IgE-binding Regions for Cross-reactivity and Fish-specific Tolerance Prediction
To determine whether sensitization to specific parvalbumin epitopes could predict cross-reactivity and clinical reactivity along the allergenicity ladder, epitope mapping was performed against parvalbumins of salmon (both β-1 and β-2), cod, grouper, and grass carp with sera from fish allergic (n=11), partial fish tolerant (n=12), and complete fish tolerant (n=5) patients based on oral food challenge outcome. Highly stringent criteria of signal intensity ≥1000 a.u., control serum, and significantly higher signal intensity of allergic samples than tolerant samples (i.e. tolerated salmon or tolerated all fishes in food challenge) based on 1% false discovery rate (FDR) were adopted to identify specific IgE-binding epitopes that can differentiate allergy and tolerance to specific fishes. Comparison between grass carp allergic and tolerant subjects revealed AA64-78 (Epi_c_64-78, LKLFLQNFSAGARAL) from grouper parvalbumin as IgE-binding epitope (Fig 4A&B, Fig S1). This epitope strongly and specifically reacted with 21/23 (91.3%, >500 fluorescence intensity) fish allergic subjects and none of the tolerant patients, and also represented the only parvalbumin epitope with such diagnostic accuracy. This epitope thus denoted a cross-reactivity biomarker to identify “general” fish allergy.
We also studied allergic and partially tolerant individuals to identify epitopes and predict fish-specific tolerance. AA19-48 of salmon parvalbumin β-1 (Sal_s_β1_19-48; CKAADTFSFKTFFHTIGFASKSADDVKKAF), AA23-37 of salmon parvalbumin β-2 (Sal_s_β2_23-37; DSFNHKAFFAKVGLA), AA34-48 of cod parvalbumin AA34-48 (Gad_m_34-48; CGLSGKSADDIKKAF) and AA16-36 of grouper parvalbumin (Epi_c_16-36; IAGCSAADSFDHKKFFKACGM) fulfilled the criteria as IgE-binding epitopes and to further identify salmon tolerance (Fig 4C-G and Fig S2-5). Among these regions, AA19-33 of salmon parvalbumin β-1 (Sal_s_β1_19-33) was exclusively bound by IgE of allergic patients at high intensity, but not by partially or completely fish tolerant individuals (Fig S2). This epitope thus represented a novel biomarker to differentiate salmon-specific tolerance in fish (grass carp) allergic patients. No major IgE-binding sites could be detected from grass carp parvalbumin based on our criteria (Figure S5). We could not detect significant correlation between sIgE level and epitope signal intensity, meaning that IgE binding to these epitopes were independent of patients’ sIgE levels to the respective fish extract (Fig S6). Most importantly, neither fish-specific nor parvalbumin-specific IgE levels distinguished between allergic and partially tolerant patients, or between salmon allergic and tolerant individuals (p > .05 and q > .16) but only the identified epitopes (Fig S7).