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\begin{document}

\title{Differential patterns of fish sensitization in Asian populations:
implication for precision diagnosis}



\author[1]{Christine Yee Yan Wai}%
\author[1]{Nicki Y.H. Leung}%
\author[1]{Agnes Sze Yin Leung}%
\author[2]{Naoko Fusayasu}%
\author[3]{Sakura  Sato}%
\author[1]{Kary J.Y. Xu}%
\author[4]{Yat S. Yau}%
\author[5]{Jaime Rosa Duque}%
\author[6]{Mike Kwan YW}%
\author[7]{James W. C. H. Cheng}%
\author[4]{Wai Hung Chan}%
\author[5]{Gilbert Chua}%
\author[6]{Qun U. Lee}%
\author[7]{David C.K. Luk}%
\author[4]{Po K. Ho}%
\author[6]{Joshua Wong SC}%
\author[6]{Ivan C.S. Lam}%
\author[1]{Gary Wong}%
\author[3]{Motohiro Ebisawa}%
\author[1]{Ting Fan Leung}%
\affil[1]{The Chinese University of Hong Kong}%
\affil[2]{Department of Pediatrics National Hospital Organization Sagamihara National Hospital Kanagawa Japan}%
\affil[3]{Department of Allergy Clinical Research Centre for Allergy and Rheumatology Sagamihara National Hospital Kanagawa Japan}%
\affil[4]{Queen Elizabeth Hospital}%
\affil[5]{The University of Hong Kong Li Ka Shing Faculty of Medicine}%
\affil[6]{Princess Margaret Hospital}%
\affil[7]{United Christian Hospital}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background} The current diagnostics of fish allergy lack
sufficient accuracy such that more reliable tests such as
component-resolved diagnosis (CRD) are urgently needed. This study aimed
at identifying fish allergens of salmon and grass carp and evaluating
the sensitization pattern towards the identified allergens in fish
allergic subjects from two distinct populations in Asia.
\textbf{Methods} One hundred and three fish allergic subjects were
recruited from Hong Kong (67 subjects) and Japan (46 subjects). Western
blot and mass spectrometry were used to identify allergens from salmon
and grass carp. Fish allergens were purified and tested against 96 sera
on ELISA to analyze patients' sensitization pattern. The protein
profiles of salmon meat prepared under different cooking methods until
core temperature reached 80\selectlanguage{ngerman}°C were evaluated by SDS-PAGE and mass
spectrometry. \textbf{Results} Three common allergens between salmon and
grass carp, namely enolase, glycerldehyde-3-phosphate dehydrogenase
(GAPDH) and parvalbumin, and two salmon-specific allergens collagen and
aldolase were identified. Parvalbumin was the major allergen for both
fishes showing an overall sensitization rate of 74.7\%, followed by
collagen (38.9\%), aldolase (38.5\%) and enolase (17.8\%). Japanese
subjects showed more diverse allergen sensitization pattern and more
frequent IgE-binding to heat-labile salmon allergens. Compared with
steaming and boiling, cooking by baking and frying retained more fish
proteins inclusive of heat-labile allergens. \textbf{Conclusions} Fish
allergic patients from different Asian populations show varying fish
allergen sensitization profiles. The relevant extracts and components
for diagnosis are population-dependent but parvalbumin and collagen are
important biomarkers. Cooking methods modify allergen composition of
salmon and appear to influence patients' allergic manifestations.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Differential patterns of fish sensitization in Asian
populations: implication for precision diagnosis}

Christine Y.Y. Wai, PhD\textsuperscript{a}\textbf{*} ; Nicki Y.H. Leung,
PhD\textsuperscript{a}\textbf{*} ; Agnes S.Y. Leung,
MBChB\textsuperscript{a,b}*; Naoko Fusayasu, MD\textsuperscript{c};
Sakura Sato, MD\textsuperscript{d}; Kary J.Y. Xu,
MPhil\textsuperscript{a}; Yat Sun Yau, MBChB\textsuperscript{e}; Jaime S
Rosa Duque, MD\textsuperscript{f,g,h}; Mike Y.W. Kwan,
MSc\textsuperscript{i}; James W. C. H. Cheng, MBBS\textsuperscript{j};;
Wai Hung Chan, MBChB\textsuperscript{e}; Gilbert T. Chua,
MBBS\textsuperscript{f,g,h}; Qun Ui Lee, MMed\textsuperscript{i}; David
C.K. Luk, MBChB\textsuperscript{j}; Po Ki Ho, MBBS\textsuperscript{e};
Joshua S.C. Wong, MBBS\textsuperscript{i}; Ivan C.S. Lam,
MBChB\textsuperscript{i}; Gary W.K. Wong, MD\textsuperscript{a};
Motohiro Ebisawa, MD\textsuperscript{d}\textbf{+} ; Ting Fan Leung, MD,
FAAAAI\textsuperscript{a,b}\textbf{+}

\textsuperscript{a} Department of Paediatrics, Prince of Wales Hospital,
The Chinese University of Hong Kong, Hong Kong SAR, People's Republic of
China

\textsuperscript{b} Hong Kong Hub of Paediatric Excellence, The Chinese
University of Hong Kong, Hong Kong SAR, People's Republic of China

\textsuperscript{c} Department of Pediatrics, National Hospital
Organization, Sagamihara National Hospital, Kanagawa, Japan

\textsuperscript{d} Department of Allergy, Clinical Research Centre for
Allergy and Rheumatology, Sagamihara National Hospital, Kanagawa, Japan

\textsuperscript{e} Department of Paediatrics, Queen Elizabeth Hospital,
Hong Kong SAR, People's Republic of China

\textsuperscript{f} Department of Paediatrics and Adolescent Medicine,
School of Clinical Medicine, The University of Hong Kong, Hong Kong SAR,
People's Republic of China

\textsuperscript{g} Department of Paediatrics and Adolescent Medicine,
The Hong Kong Children's Hospital, Hong Kong SAR, People's Republic of
China

\textsuperscript{h} Department of Paediatrics, The University of Hong
Kong Shenzhen Hospital, Shenzhen, People's Republic of China

\textsuperscript{i} Department of Paediatrics and Adolescent Medicine,
Princess Margaret Hospital and Yan Chai Hospital, Hong Kong SAR,
People's Republic of China

\textsuperscript{j} Department of Paediartics and Adolescent Medicine,
United Christian Hospital, Hong Kong SAR, People's Republic of China

\textbf{* Co-first authors: CYY Wai, NYH Leung and ASY Leung contributed
equally to this paper}

\textbf{+ Co-correspondence:}

\textbf{Motohiro Ebisawa,} Clinical Research Center for Allergy and
Rheumatology, Sagamihara National Hospital, 18-1, Sakuradai, Minami-ku,
Sagamihara, Kanagawa 252-0392, Japan

Email: m-ebisawa@sagamihara-hosp-gr.jp

ORCID: 0000-0003-4117-558X

\textbf{Ting Fan Leung,} Room 84043, Lui Che Woo Clinical Sciences
Building, Prince of Wales Hospital, Shatin, New Territories, Hong Kong

Email: tfleung@cuhk.edu.hk

ORCID: 0000-0002-6469-1926

\textbf{ORCiD ID \& E-mail contacts of all authors:}

CYY Wai: 0000-0002-2570-7745; christineyywai@cuhk.edu.hk

NYH Leung: 0000-0001-6836-6445; nyhleung@hku.hk

ASY Leung: 0000-0001-8249-4478; agnes.syl@cuhk.edu.hk

N Fusayasu: 0000-0002-9649-8135; nen19842000@yahoo.co.jp

S Sato: 0000-0003-3674-0759; ssakura8010@foodallergy.jp

KJY Xu: 0000-0002-9566-5683; karyxu9@gmail.com

YS Yau: 0000-0002-5471-9139; yysz01@ha.org.hk

JS ROSA DUQUE: 0000-0002-5292-5510; jsrduque@hku.hk

MYW Kwan: 0000-0002-1476-9625; kwanyw1@ha.org.hk

JWCH Cheng: 0000-0003-1997-5230; cch278@ha.org.hk

WH Chan: 0000-0002-9256-4799; cwhz01@ha.org.hk

GT Chua: 0000-0003-0333-0059; gtc510@gmail.com

QU Lee: 0000-0001-6297-8969; leequ@ha.org.hk

DCLK: 0000-0002-7854-0221; lukckd@ha.org.hk

PK Ho: 0000-0001-7836-5243; pollyho@ha.org.hk

JSC Wong: 0000-0003-4815-5821; wsc141@ha.org.hk

ICS Lam: 0000-0002-6909-2754; csivan15@ha.org.hk

GWK Wong: 0000-0001-5939-812X; wingkinwong@cuhk.edu.hk

\textbf{Declaration of funding sources:}

This project was supported by the Health and Medical Research Fund
(08191356), Research Grants Council Research Impact Fund (R4035-19), the
Hong Kong Institute of Allergy Research Grant and TS Lo Foundation.

\textbf{Key words}

Fish allergy; component-resolved diagnosis, parvalbumin; enolase;
aldolase; collagen

\textbf{Highlights}

Parvalbumin and collagen are major fish allergens important for
diagnosis. Grass carp parvalbumin presents higher IgE binding affinity
than salmon parvalbumin but the relevant extract and component for
diagnosis differ among populations that is partially dependent on
dietary habits.

\textbf{Word counts:}

Abstract: 254

Manuscript text: 4278

\textbf{Abstract}

\textbf{Background}

The current diagnostics of fish allergy lack sufficient accuracy such
that more reliable tests such as component-resolved diagnosis (CRD) are
urgently needed. This study aimed at identifying fish allergens of
salmon and grass carp and evaluating the sensitization pattern towards
the identified allergens in fish allergic subjects from two distinct
populations in Asia.

\textbf{Methods}

One hundred and three fish allergic subjects were recruited from Hong
Kong (67 subjects) and Japan (46 subjects). Western blot and mass
spectrometry were used to identify allergens from salmon and grass carp.
Fish allergens were purified and tested against 96 sera on ELISA to
analyze patients' sensitization pattern. The protein profiles of salmon
meat prepared under different cooking methods until core temperature
reached 80\selectlanguage{ngerman}°C were evaluated by SDS-PAGE and mass spectrometry.

\textbf{Results}

Three common allergens between salmon and grass carp, namely enolase,
glycerldehyde-3-phosphate dehydrogenase (GAPDH) and parvalbumin, and two
salmon-specific allergens collagen and aldolase were identified.
Parvalbumin was the major allergen for both fishes showing an overall
sensitization rate of 74.7\%, followed by collagen (38.9\%), aldolase
(38.5\%) and enolase (17.8\%). Japanese subjects showed more diverse
allergen sensitization pattern and more frequent IgE-binding to
heat-labile salmon allergens. Compared with steaming and boiling,
cooking by baking and frying retained more fish proteins inclusive of
heat-labile allergens.

\textbf{Conclusions}

Fish allergic patients from different Asian populations show varying
fish allergen sensitization profiles. The relevant extracts and
components for diagnosis are population-dependent but parvalbumin and
collagen are important biomarkers. Cooking methods modify allergen
composition of salmon and appear to influence patients' allergic
manifestations.

\textbf{Background}

Fish is one of the most frequent causes of food-induced allergy
alongside cow's milk, hen's egg, peanut, tree nut and shellfish, and has
been included in the European mandatory labeling legislation together
with 13 other allergens\textsuperscript{1}. Fish is a valuable source of
healthy nutrients such as omega-3 fatty acids with increasing global
demand. Fish allergy affects 0.1-0.4\% of the world's
population\textsuperscript{2,3}, has an early onset and tends to persist
throughout life\textsuperscript{4}. Allergic reactions are mainly
provoked through ingestion but also by skin contact or by inhalation of
fish steam during fish processing. The allergic reactions triggered by
fish can be life-threatening and even fatal resulting in a heavy
socioeconomic burden and negative impact on quality of
life\textsuperscript{5,6}

\selectlanguage{greek}β-\selectlanguage{english}parvalbumin represents the major fish allergen accounting for up to
90\% of sensitization and has been described and characterized in a wide
range of fish species\textsuperscript{7-10}. Parvalbumins are 8-12kDa
calcium-binding muscle proteins with extreme thermal stability and
shared 55-95\% amino acid sequence homology among various fish species.
This might account for the cross-reactivity among fish species but case
reports have shown mono-sensitization and clinical allergy to single
fish species\textsuperscript{11,12}. Alongside parvalbumin, collagen,
aldolase, enolase, pyruvate kinase, glucose-6-phosphate isomerase
(G-6-PI), creatine kinase, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), triosephosphate isomerase (TPI) and tropomyosin are other
registered fish allergens in the World Health Organization and
International Union of Immunological Societies (WHO/IUIS)
database\textsuperscript{13}. Among these allergens, collagen, aldolase
and enolase are more well-characterized. Collagen is another heat stable
protein, with reports showing anaphylaxis provoked by collagen and
illustrating IgE binding to collagen only but not
parvalbumin\textsuperscript{14,15}. Enolase and aldolase, on the other
hand, are identified as major heat-labile fish allergens in cod, salmon
and tuna\textsuperscript{16}.

The current diagnostic workup of fish allergy relies on skin prick test
(SPT), measurement of specific IgE and when indicated, an oral food
challenge\textsuperscript{17}. Although a number of fish extracts are
available on the ImmunoCAP platform, only parvalbumins from cod and
common carp are available for IgE testing and other identified fish
allergens have not been included for diagnosis due to the insufficient
understanding of their allergenic properties and clinical relevance
especially in the Asian populations with high fish consumption. Yet a
considerable number of studies have illustrated the low specificity of
SPT and specific IgE measurement while safety has always been a concern
in oral food challenges\textsuperscript{18}. Together with the reports
of monosensitization to some fish species and parvalbumin-independent
IgE sensitization\textsuperscript{19}, a testing panel inclusive of the
relevant fish allergens for precision component-resolve diagnosis (CRD)
will be of great value.

This study identified fish allergens of salmon (\emph{Salmo salar} ) and
grass carp (\emph{Ctenopharyngodon idella} ), which are among the most
commonly farmed and consumed seawater and freshwater fishes in Asia. We
compared the sensitization pattern towards the four major allergens,
\selectlanguage{greek}β-\selectlanguage{english}parvalbumin, enolase, aldolase and collagen, from both salmon and
grass carp in fish allergic subjects from Hong Kong and Japan. Based on
our results, we further characterized the allergen profiles in salmon
meat cooked at the same core temperature by different methods to dissect
the link between dietary preferences and allergen sensitization
profiles.

\textbf{Methods}

\textbf{Recruitment of fish allergic subjects}

This study recruited subjects aged 1-18 years with reported history of
IgE-mediated fish allergy from six regional hospitals in Hong Kong (HK;
Prince of Wales Hospital, Queen Mary Hospital, Queen Elizabeth Hospital,
Yan Chai Hospital, Princess Margaret Hospital and United Christian
Hospital) and Sagamihara National Hospital in Kanagawa, Japan. The
inclusion criteria include (1) subjects with convincing history of
immediate fish allergy as defined by immediate allergic symptoms within
2 hours following fish ingestion within two years before recruitment and
clinically diagnosed by an allergy specialist; and (2) subjects with
evidence of IgE sensitization to fish by blood sIgE measurement against
cod (f3), salmon (f41), tuna (f40) and/or grass carp (experimental
ImmunoCAP developed in collaboration with Thermo Fisher) and/or SPT with
commercial fish mix extract (ALK-Abell\selectlanguage{ngerman}ó). Exclusion criteria are
subjects receiving maintenance immunosuppressive treatments that cannot
be stopped for four weeks prior to study and who receive intravenous
immunoglobulin treatment within three months. Severity of allergic
symptoms to fish was calculated based on the Ordinal Food Allergy
Severity Score (oFASS)\textsuperscript{20}. Briefly, Grade 1 includes
reactions restricted to oral cavity. Grade 2 to 5 may include oral
symptoms but with other target organs affected, by which grades 2 and 3
reactions involve skin, eye/nose and/or digestive system. Involvement of
larynx and/or bronchi reactions are graded 4 while grade 5 would involve
cardiovascular and/or nervous systems. Participants and/or their legal
guardians gave written informed consent. Ethical approvals were obtained
from Joint Chinese University of Hong Kong-New Territories East Cluster
Clinical Research Ethics Committee (2019.034 \& 2019.612) and the
Sagamihara National Hospital Ethics Committee (2019-003).

\textbf{Preparation of raw and cooked fish extracts}

Frozen salmon (\emph{Salmo salar} ) and fresh grass carp
(\emph{Ctenopharyngodon idella} ) were purchased from local supermarket.
Two milligrams of raw fish meat were manually homogenized in 8mL
ice-cold phosphate-buffered saline (PBS) until a smooth paste was
achieved. Protein was then extracted overnight at 4°C with constant
stirring. The protein extract was centrifuged and supernatant was
filter-sterilized through a 0.22 \selectlanguage{greek}µ\selectlanguage{english}m polyethersulfone membrane. The fish
protein extracts were stored at -20\selectlanguage{ngerman}°C until use.

Several cooking methods including steaming (at 100°C), boiling (at
100°C), baking (at 150°C), lightwave cooking (at 180°C), frying (at
180°C) and air-frying (at 200°C) were used to prepare cooked salmon and
grass carp for comparison of its protein profile. Raw salmon meat,
weighed 20 g per pack, was cooked until the core temperature of salmon
meat reached 80°C as measured using digital thermometer. After cooking,
all samples were weighed and 1/10 of the raw and cooked fish samples
were extracted in 8mL PBS as described above. All protein extracts were
stored at -20°C until use.

\textbf{SDS-PAGE and western blotting}

Four microliters of raw or cooked fish extracts were separated on a
13.5\% SDS-polyacrylamide gel according to their molecular weights using
a Mini PROTEAN SDS-PAGE system (Bio-Rad). Protein bands were stained
with SimplyBlue SafeStain (Thermo Fisher). Western blotting was
performed using the surf-blot antibody system (Idea Scientific). One
hundred fifty microliters of raw fish extracts were resolved in 13.5\%
SDS-PAGE and transferred to PVDF membranes (Bio-Rad) with the Trans-Blot
Turbo system (Bio-Rad). Non-specific binding of the membranes was
blocked with 5\% non-fat dry milk in 0.05\% Tween-20 in Tris-buffered
saline (TBS-T, blocking solution) for 1 hour at room temperature.
Patient sera diluted at 1:10, 1:20 or 1:30 (adjusted for sIgE levels)
and HRP-conjugated anti-human IgE (Southern Biotech) diluted at 1:2000
in blocking solution were used for probing IgE-binding proteins.
IgE-binding fish proteins were visualized by incubating the membranes
with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo
Fisher), and the signal was acquired with the ChemiDoc MP Imaging System
(Bio-Rad).

\textbf{Mass spectrometry}

The IgE-reactive bands of raw fish extracts were excised from the
SimplyBlue SafeStain-stained SDS-PAGE gel, destained and digested with
trypsin following our laboratory's established
protocol\textsuperscript{21}. The tryptic digests were analyzed on the
UltrafleXtreme MALDI-ToF/ToF System (Bruker) for obtaining the peptide
mass fingerprints. Searches against the entire protein sequence database
(NCBI and UniProt) using the MASCOT search engine were then performed
for protein identification.

\textbf{Preparation of recombinant fish allergens}

The protein sequences of enolase (Sal s 2; Uniprot ID: B5DGQ7) and
aldolase (Sal s 3; Uniprot ID: B5DGM7) of \emph{Salmo salar
were}downloaded from the Uniprot database and reverse translated by MEGA
11.0. Enolase (Accession no: GBKA01005072.1) and aldolase (Accession no:
GEUQ01036650.1)

sequences of \emph{Ctenopharyngodon idella} were retrieved from the
transcriptome shotgun assembly deposited at GenBank (Accession no:
PRJNA325430) with tBLASTn search using Sal s 2 and Sal s 3 as the search
templates. The nucleotide sequences encoding the full-length allergens
were commercially synthesized and cloned into the His-tag expression
vector pET30(a)+. His-tagged recombinant allergens were then expressed
in \emph{Escherichia coli} {[}BL21 (DE3){]} by culturing in MagicMedia
(Invitrogen). Allergens were then purified using the HisPur cobalt spin
columns (Thermo Scientific) as per the manufacturer's instructions. For
parvalbumin purification, raw extracts extracted in Tris-HCl (pH 8.0)
were heated at 95\textsuperscript{o}C -- 100\textsuperscript{o}C for 15
mins, followed by centrifugation to remove precipitated heat labile
proteins. Supernatant containing soluble proteins were loaded into a
MonoQ anion exchange column (Cytiva) connected to a NGC 10 FPLC system
(Bio-Rad). Fractions containing pure parvalbumin were separated by a
gradient elution of 0 -- 1M NaCl in Tris-HCl (pH 8.0) and buffer
exchanged into PBS for downstream applications with an Amicon
centrifugal filter unit. Acid-soluble collagen was extracted from the
muscle of salmon and grass carp as described
previously\textsuperscript{22}. Briefly, diced fish meat was resuspended
in 10 volumes of 0.1mol NaOH overnight at 4°C with gentle agitation to
remove non-collagenous proteins. Upon centrifugation at 3000 rpm for 10
mins and pH neutralization with ultrapure water, fish meat was soaked in
10\% butanol and incubated for 24 h at 4°C with gentle agitation. After
centrifugation and washing in ultrapure water, samples were resuspended
in 10 volumes of 0.5M acetic acid for 4 days at 4°C with gentle
agitation. The supernatant were then salted-out by adding NaCl to a
final concentration of 2M. The resultant precipitate was dissolved in
0.5M acetic acid and dialyzed against distilled water.

The concentration and purity of purified fish allergens were determined
on the NanoDrop OneC spectrophotometer (Thermo Scientific) and SDS-PAGE,
respectively. Protein identity of the allergens was also confirmed by
mass spectrometry. All purified allergens were stored at -20°C until
use.

\textbf{Enzyme-linked immunosorbent assay (ELISA)}

Purified fish allergens diluted in coating buffer (100 mM
Na\textsubscript{2}CO\textsubscript{3}. 100 mM NaHCO\textsubscript{3},
pH 9.6) were coated onto MaxiSorp microtiter plates (Nunc) and incubated
overnight at 4°C. After washing the plates with 0.05\% Tween-20/PBS
(PBS-T) and blocking the plates with 5\% fetal bovine serum (Gibco)
diluted in PBS (blocking buffer) at room temperature for 2 hours, serum
samples diluted at 1:10 in blocking buffer were added for overnight
incubation at 4°C. Intensity of IgE binding was detected by incubating
the plates with biotinylated anti-human IgE antibodies (1:1000 dilution,
Vector Labs), HRP avidin D (1:1000 dilution, Vector Labs) and TMB
substrate (BD Biosciences). Upon terminating the reaction with 0.1M
sulfuric acid, the optical density (OD) at 450 nm was measured using a
microplate reader (BioTek). Results were considered positive only if the
OD is 3-fold higher than the mean of non-atopic negative controls.

\textbf{Statistical analysis}

GraphPad Prism (version 8.0, GraphPad Software) was used for graphical
presentation and statistical analyses. Quantitative variables were
described by median, range and interquartile range whereas qualitative
variables were described by absolute frequencies and percentages. P
\textless{} .05 was considered as statistical significance.

\textbf{Results}

\textbf{Demographics of fish allergic participants}

Sixty-seven and forty-six subjects fulfilling the inclusion criteria
were recruited from the pediatric allergy clinics in HK and the
Sagamihara National Hospital in Japan respectively (Table 1,
Supplementary Tables E1, E2). The median (range) age of fish allergic
subjects at evaluation was 6 (1-16) years and 10 (5-18) years for these
two populations, respectively. The most common clinical features shared
among the two populations were itchy mouth/throat (61.1\%, 69/113) and
urticaria (51.3\%, 58/113) while subjects from HK also commonly
displayed angioedema (59.7\%, 40/67) upon fish ingestion (Supplementary
Tables E1, E2). The two populations did not differ in terms of the
symptom severity as reflected from oFASS (p = 0.5251). SPT to fish mix
extract was only available for subjects recruited in HK with median
(range) SPT wheal size of 5 (0-11) mm, and 48 subjects (84.2\%) had
positive SPT (cutoff at 3mm). The sIgE results for cod, tuna, salmon and
grass carp are summarized in Table 1 and detailed in supplementary
Tables E1 and E2. No difference was detected in sIgE levels to these
fish species between the two populations, except for grass carp that HK
subjects had significantly higher sIgE level comparing to Japanese
subjects (Mann-Whitney test, p = 0.0020). Of note, 65/67 (97.0\%) and
34/37 (91.8\%) subjects from HK and Japan had positive sIgE to salmon
(cutoff at 0.35kUA/L), of whom 72.3\% and 70.6\% had high sIgE level and
above class III-VI, respectively. As for grass carp, 63/67 (95.5\%) and
25/30 (83.3\%) subjects from HK and Japan had positive sIgE, of whom
87.3\% and 56.0\% had high sIgE level and above class III-VI,
respectively. An overall positive correlation between sIgE levels to
salmon and grass carp was observed for both populations, by which a
stronger correlation was found among the Japanese subjects (r = 0.69, p
= 0.0002) than HK subjects (r = 0.33, p = 0.0061) based on Fisher r-to-z
transformation (z = 2.01, p = 0.04). Sera of Japanese subjects yielded
high sIgE response to salmon with an average grass carp/salmon sIgE
ratio of 0.70 (0.02-1.93) while sera of HK subjects yielded higher sIgE
response to grass carp with average ratio of 3.39 (0.02-20.8)
(Supplementary Figure 1). Pairwise comparison between grass carp- and
salmon-specific IgE levels also showed statistically higher grass
carp-specific IgE level among HK subjects but statistically higher
salmon-specific IgE level among Japanese subjects (Wilcoxon
matched-pairs signed rank test, p = 0.0008 and 0.0315 respectively).

\textbf{Identification of IgE-binding proteins in salmon and grass carp}

The extracted salmon and grass carp proteins were analyzed on SDS-PAGE
(Figure 1A). Sera from 45 subjects (30 samples from HK and 15 samples
from Japan; marked with asterisks* in Supplementary Tables E1 \& E2) and
non-atopic controls (n = 2) were used to investigate IgE-binding to
salmon and grass carp proteins by western blotting (Figure 1B \& C). No
IgE binding was observed for control sera. Serum samples from Japanese
fish allergic subjects showed more diverse IgE binding compared to those
from HK, including to grass carp extract despite of the lower IgE level
measured on ImmunoCAP. For both populations, the 11kDa band in salmon
and 10kDa band in grass carp represented the major IgE-binding protein.
In HK fish allergic subjects, 18/30 (60.0\%) and 16/30 (53.3\%) showed
IgE binding to this 10kDa protein in grass carp and salmon respectively,
while 10/15 (66.7\%) subjects in Japan had a positive reaction to the
10kDa proteins in salmon and grass carp extracts. Six (40.0\%) Japanese
subjects reacted to proteins with molecular sizes 130 kDa and 58 kDa.

Protein bands that showed IgE reactivity to at least three serum samples
were then excised from SDS-PAGE for allergen identification on mass
spectrometry. These included the 130kDa, 43kDa, 34kDa, 25kDa and 11kDa
bands from salmon extract, as well as the 43kDa, 34kDa and 10kDa bands
from grass carp extract. For salmon extract, the 130kDa protein was
identified as \selectlanguage{greek}α-\selectlanguage{english}collagen, 58kDa band as \selectlanguage{greek}β-\selectlanguage{english}enolase, 32kDa band as
aldolase A, 25kDa band as GAPDH and the 11kDa band as \selectlanguage{greek}β-\selectlanguage{english}parvalbumin
(Supplementary Table E3). Similarly, the 58kDa, 36kDa and 10kDa bands
from grass carp extract corresponded to \selectlanguage{greek}β-\selectlanguage{english}enolase, GAPDH and
\selectlanguage{greek}β-\selectlanguage{english}parvalbumin, respectively.

\textbf{Differential sensitization patterns to fish allergens between
populations}

To understand the IgE sensitization pattern, we purified the
well-characterized fish allergens including collagen, \selectlanguage{greek}β-\selectlanguage{english}enolase,
aldolase and \selectlanguage{greek}β-\selectlanguage{english}parvalbumin from both salmon and grass carp. All
allergens had \textgreater{}90\% purity as validated by SDS-PAGE
(Figures 2A \& B). IgE binding to these allergens coated onto ELISA
plates was evaluated using 46 serum samples from each of HK and Japan
subjects. The respective sensitization rates are summarized in Table 2.

For both fishes, parvalbumin was the major allergen that showed an
overall sensitization rate of 74.7\% (71/95) in fish allergic subjects
(Figures 2C-F) while enolase is the least common allergen (17.8\%
sensitization). Interestingly, the sensitization rates to salmon enolase
and aldolase, the heat-labile allergens, were higher among subjects from
Japan (15.2\% and 23.9\% respectively) than HK (4.3\% and 15.2\%
respectively). On the other hand, the sensitization rates to salmon and
grass carp collagen were higher among subjects from HK (43.5\% and
30.4\% respectively) than Japan (30.4\% and 17.8\% respectively). No
remarkable difference in parvalbumin sensitization rate could be found
between the two study cohorts. Mono-sensitization to salmon or grass
carp parvalbumin was the most common in both populations (28.3\% to
37.0\%). Only 7/96 subjects did not show IgE binding to parvalbumin but
other fish allergen, and collagen represented the leading sensitizer in
these patients (6/7 patients reacted to collagen). IgE-binding to both
parvalbumin and collagen was the most common form of co-sensitization
(13.0\% to 23.9\%).

For both populations, the sensitization rates to grass carp parvalbumin
(68.9\% for Japan and 78.3\% for HK) were higher than that to salmon
parvalbumin (63.0\% and 56.4\%) (Table 2). The IgE avidity (i.e.
OD450nm) of grass carp parvalbumin was also higher than salmon
parvalbumin as noted in the heatmap (Figure 2). Despite of the
significantly higher salmon-sIgE level than grass carp-sIgE level, IgE
avidity to grass carp parvalbumin was significantly higher than that to
salmon parvalbumin among the Japanese fish allergic subjects (Wilcoxon
matched-pairs signed rank test, p \textless{} 0.0001). However, no
difference could be detected for grass carp parvalbumin binding
intensity between the two populations regardless of the significantly
higher grass carp-sIgE level for the HK subjects. In contrast, both the
sensitization rates and IgE avidity of salmon collagen were higher than
those of grass carp collagens.

Analyses of specific sensitization were summarized in Table 3. Out of
the 36 HK subjects with positive grass carp parvalbumin-specific IgE,
ten were sensitized to grass carp parvalbumin only giving a
monosensitization rate to grass carp at 27.8\%. On the contrary, none
with positive salmon parvalbumin-specific IgE (n = 26) showed IgE
binding to salmon parvalbumin only. When taking both populations into
consideration, the overall rate of mono-sensitization to grass carp
parvalbumin (20.9\%) was higher than that to salmon parvalbumin (5.5\%).
On the contrary, mono-sensitization to salmon collagen (44.1\%) was
remarkably more common than to grass carp collagen (9.1\%). The two fish
species did not differ with respect to sensitization rate, IgE avidity
and rate of mono-sensitization to enolase and aldolase.

\textbf{Relationship between dietary habit and sensitization patterns}

Noting that Japanese subjects had higher salmon-sIgE level and a more
diverse sensitization profile to salmon fish allergens, we sought to
investigate if it might be related to different fish cooking
preferences. Salmon meat was cooked using different methods until its
core temperature reached 80\selectlanguage{ngerman}°C. Equal volumes of the extracted fish
proteins were then resolved on SDS-PAGE. A significant portion of
proteins were lost upon cooking (Figure 3). Interestingly, retention of
salmon proteins was the highest after baking followed by frying and
airfry and the lowest after steaming, boiling and lightwave cooking.
When salmon extracts were analyzed by mass spectrometry, protein bands
at 130kDa, 37kDa and 8kDa were retained following all cooking methods,
and these bands were identified as \selectlanguage{greek}α-\selectlanguage{english}collagen, GAPDH and parvalbumin,
respectively (Supplementary Table E4). On the other hand, protein bands
at 55kDa, 43kDa, 40kDa, 38kDa, and 34kDa were visualized most abundantly
in baked salmon extract, less after fry/airfry and absent in steamed or
boiled extracts. By mass spectrometry analysis, these proteins
corresponded to serum albumin, \selectlanguage{greek}β-\selectlanguage{english}enolase, actin, aldolase A and
tropomyosin, respectively.

\textbf{Discussion}

In this study, we delineated the allergen profiles of salmon and grass
carp using sera from two Asian populations. With the purified allergens
from both fish species, our study illustrates parvalbumin and collagen
as important biomarkers for fish allergy diagnosis. Our results also
indicate a wider allergen spectrum including sensitization to heat
labile allergens, as well as stronger sensitization to salmon than grass
carp among the Japanese fish allergic subjects where grass carp is
unavailable. Such differences highlights that the relevant fish species
as diagnostic marker and allergen sensitization are distinct in
different populations due to diverse dietary exposures and habits.

Ruethers et al. reported recently 10 allergens from catfish and 6
allergens from salmon\textsuperscript{13}. Specifically, tropomyosin and
TPI identified as fish allergens in their study are known shrimp
allergens\textsuperscript{23}. Despite positive shrimp-sIgE by ImmunoCAP
in six HK subjects, none showed reaction to fish tropomyosin or TPI in
our western blot. Our subjects also showed no detectable IgE binding to
allergens including creatine kinase, pyruvate kinase, L-lactate DH and
G-6-PI that were previously reported as catfish allergens. This might
partly indicate that these are only catfish-specific allergens and/or
the reactions are population-specific. Apart from the well-defined fish
allergens (i.e. parvalbumin, enolase, aldolase and collagen), we
consistently identified GAPDH as a putative fish allergen of both salmon
and grass carp\textsuperscript{12}. Such findings supported GAPDH to be
an important fish allergen.

Our present report further focuses on studying the more well-defined
fish allergens in terms of their sensitization rates. In agreement with
most studies that parvalbumin accounted for 70-95\% of allergic
reactions\textsuperscript{9,24}, parvalbumin is the major allergen in
grass carp and salmon with 74\% sensitization rate determined by western
blotting and ELISA. Collagen is the second commonest fish allergen with
37.5\% of subjects being sensitized. The reported rates of IgE binding
to collagen vary in different populations. For instance, Kalic et al.
analyzed samples from 101 fish allergic patients in Australia and showed
that 21\% of subjects displayed IgE binding to collagen from tuna,
barramundi and/or salmon\textsuperscript{14}. Kuehn et al. reported
19.3\% sensitization rate to gelatin of cod, salmon and/or tuna in 62
fish allergic patients\textsuperscript{16}. With only 21 patients,
Shimojo et al. reported a higher rate of 52.4\% sensitization to
collagen than to parvalbumin\textsuperscript{25} while Kobayashi et al.
reported also 50\% of sensitization rate to mackerel
collagen\textsuperscript{26}. IgE binding to collagen was also more
frequent in patients with more severe fish allergic symptoms than those
with mild-moderate responses while independent reports have shown
anaphylaxis provoked by gelatin/collagen\textsuperscript{15,27}. On the
other hand, Kalic et al. reported that 8/21 of collagen-IgE positive
patients showed IgE binding to collagen only but not
parvalbumin\textsuperscript{14}. Comparatively, seven of our 37
collagen-IgE positive subjects demonstrated moderate IgE binding to
collagen but absence of parvalbumin-specific IgE. Taken together,
collagen is a prevalent allergen in Asian populations. Fish allergic
patients can have IgE binding to collagen only, supporting that testing
for collagen should be included in the diagnostic workup for fish
allergy.

To our knowledge, only one study with 62 fish allergic subjects reported
the sensitization rates of 62.9\% and 50.0\% to enolase and aldolase,
respectively\textsuperscript{16}. Another smaller study by Ruethers et
al. showed that half of their 16 Australian patients showed weak to
moderate IgE reactivity to enolase in commercial tuna and salmon SPT
extracts\textsuperscript{28}. In our cohort, IgE binding to aldolase was
detected in 38.5\% of subjects while that to enolase was present in
17.8\% of subjects. We did not anticipate a major difference in the
reactivity of natural and recombinant aldolase and enolase as the reason
for lower sensitization as the recombinant enzymes were confirmed to be
reactive consistently by ELISA (data not shown) and western blots. Such
discrepancy might, on the other hand, be explained by differences in the
ethnicity and age of the studied populations: this study only recruited
children below 18 years old while many subjects studied by Kuehn et al.
were young adults (mean age 28.7, range 13-62 years). Among seven
subjects without parvalbumin-specific IgE in our cohort, only one
Japanese subject showed IgE binding to salmon and grass carp aldolase.
Testing of enolase and aldolase thus do not substantially improve the
diagnostic yield for fish allergy in Asians while detecting IgE against
parvalbumin and/or collagen might be sufficient to identify patients
with fish allergy.

This study found minimal binding of sera from HK subjects to allergens
other than parvalbumin by western blotting. A possible explanation was
the use of different serum samples for ELISA and western blot, different
sensitivity of the assays, intrinsically low abundance of these
allergens in fish extracts and the insoluble nature of collagen in
aqueous solution\textsuperscript{29}. Nevertheless, both ELISA and
western blot assays robustly indicated a wider spectrum of allergen
sensitization in Japanese than HK subjects. Japanese commonly consume
raw fishes, but this is a less likely reason for this observation given
the age-range of our recruited patients; the consumption of raw fish
might not be a major attribute to the sensitization to a wider spectrum
of allergen including the heat labile allergens. Our present study
showed, for the first time, differences in fish protein and allergen
profiles in relation to cooking methods at the same core temperature.
Despite higher cooking temperature with frying/baking compared with
steaming/boiling, frying/baking retained more heat liable allergens in
salmon meat. Steaming and boiling to make fish congee and soup are the
preferred methods of fish cooking in Hong Kong while baking and frying
of fish are more popular in Japan. For instance, 73.8\% of the
interviewed Japanese workers consumed fish by broiling (baking)
comparing to only 14.1\% for raw fish and 10\% by
stewing\textsuperscript{30}. Although we are aware that our present
study is limited by the lack of direct investigation into the dietary
habits of our studied population, our results support the importance of
dietary exposures, consumption and cooking preferences in determining
fish allergen sensitization in particular when the two populations did
not differ in terms of symptom severity. This factor should be
considered during the selection of allergens for allergy testing and the
interpretation of CRD results.

In the context of fish exposure, Japanese patients should not have eaten
grass carp due to the unavailability of this freshwater fish. Thus, IgE
binding to grass carp allergens in Japanese patients should be a result
of cross-reactivity that can be partly reflected from the stronger
positive correlation between grass carp and salmon sIgE levels than HK
subjects whom could be independently sensitized to the two fish species.
However, we observed that more Japanese patients reacted to grass carp
parvalbumin with higher OD values in ELISA, although salmon-sIgE level
was higher than grass carp-sIgE and dietary exposure to grass carp is
lacking in this population. The higher salmon-sIgE level might thus be
explained by IgE binding to other salmon allergens, particularly
collagen, when considering the lower parvalbumin content in crude salmon
extract, lower IgE affinity of salmon parvalbumin in ELISA, and the more
diverse salmon allergen binding pattern in western blots. On the other
hand, we also showed that one-fifth of subjects reacted only to grass
carp parvalbumin, which indicated that grass carp parvalbumin has higher
allergenicity than salmon parvalbumin. However, we are yet to know the
clinical reactivity to grass carp among the Japanese subjects and so the
question of improving assay sensitivity and specificity using fish
allergen (parvalbumin) with higher allergenicity remains open. The
varying allergenic potential of fish parvalbumins could be explained by
the specific sensitization of patients from their clinical
history\textsuperscript{8}. With the lack of grass carp in the local
Japanese diet, our results suggested the presence of higher diversity,
distinct and/or species-specific IgE-binding epitopes for grass carp
parvalbumin\textsuperscript{31}. It is of note that IgE sensitization
based on parvalbumin did not correlate well with patients' clinical
allergy status\textsuperscript{18}, and we postulate that the
identification of parvalbumin epitopes may help to resolve the
inconsistency in cross-reactivity of different fish species.

In summary, this study extends our understanding on the allergen profile
of two important fish species that parvalbumin and collagen are
important components for fish allergy diagnosis. We also illustrated
that the fish species extracts relevant for diagnosis and allergen
sensitization pattern differ among populations. Overall, this study
provides additional information for the implementation of relevant
extracts and components for precise fish allergy diagnosis.

\textbf{Acknowledgements}

We thank all subjects and their parents for participation.

\textbf{Conflicts of Interest}

All authors declared no competing interest exists.

\textbf{Author Contributions}

Performing laboratory assays and contributing to data analysis: CYYW,
NYHL, KJYX; Subject recruitment and sample collection: ASYL, NF, SS,
YSY, JSRD, MYWK, JWCHC, NF, WHC, GTC, QUL, DCKL, PKH, JSCW, ICSL;
Writing -- original draft: CYYW, NYHL; Funding acquisition and work
supervision: GWKW, ME, TFL. All the authors have read and approved the
manuscript.

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\textbf{Hosted file}

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\textbf{Hosted file}

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