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).