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Parvalbumin is a calcium-binding muscle protein that is present in all vertebrates. Despite being a pan-allergen in fish and frog, fish-specific IgE and antiparvalbumin IgG antibodies displayed varying cross-reactivity among fish species. In this research, the parvalbumin-binding characteristics of several antibodies were investigated, including 3 IgG antibodies against frog, carp, and cod parvalbumins, and human IgE.
By immunoassay and IgG-immunoblotting, 3 antiparvalbumin antibodies revealed inconsistent specificity among 29 raw fish muscle extracts, which may be partially attributed to the decreased levels of fish muscle parvalbumin from anterior to posterior positions. Parvalbumin-binding by antibodies was unaffected in 112 days of frozen-stored fish muscles. Anticod parvalbumin polyclonal antibody (anticod PoAb) was the most suitable for detecting parvalbumins as it reacted to the widest range, but not all fish species.
IgE-immunoblotting demonstrated intra- and inter-individual diversity in IgE-binding to fish and frog proteins. Of 39 fish-allergic individuals, >50% subjects bound to purified cod and carp parvalbumins, and proteins corresponding to parvalbumins in 21 fish extracts, whereas
Heating, calcium-depletion and Maillard reactions affected the 3 antiparvalbumin IgG antibodies binding to fish muscle extracts and cod parvalbumin, although anticod PoAb was less affected. Both Maillard and heat treatments reduced IgE binding to cod parvalbumin and these effects were more pronounced without calcium.
Parvalbumins among fish revealed higher sequence identity than non-fish species. Both calcium-binding loops representing Gad c 1 epitopes were conserved among fish and non-fish, whereas low homology in AB domain and AB/CD inter-domain junction of fish parvalbumins may contribute to variable cross-reactivity among fish. Phylogenetic analysis showed that all fish parvalbumins but zebrafish and pike were closely related. Teleost beta-parvalbumins were closely related to beta-parvalbumins of amphibians and reptiles, but divergent from alpha-parvalbumin in mammals and non-mammals.
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