FR2601781A1 - MYCOTOXIN ASSAY REAGENT AND METHOD USING THE SAME FOR DOSING MYCOTOXIN - Google Patents

Abstract

THE INVENTION RELATES TO A MYCOTOXIN ASSAY REAGENT. THE REAGENT CONSISTS OF A MONOCLONAL ANTIBODY MARKED WITH AN ENZYME DIRECTED AGAINST MYCOTOXIN. THE ENZYME MAY BE OF THE TYPE PEROXIDASE, ALKALINE PHOSPHATASE, B-GALACTOSIDASE, CATALASE, GLUCOSE OXIDASE, LACTIC ACID-OXIDASE, ALCOHOL OXIDASE OR MONOAMINE OXIDASE, AND MYCOTOXIN, GENUS-GALACTOSIDASE, GALACTOSIDASE, GLUCOSE-OXIDASE, LACTIC ACID-OXIDASE, ALCOHOL-OXIDASE OR MONOAMINE OXIDASE, AND MYCOTOXIN, GENUSERGORGANUS ORGANISIUM OR MICROGANUS ORGANISIUM IS PRODUCED BY A MICROGUS ORGANISIUM OR ASPARENILLIUM. THE INVENTION ALSO AIMS A METHOD FOR DETERMINING A MYCOTOXIN WHERE THIS REAGENT IS USED UNDER COMPETITIVE REACTION CONDITIONS. THE INVENTION APPLIES IN PARTICULAR TO THE FIELD OF THE CONTROL OF FOOD PRODUCTS.

Description

The present invention relates to an assay reagent for a mycotoxin,

  and a method of assay. More particularly, it relates to a mycotoxin assay reagent

  containing a monoclonal antibody labeled with an enzyme and a method of assaying this assay reagent.

  Mycotoxins produced by eumycetes such as Penicillium, Aspergillus and Fusarium microorganisms are sometimes carcinogenic or tumorigenic in the kidneys or liver, even if they do not exhibit acute toxicity. Among the mycotoxins, aflatoxin B1

  evidence of particularly potent carcinogenicity.

  In fact, the effect of aflatoxin B1 on humans and animals has been well studied, and it has been recognized that

  Aflatoxin B1 carcinises the liver as a target organ.

  It is therefore very important to measure the amount of mycotoxins present in food

  humans and animals before they consume them and thus avoid the ingestion of mycotoxins.

  Typical methods for the determination of myco20 toxins include, for example, thin layer chromatography, high performance liquid chromatography, gas chromatography, mass spectroscopy and animal bioassays. However, these methods are limited in their practical application because they present difficulties concerning the measurement sensitivity or the time required for measurement and analysis. New improvements are

so desirable.

  Recently, an enzyme immunoassay using enzyme labeled aflatoxin B1 has been developed.

  a polyclonal antibody specific to it [Appl. Envi.

  Microbiology, 41, 1472 (1981)]. This method is extremely practical as regards the sensitivity of the assay and the time required for the assay and analysis, but its assay sensitivity is not yet sufficient. Moreover,.

  large quantities of reagents containing the mycotoxin

  toxin must be rejected after use, it is still desirable to improve it with regard to safety and economy.

  In Proceedings of the Japanese Association of Mycotoxicology, No. 21, June 30, 1985, Ikuko Ueno describes an improved enzyme immunoassay for aflatoxin B1.

  This method allows the determination of aflatoxin B1 in the range

  from 10 to 1000 μg / 10 Vu (1 ng / ml to 100 ng / ml).

  Assaying Aflatoxin B1 with an enzyme-linked immunosorbent is also known [Bhanu P. Ram and L. Patrick Hart in J. Assoc. Off. Anal. Chem., Vol. 69, No. 5 (1986)]. According to this method, the dosage range of

  Aflatoxin B1 is also 1 ng / ml to 100 ng / ml.

  An enzyme immunoassay for the detection of mycotoxin using a monoclonal antibody against aflatoxin B1 is also known and was developed by A.A.G. Candlish, W. H. Stimson and J. E. Smith (Letters in

  Applied Microbiology, 1985, 1, 57-61). The assay range for aflatoxin B1 by this method is about 1 to 5 ng / ml.

  The abovementioned enzyme immunoassay methods are very practical because they lend themselves to simple handling and make it possible to dose many samples at a time. No enzyme immunoassay method has yet been reported which allows aflatoxin B1

less than about 1 ng / ml.

  An object of the present invention is to provide a reagent for assaying a mycotoxin such as aflatoxin B1. Another object of the present invention is to provide a reagent for assaying a mycotoxin, whose sensitivity to mycotoxin is sufficient and which reacts rapidly with it. Still another object of the present invention is to provide a reagent for assaying a mycotoxin, which is

sufficiently stable and economical.

  Yet another object of the present invention is to provide an enzyme labeled monoclonal antimycotoxin antibody which is for use in a mycotoxin assay reagent having the above properties. Yet another object of the present invention is to provide a method for assaying a mycotoxin rapidly and with high sensitivity using the reagent of

  aforementioned mycotoxin assay of the invention.

  These goals and benefits as well as others of the

  The present invention will become apparent from the following description.

  In accordance with the present invention, the goals and

  Advantages of the invention are primarily achieved by a mycotoxin assay reagent comprising a monoclonal antibody labeled with an enzyme directed against the mycotoxin.

  In the accompanying drawings: FIG. 1 is a calibration curve obtained by fixing an OTA-BSA complex (20 μg / ml) to a 96-well flat-bottomed immunoassay plate, by adding a mycotoxin assay reagent. diluted at various concentrations and a standard solution of OTA in various concentrations at the individual cells of the plate to cause an enzymatic reaction, stopping the reaction and measuring on the plate the absorbance of the reaction solution at 500 nm by means of a microplate photometer, and plotting the absorbances measured as a function of the mycotoxin concentrations: FIG. 2 is a calibration curve obtained as in the case of FIG. 1, with the exception that a complex HS-T- 2-BSA (20 μg / ml) is used in place of the OTA-BSA complex and a titrated solution of T-2 is used in place of the titrated solution of OTA; FIG. 3 is a calibration curve obtained as in the case of FIG. 1, except that an AFBi-SAB complex (0.5 μg / ml) is used in place of the OTA-BSA complex and that a standard solution of AFB is used instead of the standard solution of OTA (except that the competitive reaction is carried out for 2 hours at room temperature and the reaction with the substrate for 30 minutes at room temperature in 50% methanol , and that the absorbance is measured at 492 nm); FIG. 4 is a calibration curve obtained as in the case of FIG. 3 (except that the competitive reactions are carried out for 30 minutes at 37.degree. C. and the reaction with the substrate for 15 minutes at 37.degree. C. in methanol at 50.degree. Figure 5 shows calibration curves corresponding respectively to the 104-fold dilution of Figures 3 and 4, the absorbances being expressed in percentages; Figure 6 is a calibration curve obtained in Comparative Example 1; Fig. 7 is a calibration curve obtained in Comparative Example 2; Fig. 8 shows curves corresponding to the 105-fold and 106-fold dilutions of Fig. 7, respectively, the absorbances being percentages; and FIG. 9 shows the results of an experiment in which AFB1 is added to peanut seeds and then recovered, reference A designating peanut seeds produced in Japan to which AFB1 is not added and reference B designating peanut seeds produced in

  China to which AFB1 is not added.

  The Applicant first of all attempted to perfect the method of mycotoxin dosing by employing an enzyme immunoassay method consisting of preparing a monoclonal antibody highly specific for a mycotoxin, reacting this unlabeled monoclonal antibody (mouse immunoglobulin) with the mycotoxin. under competitive reaction conditions, and then reacting the reaction mixture with a peroxidase labeled anti-mouse immunoglobulin antibody (see Comparative Example 1 given below). As a result, the amount of mycotoxin that could be detected was of the order of ng, and its analysis required a long period of time (for example, more than about 6 hours). Since many foods and feeds were to be examined and many of them could not be stored for long periods of time, it was necessary to further develop the above method of that the mycotoxin present in the samples to be analyzed can be

  dosed simply and quickly with good sensitivity.

  Through extensive research, we have discovered that a mycotoxin assay reagent can be prepared by chemically combining an antibody with high mycotoxin specificity (hereinafter referred to as anti-mycotoxin antibody) with an enzyme. , and that the objects of the present invention can be achieved through

this reagent.

  Examples of the mycotoxin to be assayed according to the present invention are toxins produced by microorganisms of the genera Penicillium, Aspergillus and Fusarium,

  such as ochratoxin A, toxin T-2 and aflatoxin B1.

  Enzymes which can preferably be used to label the monoclonal antibody used in the present invention include, for example, enzymes of the peroxidase, alkaline phosphatase, e-galactosidase, catalase, glucose oxidase, lactic acid oxidase types, alcohol oxidase and monoamine oxidase. According to the present invention, a mycotoxin can be assayed rapidly with high sensitivity under competitive reaction conditions using a mycotoxin assay reagent containing a monoclonal antibody.

labeled with an enzyme.

  The mycotoxin assay method according to the present invention can be carried out by a method of

  By adding a mycotoxin such as aflatoxin B to a carrier such as a 96-well immunoassay, add together a sample to be analyzed and the mycotoxin assay reagent of the invention, add a solution of a substrate, for example a substrate corresponding to the enzyme used to label the antibody, and measuring the absorbance of the colored reaction solution. At the above procedure step where the mycotoxin assay reagent and the sample are added, a competitive reaction occurs between the fixed mycotoxin and the reagent and between the mycotoxin present in the sample and the reagent, so that the amount of mycotoxin present in the sample can

  be measured quickly with great sensitivity.

  For the attachment of mycotoxin, other carriers such as polyethylene beads, polystyrene beads and ABS resin beads may also be used. The following Reference Examples illustrate the preparation of an antigen for mycotoxin assay, and the

  production and purification of an antibody.

  Example. EXAMPLE 1 Preparation of mycotoxin antigen: A complex of mycotoxin and bovine serum albumin (BSA) was prepared by the following procedure as an antigen to be attached to a titration plate.

immunological to 96 cells.

  (1) A complex of ochratoxin A (OTA), produced by a microorganism of the

  Aspergillus, and SAB (hereinafter referred to as OTA-SAB).

  5 mg of OTA are dissolved in 0.12 ml of ethanol and 3 ml of 1M phosphate buffer (pH 7.0). The OTA solution is then mixed with 50 mg of BSA dissolved in 0.1M sodium chloride solution. To this mixture was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDPC), and the mixture was stirred in the dark at 20 ° C for 24 hours. The product is dialyzed against distilled water

  and freeze-dried to obtain 30 mg of OTA-BSA.

  (2) A T-2 (T-2) toxin complex produced by a microorganism of the genus Fusarium and SAB (hereinafter referred to as T-2-BSA) is prepared by the following method. The hydroxyl group of T-2 is reacted with succinic anhydride to synthesize T-2 hemisuccinate (HS-T-2). Using HS-T-2, HS-T-2-SAB is prepared in the same manner as for the preparation of OTA-BSA in (1). (3) Aflatoxin B1 (AFB1) complex produced by a microorganism of the following is prepared by the following method:

  Aspergillus, and SAB (hereinafter referred to as AFB1-SAB).

  20 mg of AFB1 (a product of Sigma Co.) was dissolved in a small amount of methanol at about 60 ° C, and 200 mg of carbomethoxyamine hydrochloride (a product of Tokyo Chemical Co., Ltd.) was added. While adjusting the pH of the solution to 7 with 5N NaOH, the solution is subjected to oximation at 60 to 70 ° C for 2 to 3 hours according to the method of Chu [F.S. Chu, M.T.S. Hsia and P. Sun, 1977, Preparation and Characterization of Aflatoxin

  B1-O-carbomethyloxime, J. Assoc. Off. Anal. Chem., 60, 791794].

  The AFB1-oxime was purified and 10 mg dissolved by addition of 1 ml of dimethylformamide. 18.0 mg of EDPC are added to a solution of 25 mg BSA in 10 ml of 0.1M buffer phosphate (pH 7.2), and the AFB1-oxime solution above is added dropwise. A further 5.2 mg of EDPC is added to the mixed solution and, while adjusting the pH of the solution to 5.5 with 0.1M hydrochloric acid, it is stirred for 24 hours at room temperature by removing the light. The product is dialyzed against water

  distilled and lyophilized to obtain 45 mg of AFBi-oxime-BSA.

  The above preparation relies substantially on the above method of Chu, and the method of Ueno [F.S. Chu 35 and I. Ueno, 1977, Production of the antibody against aflatoxin

  B1, Appl. About. Microbiol., 33, 1125-1128].

  Reference Example 2 Production and Purification of Antibodies: Monoclonal antibodies with high specificities for OTA, T-2 and AFB1 prepared by the methods described in Japanese Patent Laid-Open No. 171500/1986 are produced and purified. and 229899/1986

  and of which the Claimant became the owner.

  The monoclonal antibodies were produced by intraperitoneally administering 107 cells from an established cell line suspended in phosphate buffer to BALB / c mice (9 week old males to which 0.5 ml of pristane per ml was administered. intraperitoneal route 2 weeks previously). A marked increase in body weight of the mice is observed in about one month, and the ascites are extracted 1 to 3 weeks later. The antibody titers are from 106 to 108 for the anti-OTA monoclonal antibody, 10 to 106 for the anti-T-2 monoclonal antibody and 10 to 10 for the anti-AFB1 monoclonal antibody.

  Monoclonal antibodies from ascites are purified by the following procedure.

  The ascites are dialyzed against Tris-HCl buffer (pH 7.4) and applied to a DEAE-cellulose column equilibrated with the same buffer. A fraction which passes spontaneously through the column is salted out with 50% saturated ammonium sulfate. The resulting precipitate is dissolved in phosphate buffer (pH 7.4) and dialysis. It is determined that the resulting monoclonal antibodies directed against OTA, T-2

  and AFB1 are all of high purity by gel-plate electrophoresis using SDS polyacrylamide.

  Mycotoxins can be measured quickly

  with high sensitivity by a method (ELISA method) using mycotoxin assay reagents prepared by enzyme labeling the purified monoclonal antibodies directed against OTA, T-2 and AFB1.

  The present invention will be described hereinafter more

in detail.

  (1) Preparation of a Mycotoxin Assay Reagent Labeling of the antibody can be performed by a variety of methods such as a single step method using glutaraldehyde [Immunochemistry, 6, 43 (1969)] a two-step method using glutaraldehyde [Immunochemistry, 8, 1175 (1971)], a periodic acid oxidation method [Methods in Enzymology, 37, 133 (1975)], and a method of maleimide [Journal of the

  Biochemistry, 78, 235 (1975)]. These latter two methods are preferred.

  After conjugation with the enzyme, the antibody can be directly used for the mycotoxin assay. To further enhance the sensitivity, an enzyme-labeled antibody obtained by purifying the conjugated antibody by Sephadex gel filtration, Sephacryl, etc., is preferably used as the mycotoxin assay reagent. The fraction containing the enzyme labeled antibody is used as a mycotoxin assay reagent after being dialyzed against phosphate buffer (pH 7.4) or Tris-HCl buffer (pH 7.4) and freeze-dried or filtered through a sterile filter with no larger pore size

at 0.45 pm.

  (2) Mycotoxin assay A complex of a mycotoxin and a high molecular weight protein different from the SAB of the mycotoxin-BSA complex used as an immunogen for the production of the anti-mycotoxin antibody [e.g. Crack hemocyanin (HCF) and ovalbumin (OVA) is used as an antigen (antigen for attachment to a 96-well flat-bottomed immunoassay plate) for assaying

  mycotoxin produced by a microorganism of the genus Penicillium, Aspergillus or Fusarium.

  To assay the mycotoxin, the high molecular weight mycotoxin-protein complex is placed on the 96-well flat-bottomed immunoassay plate where it attaches. The antigen-treated cells are washed and blocked to prevent the anti-mycotoxin antibody from binding non-specifically to the alveoli to which the antigen is not bound. Then, the blocked cells are washed, and equal volumes of a sample to be analyzed (or a mycotoxin in a concentration known for the construction of a standard curve) are added and the mycotoxin assay reagent described in FIG. (1) above. The plate is left at rest for a given period of time. The cells are then carefully washed and a solution of a substrate corresponding to the enzyme used to label the antibody in the mycotoxin assay reagent (containing a substance which produces a color when a reaction occurs with the enzyme). After allowing the reaction to evolve with the enzyme for a given period of time, the absorbance of the reaction solution is measured at the wavelength at which the generated color exhibits its maximum absorbance. A calibration curve is constructed on the basis of the results obtained using a known concentration of the mycotoxin, and the concentration of the mycotoxin (eg OTA, T-2 or AFB1) in a sample to be analyzed can be determined from After the

calibration curve.

  The following nonlimiting examples illustrate more particularly the present invention. EXAMPLE 1 Preparation of mycotoxin assay reagents: Each of the three purified monoclonal antibodies directed against OTA, T-2 and AFB1 described in Reference Example 2 was scored using the known enzymatic labeling method described below to prepare reagents

mycotoxin assay.

  7.32 mg of horseradish peroxidase is dissolved in 1 ml of distilled water and 200 μl of 0.1M sodium periodate is added. The mixture is allowed to stand at room temperature for 30 minutes. The enzyme solution is dialyzed against

  1mM acetate buffer (pH 4.5) overnight at 4C.

  100 μl of 0.2M sodium carbonate buffer (pH 9.5) are then added to adjust the pH of the dialysate to 9.5. On the other hand, 8.78 mg of each monoclonal antibody (against OTA, T-2 or AFB1) dissolved in 0.1M phosphate buffer (pH 7.4) was dialyzed against 0.01M sodium carbonate buffer ( pH 9.5) overnight at 4 C. The resulting peroxidase solution and monoclonal antibody solution are mixed and the mixture is allowed to stand at room temperature for about 2.5 hours. To the reaction solution, 100 to 200 μl of 0.4% sodium borohydride are added and the mixture is allowed to stand at 4 ° C. for 2 hours. The peroxidase-labeled monoclonal antibody thus obtained is dialyzed completely against phosphate buffer (pH 7.4) at 4 ° C and used as a mycotoxin assay reagent either as it is or after freeze-drying to perform an assay.

of mycotoxin.

EXAMPLE 2

  Assay of OTA produced by a microorganism of the genus Aspergillus: OTA-BSA at 20 μg / ml is introduced into a 96-well flat-bottom immunoassay plate (manufactured by Nunc) at the rate of 100 μl per cell, and discarded overnight at 4 C to fix. The plate is washed twice with a wash solution (phosphate buffer pH 7.4 and 0.1% Tween 20). To prevent non-specific adsorption of the antibody on the plate, the plate is allowed to stand at room temperature for 30 minutes in the same washing solution as above but containing 10% of beef serum. The plate is then washed twice with the wash solution. Then, 50 μl of the mycotoxin assay reagent diluted with the same wash solution are added simultaneously to each of the cells.

3 4 4 5

  above (at 103, 3x104, 6x104, 105, and 10x, respectively) and 50μl of a standard solution of OTA (0 μg at 5μg / 50μl) prepared using the same solution. washing

  above, and leave the plate at rest at room temperature.

  ambient temperature for 30 minutes (in the case of constructing a calibration curve). The plate is washed four times with the washing solution, and a substrate solution (prepared by dissolving 20 mg of o-phenylene diamine and 10 of 35% H 2 O 2 in 50 ml of citrate buffer having a pH of 5 is added. 0) at a rate of 100 μl per cell. The plate is protected from light by means of aluminum foil and allowed to stand at room temperature for 30 minutes. Finally, 2N sulfuric acid was added at 50 μl per well to stop the enzyme reaction. After stopping the enzymatic reaction, the absorbance at 500 nm of the reaction solution was measured using a microplate photometer. The results are shown in Figure 1 of the accompanying drawings. Mycotoxin assay reagent diluted 3 x 104 times

  allows dosing with a sensitivity of a few pg.

  A sample to be assayed (50 μl of 0.1% BSA solution containing 50 μg of OTA) was examined using 50 μl of diluted 3 × 10 4 mycotoxin assay reagent. The measured amount of OTA is

  52 pg, which almost coincides with the calculated amount.

  The duration of the assay is as short as about

1 hour.

EXAMPLE 3

  Assay of T-2 Produced by a Microorganism of the Fusarium Genus: HS-T-2-BSA at 20 μg / ml was introduced into a 96-well flat-bottomed immunoassay plate (manufactured by Nunc) at a rate of 100 μg / ml. per cell, and the plate is left standing overnight at 4 ° C for the antigen to bind. The plate is washed twice with a washing solution (phosphate buffer, pH 7.4, containing 0.1% Tween 20) and, to prevent non-specific adsorption of the antibody on the plate, the plate is left behind. at rest for 30 minutes at room temperature in the same washing solution as above but containing 10% beef serum. The plate is then washed twice with the same washing solution as above, and at each time 50 μl of the mycotoxin assay reagent diluted with the same washing solution are added at the same time to each cell (FIG. 102, 103, 2 x 103, 104, 2 x 104 and 105 times respectively) and 50 μl of a standard T-2 solution (0 μg to 50 μl) prepared using the same wash solution as above. -above. The plate is then allowed to stand at room temperature for 30 minutes (in the case of construction of a calibration curve). The plate is washed four more times with the same washing solution as above, and a solution of substrate (prepared by dissolving 20 mg of o-phenylenediamine and 10 μl of 35% H 2 O 2) is added at a rate of 100 μl per well. in 50 ml of 0.1M citrate buffer having a pH of 5.0). The plate is protected from light by means of aluminum foil and allowed to stand at room temperature for 30 minutes. Finally, 2N sulfuric acid was added at 50 μl paralveole to stop the enzymatic reaction. After stopping the enzymatic reaction, the absorbance at 500 nm of the reaction solution was measured using a microplate photometer. The results are shown in Figure 2. The diluted mycotoxin assay reagent of 2 x 104 allows the

  dosage up to a few pg with great sensitivity.

  A sample to be assayed (50 μl of a 0.1% solution of BSA containing, by calculation, 50 μg of T-2) was examined according to the method described above in Example 3 using 50 μl of the reagent diluted mycotoxin

  2 x 104 times. The measured amount of T-2 is 47 μg, which almost coincides with the calculated value.

  The measurement time is as short as about

1 hour.

EXAMPLE 4

  Assay for AFB1 Produced by a Microorganism of the Aspergillus Genus: 0.5 μg / ml AFB 1-SAB was introduced into a 96-well flat-bottomed immunoassay plate (manufactured by Nunc) at a rate of 100 μl per cell. and the plate is allowed to stand overnight at 4 ° C for the antigen to bind. The plate is washed twice with washing solution (phosphate buffer, pH 7.4, containing 0.05% Tween-20) and, to prevent non-specific adsorption of the antibody on the plate, the plate is left behind. resting at room temperature for 30 minutes in the same washing solution as above but containing 10%

of beef serum.

  The plate is then washed twice with the same washing solution as above, and 50 μl of the aflatoxin B 2 5 10 (diluted 102 to 105 times) and 50 μl assay reagent are added simultaneously to each well. a titrated solution of AFB1 (0 μg to 5 ng / 50 μl) prepared using ex-traction solution for the analysis of toxins in fertilizers and feeds for humans and animals (eg 50% aqueous methanol solution) ). The plate is allowed to stand for 30 minutes at 37 ° C or for 2 hours at room temperature (in the case of constructing a calibration curve). Wash the plate four times with the same wash solution as above, and add at a rate of 1 μl. a solution of substrate (prepared by dissolving 20 mg of o-phenylenediamine and 10 μl of% H2O2 in 50 ml of 0.1M citrate buffer having a pH of 5.0). The plate is shielded from light and allowed to stand for 15 minutes at 37 ° C, or for 30 minutes at room temperature. Finally, 2N sulfuric acid is added at 50 μl per well to stop the reaction.

  enzyme. The absorbance of the reaction solution was measured at 492 nm using a microplate photometer.

  The results are shown in Figures 3 and 4. AFB can be dosed up to 6.9 μg / dosage or less with high sensitivity and the duration of the assay is also short

only 1 hour. Figure 5 shows calibration curves constructed by representing

  graphically the absorbances (expri-

  percentages) obtained through competitive reactions

  (for 2 hours at room temperature, and for 30 minutes at 37 ° C.) using the 104-fold diluted mycotoxin assay reagent.

COMPARATIVE EXAMPLE 1

  Assay of OTA produced by a microorganism of the genus Aspergillus: 20 μg / ml of OTA-BSA are introduced into a 96-well flat-bottom microtiter plate at a rate of 100 per well, and the plate is allowed to stand for a period of overnight at 4 C for the antigen to bind. The plate is washed twice with washing solution and, to prevent non-specific adsorption of the antibody on the plate, the plate is allowed to stand for 30 minutes at room temperature in a washing solution containing 10% beef serum. The plate is then washed twice with a washing solution, and 50 μl of a monoclonal antibody antimycotoxin solution having a high specificity (respectively diol) are added simultaneously to each of the cells. 102 103 104 105, 5 x 105, 10, 5 x 10, 10 and 108 times) and 50 μl of a standard solution of OTA (0 μg to 50 μl) prepared using the same wash solution. The plate is allowed to stand for 30 minutes at room temperature (in the case of construction of a calibration curve). The plate is washed three times with the wash solution, and a peroxidase labeled anti-mouse immunoglobulin antibody (diluted 103 fold with a dilution solution containing 10% beef serum) is added per 100 μl per well. The plate is allowed to stand for 2 hours at room temperature and washed four times with the wash solution. A solution of substrate (prepared by dissolving mg of ophénylènediamine and 10 μl of 35% H 2 O 2 in ml of 0.1M citrate buffer) is added at a rate of 100 μl per well. The plate is protected from light by means of aluminum foil and left

  at rest for 30 minutes at room temperature. Fina-

  2N sulfuric acid is added at a rate of 50 μl per cell to stop the enzymatic reaction. After stopping the enzymatic reaction, the absorbance is measured

  of the reaction solution at 500 nm using a microplate photo meter. The results are shown in Figure 6.

  When unlabeled anti-mycotoxin monoclonal antibody is used in the immunoassay as above, OTA can be detected only in an amount of a few ng using the 106-fold anti-OTA monoclonal antibody and putting about 6 hours to perform

the dosage.

COMPARATIVE EXAMPLE 2

  Assay of AFB1 Produced by a Microorganism of the Aspergillus Genus: 0.5 μg / ml AFB1-BSA was introduced into a 96-well, 96-well, flat-bottomed microtiter plate per well, and the plate was left at room temperature. overnight at 4 C for the antigen to settle. The plate is washed twice with washing solution and, to prevent non-specific adsorption of the antibody on the plate, the plate is allowed to stand for 30 minutes at room temperature in a wash solution containing% serum. beef. The plate is then washed twice with washing solution, and 50 μl aliquots of anti-AFB monoclonal antibody solution are added simultaneously. endowed with a high specificity (respectively

3 4 5 5

  diluted 10, 10-, 10, 5 x 105, 106 and 107 fold) and 50 μl of a standard solution of AFB1 (0 μg at 5 μg / 50 μl) prepared using an extraction solution for toxin analysis in fertilizers and food for humans and animals (eg 50% aqueous methanol solution). The plate is allowed to stand for 2 hours at room temperature (in the case of construction of a calibration curve). The plate is washed four times with the solution of

  washing and 100 μl per cell is added

  peroxidase-labeled anti-mouse immunoglobulin body (diluted 103-fold with 10% beef serum dilution solution). The plate is allowed to stand for 2 hours at room temperature and washed four times with the wash solution. A solution of substrate (prepared by dissolving mg of ophénylènediamine and 10 μl of 35% H 2 O 2 in ml of 0.1M citrate buffer having a pH of 5.0) is added per 100 μl per well. The plate is shielded from light and allowed to stand for 30 minutes at room temperature. Finally, 2N sulfuric acid is added at 50 μl per well to stop the enzyme reaction. After stopping the enzymatic reaction, the absorbance of the solution is measured

  reaction at 492 nm using a microplate photometer. The results are shown in Figure 7.

  When the absorbances are expressed as a percentage, as in Example 4, the calibration curves are as shown in FIG.

  When, as above, the unlabelled anti-AFB1 monoclonal antibody is used in the immunoassay, AFB1 can be detected only in an amount of several hundred μg / assay using the monoclonal antibody directed against AFB1 diluted by 105 times and about 6 hours to complete the assay. EXAMPLE 5 Addition of AFB1 to peanut seeds and its recovery from these seeds: By the method of Example 4, AFB was added to

  peanut seeds and recovers it.

  Peanut seeds are used as samples

  produced in Japan and peanut seeds produced in China.

  The peanut seeds are sprayed with a homogenizer after removing the pods. Particles that pass through a sieve having a 1 mm mesh opening are collected. Each of these samples is impregnated

  powdered peanut seeds with 25 μl of a methanol solution

  nolic titrated AFB1 (11.1 μg / ml). 25 ml of a 50% aqueous methanol solution are added to the impregnated sample and mixed for 3 minutes by means of a biological mixer. The mixture is then filtered through a Toyo N 1 filter paper or Whatman N 4 filter paper or equivalent filter paper. The filtrate and a sample not containing AFB1 are examined in accordance with Example 4, and the results are shown in FIG.

Figure 9.

  To the 50% methanol extract obtained, AFB1 was added in a concentration of 11.1 billion (556 μg / assay). The measured quantities of AFB1 in these

  Samples are 573 μg / assay and 625 μg / assay, on the basis of which the recovery rates are calculated to be 92% and 96%, respectively.

  As set forth above, the present invention is characterized in that the mycotoxin is attached to a carrier such as an immunoassay plate and in that the competitive reactions of the monoclonal antibody with the mycotoxin present in a sample and the mycotoxin attached to the support are performed by adding at the same time the sample to be analyzed and the mycotoxin assay reagent. Thus, the method of the present invention makes it easier to assay the mycotoxin with greater sensitivity and in a shorter period of time than conventional assay methods. The method of the present invention is also better in terms of safety

and the economy.

Claims (8)

  1. Mycotoxin assay reagent, characterized in that it comprises a monoclonal antibody labeled with a   enzyme directed against the mycotoxin.   2. Reagent according to claim 1, characterized   in that the enzyme which labels the monoclonal antibody is chosen from the enzymes of the peroxidase, alkaline phosphatase, e-galactosidase, catalase, glucose oxidase, lactic acid oxidase, alcohol oxidase and monoamine oxidase types.   3. Reagent according to claim 1, characterized in that the mycotoxin is produced by a microorganism   of the genus Penicillium, Aspergillus or Fusarium.   4. Reagent according to claim 1, characterized   in that the mycotoxin is ochratoxin A, toxin T-2 or aflatoxin B1.   5. Method for assaying a mycotoxin, characterized in that it comprises assaying the mycotoxin under competitive reaction conditions using a monoclonal antibody labeled with an enzyme directed against the mycotoxin. 20 6. Method according to claim 5, characterized   in that the enzyme which labels the monoclonal antibody is chosen from the enzymes of the peroxidase, alkaline phoAphatase, β-galactosidase, catalase, glucose oxidase, lactic acid oxidase, alcohol oxidase and monoamine oxidase types.   7. Method according to claim 5, characterized in that the mycotoxin is produced by a microorganism   of the genus Penicillium, Aspergillus or Fusarium.   8. Method according to claim 5, characterized   in that the mycotoxin is ochratoxin A, toxin T-2 or aflatoxin B1.