GB2193809A - Mycotoxin assay reagent and assay method - Google Patents

Abstract

A mycotoxin such as ochratoxin A, T-2 toxin or aflatoxin B1 is assayed under competitive reaction conditions by using an enzyme-labelled monoclonal antibody to the mycotoxin.

Description

SPECIFICATION Mycotoxin assay reagent and assay method This invention relates to an assay reagent for a mycotoxin, and an assay method. More specifically, it relates to a mycotoxin assay reagent containing a monoclonal antibody labelled with an enzyme and an assay method involving the use of the assay reagent.

Mycotoxins produced by Eumycetes such as microorganisms of the genera Penillium, Aspergillus and Fusarium are sometimes carcinogenic, or tumor-inducing in the kidneys or liver, even when they show no acute toxicity.

Aflatoxin B, shows particularly strong carcinogenicity among the mycotoxins. In fact, the effect of aflatoxin B1 on humans and animals has been well studied, and it has been known that aflatoxin B1 cancerates liver as a target organ.

It is very important therefore to measure the amount of mycotoxins in foods and feeds before they are taken by humans and animals and thus prevent in-take of the mycotoxins.

Conventional methods for quantifying mycotoxins include, for example, thin-layer chromatography, high-performance liquid chromatography, gas chromatography, mass spectroscopy, and bioassays using animals. These methods, however, are limited in practical application because they have difficulties in regard to measuring sensitivity or the time required for measuring and analysis. Further improvement is therefore desired.

Recently, an enzyme immunoassay using enzyme-labelled aflatoxin B1 and a polyclonal antibody specific for it was developed [Appl.

Envi. Microbiology, 41, 1472 (1981)]. This method is considerably practical in regard to assay sensitivity and the time required for assay and analysis, but its assay sensitivity is still not sufficient. Furthermore, since large amounts of mycotoxin-containing reagents must be discarded after use, it is still desired to be improved in regard to safety and economy.

Ikuko Ueno: Proceedings of the Japanese Association of Mycotoxicology, No. 21, June 30, 1985) discloses an improved enzyme immunoassay for aflatoxin B1. This method permits quantification of aflatoxin B1 in the range of 10 to 1,000 pg/10 pl assay (1 ng/ml to 100 ng/ml).

An enzyme-linked immunosorbent assay of aflatoxin B1 is also known [Bhanu P. Ram and L. Patrick Hart: J. Assoc. Off. Anal. Chem., vol. 69, No. 5 (1986)]. The assay range of aflatoxin B, according to this method is also 1 ng/ml to 100 ng/ml.

Enzyme immunoassay for detection of mycotoxin using a monoclonal antibody to aflatoxin B, developed by A. A. G. Candlish, W. H. Stimson and J. E. Smith is also known (Letters in Applied Microbiology, 1985, 1, 57-61). The assay range of aflatoxin B, by this method is about 1 to 5 ng/ml.

The aforesaid enzyme immunoassay methods are very practical because they lend themselves to simple handling and permit assay of many samples at a time.

No enzyme immunoassay method has yet been reported which permits quantification of aflatoxin B1 to less than about 1 ng/ml.

It is an object of this invention to provide a reagent for assaying a mycotoxin such as aflatoxin B1.

Another object of this invention is to provide a reagent for assaying a mycotoxin which has sufficient sensitivity to the mycotoxin and rapidly reacts with it.

Still another object of this invention is to provide a reagent for assaying a mycotoxin which has sufficient stability and economy.

Yet another object of this invention is to provide an enzyme-labelled monoclonal antibody to a mycotoxin, which is for use in a mycotoxin assaying reagent having the aforesaid properties.

A further object of this invention is to provide a method of quantifying a mycotoxin rapidly with high sensitivity using the aforesaid mycotoxin assaying reagent of the invention.

Other objects of this invention along with its advantages will become apparent from the following description.

According to this invention, the objects and advantages of the invention are firstly achieved by a reagent for assaying a mycotoxin, comprising an enzyme-labelled monoclonal antibody to the mycotoxin.

Figure 1 is a calibration curve prepared by fixing OTA-BSA (20 ,ul/ml) to a 96-well flatbottomed plate for immunoassay, adding a mycotoxin assaying reagent diluted to various concentrations and a standard OTA solution in various concentrations to the individual wells of the plate to induce an enzyme reaction, stopping the reaction, and measuring the absorbance at 500 nm of the reaction solution on the plate by means of a microplate photometer, and plotting the measured absorbances against the concentrations of the mycotoxin.

Figure 2 is a calibration curve prepared as in the case of Fig. 1 except using T-2-HS-BSA (20 ,lig/ml) instead of OTA-BSA and a standard T-2 solution instead of the standard OTA solution.

Figure 3 is a calibration curve prepared as in the case of Fig. 1 except using AFB,-BSA (0.5,ug/ml) instead of OTA-BSA and a standard AFB1 solution instead of the standard OTA solution (except that in 50% methanol, the competitive reaction was carried out at room temperature for 2 hours, and the substrate reaction was carried out at room temperature for 30 minutes; and the absorbance was measured at 492 nm).

Figure 4 is a calibration curve prepared as in the case of Fig. 3 (except that in 50% metha nol, the competitive reactions were carried out at 37"C for 30 minutes, and the substrate reaction was carried out at 370C for 15 minutes).

Figure 5 shows calibration curves for 104fold dilution in Figs. 3 and 4, in which the absorbances are expressed in percentage.

Figure 6 is a calibration curve prepared in Comparative Example 1.

Figure 7 is a calibration curve prepared in Comparative Example 2.

Figure 8 shows the curves for 105-fold and 106-fold dilutions in Fig. 7, in which the absorbances are expressed in percentage.

Figure 9 shows the results of an experiment in which AFB1 was added to peanuts and then recovered. In Fig. 9, A is for peanuts produced in Japan to which AFB1 was not added and B is for peanuts produced in China to which AFB was not added.

The present inventors first attempted to improve the mycotoxin assaying method by using an enzyme immunoassay method comprising preparing a monoclonal antibody highly specific for a mycotoxin, reacting this unlabelled monoclonal antibody (mouse immunoglobulin) with the mycotoxin under competitive reaction conditions, and then reacting the reaction mixture with a peroxidase-labelled antibody to mouse immunoglobulins (see Comparative Example 1 given hereinbelow). As a result, the amount of the mycotoxin could be detected to the order of ng, and its analysis required a long period of time (for example, more than about 6 hours).Since there were many foods and feeds to be assayed and many of them could not be stored for long periods of time, it was found to be necessary to improve the above method further so that the mycotoxin in assay samples can be assayed simply and rapidly with good sensitivity.

On extensive investigations, the present inventors have found that a mycotoxin assaying reagent can be prepared by chemically combining an antibody having high specificity for a mycotoxin (to be referred to as the anti-mycotoxin antibody) with an enzyme, and the objects of this invention can be achieved by this reagent.

Examples of the mycotoxin to be assayed in this invention are toxins produced by microorganisms of the general Penicillium, Aspergillus and Fusarium, such as ochratoxin A, T-2 toxin and aflatoxin BI.

Enzymes which can be preferabiy used to label the monoclonal antibody used in this invention include, for example, peroxidase, alkaline phosphatase, -galactosidase, catalase, glucose oxidase, lactic acid oxidase, alcohol oxidase, and monoamine oxidase.

According to this invention, a mycotoxin can be assayed rapidly with high sensitivity under competitive reaction conditions using a mycotoxin assay reagent containing an enzyme-labelled monoclonal antibody.

The method of assaying a mycotoxin in accordance with this invention can be carried out by a procedure comprising fixing a mycotoxin such as aflatoxin B1 to a support such as a 96-well immunoplate, adding an assay sample and the mycotoxin assay reagent of the invention together, adding a substrate solution e.g., a substrate corresponding to the enzyme used to label the antibody, and measuring the absorbance of the colored reaction solution. In the stage of adding the mycotoxin assay reagent and the sample in the above procedure, a competitive reaction occurs between the fixed mycotoxin and the reagent and between the mycotoxin in the sample and the reagent, whereby the amount of the mycotoxin in the sample can be rapidly measured with high sensitivity.

For fixation of the mycotoxin, other supports such as polyethylene beads, polystyrene beads and ABS resin beads may also be used.

The following Referential Examples illustrate the preparation of an antigen for mycotoxin assay, and the production and purification of an antibody.

REFERENTIAL EXAMPLE 1 Preparation of an antigen for mycotoxin assay: A complex of a mycotoxin and bovine serum albumin (BSA) as an antigen to be fixed to a 96-well immunoassay plate was prepared by the following procedure.

(1) A complex of ochratoxin A (OTA) produced by a microrganism of the genus Aspergillus and BSA (OTA-BSA hereinafter) was prepared by the following method.

OTA (5 mg) was dissolved in 0.12 ml of ethanol and 3 ml of 1M phosphate buffer (pH 7.0). The OTA solution was then mixed with 50 mg of BSA dissolved in 0.1M sodium chioride solution. To the 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 was dialyzed against distilled water, and lyophilized to give 30 mg of OTA-BSA.

(2) A complex of T-2 toxin (T-2) produced by a microorganism of the genus Fusarium and BSA (T-2-BSA hereinafter) was prepared by the following method.

The hydroxyl group of T-2 was reacted with succinic anhydride to synthesize T-2 hemisuccinate (T-2-HS). Using T-2-HS, T-2-HS-BSA was prepared in the same way as in the preparation of OTA-B in (1).

(3) A complex of aflatoxin B1 (AFB,) produced by a microorganism of the genus Aspergillus and BSA (AFB1-BSA hereinafter) was prepared by the following method.

AFB1 (a product of Sigma Co.; 20 mg) was dissolved in a small amount of methanol at about 60"C, and 200 mg of caerbomethoxyamine hydrochloride (a product of Tokyo Chemical Co., Ltd.) was added. While the pH of the solution was adjusted to 7 with 5N NaOH, the solution was subjected to oxime formation at 60 to 700C for 2 to 3 hours in accordance with the method of Chu [F. S.

Chu, M. T. S. Hsia and P. Sun, 1977, Preparation and Characterization of Aflatoxin B1-Ocarbomethyloxime, J, Assoc. Off. Anal.

Chem., 60, 791-794].

The AFB1-oxime was purified, and 10 mg of it was dissolved by adding 1 ml of dimethylformamide. EDPC (18.0 mg) was added to a solution of 25 mg of BSA in 10 ml of 0.1 M phosphate buffer (pH 7.2), and the above AFB,-oxime solution was added dropwise.

EDPC (5.2 mg) was further added to the mixed solution, and while adjusting the pH of the solution to 5.5 with 0.1M hydrochloric acid, it was stirred at room temperature for 24 hours under light shutting. The product was dialyzed against distilled water, and lyophilized to give 45 mg of AFB1-oxime-BSA.

The above preparation was based substantially on the aforesaid method of Chu, and the method of Ueno [F. S. Chu and I. Ueno, 1977, Production of antibody against aflatoxin BI, Appl. Environ. Microbiol. 33, 1125-1128].

REFERENTIAL EXAMPLE 2 Production and purification of antibodies: Monoclonal antibodies having high specificity for OTA, T-2 and AFB1 prepared by the methods described in Japanese Laid-Open Patent Publications Nos. 171500/1986 and 229899/1986 and owned by the inventors of the present application were produced and purified.

Monoclonal antibodies were produced by intraperitoneally administering 107 cells from an established cell line suspended in phosphate buffer to BALB/c mice (8 week-old males to which 0.5 ml of pristane had been intraperitoneally administered 2 weeks before). A marked increase in the body weight of the mice began to be observed in about 1 week, and the ascites was extracted 1 to 3 weeks later. The antibody titer was 106 to 108 for the monoclonal antibody to OTA, 104 to 106 for the monoclonal antibody to T-2, and 106 to 106 for the monoclonal antibody to AFB1.

Purification of the monoclonal antibodies from the ascites was carried out by the following procedure. The ascites was dialyzed against Tris-HCI buffer (pH 7.4), and applied to a DEAE-cellulose column equilibrated with the same buffer. A fraction which spontaneously passed through the column was salted out with 50% saturated ammonium sulfate.

The resulting precipitate was dissolved in phosphate buffer (pH 7.4) and dialyzed. The resulting monoclonal antibodies to OTA, T-2 and AFB1 were all found to have high purity by slab gel electrophoresis using SDS polyacrylamide.

Mycotoxins can be quantified rapidly with high sensitivity by a method (ELISA method) using mycotoxin assay reagents prepared by labelling the purified monoclonal antibodies to OTA, T-2 and AFB1 with enzymes.

The present invention will be further described below in detail.

(1) Preparation of a mycotoxin assay reagent Labelling of the antibody can be carried out by various methods such as a one-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 maleimide method [Journal of the Biochemistry, 78, 235 (1975)]. The latter two methods are preferred.

After conjugation with an enzyme, the antibody can be directly used for mycotoxin assay. To increase sensitivity further, an enzyme-labelled antibody obtained by purifying it by gel filtration on Sephadex, Sephacryl, etc.

is preferably used as the mycotoxin assay reagent. The enzyme-labelled antibody fraction is used as a mycotoxin assay reagent after it is dialyzed against phosphate buffer (pH 7.4) or Tris-HCI buffer (pH 7.4) and lyophilized, or filtered through a sterilized filter having a pore size of not more than 0.45 ,um.

(2) Quantification of a mycotoxin A complex of a mycotoxin and a high-molecular-weight protein different from BSA in the mycotoxin-BSA used as an immunogen for antimycotoxin antibody production [for example, keyhole limpet hemocyanin (KLH) and ovalbumin (OVA)] is used as an antigen (an antigen for fixation to a 96-well flat-bottomed plate for immunoassay) used for measurement of a mycotoxin produced by a microorganism of the genus Penicilium, Aspergillus or Fusarium.

In assaying the mycotoxin, the mycotoxinhigh-molecular-weight protein complex is placed on, and fixed to, the 96-well flat-bottomed plate for immunoassay. The wells treated with the antigen are washed and blocked to prevent the antibody to the mycotoxin from binding non-specifically to those wells to which the antigen is not bound.

Then, the blocked wells are washed, and equal volumes of an assay sample (or a mycotoxin of a known amount for preparation of a calibration curve) and the mycotoxin assay reagent described in (1) above are added. The plate is left to stand for a given period of time. The wells are then thoroughly washed, and a substrate solution corresponding to the enzyme used to label the antibody in the mycotoxin assay reagent (containing a substance which forms a color upon occurrence of an enzyme reaction) is added. After the enzyme reaction is allowed to proceed for a given period of time, the absorbance of the reaction solution is measured at a wavelength at which the color formed shows its maximum absorbance.A calibration curve is prepared on the basis of the results obtained by using a known amount of the mycotoxin, and the amount of the mycotoxin(e.g., OTA, T-2, AFB1) in the assay sample can be determined from the calibration curve.

The following examples illustrate the present invention more specifically. It should be understood that these examples are merely illustrative and do not limit the scope of the invention.

EXAMPLE 1 Prepration of mycotoxin assay reagent: Three purified monoclonal antibodies to OTA, T-2 and AFB1 described in Referential Example 2 were each labelled by the known enzyme labelling method shown below to prepare mycotoxin assay reagents.

Horse radish peroxidase (7.32 mg) was dissolved in 1 ml of distilled water, and 200 Ul of 0.1 M sodium periodate was added. The mixture was left to stand at room temperature for 30 minutes. The enzyme solution was dialyzed against 1mM acetate buffer (pH 4.5) overnight at 4"C. Then, 100 ,ul of 0.2M sodium carbonate buffer (pH 9.5) was added to adjust the pH of the dialyzate to 9.5. Separately, 8.78 mg of each monoclonal antibody (to OTA, T-2 or AFB,) 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 the monoclonal antibody solution were mixed and left to stand at room temperature for about 2.5 hours.To the reaction solution was added 100 to 200 l of 0.4% by weight sodium borohydride was added, and the mixture was left to stand at 4"C for 2 hours. The peroxidase-labelled monoclonal antibody so obtained was fully dialyzed against phosphate buffer (pH 7.4) at 4"C, and used as a mycotoxin assay reagent either as such or after lyophilization for mycotoxin assay.

EXAMPLE 2 Quantification of OTA produced by a microorganism of the genus Aspergillus: OTA-BSA (20 Ag/ml) was introduced into a 96-well flat-bottomed plate for immunoassay (made by Nunc) at a rate of 100 it1 per well, and left overnight at 40C to fix it. The plate was washed twice with a washing solution (phosphate buffer having a pH of 7.4 and containing 0.1% Tween 20). To prevent non-specific adsorption of the antibody to the plate, the plate was left to stand at room temperature for 30 minutes in the same washing solution as above but containing 10% bovine cerum. The plate was then washed twice with the washing solution.Then, 50 ,ul of the mycotoxin assay reagent diluted with the same washing solution as above (to 103-, 3X104-, 6x 104-, 105-, 10 - and 107-fold respectively) and 50 ,ul of a standard OTA solution (0 pg to 5 ,us/50 iLl) prepared by using the same washing solution as above were added simultaneously to each of the wells, and the plate was left to stand at room temperature for 30 minutes (in the case of preparing a calibration curve).The plate was washed four times with the washing solution, and a substrate solution (prepared by dissolving 20 mg of o-phenylenediamine and 10 ,ul of 35% H202 in 50 ml of 0.1 M citrate buffer having a pH of 5.0) was added at a rate of 100 ,ul per well. The plate was shut off from light with an aluminum foil, and left to stand at room temperature for 30 minutes. Finally, 2N sulfuric acid was added at a rate of 50 ,ul per well to stop the enzyme reaction. The absorbance at 500 nm of the reaction solution after stopping of the enzyme reaction was measured by using a microplate photometer. The results are shown in Fig. 1 of the accompanying drawings. The mycotoxin assay reagent diluted to 3 x 104-fold permitted quantification to a sensitivity of several pg.

An assay sample (50 ,ul of 0.1% BSA solution containing 50 pg, as calculated, of OTA) was assayed by using 50 iL1 of mycotoxin asasy reagent diluted to 3 x 104-fold. The measured amount of OTA was 52 pg which nearly agreed with the calculated amount.

The assay time was as short as about 1 hour.

EXAMPLE 3 Quantification of T-2 produced by a microorganism of the genus Fusarium: T-2-HS-BSA (20 jul/ml) was added to a 96well-flat-bottomed plate for immnoassay (made by Nunc) at a rate of 100 ,ul per well, and the plate was left to stand overnight at 4"C to fix the antigen. The plate was 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 to the plate, the plate was left to stand at room temperature for 30 minutes in the same washing solution as above but containing 10% bovine serum.The plate was then washed twice with the same washing solution as above, and 50 Al of the mycotoxin assay reagent diluted with the same washing solution as above (to 102-, 103-, 2x103-104-, 2x104- and 105-fold respectively) and 50 ,al of a standard T-2 solution [(0 pg to 5 clog)/50 jul] prepared by using the same washing solution as above were added at the same time to each of the wells.

The plate was then left to stand at room temperature for 30 minutes (in the case of preparing a calibration curve). The plate was further washed four times with the same washing solution as above, and a substrate solution (prepared by dissolving 20 mg of o-phenylenediamine and 10 it1 of 35% H202 in 50 ml of 0.1 M citrate buffer having a pH of 5.0) was added at a rate of 100 iLl per well. The plate was shut off from light with an aluminum foil, and left to stand at room temperature for 30 minutes. Finally, 2N sulfuric acid was added at a rate of 50 ,al per well to stop the enzyme reaction. The absorbance at 500 nm of the reaction solution after stoppage of the enzyme reaction was measured by using a microplate photometer. The results are shown in Fig. 2.

The mycotoxin assay reagent diluted to 2 x 1 04-fold permitted measurement to several pg with high sensitivity.

An assay sample (50 ,ul 0.1% BSA solution containing 50 pg, as calculated, of T-2) was assayed in accordance with the method described above in Example 3 using 50 ,ul of the mycotoxin assay reagent diluted to 2x 104- fold. The measured amount of T-2 was 47 pg which nearly agreed with the calculated value.

The measuring time was as short as about 1 hour.

EXAMPLE 4 Quantification of AFB, produced by a microorganism of the genus Aspergillus:- AFB1-BSA (0.5 ,ag/ml) was added to a 96well flat-bottomed plate for immunoassay (made by Nunc) at a rate of 100 iL1 per well, and the plate was left to stand overnight at 4"C to fix the antigen. The plate was washed twice with a washing solution (phosphate buffer, pH 7.4, containing 0.05% Tween 20), and to prevent non-specific adsorption of the antibody to the plate, the plate was left to stand at room temperature for 30 minutes in the same washing solution as above but containing 10% bovine serum.

The plate was then washed twice with the same washing solution as above, and 50 iL1 of the aflatoxin B1 assay reagent (diluted to 104fold) and 50 /11 of a standard AFB1 solution [(0 pg to 5 ng)/50 iL1] prepared by using an extracting solution for toxin analysis in fertilizers, feeds or foods (for example, 50% aqueous methanol solution) were added simultaneously to each of the well. The plate was left to stand at 37"C for 30 minutes or at room temperature for 2 hours (in the case of preparing a calibration curve).The plate was washed four times with the same washing solution as above, and a substrate solution (prepared by dissolving 20 mg of o-phenylenediamine and 10 ,ul of 35% H202 in 50 ml of 0.1M citrate buffer having a pH of 5.0) was added at a rate of 100 iL1 per well. The plate was shut off from light, and left to stand at 37"C for 15 minutes or at room temperature for 30 minutes. Finally, 2N sulfuric acid was added at a rate of 50 ,ul per well to stop the enzyme reaction. The absorbance of the reaction solution at 492 nm was measured by using a microplate photometer. The results are shown in Figs. 1 and 2. AFB1 could be quantified to 6.9 pg/assay or below with high sensitivity, and the assay time was as short as about 1 hour.

Fig. 5 shows calibration curves by plotting absorbances (expressed in percentage) obtained by competitive reactions (at room temperature for 2 hours, and at 37"C for 30 minutes) using the mycotoxin assay reagent diluted to 104-fold.

COMPARATIVE EXAMPLE 1 Quantification of OTA produced by a microorganism of the genus Aspergillus: OTA-BSA (20 ,ug/ml) was added to a 96well flat-bottomed microplate at a rate of 100 ,ul per well, and the plate was left to stand overnight at 4"C to fix the antigen. The plate was washed twice with a washing solution, and to prevent non-specific adsorption of the antibody to the plate, the plate was left to stand at room temperature for 30 minutes in a washing solution containing 10% bovine serum.The plate was then washed twice with a washing solution, and 50 ,ul of an anti-mycotoxin monoclonal antibody solution having high specificity and diluted to (102-, 103-, 104 104-, 105-, 5x105-, 106-, 5x106-, 107-, and 1 08-fold respectively) and 50 ,ul of a standard OTA solution [(0 pg to 5 ,us)/50 iL1] prepared by using the same washing solution were simultaneously added to each of the wells. The plate was left to stand at room temperature for 30 minutes (in the case of preparing a calibration curve).The plate was washed three times with the washing solution, and a peroxidase-labelled antibody to mouse immunoglobulin (diluted to 103-fold with 10% bovine serum-containing diluting solution) was added at a rate of 100 it1 per well. The plate was left to stand at room temperature for 2 hours, and washed four times with the washing solution. A substrate solution (prepared by dissolving 20 mg of o-phenylenediamine and 10 ,ul of 35% H202 in 50 ml of 0.1M citrate buffer) was added at a rate of 100 iL1 per well. The plate was shut off from light with an aluminum foil, and left to stand at room temperature for 30 minutes. Finally, 2N sulfuric acid was added at a rate of 50 iL1 per well to stop the enzyme reaction.The absorbance at 500 nm of the reaction solution after stoppage of the enzyme reaction was measured by using a microplate photometer. The results are shown in Fig. 6.

When the unlabelled anti-mycotoxin monoclonal antibody was used as above in immunoassay, OTA could be detected only to an amount of several ng by using the monoclonal antibody to OTA diluted to 106;fold and consuming about 6 hours for the assay.

COMPARATIVE EXAMPLE 2 Quantification of AFB1 produced by a microorganism of the genus Aspergillus: AFB1-BSA (0.5 > g/ml) was added to a 96well flat-bottomed microplate at a rate of 100 ,ul per well, and the plate was left to stand overnight at 4"C to fix the antigen. The plate was washed twice with a washing solution, and to prevent non-specific adsorption of the antibody to the plate, the plate was left to stand in a washing solution containing 10% bovine serum at room temperature for 30 minutes.The plate was then washed twice with a washing solution, and 50 Al of an anti AFB1 monoclonal antibody solution having high specificity and diluted to (103-, 104-, 106-, 5x105-, 106-, and 1 07-fold respectively) and 50 it1 of a standard AFB1 solution [(0 pg to 5 ,49)/50 iL1] prepared by using an extracting solution for toxin analysis in fertilizers, feeds or foods (for example, 50% aqueous methanol solution) were simultaneously added to each of the wells. The plate was left to stand at room temperature for 2 hours (in the case of preparing a calibration curve).The plate was washed four times with the washing solution, and a peroxidase-labelled antibody to mouse immunoglobulin (diluted to 103-fold with 10% bovine serum-containing diluting solution) was added at a rate of 100 ,41 per well. The plate was left to stand at room temperature for 2 hours, and washed four times with the washing solution. A substrate solution (prepared by dissolving 20 mg of o-phenylenediamine and 10 iL1 of 359/0 H202 in 50 ml of O.1M citrate buffer having a pH of 5.0) at a rate of 100.it1 per well. The plate was shut off from light, and left to stand at room temperature for 30 minutes.Finally, 2N sulfuric acid was added at a rate of 50 ,al per well to stop the enzyme reaction. The absorbance at 492 nm of the reaction solution after stoppage of the enzyme reaction was measured by using a microplate photometer. The results are shown in Fig. 7.

When the absorbances are expressed in percentage as in Example 4, the calibration curves were as shown in Fig. 8.

When the unlabelled anti-AFB1 monoclonal antibody was used as above in immunoassay, AFB1 could be detected only to an amount of several hundred pg/assay by using the monoclonal antibody to AFB1 diluted to 105-fold and consuming about 6 hours for the assay.

EXAMPLE 5 Addition and recovery of AFB1 to and from peanuts: By the method in Example 4, AFB1 was added and recovered to and from peanuts.

Peanuts produced in Japan and peanuts produced in China were used as samples. The peanuts were powderized by a homogenizer after the pods were removed. Particles which passed through a sieve having an opening size of 1 mm were collected. These powdery peanut samples were each impregnated with 25 Al of a standard methanol solution of AFB1 (11.1 ,ug/ml). To the impregnated sample was added 25 ml of a 50% aqueous methanol solution, and they were mixed for 3 minutes by a biomixer. The mixture was then filtered through Toyo Filter paper No. 1 or Whatman Filter Paper No. 4 or an equivalent filter paper.

The filtrate and a sample not containing AFB, were assayed in accordance with Example 4, and the results are shown in Fig. 9.

To the 50% methanol extract obtained was added AFB1 in a concentration of 11.1 ppb (556 pg/assay). The measured amounts of AFB1 in these samples were 573 pg/assay, and 625 pg/assay, from which the ratios of recovery were calculated as 92%, and 96% respectively.

As stated hereinabove, the present invention is characterized in that the mycotoxin is fixed to a support such as an immunoplate, and by adding an assay sample and the mycotoxin assay reagent at the same time, competitive reactions of the monoclonal antibody with mycotoxin fixed to the support and the mycotoxin in the sample are completed. Accordingly, the method of this invention permits quantification of the mycotoxin more easily with higher sensitivity in a shorter period of time than conventional assay methods. The method of this invention also has improved safety and economy.

Claims (7)

1. A reagent for use in the assay of a mycotoxin, which reagent comprises an enzyme-labelled monoclonal antibody to the mycotoxin.

2. A reagent according to claim 1 wherein the enzyme labelling the monoclonal antibody is selected from peroxidase, alkaline phospha tase, fi-galactosidase, catalase, glucose oxidase, lactic acid oxidase, alcohol oxidase and monoamine oxidase.

3. A reagent according to claim 1 or 2 wherein the mycotoxin is produced by a microorganism of the genus Penicillium, Aspergillus or Fusarium.

4. A reagent according to claim 1 or 2 wherein the mycotoxin is ochratoxin A, T-2 Toxin or aflatoxin B1.

5. A reagent according to claim 1 substantially as described with reference to Example 1.

6. A method of assaying a mycotoxin, which comprises assaying the mycotoxin under competitive reaction conditions using a reagent as claimed in any one of the preceding claims.

7. A method according to claim 6 substantially as described with reference to Example 2, 3 or 4.