Development and application of an enzyme immunoassay for the detection of the mycotoxin Fumigaclavine A
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The present study describes the development of specific polyclonal antibodiesagainst fumigaclavine A (FuA) in rabbits, and the development of a highly sensitiveenzyme immunoassay (EIA) for this mycotoxin. The Mannich condensationreaction with formaldehyde was used to conjugate FuA to keyhole limpethemocyanin (KLH) as the immunogen, and to bovine serum albumine (BSA) for asthe coating antigen. Conjugation of FuA to KLH with formaldehyde proved to be aneffective approach for the preparation of immunogen for anti-FuA antibodyproduction.A competitive indirect EIA was optimized using antiserum obtained from onerabbit. The EIA was very sensitive for FuA, with a 50% inhibition concentration(IC50) value of 3.3 ng/ml and a detection limit of the standard curve in buffersolutions of 0.5 ng/ml. The EIA was very specific for FuA with 1.3%, 12.6%, and0.2% cross-reactivity with FuB, FuC, and FuD, respectively. IsoFuA and severalother lysergic acid derivatives (ergonovine, ergotamine, and alpha-ergocryptine)were tested but did not cross-react in this assay.The EIA was applied to the analysis of FuA in silage and in tissue from therespiratory system of birds with aspergillosis. The detection limit for FuA in silagewas at 10 ng/g, average recoveries from artificially contaminated control sampleswere 77.8%. None of 24 analyzed silage samples contained detectable amounts ofFuA. Although the number of samples was limited, the results indicate that there isnot a widespread problem of FuA in silage.The detection limit of the assay for FuA in tissue from the respiratory system ofbirds was at 1.5 ng/g, with average recoveries from artificially contaminated of92.7%. FuA was found in 66% tissue samples of aspergillosis cases. Cultivation offungal growth on respiratory tissue samples of aspergillosis cases on malt extractagar (MEA) yielded fungal material which was typical for A. fumigatus. When themycelium was extracted and assayed by FuA EIA, high amounts of FuA was found(up to 8 mg/g mycelium). Further analysis of the mycelium extract by HPLC foundFuA, FuC, and other unidentified compounds. Hydrolysis of FuC which have beenisolated from mycelium extract of A. fumigatus gave a new fumigaclavinederivative. FuD is proposed as the name of this compound.Different specificity pattern of the FuA EIA and that of a previously developedergonovine EIA were studied. In competitive indirect EIA, the anti-FuA antibody diddetect neither ergonovine nor IsoFuA. Conversely, by competitive direct EIAformat, the anti-ergonovine antibody was able to detect both FuA and IsoFuA.Using these assays in combination with HPLC separation of cheese extracts, aseries of blue-veined cheeses from the German market were analyzed. None of 16blue-veined cheese samples contained FuA. However, the presence of IsoFuA inblue-veined cheese samples was detected using the ergonovine EIA.This is the first description of antibodies against FuA and the first development ofan EIA for FuA. This is also the first report demonstrating that FuA is correlatedwith aspergillosis in birds. However, the role of this mycotoxin in this diseaseremains to be clarified.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler