Synthesis of Protein Conjugates and Development of Immunoassays for AAL Toxins Ferenc Szurdoki,* ,† Eugene Trousdale, ‡ Barney Ward, ‡ Shirley J. Gee, † Bruce D. Hammock, †,§ and David G. Gilchrist ‡ Departments of Entomology, Plant Pathology, and Environmental Toxicology, University of California, Davis, California 95616 AAL toxins and fumonisins, produced by Alternaria alternata f. sp. lycopersici and Fusarium moniliforme, respectively, are sphinganine-analogue mycotoxins with important health implications. Highly sensitive, inexpensive, and rapid immunoassays are needed to quantify these natural toxins in food products and animal feeds. We developed novel methods for the synthesis of protein conjugates of the AAL toxin TA. Mice were immunized with the conjugates, and the resulting polyclonal antisera were used to devise class-selective ELISAs for AAL compounds. The most selective assays had detection limits for the AAL toxins in the low parts per billion range with no significant cross-reactivities for a number of structurally similar compounds, including fumonisin B 1 and sphinganine. Our selected conjugates are candidate immunogens to produce monoclonal antibodies and further polyclonal antisera for the detection of the AAL toxins. Keywords: AAL toxins; fumonisins; synthesis of conjugates of AAL toxin TA; polyclonal mouse antisera; enzyme immunoassay; ELISA; cross-reactivity INTRODUCTION AAL phytotoxins, produced by the fungal pathogen Alternaria alternata f. sp. lycopersici, are tricarballylic esters of a series of long-chain aminopolyols structurally related to sphinganine (Bottini et al., 1981; Caldas et al., 1994, 1995; Boyle et al., 1994; Oikawa et al., 1994; Boyle and Kishi, 1995) (Figure 1). Each of the five major toxin fractions (TA, TB, TC, TD, and TE) consists of a pair of regioisomer esters (Figure 1). Fumonisins (e.g., FB 1 , Figure 2), mycotoxins structur- ally related to AAL toxins, originally were isolated from maize colonized by Fusarium moniliforme, a common pathogen of maize and other grains (Nelson et al., 1993). Consumption of fumonisin-contaminated maize has been reported to induce maladies ranging from cancer to renal, neural, and hepatic necrosis in several animal species and may be responsible for human esophageal cancer (Thiel et al., 1992; Riley et al., 1994; Norred and Voss, 1994). FB 1 and AAL-toxin TA induced genotype- specific cell death in tomato lines isogenic for the Asc gene (Gilchrist et al., 1992), and both compounds were found to be cytotoxic to cultured mammalian cells (Mirocha et al., 1992). Recent evidence indicates that cell death in both plant and animal systems triggered by both TA and FB 1 toxins shows stereotypical hall- marks of apoptosis (Wang et al., 1996a,b). While the signal transduction pathways leading to either cell death or cell proliferation induced by these toxins are unknown, current studies suggest that interference with early steps in sphingolipid biosynthesis (Riley et al., 1994; Abbas et al., 1994; Wu et al., 1995) may play an important role in initiating the cellular responses observed. Both AAL toxins and fumonisins are specific inhibitors of the sphinganine (sphingosine) N-acyltrans- ferase (ceramide synthase) enzyme in animals (Merrill et al., 1993; Wang et al., 1996a) and plants (Abbas et al., 1994). The structural and toxicological similarity of the AAL toxins and fumonisins and the presence of both fungal pathogens in harvested plant products have raised concern about the contamination of human foods and * Author to whom correspondence should be ad- dressed [telephone (916) 752-5109; fax (916) 752-1537; e-mail fqszurdoki@ucdavis.edu]. † Department of Entomology. ‡ Department of Plant Pathology. § Department of Environmental Toxicology. Figure 1. Structures of AAL toxins. Each major toxin fraction is composed of a pair of regioisomers, e.g., TA is a mixture of TA1 and TA2. Figure 2. Structures of fumonisins FB1-3. 1796 J. Agric. Food Chem. 1996, 44, 1796-1803 S0021-8561(95)00726-6 CCC: $12.00 © 1996 American Chemical Society + +