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Studies on the immunosuppressive effects and detection of naturally-occuring toxins

Stack, Edwina C. (2012) Studies on the immunosuppressive effects and detection of naturally-occuring toxins. PhD thesis, Dublin City University.

Abstract
Episodes of toxin-producing phytoplankton occur worldwide, causing both animal and human fatalities. Toxicity occurs through consumption of phycotoxins, including azaspiracid, which accumulate in filter-feeding shellfish. Microcystins are hepatotoxins, produced mainly by freshwater cyanobacteria. Aflatoxins are potent, fungal hepatocarcinogens, which occur mainly in food and feed products. The purpose of this research was to examine the cytotoxic and immunosuppressive effects of aflatoxins (B1, B2 and G1), azaspiracid-1 and microcystin-LR in vitro, using the murine macrophage cell line, J774A.1. The results clearly demonstrated that azaspiracid and microcystin had a significant effect on host defence functions, through deregulation of IL-6, IL-10, IL12p40 and TNF-α cytokine expression. Microcystin exposure significantly decreased IL-1β expression. ‘Toll-like’ receptor (TLR2 and CD14) expression was altered following aflatoxin exposure, while apoptotic marker (caspase-1) expression was affected following microcystin exposure. This knowledge should be taken into consideration in the implementation of detection limits, aimed at minimising risks to human health through toxin exposure. Increased awareness of the hazards presented by toxins led to the requirement for recombinant antibodies for these targets, for incorporation into sensitive detection immunoassays. This thesis describes the production of leprine and avian immune libraries for azaspiracid and microcystin, respectively. Attempts were made to isolate azaspiracid-specific antibodies with little success. Phage display was utilised to successfully isolate two single chain antibody fragments (scFvs) to microcystin from the avian library. Error-prone PCR resulted in the isolation of a mutant clone which displayed a 2.3-fold improvement in sensitivity by ELISA, with an LOD of 1.4 ng/mL. The mutant scFv displayed an altered cross-reactivity profile to the microcystin variants tested using Biacore™ inhibition analysis. The recombinant antibodies were successfully applied to the development of fluorescence-based immunoassay formats. The biotinylated mutant scFv was incorporated into a slide-based assay format on a functionalised glass substrate (IC50 ~ 1 µg/L). This assay had the potential to accurately detect microcystin and its variants, below the regulatory limit of 1 µg/L. The application of these highly-sensitive recombinant antibodies into rapid and inexpensive fluorescence detection systems could aid in the development of an early warning system for toxin outbreaks.
Metadata
Item Type:Thesis (PhD)
Date of Award:November 2012
Refereed:No
Supervisor(s):O'Kennedy, Richard
Uncontrolled Keywords:aflatoxins; Microcystins; immunosuppressive effects; toxicity; consumption of phycotoxins
Subjects:Biological Sciences > Biotechnology
Humanities > Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:17096
Deposited On:20 Nov 2012 11:56 by Richard O'Kennedy . Last Modified 30 Jul 2021 14:50
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