Feb 21, 1985 - radioimmunoassay (RIA) was developed as an alternative for detecting zearalenone in clinical samples (26). The RIA employed zearalenone ...
Vol. 50, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1985, p. 332-336
0099-2240/85/080332-05$02.00/0 Copyright © 1985, American Society for Microbiology
Indirect Enzyme-Linked Immunosorbent Assay for the Mycotoxin Zearalenonet MING-TSUNG LIU,' BHANU P. RAM,' L. PATRICK HART,2 AND JAMES J. PESTKAl* Departments of Food Science and Human Nutrition' and Botany and Plant Pathology,2 Michigan State University, East Lansing, Michigan 48824-1224 Received 21 February 1985/Accepted 13 May 1985
A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for a-zearalenol, P-zearalenol, a-zearalanol, and P-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.
MATERIALS AND METHODS
Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-Presorcylic acid lactone] is a secondary metabolite produced by members of the genus Fusarium (18) after infection of corn and small grains (11, 17). Although zearalenone has a low order of toxicity, it can exert estrogenic effects on the mammalian reproductive system (16). Zearalenone-induced hyperestrogenism in female swine results in enlargement of the uterus and nipples, vulvar swelling, vaginal prolapse, and infertility, while exposure of male swine to the compound can result in testicular atrophy and enlarged mammary glands. Two in vivo metabolites of zearalenone, a- and ,B-zearalenol, are also estrogenic and have been detected in bovine milk (19). The extensive biological effects of zearalenone require that its presence be routinely monitored in human foods and animal feeds. Current analytical methods for zearalenone such as thin-layer chromatography (TLC) (8, 9, 23, 24), gas-liquid chromatography (23, 25), and high-pressure liquid chromatography (13, 23, 27) necessitate time-consuming extraction and sample clean-up and are thus unsuitable for the routine screening of large sample numbers. Recently, a radioimmunoassay (RIA) was developed as an alternative for detecting zearalenone in clinical samples (26). The RIA employed zearalenone antiserum produced in swine and had a detection limit of 0.25 ng per assay. We have investigated this approach further with the intent of devising a simple enzyme-linked immunosorbent assay (ELISA) for zearalenone in foods and feed. In this report we describe: (i) the production of zearalenone antibodies in the rabbit; (ii) the development of a competitive indirect ELISA for zearalenone; and (iii) preliminary application of this immunoassay to the detection of zearalenone in corn, wheat, pig rations, and milk.
Materials. All inorganic chemicals and organic solvents were reagent grade or better. Bovine serum albumin (BSA), Tween 20, 2,2-azino-di-3-ethylbenzthiazoline-6-sulfonate
(ABTS), hydrogen peroxide, horseradish peroxidase (type VI), N,N-dimethylformamide, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide, poly-L-lysine (molecular weight, 57,000), and chicken egg albumin (ovalbumin, grade III) were purchased from Sigma Chemical Co., St. Louis, Mo. Isobutyl chloroformate, tetrahydrofuran, triethylamine, and carboxymethoxylamnine hemihydrochloride were purchased from Aldrich Chemical Co., Inc., Milwaukee, Wis. Goat anti-rabbit immunoglobulin G peroxidase was purchased from Cappel Laboratories, West Chester, Pa. Zearalenone, cx-zearalenol, 3-zearalenol, a-zearalanol, and ,B-zearalanol were gifts from International Minerals & Chemical Corp., Terre Haute, Ind. Complete and incomplete Freund adjuvants were purchased from Difco Laboratories, Detroit, Mich. Rabbits (New Zealand White does) were obtained from the Bailey Rabbitry, Alto, Mich. Preparation of immunogen. Since zearalenone has no reactive group for coupling reactions, it was first converted to zearalenone-6'-carboxymethyloxime (Z-oxime) by the method of Thouvenot and Morfin (26). Z-oxime was then conjugated to BSA by a mixed anhydride procedure for use as an immunogen (15). Briefly, Z-oxime (15 mg) was dissolved in 5 ml of dry tetrahydrofuran and cooled to -5°C in an ice-salt bath. Trimethylamine (8 ,ul) and isobutyl chloroformate (10 pI) were added, and the solution was stirred for 20 min. This reaction mixture was slowly added to a solution of BSA (75 mg) dissolved in water (15 ml) and pyridine (7.5 ml) at 4°C. The mixture was stirred for 30 min at 4°C and then overnight at roomn temperature. The resulting BSA conjugate was dialyzed against 4 liters of distilled water (three changes) for 3 days. An approximate molar ratio of zearalenone to BSA of 20:1 was determined spectrophotometrically for the resulting conjugate (26).
* Corresponding author. t Article no. 11566 from the Michigan State Agricultural Experiment Station. 332
INDIRECT ELISA FOR ZEARALENONE
VOL. 50, 1985
Rabbit immunization. Rabbits were immunized by two procedures. In procedure A, a modification of the method of Dawson et al. (4), two rabbits (A-1 and A-2) were injected intramuscularly at weeks 0, 2, and 4 with Z-oxime BSA conjugate (250 ,ug) in 2 ml of saline-Freund complete adjuvant (1:1). Intravenous boosters of Z-oxime BSA (250 ,ug) dissolved in 1 ml of saline were given at weeks 6, 9, 11, 14, and 17. In procedure B, two rabbits (B-1 and B-2) were injected intradermally with Z-oxime BSA (2 mg) dissolved in 1 ml of saline and emulsified with 1 ml of Freund complete adjuvant at 30 to 40 sites on a shaved back area. A booster of conjugate (0.5 mg) in 2 ml of saline-Freund complete adjuvant (1:1) was injected intramuscularly at week 7, and boosters of conjugate (0.5 mg) in 2 ml of saline-Freund incomplete adjuvant (1:1) were injected intramuscularly at weeks 11 and 17. Blood samples were drawn via marginal ear vein at regular intervals, and the antiserum was purified by 35% ammonium sulfate precipitation (12). Preparation of Z-oxine poly-L-lysine conjugate. Z-oxime was conjugated to poly-L-lysine, for use as an ELISA solid phase, by an N-hydroxysuccinimide ester method (14). Z-oxime (10 mg) was dissolved in dimethylformamide (0.2 ml), and equimolar amounts of N,N'-dicyclohexylcarbodiimide (7.1 mg) and N-hydroxysuccinimide (4 mg) were added to the solution. This was mixed for 30 min at 25°C and then added slowly to poly-L-lysine (20 mg) dissolved in 0.5 ml of 0.13 M NaHCO3. The reaction mixture was stirred for 30 min at 25C and then dialyzed against 0.1 M NaHCO3 (4 liters) for 1 day and distilled water (4 liters) for 2 days at 4°C. The ratio of zearalenone conjugated to poly-L-lysine was determined spectrophotometrically to be 19:1. Antibody titration by indirect ELISA. One hundred microliters of Z-oxime poly-L-lysine (2 ,ug/ml in 0.1 M sodium bicarbonate buffer [pH 9.6]) was added to each well of a 96-well microtiter plate (Immulon Removawells; Dynatech Laboratories, Alexandria, Va.) and incubated overnight at 4°C or for 2 h at 37°C. Wells were washed three times with 0.25 ml of 0.1 M phosphate buffer in 0.15 M saline (PBS) (pH 7.5) containing 0.05% (vol/vol) Tween 20 (PBS-Tween). To block unbound solid-phase sites and minimize nonspecific binding, 200 ,ul of 1% (wt/vol) ovalbumin in PBS was added to each well and incubated for 30 min at 37°C. Wells were washed two more times in PBS-Tween and then incubated for 1 h at 37°C with 50 1,u of rabbit zearalenone antiserum diluted serially in 1% ovalbumin in PBS-Tween. Wells were washed five times and then reacted for 30 min at 37°C with 50 ,ul of goat anti-rabbit peroxidase conjugate, diluted 1:1,000 in 1% ovalbumin in PBS-Tween. Wells were washed six more times with PBS-Tween, and bound peroxidase was determined by the addition of ABTS substrate (20 min) as described by Pestka et al. (21). Competitive ELISA. The indirect competitive ELISA was identical to the indirect titration procedure except that 50-pd samples of zearalenone (or zearalenone analog) dissolved in 10% (vol/vol) methanol in PBS-Tween were simultaneously incubated for 1 h at 37°C with 50 pul of diluted zearalenone antiserum (1:50 to 1:200) in 1% ovalbumin in PBS-Tween. Bound antibody was then determined as described above. To ensure solubility, standard zearalenone and zearalenone analogs were kept in methanol (1 mg/ml) and then diluted to 10% methanol with PBS-Tween on the day of assay. ELISA of spiked samples. Whole corn and wheat kernels and pig feed (containing ground shell corn and soybean meal) were initially extracted by blending samples (50 g) for 5 min in 200 ml of methanol-water (60:40) (8). The mixture was filtered through Whatman no. 4 filter paper, and the filtrate
333
0
1.8-
6
1.2w
z
0)8 co
m
0.41%
0
.1'01
1b2
1os
'04
SERUM DILUTION
FIG. 1. ELISA titration of rabbit anti-zearalenone antibody. Symbols: 0, rabbit B-1 (18 weeks) antiserum; 0, preimmune control serum.
was diluted in PBS-Tween to give a final methanol concentration of 10% (vol/vol). Diluted filtrates, unspiked and spiked (0.5 to 50 ng/ml) with zearalenone, were subjected to indirect competitive ELISA. For pasteurized whole milk, the sample was diluted 1:1 with 20% (vol/vol) methanol in PBS-Tween. Unspiked and spiked (0.5 to 50 ng of zearalenone per ml) samples of diluted milk were also subjected to indirect competitive ELISA. For semiquantitative TLC detection of zearalenone in corn, wheat, and feed, methanol was evaporated from methanol-water (60:40) extracts, and the samples (50 g equivalent) were then extracted three times with equal volumes of ethyl acetate. Ethyl acetate fractions were dried over sodium sulfate and evaporated to dryness. Extracts were redissolved in 100 pul of ethyl acetate, and 1 to 5 p.l was spotted onto silica gel plates. Plates were developed in toluene-ethyl acetate (1:3). Zearalenone content in sample extracts was estimated visually by comparison with standard under short-wave UV light.
RESULTS Production of antibody against zearalenone. Two different immunization procedures were used to elicit zearalenone antibody responses in rabbits. An indirect ELISA was devised to monitor titers of zearalenone antiserum whereby rabbit antiserum was incubated over a microtiter plate solid phase coated with Z-oxime poly-L-lysine and total bound antibodies were subsequently detected with goat anti-rabbit peroxidase conjugate. The serum dilution visually distinct in color from preimmune serum control at the same dilution was arbitrarily designated as the titer. Figure 1 shows the titration curve for rabbit B-1 (18 weeks) serum. Preimmune serum controls showed negligible absorbance, whereas this antiserum had a titer of 1,280.
APPL. ENVIRON. MICROBIOL.
LIU ET AL.
334
TABLE 1. Zearalenone antibody titers in rabbits immunized by procedures A and B' ELISA titer Wk after immunization A-2 B-2 B-1 A-1 7 9 11 14 18 23
1,280
1,280
5,120 5,120 1,280
1,280 20,480 5,120 5,120 5,120
5,120 5,120
1,280 5,120 5,120 1,280 1,280 5,120
1,280 5,120 20,480 20,480 5,120 5,120
a Procedure A consisted of three intramuscular injections of Z-oxime BSA at 2-week intervals followed by intravenous boosters. Procedure B consisted of initial multiple intradermal injections of Z-oxime BSA followed by two intramuscular boosts.
A summary of titers from the four rabbits over 23 weeks is shown in Table 1. Titers of 1,280 were obtained in all rabbits at week 7. The highest titers, 20,480, were detected in rabbits A-2 and B-2 at week 11. Repeated boosters after week 11 did not significantly increase zearalenone titers. In general, both procedures A and B appeared equally suitable for eliciting zearalenone antibody in rabbits. Competitive indirect ELISA. A competitive indirect ELISA was carried out by simultaneously incubating zearalenone with an appropriate dilution of rabbit zearalenone antiserum over a Z-oxime poly-L-lysine solid phase and then determining bound rabbit antibody with a goat anti-rabbit peroxidase conjugate. A typical competition curve is shown in Fig. 2. The response range for this curve was between 0.5 and 50 ng/ml. A level of 5 ng/ml could be readily determined without the aid of a spectrophotometer. Two essential components in our indirect ELISA were the use of 1% (wt/vol) ovalbumin as a blocking protein and the preparation of Z-oxime poly-L-lysine solid phase by the N-hydroxysuccinimide procedure. Reproducible competition curves were not obtained when 1% (wt/vol) BSA or 0.02% (wt/vol) gelatin were used as blocking agents, or when the Z-oxime poly-L-lysine solid phase was prepared by the mixed anhydride method. Specificity of zearalenone antibody. To ascertain the specificity of the rabbit zearalenone antiserum, the ability of zearalenone analogs (26) to compete in the indirect ELISA
was evaluated. Concentrations which resulted in 50% inhibition of zearalenone antibody binding to the solid phase were 3, 6, 25, 55, and 120 ng/ml for zearalenone, azearalenol, 13-zearalenol, a-zearalanol, and ,3-zearalanol, respectively. ELISA detection of zearalenone in spiked samples. We tested the feasibility of using the indirect competitive ELISA for screening of zearalenone in foods and feeds by assaying for zearalenone in unspiked diluted methanol-water extracts (1:24) of corn, wheat, and pig rations and in diluted whole milk (1:1). Maximum absorbances (0 ng of toxin per ml; n = 3) for buffer and unspiked feed, wheat, corn, and milk were 0.51 ± 0.02, 0.34 ± 0.01, 0.47 ± 0.06, 0.43 ± 0.03, and 0.46 ± 0.05, respectively, suggesting that low levels of zearalenone might have been present in some of the samples. Extrapolation of sample extract absorbances on the ELISA buffer standard curve yielded zearalenone estimates for unspiked feed, wheat, corn, and milk of 61 ± 25, 16 ± 12, and 37 ± 21 ,ug/kg, and 1 ± 0.5 ng/ml, respectively. Semiquantitative TLC analysis (without cleanup) indicated that the approximate levels of zearalenone in unspiked feed, wheat, and corn were 200, 40, and 100 ,ug/kg, respectively. No attempts were made to confirm the presence of zearalenone or its metabolites in the milk sample by TLC. Zearalenone antibody competes with spiked zearalenone in the presence of these diluted methanol-water extracts of corn, wheat, and pig feed or in diluted milk (Fig. 3). Differences existing among the slopes of the absorbance responses for these commodities could have been due to interfering substances in the respective extracts or to the presence of zearalenone in the samples before spiking, as was confirmed by TLC. Verification of potential extract interference in the ELISA curve will require further recovery testing with zearalenone-free and naturally contaminated samples. Should sample maxtrix problems exist for certain commodities, it might be necessary to include clean sample extracts in comparative standard curves. Nevertheless, our preliminary results indicate that it should be possible to screen for zearalenone in corn, wheat, and pig feed by ELISA after only methanol-water extraction. --
0.5
-
buffer
pig feed wheat
0.4-
*....
corn milk
w
C:) 0
w
80-
z
60-
cc 0
co
(I)
0.30.2.
x :
z C.)
40-
w
0-
a:
20-
0O
ZEARALENONE (ng/ml)
100
1o2
101 ZEARALENONE
103
(ng/ml)
FIG. 2. Competitive indirect ELISA standard curve to zearalen(one. Each data point represents triplicate determinations in a single microtiter plate. In this standard curve, absorbance for assay at 1 ng of zearalenone per ml was significantly different (95% confident ce) than absorbance obtained in the absence of toxin.
FIG. 3. Effect of sample in competitive ELISA standard curve. Triplicate assays were conducted on sampks spiked with zearalenone at 0.5, 1.0, 2.5, 5.0, 10 and 50 ng/ml as indicated in the abscissa. Power function best-fit linear regressions were constructed with Omicron Plotrax 2 software (Engineering-Science, Inc., Atlanta, Ga.). Correlation coefficients for buffer, feed, wheat, corn, and milk regressions were 0.987, 0.926, 0.905, 0.955, and 0.946, respectively.
VOL. 50, 1985
INDIRECT ELISA FOR ZEARALENONE
DISCUSSION ELISAs are alternatives to conventional chemical methods for screening mycotoxins in agricultural commodities. Procedures have been described for the production of antibodies to aflatoxin (3), ochratoxin (1), and T-2 toxin (2) in rabbits and for the use of these antibodies in ELISAs for detecting these toxins in foods, feeds, and biological fluids (5-8, 20-22). This is the first report of production of zearalenone antibody in rabbits. Although Thouvenot and Morfin (26) described the production of zearalenone antibodies in swine, they were unable to obtain a similar response in rabbits. Inability to produce these antibodies in rabbits was attributed to the observation that rabbit serum albumin binds to zearalenone with a high capacity and a low affinity. These reported difficulties led us initially to compare two immunization procedures. Procedure A consisted of three intramuscular injections given at 2-week intervals with subsequent intravenous boosters, and procedure B consisted of initial multiple intradermal injections followed by two intramuscular boosts. Although procedure B is the approach typically used for mycotoxins and other haptens (1-3), Dawson et al. (4) have reported that procedure A is a simpler and more efficient method for eliciting hapten antibodies. Our results suggest little difference existed between these two immunization procedures in maximal titers obtained or duration of antibody response. Our ability to produce zearalenone antibody in rabbits while other attempts have been unsuccessful might be attributable to several factors. First, we utilized a mixed anhydride conjugation procedure which resulted in a 20:1 molar ratio of zearalenone to BSA as compared with the carbodiimide procedure of Thouvenot and Morfin which yielded a 7:1 molar ratio of zearalenone to BSA. Secondly, we used an ammonium sulfate precipitation purification step in our rabbit serum which should have greatly decreased rabbit serum albumin present in the ELISA. Lastly, the indirect ELISA titration procedure may have greater sensitivity than titration by RIA. The latter possibility is further supported by the observation of Fan et al. (7) that indirect ELISA titers for T-2 antisera were 30 times higher than RIA titers of the same sera. Both direct and indirect competitive ELISAs have been used in mycotoxin detection. In the direct assay, a toxinperoxidase conjugate is simultaneously incubated with free toxin over a solid-phase toxin antibody to develop a competition curve (5, 8, 20). In the indirect assay, toxin antibody competes with free toxin for binding to a solid-phase toxinprotein conjugate (6, 7). Although we have successfully made Z-oxime peroxidase conjugates, we have been unsuccessful in developing a direct ELISA for zearalenone (unpublished data). However, the indirect approach described here had a detection limit for zearalenone at the 1-ng/ml level which was equivalent to 0.05 ng per assay. This compares favorably with the minimum detection level of 0.25 ng per assay obtained by RIA (26). Based on the concentrations required to decrease maximal absorbance in the competitive ELISA by 50%, the reactivities of our rabbit antiserum for zearalenone, a-zearalenol, 1-zearalenol, a-zearalanol, and 1-zearalanol were 100, 50, 12, 6, and 3% respectively. This suggests that the antibody preferentially recognizes the double bond at-the Cl' to C2' position. Furthermore, the a-configuration is recognized over the ,3-configuration at C6' of zearalenol. The ability of this antiserum to cross-react with the zearalenols is useful since these compounds have been shown to occur naturally (10) and are in vivo metabolites of zearalenone (19).
335
Thouvenot and Morfin (26) determined by RIA that the relative reactivities of porcine zearalenone antibody for zearalenone, a-zearalenol, ,B-zearalenol, a-zearalanol, and ,B-zearalanol were 100, 100, 44, 53, and 44%, respectively. Thus, while the rabbit and pig antisera followed a similar rank order of cross-reactivity for these analogs, the rabbit antibody exhibited greater apparent specificity for zearalenone. Besides species variation, these differences might be attributable to immunogen conjugation methods (mixed anhydride versus carbodiimide) or immunoassay (ELISA versus RIA) procedures used in these two studies. In conclusion, we have described procedures for the production of zearalenone antibody in rabbits and for the use of this antibody in the competitive indirect ELISA for zearalenone. Indirect competitive ELISA would have significant advantages over existing methods which require extensive cleanup of the initial extract (9, 13, 23-25, 27). Gendloff et al. (8) have shown that ELISA estimates of T-2 toxin in methanol extracts of corn correlate with gas-liquid chromatography. In preliminary experiments, we have observed that the ELISA can be used to semiquantitatively screen for zearalenone in methanol extracts of corn, wheat, and pig feed and that these results correlated with TLC estimates. We are currently conducting a collaborative study to verify the applicability of indirect ELISA for quantitating zearalenone in naturally contaminated grain samples. Although our preliminary study indicated that our unspiked milk sample might contain trace levels of zearalenone (1 ng/ml), suitable extraction and analytical methods for confirming this observation are not currently available. The feasibility of using indirect ELISA to detect zearalenone in milk merits further investigation since the method might be applicable to the investigation of carryover of zearalenone from contaminated feed to milk and in market screening of the toxin in dairy products. ACKNOWLEDGMENTS This work was supported by U.S. Department of Agriculture Animal Health grant 83-CRSR-2-2257, the Michigan State University Center for Environmental Toxicology, and the Michigan Food Industry. We thank Janeen Hunt for her able assistance in manuscript preparation.
LITERATURE CITED 1. Chu, F. S., F. C. C. Chang, and R. D. HinsdiH. 1976. Production 2. 3. 4. 5.
6. 7. 8.
of antibody against ochratoxin A. Appl. Environ. Microbiol. 31:831-835. 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. Chu, F. S., and I. Ueno. 1977. Production of antibody against aflatoxin Bl. Appl. Environ. Microbiol. 33:1125-1128. Dawson, E. E., E. H. C. Denissen, and B. K. Weemen. 1978. A simple and efficient method for raising steroid antibodies in rabbits. Steroids 31:357-365. El-Nakib, O., J. J. Pestka, and F. S. Chu. 1981. Analysis of aflatoxin B1 in corn, wheat, and peanut butter by an enzymelinked immunosorbent microassay and a solid-phase radioimmunoassay. J. Assoc. Off. Anal. Chem. 64:1077-1082. Fan, T. S. L., and F. S. Chu. 1984. Indirect enzyme-linked immunosorbent assay for detection of aflatoxin B1 in corn and peanut butter. J. Food Prot. 47:263-266. Fan, T. S. L., G. S. Zhang, and F. S. Chu. 1984. An indirect enzyme-linked immunosorbent assay for T-2 toxin in biological fluids. J. Food Prot. 47:964-967. Gendloff, E. H., J. J. Pestka, S. P. Swanson, and L. P. Hart. 1984. Detection of T-2 toxin in Fusarium sporotrichioides-
336
9.
10.
11. 12.
13. 14. 15.
16.
17.
18.
APPL. ENVIRON. MICROBIOL.
LIU ET AL.
infected corn by enzyme-linked immunosorbent assay. Appl. Environ. Microbiol. 47:1161-1163. Gimeno, A. 1983. Rapid thin layer chromatographic determination of zearalenone in corn, sorghum, and wheat. J. Assoc. Off. Anal. Chem. 66:565-569. Hagler, W. M., C. J. Mirocha, S. V. Pathre, and J. C. Behrens. 1979. Identification of the naturally occurring isomer of zearalenol produced by Fusarium roseum "Gibbosum" in rice culture. Appl. Environ. Microbiol. 37:849-853. Hart, L. P., W. E. Braselton, and T. C. Stebbins. 1982. Production of zearalenone and deoxynivalenol in commercial sweet corn. Plant Dis. 66:1133-1135. Hebert, G. A., P. L. Pelham, and B. Pittman. 1973. Determination of the optimal ammonium sulfate concentration for the fractionation of rabbit, sheep, horse, and goat antisera. Appl. Microbiol. 25:26-36. James, L. J., L. G. Mcgirr, and T. K. Smith. 1982. High pressure liquid chromatography of zearalenone and zearalenols in rat urine and liver. J. Assoc. Off. Anal. Chem. 65:8-13. Kohen, F., J. B. Kim, G. Barnard, and H. R. Lindner. 1980. An assay for urinary estriol-16-glucuronide based on antibodyenhanced chemiluminescence. Steroids 36:405-419. Lau, H. P., P. K. Gaur, and F. S. Chu. 1981. Preparation and characterization of aflatoxin B,-hemiglutarate and its use for the production of antibody against aflatoxin B,. J. Food Safety 3:1-13. Mirocha, C. J., and C. M. Christensen. 1974. Oestrogenic mycotoxins synthesized by Fusarium, p. 129-148. In I. F. H. Purchase (ed.), Mycotoxins. Elsevier Scientific Publishing Co., Amsterdam. Mirocha, C. J., C. M. Christensen, and G. H. Nelson. 1971. F-2 (zearalenone) estrogenic mycotoxin from Fusarium, p. 107-138. In S. Kadis, A. Ciegler, and S. J. Ayl (ed.), Microbial toxins, vol. 7. Academic Press, Inc., New York. Mirocha, C. J., S. V. Pathre, and C. M. Christensen. 1977. Zearalenone, p. 345-364. In J. V. Rodricks, C. W. Hesseltine,
19.
20.
21. 22.
23.
24.
25.
26.
27.
and M. A. Mehlman (ed.), Mycotoxins in human and animal health. Pathotox Publishers, Inc., Park Forest South, Ill. Mirocha, C. J., S. V. Pathre, and T. S. Robison. 1981. Comparative metabolism of zearalenone and transmission into bovine milk. Food Cosmet. Toxicol. 19:25-30. Pestka, J. J., P. K. Gaur, and F. S. Chu. 1980. Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay. Appl. Environ. Microbiol. 40:1027-1031. Pestka, J. J., Y. K. Li, and F. S. Chu. 1982. Reactivity of aflatoxin B2X, antibody with aflatoxin Bl-modified DNA and related metabolites. Appl. Environ. Microbiol. 44:1159-1165. Pestka, J. J., Y. Li, W. 0. Harder, and F. S. Chu. 1981. Comparison of radioimmunoassay and enzyme-linked immunosorbent assay for determining aflatoxin M, in milk. J. Assoc. Off. Anal. Chem. 64:294-301. Scott, P. M., T. Panalaks, S. Kanhere, and W. F. Miles. 1978. Determination of zearalenone in cornflakes and other cornbased foods by thin layer chromatography, high pressure liquid chromatography, and gas-liquid chromatography. J. Assoc. Off. Anal. Chem. 61:593-600. Swanson, S. P., R. A. Curley, D. G. White, and W. B. Buck. 1984. Rapid thin layer chromatographic method for determination of zearalenone and zearalenol in grains and animal feeds. J. Assoc. Off. Anal. Chem. 67:580-582. Thouvenot, D. R., and R. F. Morfin. 1979. Quantitation of zearalenone by gas-liquid chromatography on capillary glass columns. J. Chromatogr. 170:165-173. Thouvenot, D. R., and R. F. Morfin. 1983. Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies. Appl. Environ. Microbiol. 45:16-23. Turner, G. V., T. D. Phillips, N. D. Heidelbaugh, and L. M. Russell. 1983. High pressure liquid chromatographic determination of zearalenone in chicken tissues. J. Assoc. Off. Anal. Chem. 66:102-104.
