APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, Feb. 1994,
p.
729-731
Vol. 60, No. 2
0099-2240/94/$04.00+0 Copyright C) 1994, American Society for Microbiology
Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay of Fusarium T-2 and Zearalenone Toxins in Cereals ILDIKO BARNA-VETRO, AGNES GYONGYOSI, AND LASZL6 SOLTI* Agricultural Biotechnology Center, Institute for Animal Sciences, H-2101 Godoll6, Hungary Received 23 February 1993/Accepted 31 October 1993
Direct, competitive enzyme-linked immunosorbent assays (ELISAs) with monoclonal antibodies have been developed for quantitative determination of trichothecene T-2 toxin (T-2), and zearalenone (F-2) from different cereals. Among the several extraction solvents tried, 89%v acetonitrile with additives was chosen. The extracts were then used without cleanup in the ELISA. With appropriate dilution of the samples (1:25 or 1:50), the matrix effects caused by lipid and/or protein content of the samples can be diminished to the extent that the assay is no longer impaired. The mean recoveries from cereals infected with 100 to 2,000 ng of T-2 and 50 to 500 ng of F-2 per g were 85 and 91%, respectively. The measuring range of the T-2 test is 100 to 2,000 ng/g, and that of the F-2 test is 25 to 400 ng/g. The mean within-assay and interassay coelficients of variation of standard curves are both less than 10%. According to recovery results with artificially infected cereals, our tests proved to be suitable for rapid screening of food and feed samples for the presence of T-2 and F-2 toxins.
The climatic situation in Europe, and particularly in Hungary, is favorable for occurrence of toxin-producing Fusarium fungi and their metabolites. T-2 toxin and zearalenone (also referred to as F-2 toxin) are frequently contaminants of cereal grains and other corn-based food and feedstuff (7). These compounds can cause serious health problems in humans and other animals in concentrations as low as micrograms or nanograms of toxin per gram of food or feed; therefore, it is necessary to use sensitive methods for their detection. Chemical methods for measuring mycotoxins in cereals, e.g., thinlayer chromatography, liquid chromatography, or gas chromatography-mass spectroscopy, are time-consuming and quite expensive and require complicated sample cleanup. The recent development of enzyme-linked immunosorbent assays (ELISAs) has proved that immunoassays based on antigenantibody reaction are fast, specific, sensitive, and inexpensive methods for mycotoxin analysis (1, 4). Our laboratory developed monoclonal antibody (MAb)-based, single-extraction, direct competitive ELISAs for the determination of T-2 and F-2 toxins in different cereals. We report the setup and validation of these tests, needed before the tests can be used routinely. Toxin labels. T-2 (Sigma Chemical Co.) was first converted to T-2-hemisuccinate (2) and conjugated by an active ester method to horseradish peroxidase (Reanal) as described by Esgin et al. (3). Zearalenone (Sigma) was first modified to F-2-oxim (9) and then coupled to horseradish peroxidase (3). The working dilutions of the conjugates were determined in a direct ELISA. Antibodies. Hybridoma cell lines secreting monoclonal antiT-2 and anti-F-2 were produced as published elsewhere (5). The MAbs were used in our ELISAs as ascitic fluids without purification. The specificity of the antibodies towards T-2 and F-2 metabolites was tested in a direct ELISA. The crossreactions with acetyl-T-2, HT-2, and iso-T-2 were 12.8, 3.4, and 2.5%, respectively. The specificities of F-2 antibody were 100,
138, 69, 6, 91, and 21% for zearalenone, zearalanone, a-zearalanol, ,B-zearalanol, ot-zearalenol, and P-zeralenol, respectively. Direct competitive ELISA. (i) Coating. Microplate wells (Dynatech M 129) were sensitized with 100 ,ul of MAb T-2 (1:500) or MAb F-2 (1:15,000) ascitic fluid and incubated at room temperature for 18 h. Afterwards, the plates were washed three times with distilled water-Tween 20 (0.05%). (ii) Immune reaction. Fifty-microliter toxin standards or extracted samples were coincubated in wells with 50 ,ul of peroxidase conjugate for 1 h at room temperature. T-2hemisuccinate-horseradish peroxidase label was routinely
B/Bo %
0L
0.1
Corresponding author. Mailing address: Agricultural Biotechnology Center, Institute for Animal Sciences, Szent-Gyorgyl Albert u. 4., P.O. Box 411, H-2101 Godoll6, Hungary. Phone: 36 28 330-617. Fax: 36 28 330-647. Electronic mail address:
[email protected]. *
1
I I11111
1
10
Toxin concentration (ng/ml) FIG. 1. Dose-response curves of T-2 and F-2 toxins. 729
730
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Effect of different extraction solvents on recovery of T-2 toxin from artificially infected maize
(ng/g)
(ng/g)
% Recovered
300 300 300
607 253 255
200 84 85
Amt added Amt recovered
Solvent
Ethanol-water (80:20) Acetonitrile-water (80:20) Acetonitrile-KCl-H2SO4 (89:10:1)
2.6
OD 450 nm Ethanol Aoetonitrile
used in 1:10,000 dilution, while F-2-oxim-horseradish peroxidase was used in a dilution of 1:30,000 with phosphate-buffered saline (PBS)-Tween. (iii) Enzyme reaction. After four washing steps, the wells were incubated with 150 ,ul of tetramethylbenzidine-H202 substrate per well for 15 min. The color reaction was terminated with 50 ,ul of 6 N sulfuric acid, and the A4st) was measured. (iv) Evaluation. Standard curves of T-2 and F-2 were obtained by plotting log10) concentration (x axis) against B/Bo (y axis): BIBo = (OD of standard or sample)/(OD of blank [no toxin added]), where optical density (OD) is the mean A450. The concentrations of T-2 and F-2 in sample extracts were assessed by using calibration curves and are expressed in nanograms per gram by multiplying the nanogram-per-milliliter value by 100 (at 1:25 sample dilution) or 200 (at 1:50 sample dilution). The slope of the ELISA standard curve is the color change per concentration unit. Recovery of T-2 and F-2 toxins from artificially infected cereals. To 5 g of finely ground cereals (wheat, maize, or barley), 100 to 2,000 ng of pure T-2 or 25 to 500 ng of F-2 per g was added 1 day prior to extraction. Thereafter, the samples were shaken for 1 h with 20 ml of extraction solvent (composed of 89 parts acetonitrile, 10 parts 0.5% KCl, and 1 part 1% H2S04). The extracts were then left to sediment, and 100-,ul aliquots were diluted 1:25 or 1:50 with PBS-Tween and used directly in the assay. The first step in developing a direct competitive ELISA is to determine the optimal antibody and mycotoxin-peroxidase dilution. In our experiments, the MAbs were used as ascitic fluid without purification, because they did not cause any nonspecific reaction in the tests. Figure 1 shows the doseresponse curves of T-2 and F-2. Detection limits in buffer solutions (0 ± 2 standard deviations) were 0.43 and 0.2 ng/ml for T-2 and F-2, respectively. The most accurate measurements were obtained in the middle of the range. The 50% values of B/B() were 2.3 and 0.6 ng/ml for T-2 and F-2, respectively. Sensitivity is defined as the slope of the curve at the inflection points (i.e., middle of the test) (6). These values, estimated with the Statgraph program, proved to be 0.98 for T-2 and 1.02 for F-2 tests, near the optimal. The correlation coefficient (r) of
0.5e
0
.
5
......-.........
10 16 20 26 30 35 40 45 50 % of solvent
55
60 65 70 75 80
FIG. 2. Nonspecific inhibitory effects of ethanol and acetonitrile in increasing concentrations. OD, optical density.
the linear part of the calibration curves was 0.98 in both tests. The within-assay and interassay coefficients of variation of the standard points of T-2 (0.5 to 25 ng/ml) and F-2 (0.25 to 2.0 ng/ml) were each less than 10%. A very important step of validation is the detection of matrix effects, which in most cases should be avoided by cleaning the samples (6). To extract mycotoxins from different cereals, methanol- or acetonitrile-based organic solvents are generally used in various concentrations, combined with different cleanup steps (8). The aim of our work was to eliminate sample cleanup before the assay and to simplify the procedure. In the present study, ethanol was used instead of methanol. Our experience with ethanol- or acetonitrile-based extraction solvents was very similar to those of others (8). Cereal extracts containing ethanol-water (80:20) showed significantly more interference in ELISA as confirmed by a recovery rate of the added toxin that was too high (200%) (Table 1). The supernatants were turbid because of unsoluble lipid components. The acetonitrile-based solvent, however, was acceptable; the extracts remained clear, and by appropriate dilution of the samples with PBS-Tween, the interference could be minimized. This result was due to the use of highly sensitive MAbs. The nonspecific inhibition caused by solvents containing different acetonitrile and ethanol concentrations was also checked in direct competitive ELISA. In the range of 10 to 100%, both solvents inhibited the binding of toxin-peroxidase
TABLE 2. Recovery of T-2 mycotoxin from artificially contaminated cerealsa Maize
Barley
Wheat T-2 added
(ngag)
Amt detected
Amt recovered
Amt detected
Amt recovered
Amt detected
Amt recovered
(ng/g)
(%)
(ng/g)
(%)
(%)
100 300 500 1,000
83 235 391 830 1,347
83 78 78 83 67
81 263 453 891 1,464
81 87 90 89 73
(ng/g) 93 276 446 902 1,382
2,000
a Each sample was infected separately in three parallel experiments and then extracted and assayed in three replicates.
93 92 89 90 69
731
NOTES
VOL. 60, 1994 TABLE 3. Recovery of F-2 mycotoxin from artificially contaminated cereals"
Maize
Barley
Wheat F-2 added
(ng/g)
Amt detected
Amt recovered
Amt detected
Amt recovered
Amt detected
Amt recovered
(ng/g)
(%)
(ng/g)
(%)
(ng/g)
(%)
50 100 250 500
43 84 212 455
85 84 85 91
52 98 203 401
105 98 81 82
56 87 235 457
111 87 94 91
"Each sample was infected in three parallel experiments and then extracted and assayed in three replicates.
conjugate to the solid-phase antibody (Fig. 2), but this effect could be avoided by decreasing the acetonitrile or ethanol concentration to less than 10%. This observation was not confirmed by others (8), who obtained insensitive and flat calibration curves with decreasing acetonitrile concentration. Recoveries of T-2 and F-2 toxins from artificially infected cereals are summarized in Tables 2 and 3. The average recoveries were 82% for T-2 and 91 % for F-2. The measuring ranges of our tests are 100 to 2,000 ng/g for T-2 and 25 to 400 ng/g for F-2. Finally, it should be emphasized that the deterioration and ending of the test may be due to the limited stability of the antibody and that of the enzyme tracer. In our tests the antibodies are precoated on the surface of polystyrene plates, and these were stable for more than 6 months. The enzymelabeled toxins prediluted with ammonium sulfate solution were stable for 6 months as well. Our results indicate that these immunoassays can be applied to quantitative measurement of mycotoxins from single extracts of cereals. Because sample cleanup is not necessary, these tests can be completed in 3 h. This study was partly supported by a grant of the Hungarian Committee of Technical Development (OMFB 91-97-07-0140). We are indebted to Anna Wolfling and Erzs6bet Szab6 for their excellent technical skills.
REFERENCES 1. Chu, F. S. 1990. Immunoassays for mycotoxins: current state of the art. Commercial and epidemiological applications. Vet. Hum. Toxicol. 32:42-50. 2. Chu, F. S., S. Grossman, R.-D. Wei, and C. J. Mirocha. 1979. Production of antibody against T-2 toxin. Appl. Environ. Microbiol. 37:104-108. 3. Esgin, V. S., E. Martlbauer, and G. Terplan. 1989. Entwicklung und Anwendung eines enzymimmunologischen Verfahrens zum Nachweis von T-2 Toxin in Milch. Arch. Lebensmittelhyg. 40:97120. 4. Gazzaz, S. S., B. A. Rasco, and F. M. Dong. 1992. Application of immunochemical assays to food analysis. Crit. Rev. Food Sci. Nutr. 32:197-229. 5. Gyongyosi, H. A., I. Barna-Vetr6, and L. Solti. 1992. Monoclonal antibody for determination of fusarium T-2 toxin by ELISA. Anim. Breed. Feed. 41:329-336. 6. Hock, B., T. Giersch, and K. Kramer. 1992. Immunoassays for environmental analysis. Anal. Magazine 20:29-33. 7. Kegl, T., and A. Vinyi. 1991. T-2 fusariotoxicosis in a cattle stock. Hung. Vet. J. 46:467-491. 8. Ramakrishna, N., J. Lacey, A. A. G. Candlish, J. E. Smith, and I. A. Goodbrand. 1990. Monoclonal antibody-based enzyme linked immunosorbent assay of aflatoxin B,, T-2 toxin, and ochratoxin A in barley. J. Assoc. Off. Anal. Chem. 73:71-76. 9. Thouvenot, D., and R. F. Morfin. 1983. Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies. Appl. Environ. Microbiol.
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