Doyle, Sean and Fox, M. and Gray, G. and Kavanagh, Kevin (2004) Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development. Journal of Microbiological Methods, 56 (2). pp. 221-230. ISSN 0167-7012

Download (552kB)

Abstract

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.

Item Type:	Article
Keywords:	Gliotoxin; ELISA; MALDI-TOF; Hapten; Imminocompromised;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	166
Identification Number:	https://doi.org/10.1016/j.mimet.2003.10.009
Depositing User:	Dr. Sean Doyle
Date Deposited:	22 Dec 2004
Journal or Publication Title:	Journal of Microbiological Methods
Publisher:	Elsevier B.V.
Refereed:	Yes
Funders:	Enterprise Ireland (EI), Higher Education Authority (HEA)
URI:	http://www.sciencedirect.com
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

Repository Staff Only(login required)

Item control page

Downloads

Origin of downloads