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 |
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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