Introduction
Fish is one of the “Big 8” food allergens, with IgE-mediated fish allergy affecting approximately 1% of the world population and a much higher prevalence in pediatric cohorts at up to 7%1-3. Patients with fish allergy suffer from symptoms ranging from mild skin rashes to life-threatening anaphylaxis. Conventionally, fish allergy has been considered an umbrella term, and patients who are diagnosed to be allergic to one kind of fish are often advised to avoid all fish. Clinical reactions to multiple fish species have been mainly attributed to the cross-reactive major fish allergen, parvalbumin 4 . However, there are also reports of mono-allergy to single fish while tolerating others. Our team neatly demonstrated that a significant proportion of patients are tolerant to salmon despite experiencing severe allergic reactions to grass carp at double-blind, placebo-controlled food challenges (DBPCFC) 5 . Selection of appropriate fish for IgE testing and dietary reintroduction remains a major clinical gap due to the wide range of fish species from evolutionary distant classes consumed worldwide.
Parvalbumin is a 12-kDa sarcoplasmic calcium-binding protein for muscle contraction. It accounts for up to 95% of sensitization in patients with IgE-mediated fish allergy 6,7 . The capability of recombinant carp parvalbumin to induce the release of histamine from basophils of patients allergic to fish has long been reported 8 . Its high allergenicity (the ability to stimulate allergic reactions) can be attributed to its supreme stability even after cooking at high temperatures9 . The activity of parvalbumin was also retained after treatment of denaturing agents, meaning that its allergenicity depends primarily on its amino acid sequence instead of higher-level structures. Parvalbumin has been shown to be involved in fast muscle relaxation, in which parvalbumin level and relaxation speed of muscles are closely and positively correlated10 . It can mainly be found in fast-twitching white muscles, while endurable dark muscles in fish contain less parvalbumin. Therefore, migratory fish, such as tuna, have comparatively more dark muscle and hence less parvalbumin than sedentary fish, which have more white muscle instead 11 .
Differences in the protein levels of parvalbumin correlated with the difference in IgE reactivity to fish12 . Clinical studies have indicated that sera of fish-allergic patients were more reactive to white muscle extract than to dark muscle extract, in which parvalbumin level was four to eight times lower 13 . However, a comprehensive large-scale study detailing the clinical reactions to multiple fish species, analysis of serological IgE reactivity and their correlation to the parvalbumin level of the corresponding fish, and the identification of fish-specific IgE-binding epitopes for clinical cross-reactivity prediction is lacking. Herein, we report the clinical results of 200 fish-allergic subjects (specific IgE distribution, self-reported reactions to fishes, and DBPCFC data) and molecular analysis (parvalbumin levels determined by both protein and transcriptomic approaches and IgE reactivity of recombinant parvalbumins) for the construction of fish allergenicity ladder, as well as IgE-binding epitopes for clinical cross-reactivity prediction. Such ladder and the epitope results can stratify fish-allergic patients and the allergenicity difference of fishes to direct the selection of fishes for improving diagnosis and the gradual reintroduction of fishes in patients with IgE-mediated fish allergy.