performed under anaerobic conditions such as a carbon dioxide atmosphere. The specimen and the T cell can be contacted in an appropriate container, and examples of the container include a 96-well microplate. In particular, the 96-well microplate is preferable in that it can easily cope with measurement automation.

  As a method for detecting apoptosis in the step [2], for example, a method for measuring the activity of caspase activated by apoptosis induction of a cell, a method for detecting a DNA ladder by electrophoresis of DNA isolated from a cell, annexin Examples include a method of staining cells with V, and a method of measuring the activity of caspases activated by inducing apoptosis of cells is preferable. The method for measuring caspase activity is described in detail below.

  The activity of caspase can be measured, for example, by producing a dye, fluorescence, luminescence or the like by the reaction between the caspase substrate and caspase, and measuring the intensity of the produced dye, fluorescence, luminescence or the like. The caspase substrate is not particularly limited as long as it is cleaved by caspase to generate a dye, fluorescence, luminescence, etc. For example, the catalog “Apoptosis” (2000 version) of the Institute for Medical Biology (MBL), Inc. And the caspase substrate described in the catalog “Reagent Catalog for Apoptosis Research” of Funakoshi Co., Ltd., and the like. Examples of caspase 3 substrates include peptide derivatives having a partial sequence of aspartic acid-glutamic acid-valine-aspartic acid. In the peptide derivative, the N-terminal side may be protected with an acyl group or the like, and the C-terminal side is bound with a substrate that is cleaved by caspase 3 to generate a dye, fluorescence, luminescence, or the like. It is preferable.

Examples of the substrate for generating the dye include 4-nitroaniline. Examples of the substrate that generates fluorescence include rhodamine, 7-amino-4-trifluoromethylcoumarin (AFC), and 7-amino-4-methylcoumarin (AMC).
In the case of a substrate that produces a dye, the activity of caspase can be measured by measuring the absorbance of the reaction solution with a spectrophotometer. In the case of a substrate that generates fluorescence, the activity of caspase can be measured by measuring the intensity of the generated fluorescence with a fluorescence intensity meter. In the case of a substrate that generates luminescence, the activity of caspase can be measured by measuring the intensity of the generated luminescence with a luminescence intensity meter.

(Method for quantifying trichothecene mycotoxins)
The method for quantifying trichothecene mycotoxins in a sample is not particularly limited as long as it is a method capable of quantifying trichothecene mycotoxins in a sample, but for example, various trichothecene mycotoxins contained in a sample may be analyzed for each trichothecene mycotoxin. A method for quantification, a method for quantification as an amount converted to a specific trichothecene mycotoxin, and the like can be mentioned. A method of quantifying the amount as a specific trichothecene-based mycotoxin is preferable. The specific trichothecene-based mycotoxin is not particularly limited as long as it is a trichothecene-based mycotoxin, and examples thereof include DON, NIV, T-2, and Velkaline A.

Examples of the quantitative method of the present invention include a method comprising the following steps.
[1] Contact a T cell with a standard solution of a specific concentration of a specific trichothecene mycotoxin (M ₁ ) to induce apoptosis of the T cell, and measure the activity of caspase activated by the induced apoptosis. A step of preparing a calibration curve showing a correlation between the concentration of the trichothecene mycotoxin (M ₁ ) and the activity of caspase;
[2] A step of preparing a calibration curve showing a correlation between the concentration of each trichothecene mycotoxin and the activity of caspase for other types of trichothecene mycotoxins (M _{2 to} M _n ; n is an arbitrary integer);
[3] A step of calculating a relative value of the toxicity intensity of other trichothecene mycotoxins (M _{2 to} M _n ) with respect to the toxicity intensity of a specific trichothecene mycotoxin (M ₁ ) based on the caspase activity;
[4] A step of bringing a T cell into contact with a specimen to induce apoptosis in the T cell and measuring the activity of caspase activated by the induced apoptosis;
[5] Using the caspase activity measured in the step [4] and the calibration curve prepared in the step [1], the trichothecene mycotoxin content in the sample is converted into a specific trichothecene mycotoxin (M ₁ ) Expressing as content;

There is no restriction | limiting in particular as long as it is an aspect which enables the said process as a specific aspect of each process.

  As a method for measuring the activity of caspase activated by induction of apoptosis of T cells for the trichothecene mycotoxin in the step [1], for example, a standard solution having a known concentration of the specific trichothecene mycotoxin was used as a specimen. Examples include the aforementioned method for detecting trichothecene mycotoxins. Induction of T cell apoptosis and measurement of caspase activity in the step [1] can be carried out, for example, by the method used in the aforementioned method for detecting a trichothecene mycotoxin.

  Examples of other trichothecene mycotoxins in the step [2] include NIV and T-2 when the specific trichothecene mycotoxin is DON, and DON when the specific trichothecene mycotoxin is NIV. T-2 etc. are mentioned, and when a specific trichothecene system mycotoxin is T-2, DON, NIV, etc. are mentioned. As a method for measuring the activity of caspase activated by apoptosis of T cells for other trichothecene mycotoxins, for example, the above-mentioned trichothecene mycotoxins using standard solutions of known concentrations of the other trichothecene mycotoxins are used as specimens. Examples include detection methods.

As a method for calculating the relative value of the toxicity intensity of other trichothecene mycotoxins (M _{2 to} M _n ) relative to the toxicity intensity of a specific trichothecene mycotoxin (M ₁ ) in the step [3], for example, [1] and [ 2], a method of calculating from the slope of the calibration curve based on the calibration curve for each trichothecene mycotoxin (M _{1 to} M _n ) prepared in the step of 2], a specific trichothecene mycotoxin (M ₁ ) at a certain concentration And a method of calculating from the ratio of caspase activity values of trichothecene mycotoxins (M _i ) to be compared.

Examples of a method for calculating the relative value of the toxicity intensity from the slope of the calibration curve include the following methods. _{A 1} the slope of the calibration curve at a particular trichothecene mycotoxins _{(M 1),} when the slope of the calibration curve in the other trichothecene mycotoxins _(M 2 ~M _n) and _a 2 ~a _n, particular trichothecene mycotoxins ( the relative values of the toxic intensity of trichothecene mycotoxins _(M 1 ~M _n) to the toxic intensity of M _{1) (f} 1 ~f _n), respectively formula (1)
(Equation 1)

f _i = a _i / a ₁ (i: _{1 to} nn are integers) (1)
(F ₁ = 1).
Examples of the method for calculating the relative value of the toxicity intensity from the ratio of caspase activity include the following methods. From a calibration curve for a specific trichothecene mycotoxin (M ₁ ), a toxicity intensity (Y ₁ ) at a specific concentration (X) (the concentration is preferably a concentration that is quantitatively recognized in the calibration curve). To decide. Similarly, determined from the calibration curve in the other trichothecene mycotoxins _(M 2 ~M _n), toxic intensity at the concentration (X) a _(Y 2 _{~Y n)} for each trichothecene mycotoxins _(M 2 ~M _n) To do. The relative value (f _{1 to} f _n ) of the toxicity intensity of the trichothecene mycotoxins (M _{1 to} M _n ) with respect to the toxicity intensity of the specific trichothecene mycotoxins (M ₁ ) is represented by the formula (2)
(Equation 2)

f _i = Y _i / Y ₁ (i: _{1 to} nn are integers) (2)
(F ₁ = 1).
Examples of the method for measuring caspase activity in the step [4] include the method used in the aforementioned method for detecting trichothecene mycotoxins.

As a method for converting the trichothecene mycotoxin content in the sample in the step [5] into the amount of a specific trichothecene mycotoxin, for example, the caspase activity (F) obtained in the step [4] and the method prepared in [1] and a calibration curve for the trichothecene mycotoxins (M _1), a method for calculating the trichothecene mycotoxins (M ₁₎ the amount corresponding to caspase activity value (F), and the like. In this case, the calculated value is the trichothecene mycotoxin (M ₁ ) equivalent content of the trichothecene mycotoxin content in the specimen. A value obtained by multiplying the calculated value by the relative value f _i becomes the trichothecene mycotoxin (M _i ) equivalent content of the trichothecene mycotoxin content in the specimen.

The validity of the quantification method of the present invention can be verified by performing the following steps [6] and [7] in addition to the above [1] to [5].
[6] A step of measuring M _{1 to} M _n in a sample by another quantitative method (for example, an immunological assay such as ELISA); and
[7] The sum of numerical values obtained by multiplying the various trichothecene mycotoxin contents in the sample quantified in the step [6] by the relative value obtained in the step [3] is used as the specific trichothecene mycotoxin amount, 5] A step of examining the correlation with the specific trichothecene mycotoxin content calculated in the step of 5].

  Examples of the quantification method other than the present invention in the step [6] include immunological measurement methods such as ELISA. Specifically, based on the immunological measurement method disclosed in WO 01/018196, a monoclonal antibody that specifically recognizes DON and NIV, a monoclonal antibody that specifically recognizes NIV, T-2 By using a monoclonal antibody that specifically recognizes DON, NIV, and T-2 in a sample, respectively.

  In the step [7], the sum of the values obtained by multiplying the various trichothecene mycotoxin contents in the sample quantified in the step [6] by the relative value obtained in [3] is the specific trichothecene mycotoxin in the sample. It will represent the content. Therefore, for a plurality of samples, the specific trichothecene mycotoxin content obtained by the above method and the specific trichothecene mycotoxin content calculated by the method of the present invention are respectively calculated, and the correlation between these values is calculated. By examining, the validity of the numerical value of the quantification method of the present invention can be verified.

  In the method of the present invention, a buffer solution, a surfactant, a stabilizer and the like can be appropriately used. The buffer used in the buffer is not particularly limited as long as it has a buffer capacity, but has a pH of 1 to 11, for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphorus Acid buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer , Borate buffer, carbonate buffer, glycine buffer, 2-morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamide) Iminodiacetic acid (ADA), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES) N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl Ethanesulfonic acid (HEPES), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), N- [tris (hydroxymethyl) methyl] -2-hydroxy- 3-aminopropanesulfonic acid (TAPSO), piperazine-N, N′-bis (2-hydroxypropyl) Pansulfonic acid) (POPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] -2-hydroxypropanesulfonic acid (HEPPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl ] Propanesulfonic acid [(H) EPPS], N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N-bis (2-hydroxyethyl) glycine (Bicine), N-tris (hydroxymethyl) methyl -3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), N-cyclohexyl-3-amino Good buffer such as propanesulfonic acid (CAPS) It is. The concentration of the buffer solution is not particularly limited as long as it is suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L, and 0.01 to 0.00. 1 mol / L is particularly preferable. Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. Examples of the stabilizer include dithiothreitol (DTT).

(Reagent for detection of trichothecene mycotoxins)
The reagent for detecting a trichothecene mycotoxin of the present invention is not particularly limited as long as it contains a T cell that induces apoptosis by contact with the trichothecene mycotoxin. Examples of the T cells include the T cells mentioned above (T cells).

  Examples of the apoptosis detection reagent include a caspase activity measurement reagent, a DNA ladder detection reagent, a cell staining reagent, and the like, and a caspase activity measurement reagent is preferable. Examples of the caspase activity measuring reagent include a reagent containing a caspase substrate, and examples of the cell staining reagent include annexin V and the like. Examples of the caspase substrate include the aforementioned caspase substrates.

(Tricotene-based mycotoxin determination reagent)
The reagent for quantifying trichothecene mycotoxins of the present invention is not particularly limited as long as it is a reagent containing a T cell and a trichothecene mycotoxin standard solution in which apoptosis is induced by contact with the trichothecene mycotoxin. Examples of the T cells include the T cells mentioned above (T cells).

  Examples of the trichothecene-based mycotoxin standard solution include a known concentration of DON reagent, a known concentration of NIV reagent, a known concentration of T-2 reagent, and a known concentration of Verkaline A reagent. In the quantification of trichothecene mycotoxins, these standard solutions are used as a solution prepared in advance, but immediately before the quantification, solid state trichothecene mycotoxins (known amounts) are dissolved in a known volume of solvent, or It is also used as a solution prepared by adding a known volume of a solvent to a solid state trichothecene mycotoxin (known amount).

  Examples of the apoptosis detection reagent include a caspase activity measurement reagent, a DNA ladder detection reagent, a cell staining reagent, and the like, and a caspase activity measurement reagent is preferable. Examples of the caspase activity measuring reagent include a reagent containing a caspase substrate, and examples of the cell staining reagent include annexin V and the like. Examples of the caspase substrate include the aforementioned caspase substrates.

The reagent of the present invention may appropriately contain a buffer, a surfactant, a stabilizer and the like. Examples of the buffer used in the buffer include the aforementioned buffer. Examples of the surfactant and the stabilizer include the aforementioned surfactant and stabilizer, respectively. The reagent of the present invention may be stored and transported in the form of a kit.
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, these do not limit the scope of the present invention at all. In this example, reagents and equipment from the following manufacturers were used.

  DON (Sigma), NIV (Sigma), T-2 (Sigma), Belkaline A (Sigma), fetal calf serum (Sigma), penicillin (Sigma), streptomycin (Sigma) ), RPMI-1640 medium (ICN Biomedicals), HEPES (Wako Pure Chemicals), Sucrose (Wako Pure Chemicals), DTT (Wako Pure Chemicals), Nonidet P-40 Sigma), CHAPS (Wako Pure Chemical Industries), Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin (Caspase 3 substrate; Pharmingen International), 96-well microplate (Falcon) Fluoroscan Ascent (manufactured by Life Sciences International Company).

Method for measuring caspase 3 activity (1) Culture of LMAT cells Using an RPMI-1640 medium containing 10% fetal calf serum, 100 IU / mL penicillin and 0.1% streptomycin, LMAT cells [“Ryuchemia (Leukemia) ", 1996, Vol. 10, p. 102-105] was cultured at 37 ° C. in an atmosphere of air-carbon dioxide (95: 5).

(2) Method for quantifying caspase 3 activity LMAT cells were suspended in RPMI-1640 medium without serum, seeded in a Petri dish, and cultured overnight at 37 ° C. in a carbon dioxide incubator. Cells were collected by centrifugation (900 rpm / min; 5 min) and suspended in RPMI-1640 medium without serum to 1 × 10 ⁶ / mL. After dispensing 100 μL of suspension (containing 1 × 10 ⁵ cells) to each well of a 96-well microplate (model number: 7033), the specimen was added to the well, and the plate was carbon dioxide gas at 37 ° C. for 12 hours. Incubated in incubator. Immediately after the incubation, the medium (75 μL) was extracted from each well, and the cells remaining in the well after extraction were frozen at −80 ° C. for 30 minutes. After thawing the frozen cells on ice, the pH 7.25 lysis buffer (50 μL) [HEPES (100 mmol / L), sucrose (10%), DTT (2 mmol / L), Nonidet P-40 ( 10 ⁻⁶ v / v%) and CHAPS (0.1%)] was added to the thawed cells and mixed gently [“FEBS Letters”, 1997, Vol. 414, p. 552-556, and “FEBS Letters”, 1998, Vol. 429, p. 351-355]. Subsequently, Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin [“Nature”, 1995, vol. 376, p. 37-43] 2.5 mmol / L aqueous solution (1 μL) was added to each well, and the reaction was immediately started at 37 ° C. Fluorescence intensity derived from 7-amino-4-methylcoumarin liberated by the reaction was quantified using a fluoroscan ascent at an excitation wavelength of 355 nm and a fluorescence wavelength of 460 nm ["Journal of Biological Chemistry". 1997, Vol.272, p. 1965-1969], and the activity of caspase 3 was expressed as the initial reaction rate (pmol / min / 10 ⁶ cells).

Apoptosis of LMAT cells by DON LMAT cells were prepared in the same manner as in Example 1 except that 1 μL of PBS, 500 μmol / L, 1000 μmol / L, 1500 μmol / L, and 2000 μmol / L of DON in PBS was used as a specimen. The activity of caspase 3 in apoptosis was quantified. As a result, as shown in FIG. 1, it was found that the activity of caspase 3 increases as the concentration of DON increases. Between the concentration (x) of DON and the activity (y) of caspase 3, there is a formula (3)
(Equation 3)

y = 2228 * exp (0.0222 * x) (3)
The relationship was recognized.

Apoptosis of LMAT cells by NIV Example 1 except that 1 μL of 1% DMSO-PBS, 1% DMSO-PBS solution of each concentration of 1% DMSO-PBS, 250 μmol / L, 500 μmol / L, 750 μmol / L and 1000 μmol / L In the same manner as described above, the activity of caspase 3 in apoptosis of LMAT cells was quantified. As a result, as shown in FIG. 2, it was found that the activity of caspase 3 increases as the concentration of NIV increases. Between the concentration (x) of NIV and the activity (y) of caspase 3, there is a formula (4)
(Equation 4)

y = 1311 * exp (0.0212 * x) (4)
The relationship was recognized.

Apoptosis of LMAT cells by T-2 The same method as in Example 1 except that 1 μL of DMSO solution of T-2 at each concentration of DMSO, 5 μmol / L, 10 μmol / L, 15 μmol / L and 20 μmol / L was used as a specimen. Was used to quantify the activity of caspase 3 in apoptosis of LMAT cells. As a result, as shown in FIG. 3, it was found that the activity of caspase 3 increases as the concentration of T-2 increases. Between the concentration of T-2 (x) and the activity of caspase 3 (y), the formula (5)
(Equation 5)

y = 20.885 * exp (0.0235 * x) (5)
The relationship was recognized.

Apoptosis of LMAT cells by vercarin A 1 μL of 1% DMSO-PBS, 1% DMSO-PBS solution of 1% DMSO-PBS, 2.5 μmol / L, 5 μmol / L, 10 μmol / L and 15 μmol / L of each concentration of Velcarin A Quantified and evaluated the activity of caspase 3 in apoptosis of LMAT cells by the same method as in Example 1. As a result, as shown in FIG. 4, it was found that the activity of caspase 3 increases as the concentration of verkaline A increases. Between the concentration (x) of verkaline A and the activity (y) of caspase 3, there is a formula (6)
(Equation 6)

y = 2.24043 * exp (0.0417 * x) (6)
The relationship was recognized.

Relative toxicity intensity of each trichothecene mycotoxin against DON From the calibration curves showing the relationship between the concentration (x) for each trichothecene mycotoxin and the activity (y) of caspase 3 in Examples 2 to 5, DON, NIV, T- 2. From the proportionality constants for Velkaline A, the relative values of NIV, T-2, and Velkaline A relative to DON were calculated as 1.83, 102, and 400, respectively. Moreover, the relative values of NIV and T-2 with respect to DON calculated from the caspase activity in the case of various trichothecene mycotoxins 50 nM were 1.6 and 40, respectively. From this result, it was found that the toxicity of trichothecene mycotoxins became stronger in the order of DON <NIV <T-2 <Bercalin A, which was consistent with the generally known order of toxicity of trichothecene mycotoxins.

Determination of trichothecene mycotoxin content in pulverized wheat (DON equivalent content)
The sample for quantification was prepared by the following procedure. 85% (v / v) acetonitrile (40 mL) was added to the wheat pulverized product (5 g), shaken for 90 minutes, a part of the extract (8 mL) was dried to dryness, and the dried product was DMSO (100 μL). The sample was dissolved in
The samples 1 to 9 thus prepared from the pulverized wheat flour 1 to 9 were each diluted 5-fold with 1% DMSO-PBS, and the obtained 5-fold diluted specimen was used in an amount of 0.2 μL. The method of Example 1 Was used to quantify caspase 3 activity. From the quantified caspase 3 activity value and the calibration curve for DON prepared in Example 2, the DON equivalent concentration of trichothecene mycotoxin in each sample was determined. Table 1 shows the DON equivalent content of trichothecene-based mycotoxins in the pulverized wheat flour that was the basis of sample preparation.

Here, from the DON equivalent concentration [x (μmol / L)] of the trichothecene mycotoxin in the sample, the DON equivalent content [y (ppm)] of the trichothecene mycotoxin in the pulverized wheat flour from which the sample was prepared is represented by the formula ( 7)
(Equation 7)

y = x * 100 * 296.3 * 2500 * 10 ⁻⁶ (7)
Is calculated by
On the other hand, the concentrations of DON, NIV and T-2 in each sample (samples 1 to 9) were measured using FA / mycotoxin [DON + NIV] (manufactured by Kyowa Medex; DON + NIV comprehensive quantification kit), FA / mycotoxin [NIV] (Kyowa). Quantification by enzyme immunoassay (ELISA) using Medex; NIV quantification kit) and FA Mycotoxin [T-2 + HT-2] (Kyowa Medex; T-2 + HT-2 comprehensive quantification kit) As a result, the results shown in Table 2 were obtained.

From the results of Table 2 obtained by the ELISA method and the relative values of NIV and T-2 with respect to DON determined in Example 6 (1.6 and 40, respectively), the wheat pulverized product from which each specimen was prepared DON equivalent content of trichothecene mycotoxin in the formula (8)
(Equation 8)

w = x + 1.6 * y + 40 * z (8)
[Wherein, w represents the DON equivalent content (ppm) of trichothecene-based mycotoxin in the pulverized wheat, x, y, and z are DON, NIV, and T-2 + HT-2 in the sample determined by the ELISA method, respectively. When the concentration was calculated, the results shown in Table 2 were obtained.

  Comparison between Table 1 and Table 2 shows that the DON equivalent content of trichothecene mycotoxins in the pulverized wheat flour calculated by the LMAT method and the DON equivalent content of trichothecene mycotoxins in the pulverized wheat flour calculated by the ELISA method Although an 84-fold difference was observed, as shown in FIG. 5, a good correlation was observed between the two quantitative methods. This means that the quantitative value obtained by the LMAT method is converted into the quantitative value obtained by the ELISA method by multiplying the quantitative value by a constant. That is, it is shown that quantification by the LMAT method is effective for pricing by the ELISA method. In addition, in the quantification of environmental pollutants such as aflatoxin and dioxin, the LMAT method determines the content of trichothecene mycotoxins in the sample, taking into account the current situation where differences in the values between the quantification methods are allowed in some cases. It can be said that it is a simple method suitable for quantification.

  According to the present invention, methods and reagents useful for the detection and quantification of trichothecene mycotoxins contained in specimens such as food and feed are provided.

It is a figure showing the relationship between the density | concentration of DON and the activity of caspase 3 in the apoptosis of the LMAT cell by DON. It is a figure showing the relationship between the density | concentration of NIV and the activity of caspase 3 in the apoptosis of the LMAT cell by NIV. It is a figure showing the relationship between the density | concentration of T-2 and the activity of caspase 3 in the apoptosis of the LMAT cell by T-2. It is a figure showing the relationship between the density | concentration of verkaline A and the activity of caspase 3 in the apoptosis of the LMAT cell by verkaline A. It is a figure showing the correlation with the LMAT method and ELISA method regarding DON conversion content determination of the trichothecene system mycotoxin in pulverized wheat.

Claims (15)

A method for detecting trichothecene-based mycotoxins in a specimen, which comprises inducing apoptosis in the cell by bringing the T cell into contact with the specimen and quantifying the induced apoptosis. A method for quantifying a trichothecene mycotoxin content in a specimen, which comprises inducing apoptosis in the cell by bringing the T cell into contact with the specimen and quantifying the amount of induced apoptosis. The amount of trichothecene mycotoxin in the sample is converted as the amount of any trichothecene mycotoxin selected from the group consisting of deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T-2), and vercarin A. The method according to claim 2, wherein the content is as follows. The method according to any one of claims 1 to 3, wherein the measurement of apoptosis is a method of measuring the activity of caspase activated by induction of apoptosis. The method according to claim 4, wherein the caspase is caspase-3. The method according to any one of claims 1 to 5, wherein the T cell is a T cell derived from a leukemia patient. The method according to any one of claims 1 to 6, wherein the T cells are CD4 positive and CD8 positive T cells. The method according to any one of claims 1 to 7, wherein the T cells are LMAT cells (FERM BP-08598). A reagent for detecting trichothecene-based mycotoxins in a specimen, comprising a T cell. A reagent for quantifying trichothecene mycotoxins in a specimen, comprising a T cell and a trichothecene mycotoxin standard solution. The reagent according to claim 9 or 10, further comprising a caspase substrate. The trichothecene mycotoxin is any trichothecene mycotoxin selected from the group consisting of deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T-2), and vercarin A. The reagent according to Item 1. The reagent according to any one of claims 9 to 12, wherein the T cell is a T cell derived from a leukemia patient. The reagent according to any one of claims 9 to 13, wherein the T cells are CD4 positive and CD8 positive T cells. The reagent according to any one of claims 9 to 13, wherein the T cells are LMAT cells (FERM BP-08598).

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