Parvalbumin: characterisation of the cross-reactive major fish allergen

Fish are the largest and most diverse group of vertebrates. Fish are also a part of the eight food groups that cause the majority of IgE mediated food reactions. Detection tools for fish allergens and fish allergy diagnostics are however limited due to the great diversity of fish species, despite fish allergy and its major allergen parvalbumin being well documented. Currently the best treatment strategy for fish allergy is avoidance. The most commonly studied fish are cod, carp and Atlantic salmon as they are frequently consumed in North America and Europe. However much less is known about fish allergens in the Australasian region, although fish is widely consumed in this region.\ud \ud The major fish allergen is parvalbumin, a small calcium binding protein found in the muscles of vertebrates which are the biggest group of animal derived food allergens, part of the EF Hand domain protein family. Fish can express multiple parvalbumin isoforms which may differ greatly in amino acid sequence that further complicates the diagnosis of fish allergy and the detection of these allergens. In this PhD thesis, fish allergen detection methods will be evaluated in addition to the characterisation of novel fish allergens from the Australasian region to improve current diagnostic and detection methods and future development of immunotherapies for fish allergy sufferers.\ud \ud Firstly, the evaluation of the cross-reactivity of parvalbumin from a variety of bony and cartilaginous fish, from the Asia-Pacific region was performed with the monoclonal anti-parvalbumin antibody PARV-19. The presence of monomeric and oligomeric parvalbumin was demonstrated in all fish analysed, except for gummy shark, which is a cartilaginous fish. Heat processing of this allergen greatly affected its antibody reactivity. While heating caused a reduction in antibody reactivity to multimeric forms of parvalbumins for most bony fish, a complete loss of reactivity was observed for all cartilaginous fish except for the elephant shark. Molecular analysis demonstrated that the observed cross-reactivity between parvalbumin from diverse fish species is due to the molecular phylogenetic association of this major fish allergen.\ud \ud A more comprehensive phylogenetic analysis was performed with all currently known parvalbumin sequences to determine possible candidate antigens for new cross-reactive antibodies. Polyclonal rabbit antibodies were raised against parvalbumins from frequently consumed barramundi (Lates calcarifer), basa (Pangasius bocourti), pilchard (Sardinops sagax) and Atlantic salmon (Salmo salar). These were evaluated for crossreactivity against a panel of 45 fish extracts, including raw, heated and canned fish. Anti-barramundi parvalbumin proved to be the most cross-reactive antibody followed by anti-pilchard and anti-basa antibody. In contrast the anti-salmon antibody was very specific and only reacted to salmonidae and very few other fish. All analysed fish species, except mahi mahi, swordfish, yellowfin tuna and all five canned fish had parvalbumin detected in raw extracts. However antibody reactivity to many species was heat liable or susceptible to denaturation, demonstrating that these parvalbumins have most likely conformational epitopes, which lose antibody reactivity after heat treatment.\ud \ud Frequent allergic reactions to ingested barramundi among adults and children initiated further characterisation of allergenic proteins from this frequently ingested barramundi. Serum samples from 17 fish allergic adults and children from Australia were analysed by ELISA and immunoblotting for IgE antibody reactivity to raw and heated barramundi proteins. The molecular analysis of the identified allergens included mass spectrometric analysis, genetic sequencing and generation of recombinant allergens. Two novel parvalbumin isoforms (isoallergens) of the β-type were identified as the only allergens in barramundi and subsequently designated as Lat c 1.0101 and Lat c 1.0201 after registration with the International Union of Immunological Societies (IUIS). These two isoallergens are differentially expressed in barramundi tissue but bind IgE from the same patients. However these heat stable parvalbumin allergens from barramundi seem to have differential IgE binding capacity between adults and paediatric patients. IgE and IgG₄ epitopic regions of Lat c 1.0101 were elucidated by using 7 overlapping peptides, which were analysed by immunoarray with serum of fish allergic patients. IgE and IgG₄ binding epitopes were compared to assess to the possibility of designing future novel immunotherapeutics for fish allergy.\ud \ud In conclusion this dissertation has demonstrated the generation of highly cross-reactive anti-parvalbumin antibodies that could be used for the detection of allergenic fish parvalbumin in contaminated food products. These cross-reactivity studies also highlight the limitations of using antibodies for parvalbumin detection in processed fish. The in depth molecular and immunological characterisation of novel isoallergens from barramundi has widened the knowledge of fish allergy in the Asian Pacific region and improves current diagnostic approaches and the information gained from elucidating antibody epitopes can be used in the future development of specific immunotherapies for fish allergy sufferers