Characterization of new Alternaria alternata--specific rat monoclonal antibodies.

In this study, three different rat hybridoma cell lines secreting monoclonal antibodies (mAbs) recognizing the spores from Alternaria alternata, a plant pathogenic fungus, contaminant of food products and important cause of both allergic rhinitis and asthma, have been characterized. These three mAbs are all of IgM isotype. Two antibodies, A1 and F10, were cross-reactive antibodies recognizing spores from Alternaria, Cladosporium, Penicillium, Aspergillus and Stachybotrys genera, but not the yeasts Saccharomyces cerevisiae or Candida albicans. Competitive and sandwich assays demonstrated that these two mAbs were directed against the same or very close repetitive(s) epitope(s). A1-based sandwich ELISA efficiently detected this epitope in various mould (but not yeast)-soluble extracts prepared from strains grown in the laboratory. Moreover, this A1-based sandwich ELISA detected its cognate epitope in air and dust samples obtained from dwellings. The third antibody, E5, recognized only the spores of Alternaria and the phylogenetically very close Ulocladium botrytis. This E5 antibody is directed against a repetitive epitope found in Alternaria and Ulocladium laboratory extracts and can be used in a sandwich assay for the quantification of these moulds. Therefore, E5 antibody is a promising tool for the development of Alternaria-Ulocladium-specific immunoassays, while A1 and F10 could be interesting tools for the quantification of the total mould biomass.

Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays.

An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17beta-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4-tert-octylphenol (t-OP), bisphenol A (BPA), using both liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC-MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.

Androgenic and antiandrogenic activities in water and sediment samples from the river Lambro, Italy, detected by yeast androgen screen and chemical analyses.

The river Lambro is the most polluted tributary of the river Po in North Italy and was chosen as a representative water course discharging industrialized areas. Water and sediment samples of the river Lambro were investigated regarding the presence of endocrine disrupting compounds. A combined procedure was used consisting of solid-phase extraction and HPLC based fractionation of samples, of screening for (anti)androgenic activity using the yeast androgen screen (YAS) and of chemical analysis using HPLC-MS/MS and GC-MS. Androgenic and antiandrogenic activities were found in specific fractions of the water and sediment while the total extracts showed antiandrogenic activity only. The chemical analysis of the fractions and total extracts with antiandrogenic activities revealed the presence of compounds with suspected antiandrogenic potency such as bisphenol A, iprodione, nonylphenol, p,p'-DDE and tert-octylphenol but other unknown compounds contributed mainly to the observed antiandrogenic activities. The antiandrogenic load of the river Lambro ranged between 1.34 and 17.1 microM flutamide-equivalents and may pose a risk to aquatic environments. Future screenings for EDC in the environment that have the potential to interfere with reproduction of aquatic organisms should be extended to different modes of actions including (anti)androgenic ones.