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Development of a Protein Microarray System for the Diagnosis of Severe Equine Asthma and the Effect of Forage Treatment on IgE-Protein Binding

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White, Samuel James (2019) Development of a Protein Microarray System for the Diagnosis of Severe Equine Asthma and the Effect of Forage Treatment on IgE-Protein Binding. PhD thesis, University of Gloucestershire. doi:10.46289/9S5W22HR

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Official URL: https://eprints.glos.ac.uk/13993/

Abstract

Severe equine asthma (sEA) is a prevalent, performance limiting disease which occurs in genetically susceptible horses. Severe equine asthma is associated with increased levels of allergen specific immunoglobulin E (IgE), which along with intradermal and basophil activation tests, lead many to speculate the disease may be IgE mediated. Historically, a plethora of cumbersome and expensive techniques have been adopted to characterise antibody–allergen interaction, rendering widespread allergen assessment problematic. The aim of this thesis is to utilise recent advances in computational and robotic technologies to “print” hundreds of environmental allergens and monitor their reactions with specific immunoglobulins in sera/bronchoalveolar lavage fluid (BALF) from sEA/non-affected horses, and explore the effect of hay preparation methods on IgE-protein binding. In paper I, conditions were optimised for printing, incubation, immunolabeling, biological fluid sources, concentration techniques, reproducibility and specificity. The developed platform identified novel allergens, an association between sEA and pollen sensitisation, and a positive correlation between sera/BALF specific IgE profile. Applying this technique with mathematical modeling, paper II demonstrated the high discriminatory power of this approach, enabling the accurate diagnosis of sEA using serological IgE as a biomarker. The platform identified environmental influences on IgE profiles, discrimination between differing IgE-mediated conditions, and revealed novel pollen, bacteria, mould and arthropod proteins associated with sEA. Latex proteins, a genus previously untested in the horse, were the most influential variables. Further developing this platform into a competition technique, Paper III demonstrated the ability to identify sEA-associated allergens in hay samples, and assess the effect soaking and steaming have on IgE-protein binding using specific horse/hay combinations. Forage preparation techniques had no impact on hay allergenicity. This work provides a new, rapid and more accurate approach to sEA diagnosis, providing a platform for tailored management and the development of allergen-specific immunotherapy.

Item Type:

Thesis (PhD)

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