Anoplocephala perfoliata is a species of parasitic worm that belongs to a group
known as cestodes, which specifically target equine animals. As with all types of
tapeworms, these parasites infect the gastrointestinal tract of their host, with
devastating and potentially fatal consequences. The current lack of a sensitive and
specific test for this parasite means that it continues to go undetected, hense this
project aims to develop a novel and rapid diagnostic test with high sensitivity and
specificity to help increase detection, thus precluding economic loss in the equine
industry.
The project details the development of three unique detection platforms; an
ELISA, a lateral flow assay and an impedimetric immunosensor, aimed to detect IgA in
saliva, since IgA is the dominant immunoglobulin of the mucosal immune system. IgA
was therefore believed to be the ideal marker for rapid, specific and early indication of
infection with A. perfoliata. Diagnosis using saliva samples was an integral part of this
project, since it would allow for non-invasive sampling, by non-skilled personnel.
A highly sensitive ELISA-based detection system was developed in this project
for the detection of 3 different types of IgA. The first ELISA was developed to detect
non-specific or ‘total’ IgA levels. Using a sandwich ELISA format, IgA was detectable
with a LoD of ~0.04 ng/ml. A second ELISA was developed using the crude
excretory/secretory (E/S) antigen, cultured from A. perfoliata worms, which were
obtained by a vet during post-mortem examination of infected horses. The crude
antigen mix was then used to fabricate an ELISA to detect specific IgA in saliva,
produced against the E/S antigens. The crude antigen was then employed in a series of
SDS PAGE and western blot experiments, which revealed the 12/13 kDa antigen as the
main antigen detected by IgA in saliva. The 12/13 kDa was then electroeluted and used
to immunise rabbits, in order to obtain anti-12/13 kDa antibodies, which were later
used to purify large quantities of the 12/13 kDa antigen from the crude antigen mix.
This allowed for the fabrication of the third and final ELISA, to detect IgA specific to the
12/13 kDa antigen. The 3 ELISAs were optimised throughout this project to ensure the most ideal conditions, such as antibody concentrations, sample dilutions, sample
diluents, incubation temperatures and times were employed to obtain maximum assay
sensitivity, specificity and productivity in a commercial setting.
Testing samples (n = 24) using all 3 ELISAs and then standardising the specific
IgA levels against the non-specific IgA, allowed for a novel and reliable detection
method for A. perfoliata to be developed. This diagnostic test was developed in
partnership with Austin Davis Biologics Ltd., who in April 2014 launched a screening
programme which now offers horse owners an accurate means of testing their horses
for A. perfoliata infections accurately.
The second detection platform developed during this project was a lateral flow
assay, whereby an immunochromographic strip was used to measure IgA levels in
saliva. The studies performed determined the optimal conditions as using 40 µl of a
1:1,000 dilution of saliva using PBS(T) 1% as the sample diluent. The capture and
control antibody were used at a concentration of 0.2 mg/ml, which were coated on the
nitrocellulose membrane using an automated dispensing system (BioDot). The
conjugate was labelled using gold nanoparticles, since it does not require any
substrates or wash steps and its aggregation allows for immediate visual detection. A
LoD of ~47 ng/ml was obtained for this assay.
The final detection system investigated as part of this project was a label-less
impedimetric immunosensor, whereby IgA was detected by means of electrochemical
impedance spectroscopy (EIS). Polyaniline was the conductive polymer chosen to coat
the surface of the screen printed carbon electrode, since the amine groups could be
utilised to immobilise biotin molecules. A biotin-avidin complex was employed to
ensure the uniform immobilisation of the capture antibody. Using the capture and
control antibody at a concentration of 50 µg/ml and 10 mM ferri-ferrocyanide as the
redox solution, IgA concentrations over a range of 100 – 0 ng/ml were investigated by
Electrochemical Impedance Spectroscopy (EIS).
| Identifer | oai:union.ndltd.org:CRANFIELD1/oai:dspace.lib.cranfield.ac.uk:1826/9235 |
| Date | 12 1900 |
| Creators | Carr, Sinead |
| Contributors | Higson, Seamus P. J., Davis, Frank |
| Publisher | Cranfield University |
| Source Sets | CRANFIELD1 |
| Language | English |
| Detected Language | English |
| Type | Thesis or dissertation, Doctoral, PhD |
| Rights | © Cranfield University 2014. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner. |
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