Doyle, Sean and Fox, M. and Gray, G. and Kavanagh, K.
Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development.
Journal of Microbiological Methods, 56 (2).
Immunological detection of secreted low molecular weight toxins represents a potentially novel means of diagnosing
infection by the fungus Aspergillus fumigatus. Two such metabolites, gliotoxin and helvolic acid, were selected and conjugated
to thyroglobulin for antisera generation in rabbits. Gliotoxin was initially activated using N-[ p-maleimidophenyl] isocyanate
(PMPI) and subsequently conjugated to S-acetyl thioglycolic acid N-hydroxysuccinimide-activated thyroglobulin, whereas
helvolic acid was activated with N-(3-Dimethylaminopropyl)-NV-ethylcarbodiimide (EDC) in the presence of thyroglobulin
prior to immunisation. To facilitate subsequent antisera evaluation, both toxins were similarly conjugated to bovine serum
albumin (BSA). Matrix-Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) mass spectrometry and SDSPAGE
analysis confirmed covalent attachment of toxins to BSA in the ratios of 15 and 2.4 mol per mol BSA for gliotoxin and
helvolic acid, respectively. Resultant high titer antisera were capable of detecting both BSA-conjugated toxins (inhibitory
concentration (IC)50: 4–5 Ag/ml). Free toxins were also detectable by competitive immunoassay, whereby 10 Ag/ml free
gliotoxin (30 AM) and helvolic acid (17 AM), respectively, inhibited antibody binding to cognate toxin–BSA previously
immobilised on microwells. This work confirms that sensitive and specific antisera can be raised against fungal toxins and may
have an application in diagnosing fungal infection.
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