Richard A. Slawecki

Development of a magnetic particle immunoassay for polybrominated diphenyl ethers and application to environmental and food matrices

A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, and soil samples. The assay was rapid and can be used to analyze fifty samples in about 1h after sample cleanup. The assay has a limit of detection (LOD) below 0.1 ppb towards the following brominated diphenyl ether (BDE) congeners: BDE-47, BDE-99, BDE-28, BDE-100, and BDE-153, with the LOD approximately the same as GC-NCI-MS. The congeners most readily recognized in the ELISA were BDE-47 and BDE-99 with the cross-reactivities of BDE-28, BDE-100, and BDE-153 being less than 15% relative to BDE-47. As anticipated, the sensitivities are proportional to the similarities between the hapten structure and the BDE congener structure. Some oxygenated congeners with structural similarity to the hapten showed high to moderate cross-reactivities. Very low cross-reactivity was observed for other PBDEs or chlorinated environmental contaminants. The assay gave good recoveries of PBDEs from spiked water samples and a very small within and between day variance. Comparison with GC-NCI-MS demonstrated the ELISA method showed equivalent precision and sensitivity, with better recovery. The lower recovery of the GC-NCI-MS method could be caused by the use of an internal standard other than an isotopically substituted material that could not be used because of the fragmentation pattern observed by this method. The cleanup methods prior to ELISA were matrix dependent, no pretreatment was needed for environmental water samples, while fish, milk, and soil samples required various degrees of cleanup. Analysis of this wide variety of environmental samples by both ELISA and GC-MS demonstrated ELISA provides a timely and cost-effective method to screen for PBDEs in a variety of samples.

Novel Fungitoxicity Assays for Inhibition of Germination-Associated Adhesion of Botrytis cinerea and Puccinia recondita Spores

ABSTRACT Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC50s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC50s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi.

Identification of ceramide-phosphorylethanolamine in Oomycete plant pathogens: Pythium ultimum, phytophthora infestans, and Phytophthora capsici

Cellular lipids were extracted from three species of Oomycete plant pathogens (Pythium ultimum, Phytophthora infestans, and Ph. capsici) and analyzed via normal-phase high-performance liquid chromatography with flame-ionization detection. The most abundant polar lipids in each of the three species were the polar membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine, and a phosphosphingolipid that eluted soon after PE. Structural analysis via mass spectrometry and nuclear magnetic resonance spectrometry revealed that the phosphosphingolipid was ceramide phosphorylethanolamine (Cer-PE). The most abundant molecular species of Cer-PE in P. ultimum had a molecular weight of 670.5, contained an unusual 19-carbon branched triunsaturated sphingoid (C19-Δ4, 8, 10, 9-methyl long-chain base) and palmitic acid as the amidelinked fatty acid. The most abundant molecular species of Cer-PE in Ph. infestans has a molecular weight of 714.5, contained a common 16-carbon 1,3 di-OH sphingoid, and erucic (cis 13-docosenoic, C22-Δ13) acid as the amide-linked fatty acid. The Cer-PE in Ph. capsici comprised a mixture of each of the two molecular species found in P. ultimum and Ph. infestans.