Developing new methods to identify airborne proteins responsible for causing allergic asthma

Abstract

Allergic diseases such as asthma and hayfever are the most common chronic disease in European children and adults and occur in response to substances present in the environment, referred to as allergens. Allergens are present in indoor, outdoor and occupational environments. In most cases, the allergen responsible for allergic symptoms will be identified using diagnostic assessments such as skin prick testing; however, there are examples of individuals experiencing allergic symptoms where the allergen responsible has not yet been identified. This is particularly common in the occupational environment. In this PhD I aimed to explore methods used to identify proteins highly likely to be allergens and responsible for causing allergic asthma. I conducted a systematic review on asthma outbreaks to describe which laboratory methods have been used to identify airborne allergens. I then developed and optimised my own laboratory methods to identify airborne proteins that could be responsible for causing allergic asthma. I found that air sampling, skin prick testing and detection of serum specific-IgE binding were the most frequent methods used to identify allergens associated with outbreaks of asthma. Using known allergens from mouse urine, I developed and optimised a method to identify unknown allergens. The method consists of an enhanced chemiluminescence western blot and in-gel excision of serum specific-IgE binding regions, for protein identification by nano liquid chromatography coupled to tandem mass spectrometry. Application of this method identified potentially novel allergenic proteins existing within different occupational settings. In animal testing facilities located in London and Salzburg, allergenic proteins were identified from mouse urine, mouse epithelium and the common fruit fly (Drosophila melanogaster). In supermarket scratch bakeries based in the UK allergenic proteins were identified from ‘improver’ enzymes. I also developed methods for detecting outdoor airborne protein from Teflon and polycarbonate filters used for air sampling. The registration of allergens on online databases relies on researcher-based identifications and the increase in allergen component-resolved diagnosis means this field is likely to develop rapidly in the coming years. Further work is needed to confirm the allergenicity of the proteins identified in this thesis, that may be responsible for causing allergic asthma.