comparison of sterigmatocystin production by Aspergillus versicolor and ... cystin/ml culture and 5 #g extracellular sterigmatocystin/ml broth, respectively.
Mycopathologia 107: 93-100, 1989. 9 1989KluwerAcademic Publishers. Printedin Belgium.
Immunochemical assay applied to mycotoxin biosynthesis: ELISA comparison of sterigmatocystin production by Aspergillus versicolor and Aspergillus nidulans Duck-Hwa Chung,* Mohammed M. Abouzied & James J. Pestka Department of Food Science and Human Nutrition, Michigan State University, East Lansing, M I 48824, USA; * Visiting Professor from the Dept. of Food Science and Technology Gyeongsang National University, Gyeongham, Korea Accepted 12 December 1988
Conventional thin layer and instrumental methods for analyzing mycotoxins and their precursors are time-consuming and make the investigation of mycotoxin biosynthesis particularly difficult. As an alternative, sensitive enzyme-liked immunosorbent assays (ELISAs) can be utilized to analyze for these compounds. In this report, sterigmatocystin production in test tube cultures of Aspergillus versicolor ATCC 18643 and Aspergillus nidulans ATCC 32610 were compared using competitive ELISA. Polyclonal antiserum that was prepared against a sterigmatocystin hemiacetal-bovine serum albumin conjugate exhibited greatest specificity for sterigmatocystin hemiacetal and sterigmatocystin with less reactivity for O-methylsterigmatocystin. The antiserum could be used to detect as little as 50 ng/ml sterigmatocystin in ELISA. Direct ELISA could be performed on diluted culture broth and on mycelial extracts solubilized with N,N-dimethylformamide.Aspergillus versicolor ATCC 18643 produced more sterigmatocystin in SLS medium than in YES medium, and showed maximal levels at between 9 to 12 days incubation. Approximately 75 ~o of sterigmatocystin was detectable in mycelium (254 #g/ml culture) compared to the extracellular fraction (87 #g/ml culture). Aspergillus nidulans exhibited qualitatively similar patterns of growth and toxigenesis in SLS medium but accumulated maximal levels of only 15/~g mycelial sterigmatocystin/ml culture and 5 #g extracellular sterigmatocystin/ml broth, respectively.
Mycotoxins are a chemically diverse group of toxic secondary metabolites that are produced by fungi and often occur in agricultural commodities. Because of their wide range of toxic effects, mycotoxins cause severe economic losses to farmers and livestock producers and pose a health threat to humans consuming contaminated foods. Long term prospects for biotechnological control of mycotoxins require elucidation of the specific steps and regulation of their biosynthetic path-
ways. Analytical methods for detection of mycotoxins and their biosynthetic precursors typically employ thin layer chromatography, high performance liquid chromatography, gas chromatography, or mass spectrometry. While it is relatively simple to resolve a mixture of pure mycotoxins using these techniques, problems occur when analyses are conducted against the complex background of pigments, lipids and other components found in culture extracts. Overcoming interference involves extensive clean-up and may require liquid-liquid partition, column chromato-
94 graphy, precipitation, and evaporation steps. Instrumentation requirements can range from a ultraviolet viewing box to a mass spectrometer. The end result is a lengthy and costly procedure that severely limits the number of samples that can be routinely analyzed and thus ability to study mycotoxin biosynthetic pathways [5, 26, 27]. As was proposed over a decade ago , it is now possible to use immunochemical assays, which are procedures common in the clinical laboratory, for detecting mycotoxins [ 14]. The basis for immunoassay involves competition between a free mycotoxin and a labeled mycotoxin for an antibody binding site. Antibodies exhibit exquisite specificity and can discriminate minor differences in chemical structure of an immunogen, such as the location or presence of a hydroxyl moiety. Affinity constants for antibodies typically exceed 10-8. Libraries of polyclonal and monoclonal antibodies to aflatoxins, zearalenones, trichothecenes, ochratoxins and their metabolites are now available for either direct analysis of liquids or solid samples after a simple solvent extraction. The two most common immunoassays used for mycotoxins are radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA). ELISA has several advantages over RIA and conventional chemical methods that include simplicity, ease of sample preparation, use of stable reagents and absence of radiation hazard. In the competitive direct ELISA, mycotoxin-enzyme conjugate (usually horseradish peroxidase) is simultaneously incubated with unconjugated toxin over solid phase-bound mycotoxin antibody [ 14-16]. Because this assay is based on competition for antibody binding sites, free toxin concentration is inversely related to antibody-bound enzyme conjugate, and thus can be calculated based on the development of endproduct absorbance obtained after addition of an appropriate enzyme substrate. Research in our laboratory is currently focused on aflatoxin biosynthesis. Sterigmatocystin is a secondary metabolite that is a biogenetic precursor of aflatoxin B 1 [6, 7, 10] and is produced by Aspergillus versicolor, Aspergillus nidulans, Asper-
gillus rugulosum, and Bipolaris sp. [9, 17, 22]. Insight into the pathway leading to sterigmatocystin and its control will be fundamental in understanding regulation of the aflatoxin biosynthesis. Investigation of sterigmatocystin biosynthesis by A. nidulans would be particularly valuable because the molecular biology of this organism is well-advanced relative to other fungi. However, because yields of sterigmatocystin are low compared to A. versicolor it is difficult to monitor mycelial and extracellular accumulation of this compound by conventional methods [17, 19-21, 23, 24]. ELISA potentially has advantages for monitoring sterigmatocystin in culture and mutant isolation because of its simplicity and sensitivity [11, 13]. The objectives of the present study were therefore to adapt ELISA for assessing sterigmatocystin production in Aspergillus cultures and to utilize this approach for comparing sterigmatocystin production by Aspergillus versicolor ATCC 18643 and Aspergillus nidulans ATC 32610.
Materials and methods
Materials. All inorganic chemicals and organic solvents were reagent grade or better. Sterigmatocystin, o-methylsterigmatocystin, aflatoxin B1, aflatoxin M1, Tween-20, bovine serum albumin (B SA), 2,2'-azino-di-(3-ethylbenzenthiazoline (sulfonic acid) (ABTS), and horseradish peroxidase type VI (HRP) were purchased from Sigma Chemical Co. (St. Louis, MO.). Complete and incomplete Freund's adjuvant were purchased from Difco Laboratories (Detroit, MI). Rabbits (New Zealand White female) were purchased from Bailey Rabbitry (Alto, MI). For preparation of protein conjugates, sterigmatocystin was converted to sterigmatocystin hemiacetal and conjugated to B SA for use as immunogen or to HRP for use as enzyme marker by the method of Li and Chu . Conjugates (1.0 mg/ml)were stored in 0.5 ml aliquots at - 2 0 ~ Antisera preparation. Antiserum specific for free sterigmatocystin was produced against sterigma-
95 tocystin hemiacetal-B SA conjugates in rabbits as described by Pestka et al. [ 15 ]. Briefly, 1500 ~g of sterigmatocystin hemiacetal-B SA in 1 ml of saline was emulsified with 3 ml of Freund's complete adjuvant. The emulsion (1.3 ml) was intradermally injected at multiple sites into the back of each rabbit. Bleeding and booster injections were made at intervals after the initial injection. The serum was purified by precipitation with 35~o saturated ammonium sulfate  and reconstituted to the original volume with 0.01 M phosphate buffered saline (PB S, pH 7.2). Antisera was dialyzed against the same buffer for 48 hrs. at 4~ Direct competitive ELISA. Pooled antiserum was diluted (1:75) in PBS containing 0.001~o BSA, and 100 ul aliquots air-dried to polystyrene microtiter wells (Immunon Removawells, Dynatech Laboratories, Alexandria, VA) . Antiseracoated plates were washed three times by filling each well with 300/~1 of 0.02 ("/v) Tween-20 in PB S (PB S-Tween) and aspirating the contents. Sterigrnatocystin standard or samples were mixed in equal volumes with sterigmatocystin hemiacetal-HRP conjugate (diluted 50-fold in 2 ~o ovalbumin in PB S) and 100/~1 of this mixture was added to each well. Plates were incubated for 40 min at 28 ~ and wells washed 6 times as described above. Bound sterigmatocystin hemiacetal-HRP was determined by incubating ABTS substrate for 30-60 min as described previously . Sterigrnatocystin content was calculated from standard competition curves comparing log sterigmatocystin concentration vs absorbance. Maximal absorbance under these conditions was typically between 0.7 to 1.0. Fungal cultures. Aspergillus versicolor ATCC 18643 and Aspergillus nidulans ATCC 32610 were purchased from American Type Culture Collection (Rockville, Maryland) and maintained on potato dextrose agar (PDA) (Difco) containing 0.5~o yeast extract. Fungi were grown on PDA slants for 15 days at 28 ~ Spores were harvested by adding 10 ml of sterile 0.01 M phosphate buffer (pH 7.0) to the slant and gently dis-
persing conidia with a sterile inoculating loop. This procedure was repeated three times and the harvested spore suspension pooled. The spore suspension then filtered through 2 layers of sterile cheese cloth and diluted with sterile phosphate buffer to a concentration of 107 spores/ml as determined by plate count on PDA. Media and culture conditions. Modified synthetic low salt (SLS) medium [4, 12] contained in 1 liter: sucrose, 85 g; L-asparagine, 10 g; (NH4) 2 SO4, 2g, KH2PO4, 2g; M g S O 4 : 6 H 2 0 , lg, CaCI" 2H20, 75 mg; ZnSO4, 10 mg; NazB407, 2 mg, FeSO4" H20, 2 mg; 2 mg, MnC12 94H20, 5 mg; and ammonium molybdate, 2 mg. Yeast extract-sucrose (YES) medium contained in 1 liter: yeast extract, 20g; and sucrose, 150g. Medium (6 ml) was dispensed into 18 x 150 culture tubes, autoclaved and inoculated with 105 spores. Culture were incubated without shaking at 28 ~ in the dark, and analyzed at three days intervals for 15 days. The mycelial mat was separated from the broth by filtering through Whatman No. 4 filter paper, and dried at 50 ~ for 24 hrs. The culture filtrate was assayed for pH and for sterigmatocystin by ELISA. Dried mycelium (0.01-0.05 g) was extracted twice with 6 ml of chloroform and evaporated. Dried extracts were dissolved in a minimal aliquot of N, N-dimethylformamide-4~ KCL (48 + 2), and analyzed by sterigmatocystin ELISA after appropriate (104-fold) dilution in PB S.
Sterigmatocystin ELISA. Since sterigmatocystin is extremely insoluble in aqueous solution, ELISA has been previously attempted only after quantitative conversion to the hemiacetal form [ 11, 13]. We were able to eliminate this step and used free sterigmatocystin for determination in both culture broth and mycelium. When free sterigmatocystin was dissolved in various solvents and diluted in assay buffer to a final solvent concentration of 0.01 ~o and the effects on direct ELISA evaluated, DMF-4~o KCL (48 + 2) was found more suitable