# Optimization of the diagnosis and characterization of gibberellin-regulated protein sensitization

Lorenzo Cecchi, Pascal Poncet, Lucia Maltagliati, Giulia Carli, Donatella Macchia, Laura Maggi, Elisa Meucci, Paola Parronchi, Alessio Mazzoni, Lorenzo Salvati, et al.

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## To cite this version:

Lorenzo Cecchi, Pascal Poncet, Lucia Maltagliati, Giulia Carli, Donatella Macchia, et al. Optimization of the diagnosis and characterization of gibberellin-regulated protein sensitization. Annals of Allergy, Asthma and Immunology, 2023, 132 (1), pp.82-90.e1. 10.1016/j.anai.2023.09.011. pasteur-04675298

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# Optimization of the diagnosis and characterization of gibberellin-regulated protein sensitization: an Italian cohort study

Cecchi Lorenzo MD\*¹, Poncet Pascal PhD\*2,3, Maltagliati Lucia MD⁴, Carli Giulia MD¹, Macchia Donatella MD⁴, Maggi Laura PhD⁵, Meucci Elisa MD⁴, Parronchi Paola PhD⁵,6, Mazzoni Alessio PhD⁵, Salvati Lorenzo MD⁵, Scala Enrico MD⁷, Sénéchal Hélène PhD², Aizawa Tomoyasu PhD⁸, Villalta Danilo MD⁹, Annunziato Francesco PhD°5,10, Cosmi Lorenzo PhD°5,11, Farsi Alessandro MD°1

1. SOSD Allergology and Clinical Immunology, Ospedale S. Stefano, Prato, USL Toscana Centro, Italy

2. Armand Trousseau Children Hospital, APHP, Biochemistry department, Allergy & Environment research team, Paris France

3. Institut Pasteur, Immunology department, Paris, France

4. SOSD Allergology and Clinical Immunology, Ospedale S. Giovanni di Dio, Firenze, USL Toscana Centro, Italy

5. Department of Experimental and Clinical Medicine, University of Florence, 50134 Firenze, Italy

6. Immunology and Cell Therapy Unit, Careggi University Hospital, 50134 Firenze, Italy

7. Clinical and Laboratory Molecular Allergy Unit, IDI - IRCCS, Rome, Italy

8. Hokkaido University, Protein Science Laboratory, Sapporo, Japan

9. Immunologia e allergologia, Ospedale S. Maria degli Angeli, Pordenone, Italy

10. Flow Cytometry Diagnostic Center and Immunotherapy (CDCI), Careggi University Hospital, 50134 Firenze, Italy

11. Immunoallergology Unit, Careggi University Hospital, 50134 Florence, Italy

**Corresponding author:**

Cecchi Lorenzo

SOSD Allergology and Clinical Immunology, Ospedale S. Stefano, via Cavour 87/89, 59100, Prato, USL Toscana Centro, Italy

Tel. 0039 057480732; lorenzo.cecchi@unifi.it

**Conflict of interest:** L.C. has received consultant arrangements and personal fees for lectures from Thermo Fisher Scientific, GSK, ALK Abello, Sanofi, Novartis, Astra Zeneca outside the submitted work. P.P. has received consultant arrangements/personal fees for lectures from GSK, Novartis, LeoPHARMA. E.S. has received consultant arrangements and speakers' bureau participation from Stallergenes, Thermo Fisher Scientific, and DASIT S.p.A. and non-financial support from Macroarray Diagnostics, Vienna, all outside the submitted work. All other authors declare no conflict of interest.

**Funding source:** Funds of Dept. of Experimental and Clinical Medicine of University of Florence (ex-60%) derived from Ministero dell'Istruzione, dell'Università e della Ricerca (Italy); Italian Ministry of Health, Current Research Program 2021-2023.

**Keywords:** Cup s 7, diagnosis, gibberellin-regulated protein, pollen food allergy, Pru p 7.

**Word Count:** 3790.

**Figures:** 4.

**Tables:** 2.

<footnote>* joint first authors</footnote>

<footnote>° joint senior authors</footnote>
1 Introduction

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Pru p 7 is an allergen belonging to the Gibberellin-Regulated Protein (GRP) also known as Snakin/GASA (Gibberellic Acid-Stimulated Arabidopsis) protein family. Members of the GRP family are widely distributed within the plant kingdom. They exhibit antimicrobial properties and the amino-acid sequence shows 12 cysteines, at conserved positions, engaged in 6 disulfide bridges, that confer the protein thermostability and proteolysis resistance¹. Pru p 7 was the first GRP allergen isolated and described in 2013 in peach² and to date, seven other plant food allergens belonging to this allergenic family have been identified: Pru m 7 in Japanese apricot³, Pun g 7 in pomegranate⁴, Cit s 7 in orange⁵, Pru av 7 in cherry⁶, Cap a 7 in bell pepper⁷, Fra a GRP in strawberry⁸ and Vit v GRP in grape⁹. The first description of an association between a respiratory allergy to cypress pollen and allergy to peaches dates back to 2006¹⁰ and the involvement of cross-reactive GRPs in this association was then demonstrated in larger studies in France¹¹⁻¹⁴, Japan¹⁵ and Italy¹⁶. Currently, cross-reactive GRPs in pollen are only reported in Cupressaceae from Mediterranean cypress (Cup s 7, *Cupressus sempervirens*), mountain cedar (Jun a 7, *Juniperus ashei*), Japanese cedar (Cry j 7, *Cryptomeria japonica*) and Arizona cypress (Cup a GRP, *Hesperocyparis arizonica*). Comparative analysis of the primary structure of GRPs from plant food and Cupressaceae pollen shows high sequence conservation between these molecules, although they belong to different plant taxonomic orders¹⁷.

The real prevalence of food allergy due to sensitization to proteins belonging to the GRP family is not fully known and the association with pollen GRP sensitization is rarely studied. In Japan, the prevalence of food allergy caused by GRPs was 65% in 100 patients with reactions to peach who were not sensitized to Bet v 1 and Profilin¹⁸ while about 20% of Japanese cedar allergic patients were shown to be Pru p 7 sensitized¹⁹. However, in these two studies, no data were reported on the sensitization to Cry j 7, the pollen GRP from Japanese cedar. The only Japanese study where plant food and Japanese cedar pollen GRP sensitizations were assessed shows that 46% of young patients
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suffering from pollen food syndrome were sensitized to GRP while no GRP sensitization was found
in Japanese cedar pollen-only allergic young patients¹⁵. In France, it has been reported that Pru p 7
may represent a major allergen of peach in the Southern regions, with a prevalence of sensitization
towards Pru p 7 greater than 60% in patients with food allergy induced by the consumption of peach²⁰.

Although a positive correlation with the amount of Cupressaceae pollen counts in the atmosphere
was mentioned, Cup s 7 IgE reactivity data were lacking. In Italy, sensitization to Pru p 7 appears to
be uncommon, with approximately 2% of cypress pollen allergic patients showing a Pru p 7
sensitization, independently of different national geographical areas²¹ in keeping with previously
published results on Cup s 7 sensitization (formerly BP14)²².

In Japan as well as in France, Pru p 7 sensitization was shown to trigger systemic reactions,
including anaphylaxis, in both adults and young adults²³ as well as in pediatric patients²⁴.
Furthermore, Pru p 7-specific IgE levels were reported to be positively correlated to the severity of
the reactions¹⁸,²⁰,²⁵. Clinically, symptoms are similar to other types of food allergies, but an
association has been described between allergy to Pru p 7 and facial angioedema, especially of the
periorbital region, as well as a sensation of laryngeal tension²⁶. The association between the onset of
clinical manifestations and cofactors such as physical exercise or the intake of non-steroidal anti-
inflammatory drugs (NSAIDs), was reported with variable frequency, ranging between 61.5%¹⁸ and
16.7%³.

The study aimed to optimize the diagnostic work-up of a group of patients with suspected
allergy to Pru p 7 by evaluating the specific IgE profiles of patients to cypress pollen allergens and
various GRPs using western blot, in addition to singleplex (ImmunoCap) and multiplex (ALEX2)
assays, and analysing the ability of Pru p 7 and peach extract to induce basophil activation. The results
lead to an optimal characterization of Pru p 7 sensitized patients, by identifying within the same
cohort culprit foods and tree pollen allergens involved, assessing the severity of reactions and the
requirement of cofactors for symptom onset.
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**Methods**

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*Study Populations*

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Consecutive adult patients referring to SOS Allergology and Clinical Immunology of Prato and the San Giovanni di Dio Hospital, Florence (Azienda USL Toscana Centro, Firenze, Italy) with suspected allergy to Pru p 7 were enrolled in the study between June 2018 and February 2020. Two control populations with food allergy due to sensitization to Pru p 3 and respiratory allergy to cypress pollen were also included in the study.

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Inclusion criteria for patients with suspected allergy to Pru p 7 were: (i) systemic immediate-type allergic reaction, following the ingestion of plant food, (ii) positive skin prick tests (SPT) for LTP-enriched peach extract (B717 Soluprick by Alk Abelló, Milan, Italy) and cypress extract (SPT 613, *Cupressus sempervirens*, Lofarma, Milan, Italy), and (iii) absence of specific IgE directed towards the recombinant Pru p 3, the LTP allergen from peach. The determination of specific IgE for food was performed before the enrolment (ImmunoCAP system, Thermo Fisher Scientific, Uppsala, Sweden).

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The control population of patients with food allergy sensitized to Pru p 3 was enrolled according to the following inclusion criteria: (i) systemic immediate-type allergic reaction following the ingestion of plant food, (ii) positive SPT to LTP-enriched peach extract (ALK) and negative to cypress extract (Lofarma) and (iii) presence of specific IgE directed against the recombinant Pru p 3 allergen from peach. Similarly to the study cohort, the determination of food-specific IgE was performed before study enrolment.

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The control population of patients with respiratory allergy (rhinoconjunctivitis or allergic asthma) associated with cypress pollen sensitization comprised 9 patients, selected according to the following inclusion criteria: (i) respiratory symptoms (rhinoconjunctivitis or allergic asthma) occurring during the *Cupressaceae* pollination season, (ii) absence of adverse reactions following ingestion of food, and (iii) negative SPT to LTP-enriched peach extract and positive to cypress extract.
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Enrolled populations are summarized in Figure 1.

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The severity of reactions was graded according to the Mueller grading system. Patients enrolled in the study provided written informed consent to participate in the study. All the enrolled patients were evaluated for medical history, *in vivo* diagnostic tests (SPT and prick by prick with fresh food), and *in vitro* tests (basophil activation test, specific IgEs and determination of IgE directed against GRPs by western-blot). The study was approved by the local Ethical Committee (ID 13721 – 19/18 PO) and by the Ethical Committee of IDI-IRCCS (IDI-IRCCS CE 579/2021).

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### *In vivo* skin tests

Skin allergy tests were performed by skin scarification (SPT) by applying a drop of allergen extract to the volar surface of the forearm. SPTs were performed with LTP-enriched peach extract (B717 Soluprick by Alk Abelló, Milan, Italy) and with cypress extract (SPT 613, *Cupressus sempervirens*, Lofarma, Milan, Italy), as well as a standardized panel of inhalant and food allergens. The examination with fresh food (prick by prick, PBP) was performed using the following foods: peach jam (peach jam, Hero, Verona, Italy), apricot jam (apricot jam, Hero, Verona, Italy), lemon jam (organic Sicilian lemon jam, Sicilian, Italy), fig jam (organic fig jam, Vivi Verde COOP, Italy or organic fig extra jam, Biosicilia, Italy), orange juice (Orange Nectar, Esselunga, Italy).

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### Basophil Activation Test

Basophil activation tests were performed on the whole blood of all patients using a commercially available kit and following the manufacturer's protocol (Basotest™, Glycotope Biotechnology, Berlin, Germany). Peach and cypress extracts, and recombinant Pru p 7 were used as allergen reagents (see Patients and Methods, protein reagents). Extract and molecular allergens were used at final concentrations of 1 µg/mL. Allergen reagents were initially tested at 1000, 100, 10, and 1 ng/mL (Supplementary eFigure 1). Negative and positive controls (patient background and formyl-methionyl-leucyl-phenylalanine [fMLP] activation) were assayed for each patient. Stimulation and
106 washing buffer solutions used were those provided with the commercial kit. A tricolour flow
107 cytometric assay was applied to detect basophil activation²⁰,²⁷. Fluorochrome-conjugated monoclonal
108 antibodies used were anti-CD63 and anti-IgE (FITC and PE respectively as reagent F of BasotestTM
109 Kit), and anti-CD45 (PerCP, by BD Biosciences). Samples were acquired on a BD LSR II flow
110 cytometer with FACSDiva Software (BD Biosciences). Five hundred basophils or more were
111 analyzed for each stimulation condition. BAT was considered positive for a specific allergen
112 concentration when CD63 expression (percentage of CD63+ cells among basophils) was above 15%
113 subtracting background. The gating strategy is represented in Supplementary eFigure 2.
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Protein reagents

116 Protein extraction

117 Proteins from Mediterranean cypress *Cupressus sempervirens* pollen grains (10%, w:v)
118 (Allergon AB, Angelholm, Sweden) or peach were extracted by grinding on a multidirectional grinder
119 FastPrep 24 (MP-Biomedicals, Illkirch, France) in phosphate buffer saline (PBS) in presence of 1mm
120 glass beads. After centrifugation and filtration, the supernatant was dialyzed against 1:10 diluted PBS,
121 lyophilized, dissolved in distilled water and stored as aliquots at -20°C.

122 Recombinant proteins

123 The GRPs from Mediterranean cypress (Cup s 7), Japanese cedar (Cry j 7), peach (Pru p 7),
124 and potato (snakin-1) were produced as recombinant proteins in *Pichia pastoris* and purified
125 essentially as described previously¹⁵,²⁸. Correct folding and purity were assessed by mass
126 spectrometry, chromatography, circular dichroism and nuclear magnetic resonance. IgE reactivity
127 was assessed by immunoassay using different sera originating from patients diagnosed allergic to the
128 respective plant foods or pollen grains.

IgE immunoreactivity
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IgE dosage
---
Specific IgEs to Pru p 3, Bet v 1, Bet v 2 and Pru p 7, were evaluated using ImmunoCAP, (Thermofisher, Uppsala, Sweden), following the manufacturer's instructions. Values > 0.15 kUA/L were considered positive. The serum IgE reactivity profile was also analyzed for each patient using Allergy Explorer-ALEX²® (Macro-ArrayDX, Vienna, Austria) platforms following the manufacturer's instructions. The microchip allows the simultaneous IgE testing of 300 allergens (molecules and extracts) including Pru p 7. Results are expressed in arbitrary units and a value of ≥ 0.3 kUA/L was considered positive.

### *Western blot*

Western blot (WB) was essentially performed as described elsewhere⁵. Briefly, proteins from pollen extract were first separated by SDS-PAGE in the unreduced condition in an 8-18% gradient polyacrylamide gel. They have then transferred onto cyanogen bromide-activated nitrocellulose membranes (NCa). Membrane strips were then individually incubated with patient serum and IgE binding was revealed with alkaline phosphatase coupled antibodies directed against human IgE (ε specific, Sigma-Aldrich, Saint Louis, USA) followed by appropriate substrate (mixture of Nitro Blue Tetrazolium and Bromo Chloro Indolyl Phosphate).

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147 **Results**

148 *Patients' characteristics*

149 Between June 2018 and February 2020, a total of 37 patients were enrolled at the Allergology
150 and Clinical Immunology Unit in Prato and at the Allergology and Immunology Unit, San Giovanni
151 di Dio Hospital, Florence (Azienda USL Toscana Centro), in Italy. Twenty-three patients had
152 suspected allergy to Pru p 7, 5 patients were enrolled in the control group with food allergy sensitized
153 to Pru p 3 and 9 patients in the control group with cypress pollen allergy. Table 1 shows the basic
demographics and clinical features of the enrolled patients.
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155 Co-sensitization to plant panallergens was assessed by measuring IgE to Bet v 1 and Bet v 2
156 (ImmunoCAP). Of the 23 patients with suspected allergy to Pru p 7, 5 patients (out of 23, 21.7%) had
157 IgE to Bet v 1. None of the patients showed sensitization to profilin, evaluated by both skin prick
tests and ImmunoCAP assay.
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159 *Culprit foods*

160 All patients from group 1 experienced immediate systemic adverse reactions following the
161 ingestion of food(s) of plant origin. The food most commonly implicated as a cause of adverse
162 reaction was peach, representing the culprit food in 21/23 patients (91.3%), followed by fig in 5/23
163 patients (21.7%), orange in 5/23 patients (21.7%), apple in 5/23 patients (21.7%), plum in 5/23
164 patients (21.7%) and lemon in 4/23 patients (17.4%) (Table 2). From a botanic point of view,
165 reactions were mainly triggered by Rosaceae in 59% of plant food allergy cases dominated by peach,
166 Rutaceae in 14.8% of cases (5 orange and 4 lemon); Moraceae (5 fig); Lythraceae (2 pomegranate);
167 Cucurbitaceae (1 melon and 1 watermelon); Solanaceae (1 bell pepper and 1 eggplant); Fabaceae (1
168 peanut) and berries (in one patient). In one patient (Pt 005), the food triggering the adverse reaction
169 was not identified.
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170 The majority of patients presented a reaction following the ingestion of multiple foods (Figure
171 2a). On the contrary, 7 patients (30.4%) presented an adverse reaction following the ingestion of a
172 single food which in 85.7% of cases was peach, while in one patient the only culprit food was
watermelon. Sixteen patients reacted with more than one food of plant origin, of which seven patients with 2 foods (30.4%), 2 patients with three foods (8.7%), and the remaining 7 patients with 4 or more foods (30.4%).

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## Severity of Reactions

Given our inclusion criteria, all studied patients presented an immediate systemic adverse reaction following the ingestion of the culprit food. Out of the 21 patients reacting to peach, 6 had Mueller grade I reactions (28.6%), 7 grade II (33.3%), 6 grade III (28.6%) and 2 patients experienced grade IV (9.5%) reactions. Apple, plum, fig, cherry, apricot, peanut, watermelon, melon, eggplant and bell pepper induced grade I or II reactions depending on the patient. In addition to peach, grade III and IV reactions were also observed with the two Rutaceae fruits (orange and lemon) and pomegranate.

Only 2 patients (8.7%) had a highest Mueller grade IV with peach or lemon whereas grade III, II and I were observed for 8 (34.8%), 7 (30.4%) and 6 (26.1%) patients, respectively (Figure 2b).

Table 2 summarizes offending food(s) and respective reaction severity, for each patient with suspected Pru p 7 allergy.

In the group with suspected allergy to Pru p 7, 6 patients (26.1%) experienced an adverse reaction, at least on one occasion, in the presence of a cofactor. Exercise-induced anaphylaxis was a co-factor for 4 patients (17.4%), 2 with peach (Mueller grade 3 or 2) and 2 with Nutella or berries (Mueller grade 1). Concomitant administration of NSAIDs was involved as a cofactor for 2 patients (8.7%), one with peach (Mueller grade 2) and the other one with apple (Mueller grade 1).

## Basophil Activation Test and Pru p 7 IgE detection

In the population of subjects with suspected allergy to Pru p 7, BAT was found positive for rPru p 7 in 20 patients (87%) that showed significant basophil activation also for peach extract (Figure 3a). Pt036 showed negative results with Pru p 7 and a very low positivity with peach extract. In addition,

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BAT was positive for cypress extract in 22 patients (95.7%) (Figure 3a). Pt032 was found negative for the three reagents.

sIgE to Pru p 7 were also tested by Singleplex (immunoCAP) and multiplex (ALEX²) assays in group 1 and 22/23 (95.7%) and 17/23 (73.9%) resulted in a positive test, respectively.

The control population with LTP-mediated food allergy without sensitization towards cypress pollen included 5 patients. BAT was positive for peach extract in all the cases and negative for rPru p 7 in all 5 subjects (Figure 3b).

The control population with respiratory sensitization towards cypress pollen in the absence of food allergy included 9 patients. Similarly, in this group, BAT were found negative for rPru p 7 in all the patients and positive for cypress pollen extract in 8 patients (88.9%). However, one patient (11.1%), despite a negative history of food adverse reactions, had weak positive BAT results towards peach extract.

### Western-blot

Out of 23 patients with suspected Pru p 7 allergy (group 1), 17 exhibited a reactivity at 14 kDa corresponding to the reactivity area of cypress pollen GRP as previously demonstrated¹⁵,²⁹. No Cup s 7 reactivities were found in the LTP<sup>pos</sup> control group (group 2), as expected from the study design. In the cypress<sup>pos</sup> peach<sup>neg</sup> control group (group 3), 2 patients were IgE reactive to Cup s 7 (Figure 4a). These results mean that pollen GRP sensitization may not be systematically associated with the known GRP-containing fruit.

Five patients from group 1, 2 from group 2 and 2 from group 3 were further tested against three other recombinant GRPs by western blots, Pru p 7 from peach, Cry j 7 from Japanese cedar pollen and snakin-1 from potato, a GRP not yet reported allergenic and sharing 82% sequence identity with Pru p 7 (Figure 4b). The GRP reactivity profile of patients is diversified. Pt001 is very low in reactivity against Cup s 7, Pru p 7 and Cry j 7 but high against snakin-1. This is not the case for Pt003 or Pt006 unable to recognize snakin-1 while highly positive on the other GRPs. None of the patients from groups 2 and 3 recognized GRPs.
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Additional information about in vitro test results may be found as Supplementary Text and

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Supplementary eTable 1.

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228 **Discussion**

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230 Since the first isolation and description of Pru p 7 in peach², numerous data about food allergy
231 due to sensitization to highly homologous and conserved proteins belonging to the GRP family have
232 been collected, leading to the definition of a novel plant-food allergy syndrome (PFAS)¹⁷. Food and
233 pollen GRP sensitizations were assessed by few studies¹¹⁻¹⁴ and clinically and biologically
234 documented by Iizuka et al in Japanese cedar pollen allergic cohorts¹⁵. Despite the observation of a
235 positive correlation with the geographical distribution of *Cupressaceae* in Europe²⁰, cypress pollen
236 and peach fruit GRP sensitization has never been biologically and clinically evaluated in a same
237 European cohort.

238 Pru p 7 sensitization has already been associated with systemic reactions, including
239 anaphylaxis, in adults²⁰, adolescents²³ and children²⁴ but it may also be asymptomatic or associated
240 with oral allergy syndrome²¹. Cofactors like physical exercise and NSAIDs intake as well as Pru p 7-
241 specific IgE levels have been reported to influence the occurrence and severity of reactions³,¹⁸,²⁰,²⁵.

242 Our study demonstrated that a combination of several analytical methods associated to an
243 exhaustive clinical analysis is required to improve and ascertain GRP hypersensitivity detection in a
244 population of atopic patients. We proposed that the three initial selection criteria should include (i)
245 positive SPT to LTP-enriched peach extract, (ii) negative immunoassay to LTP (Pru p 3) and (iii)
246 positive SPT to cypress pollen extract. In the so-selected population, sensitization to Pru p 7 was
247 confirmed in 96% and 74% of patients by singleplex (ImmunoCAP) and multiplex IgE detection
248 assays (ALEX²), respectively. By western blot, 74% of selected patients reacted to Cup s 7 either in
249 total pollen extract or on recombinant allergen (data not shown), the cypress pollen GRP. Basophil
250 activation test exhibited 87% response to Pru p 7 in the selected population, while none of LTP
251 positive patients was reactive to Pru p 7 with this assay.

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253 Taken individually each method has its own sensitivity and specificity and, when combined,
despite a few discrepancies, they were revealed to be complementary. Differences between the
various assays can be related to the different nature of reagents, raw extracts or recombinant
molecules as well as to possible repertoire differences between FcεRI-bound IgE or circulating
specific IgEs. Moreover differences in chemical coupling procedures to solid phase in singleplex or
multiplex assays may impact the epitope presentation of the allergen and consequently specificity
and sensitivity.

Whether Pru p 7 assay is able to detect any GRP sensitization remains to be more thoroughly
studied. GRPs display a conserved primary structure with cystein residus at same location conferring
to the molecule very similar 3D structures. However *in silico* IgE epitope mapping show some
differences between different GRPs¹ and monoclonal antibodies raised against Pru p 7 are able to
discriminate pollen GRP from peach GRP³⁰.

As already reported in the literature (reviewed in¹), GRP sensitization encompasses several
botanical varieties of culprit food, Rosaceae, Rutaceae, Solanaceae, Lythraceae and Vitaceae. The
Pru p 7-positive patients studied herein, were also found multi food-reactive and almost all of them
presented a reaction to peach. Botanical species more frequently implicated in adverse reactions in
our study, were Rosaceae, Rutaceae and Moraceae. Interestingly no GRP was reported, up to now, in
Moraceae. Given the ubiquity of the GRP and their crucial role in plant defence and redox
homeostasis, a large number of plant food should be expected to contain allergenic GRP. Obviously,
this is not the case as exemplified by the potato GRP, snakin-1, sharing 82% sequence identity with
Pru p 7 and not, as yet, described as an allergen although Cap a 7, a member of the same Solanaceae
family, was shown to be allergenic⁷. As well, apple GRP, while thoroughly studied, was not found
allergenic³¹. The allergenicity of GRPs probably relies on circumstantial or molecular cofactors.
Cofactors like physical exercise or NSAIDs were shown in this study to be associated with the adverse
reaction in 26% of cases, a percentage in agreement with published data³,¹⁸.

GRPs, as indicated by their name, are produced in response to the endogeneous phytohormone
gibberellin whose structure and role are very well known, particularly in plant and fruit growth³².
Exogenous gibberellin treatment is nowadays widely used in modern agriculture to increase yield
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and/or quality of many plant food species³³⁻³⁵. This treatment may account for an over expression of GRP and consequently an increase of their allergenicity. Alternatively diverse dietary habits in distinct geographical areas may also impact the GRP exposure and its sensitizing ability.

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Taken together, our data demonstrate the reliability of combined diagnostic methodologies to optimize the characterization of GRP allergy. Clinical suspicion should rise when a patient reporting an adverse reaction to food presents LTP-enriched peach together with cypress pollen skin reactivity. The availability of Pru p 7 immunoCAP assay will confirm GRP sensitization and also give insights about the frequency of a possible co-sensitization in LTP-positive patients. Clinicians should be aware of the weaker sensitivity of multiplex assays compared to singleplex IgE detection. Western blot is mainly useful for research purposes and is crucial to help identifying novel GRP proteins from food extracts. Our study supports and validates the diagnostic algorithm proposed in the chapter GRP of the second edition of the Molecular Allergology User's Guide (https://hub.eaaci.org/resources_guidelines/molecular-allergology-users-guide-2-0/).

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In conclusion, our cohort of Pru p 7-Cup s 7 allergic patients confirms a clinical syndrome characterized by respiratory allergy and food allergy, where the most frequent culprit is peach fruit and the severity of reaction may range from mild to grade 4 anaphylaxis. Patients and clinicians should be alert about cofactors determining the onset or intensity of symptoms. Diagnosis can rely on Pru p 7-specific IgE assays, which have shown good sensitivity and specificity.

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300 References

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302 303 1. Iizuka T, Barre A, Rougé P, Charpin D, Scala E, Baudin B, et al. Gibberellin-regulated proteins: Emergent allergens. *Front Allergy*. 2022;3(Sept 9):877553.

304 305 306 2. Tuppo L, Alessandri C, Pomponi D, Picone D, Tamburrini M, Ferrara R, et al. Peamaclein--a new peach allergenic protein: similarities, differences and misleading features compared to Pru p 3. *Clin Exp Allergy*. 2013;43(1):128-40. Epub 2013/01/03.

307 308 3. Inomata N, Miyagawa M, Aihara M. Gibberellin-regulated protein in Japanese apricot is an allergen cross-reactive to Pru p 7. *Immun Inflamm Dis*. 2017;5:469-79.

309 310 311 4. Tuppo L, Alessandri C, Pasquariello MS, Petriccione M, Giangrieco I, Tamburrini M, et al. Pomegranate cultivars: identification of the new IgE-binding protein pommaclein and analysis of anti-oxidant variability. *J Agric Food Chem*. 2017;65(13):2702-10.

312 313 5. Inomata N, Miyakawa M, Ikeda N, Oda K, Aihara M. Identification of gibberellin-regulated protein as a new allergen in orange allergy. *Clin Exp Allergy*. 2018;48(11):1509-20. Epub 2018/08/14.

314 315 6. Inomata N. [IUIS/WHO Pru av 7 description]. http://wwwallergenorg/searchphp?allergensource=sweet+cherry&searchsource=Search. 2019.

316 317 318 319 7. Takei M, Nin C, Iizuka T, Pawlikowski M, Selva M-A, Chantran Y, et al. Capsicum allergy: involvement of Cap a 7, a new clinically relevant Gibberellin-regulated protein cross-reactive with Cry j 7, the Gibberellin-regulated protein from Japanese cedar pollen. *Allergy Asthma Immunol Res*. 2022;14(4):(in press).

320 321 322 8. Inuo C, Okazaki F, Shiraki R, Tanaka Y, Momma K, Kondo Y, et al. Generalized allergic reaction in response to exercise due to strawberry gibberellin-regulated protein: a case report. *Allergy Asthma Clin Immunol*. 2022;18(1):49.

323 324 325 326 9. Kobayashi T, Shimojo N, Nakamura M, Matsunaga K, Nakata J, Tagami K, et al. A case of food-dependent exercise-induced anaphylaxis due to grape gibberellin-regulated protein. *Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology*. 2022;33(9):e13850.

327 328 10. Hugues B, Didierlaurent A, Charpin D. Cross-reactivity between cypress pollen and peach: a report of seven cases. *Allergy*. 2006;61(10):1241-3.

329 330 331 11. Sénéchal H, Santrucek J, Melcova M, Svoboda P, Zidkova J, Charpin D, et al. A new allergen family involved in pollen food associated syndrome: snakin/gibberellin regulated proteins *J Allergy Clin Immunol*. 2018;141(1):411-4.

332 333 12. Sénéchal H, Charpin D, Couderc R, Poncet P. Gibberellin-regulated proteins and the enigma of the « missing link ». *Rev Fr Allergol*. 2018;58:63-7.

334 335 336 13. Poncet P, Aizawa T, Senechal H. The subtype of Cupressaceae pollinosis associated with Pru p 7 sensitization is characterized by a sensitization to a cross-reactive gibberellin-regulated protein in cypress pollen: BP14. *Clin Exp Allergy*. 2019;49(8):1163-6.

337 338 339 14. Senechal H, Keykhosravi S, Couderc R, Selva MA, Shahali Y, Aizawa T, et al. Pollen/Fruit Syndrome: Clinical Relevance of the Cypress Pollen Allergenic Gibberellin-Regulated Protein. *Allergy Asthma Immunol Res*. 2019;11(1):143-51.

340 341 15. Iizuka T, Takei M, Saito Y, Rumi F, Zheng J, Lu X, et al. Gibberellin-Regulated Protein sensitization in Japanese cedar (*Cryptomeria japonica*) pollen allergic Japanese cohorts. *Allergy*. 2021;76:2297-302.

342 343 344 16. Tuppo L, Alessandri C, Giangrieco I, Ciancamerla M, Rafaiani C, Tamburrini M, et al. Isolation of cypress gibberellin-regulated protein: Analysis of its structural features and IgE binding competition with homologous allergens. *Mol Immunol*. 2019;114:189-95.

345 346 17. Poncet P, Senechal H, Charpin D. Update on pollen-food allergy syndrome. *Expert Rev Clin Immunol*. 2020;16(6):561-78. Epub 2020/07/22.
347 18. Inomata N, Miyagawa M, Aihara M. High prevalence of sensitization to gibberellin-regulated protein (peamaclein) in fruit allergies with negative immunoglobulin E reactivity to Bet v 1 homologs and profilin: Clinical pattern, causative fruits and cofactor effect of gibberellin-regulated protein allergy. *J Dermatol* 2017;44(7):735-41.

351 19. Koga T, Tokuyama K, Ogawa S, Morita E, Ueda Y, Itazawa T, et al. Surveillance of pollen-food allergy syndrome in elementary and junior high school children in Saitama, Japan. *Asia Pacific allergy*. 2022;12(1):e3. eCollection 2022 Jan.

354 20. Klingebiel C, Chantran Y, Arif-Lusson R, Ehrenberg AE, Ostling J, Poisson A, et al. Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy. *Clin Exp Allergy*. 2019;49(4):526-36.

357 21. Asero R, Abbadessa S, Aruanno A, Barilaro G, Barzaghi C, Bignardi D, et al. Detection of Gibberellin-Regulated Protein (Peamaclein) Sensitization among Italian Cypress Pollen-Sensitized Patients. *J Investig Allergol Clin immunol*. 2022;32(1):40-7. Epub 2020/08/01.

360 22. Shahali Y, Nicaise P, Brazdova A, Charpin D, Scala E, Mari A, et al. Complementarity between Microarray and Immunoblot for the Comparative Evaluation of IgE Repertoire of French and Italian Cypress Pollen Allergic Patients. *Folia Biol (Praha)*. 2014;60(4):192-201. Epub 2014/08/26.

363 23. Ando Y, Miyamoto M, Kato M, Nakayama M, Fukuda H, Yoshihara S. Pru p 7 Predicts Severe Reactions after Ingestion of Peach in Japanese Children and Adolescents. *Int Arch Allergy Immunol*. 2020;181(3):183-90. Epub 2019/12/11.

366 24. Biagioni B, Tomei L, Valleriani C, Liccioli G, Barni S, Sarti L, et al. Allergy to Gibberellin-Regulated Proteins (Peamaclein) in Children. *Int Arch Allergy Immunol*. 2021;182(12):1194-9.

368 25. Inomata N, Okazaki F, Moriyama T, Nomura Y, Yamaguchi Y, Honjoh T, et al. Identification of peamaclein as a marker allergen related to systemic reactions in peach allergy. *Ann Allergy Asthma Immunol*. 2014;112(2):175-7 e3. Epub 2014/01/29.

371 26. Inomata N, Miyakawa M, Aihara M. Eyelid edema as a predictive factor for sensitization to Pru p 7 in peach allergy. *J Dermatol*. 2016;43(8):900-5. Epub 2016/02/27.

373 27. Monneret G, Gutowski M, Bienvenu J. Detection of allergen-induced basophil activation by expression of CD63 antigen using a tricolour flow cytometric method. *Clin Exp Immunol*. 1999;115(3):393-6.

375 28. Kuddus R, Rumi F, Tsutsumi M, Takahashi R, Yamano M, Kamiya M, et al. Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in *Pichia pastoris*. *Protein Expression and Purification*. 2016;122:15-22.

378 29. Shahali Y, Sutra JP, Charpin D, Mari A, Guilloux L, Sénéchal H, et al. Differential IgE sensitization to cypress pollen associated to a basic allergen of 14 kDa. *FEBS J*. 2012;279(8):1445-55. Epub 2012/02/22.

380 30. Okazaki F, Momma K, Hirakawa Y, Kawai N, Yamaguchi-Murakami Y, Adachi R, et al. Determination of Severe Peach Allergens, Gibberellin-Regulated Protein, and Lipid Transfer Protein, Using Monoclonal Antibodies. *J Nutr Sci Vitaminol (Tokyo)*. 2022;68(3):221-7.

383 31. Tuppo L, Alessandri C, Giangrieco I, Tamburrini M, Arriaza R, Chruszcz M, et al. When the Frequencies of Sensitization and Elicitation of Allergic Reaction Do Not Correlate-The Case of Apple Gibberellin-Regulated Protein Tested in an Italian Population. *Front Allergy*. 2021;2(Oct21).

386 32. Castro-Camba R, Sánchez C, Vidal N, Vielba JM. Plant Development and Crop Yield: The Role of Gibberellins. *Plants (Basel)*. 2022;11(19):2650.

388 33. Nothmann J, Koller D. Non-transmissible and long-lasting effects of exogenous gibberellin on floral morphology in the eggplant (Solanum melongena L.). *Planta*. 1975;123(2).

390 34. Gao S, Chu C. Gibberellin Metabolism and Signaling: Targets for Improving Agronomic Performance of Crops. *Plant Cell Physiol*. 2020;61(11):1902-11. Epub 2020/08/08.

392 35. Mushtaq S, Amjad M, Ziaf K, Afzal I. Gibberellins application timing modulates growth, physiology, and quality characteristics of two onion (Allium cepa L.) cultivars. *Environ Sci Pollut Res Int*. 2018;25(25):25155-61.
<table><thead><tr><th>Variable</th><th>Patients with suspected Pru p 7 allergy</th><th>Patients with LTP food allergy</th><th>Patients with respiratory allergy</th></tr></thead><tbody><tr><td>Total no. cases</td><td>23</td><td>5</td><td>9</td></tr><tr><td>Males, n (%)</td><td>7 (30.4%)</td><td>3 (60%)</td><td>2 (22.2%)</td></tr><tr><td>Females, n (%)</td><td>16 (69.6%)</td><td>2 (40%)</td><td>7 (77.8%)</td></tr><tr><td>Age, median (IQR)</td><td>47 (13.5)</td><td>40 (25)</td><td>50 (23.3)</td></tr><tr><td>Rhinoconjunctivitis, n (%)</td><td>23 (100%)</td><td>3 (60%)</td><td>9 (100%)</td></tr><tr><td>Asthma, n (%)</td><td>7 (30.4%)</td><td>2 (40%)</td><td>3 (33.3%)</td></tr><tr><td>Atopic dermatitis, n (%)</td><td>3 (13%)</td><td>0 (0%)</td><td>1 (11.1%)</td></tr></tbody></table>

<caption>**Table 1.** Patients' main demographic and clinical features.</caption>
<table><thead><tr><th></th><th>Peach</th><th>Apple</th><th>Plum</th><th>Fig</th><th>Orange</th><th>Lemon</th><th>Cherry</th><th>Pomegranate</th><th>Apricot</th><th>Almond</th><th>Peanut</th><th>Watermelon</th><th>Melon</th><th>Eggplant</th><th>Sweet pepper</th><th>Others</th><th>Total n. culprits per case</th></tr></thead><tbody><tr><td>Pt 001</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>5</td></tr><tr><td>Pt 003</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Nutella Pesto</td><td>4</td><td></td></tr><tr><td>Pt 005</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Unknow</td><td>2</td><td></td></tr><tr><td>Pt 006</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>7</td></tr><tr><td>Pt 008</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Pt 011</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Pt 012</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Berries</td><td>2</td><td></td></tr><tr><td>Pt 013</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td></tr><tr><td>Pt 014</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Pt 015</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td></tr><tr><td>Pt 016</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Pt 017</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td></tr><tr><td>Pt 021</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>6</td></tr><tr><td>Pt 022</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Pt 024</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td></tr><tr><td>Pt 025</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Pt 026</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td></tr><tr><td>Pt 027</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Pt 031</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td></tr><tr><td>Pt 032</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Pt 034</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td></tr><tr><td>Pt 035</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Pt 036</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td></tr><tr><td>Total n. cases per culprit</td><td>21</td><td>5</td><td>5</td><td>5</td><td>5</td><td>4</td><td>3</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>4</td><td>61</td></tr><tr><td>% of cases</td><td>91.3</td><td>21.7</td><td>21.7</td><td>21.7</td><td>21.7</td><td>17.4</td><td>13.0</td><td>8.7</td><td>4.3</td><td>4.3</td><td>4.3</td><td>4.3</td><td>4.3</td><td>4.3</td><td>4.3</td><td>4.3</td><td>13.0</td><td></td></tr></tbody></table>

Severity (Mueller)

1

2

3

4

<caption>**Table 2.** Concise view of each patient's culprit food(s) and relative reaction severity.</caption>
406 **Figures**

407

408 **Figure 1.** Flowchart summarizing the characteristics of the enrolled populations.

409 SPT skin prick test; LTP lipid-transfer protein; Pru p Prunus persica

<figure><img src="image_3.png" /></figure>

410

411

412
<figure><img src="image_4.png" /><figcaption>**Figure 2.** a) Proportion (%) of patients from group 1 with allergy to 1 ■ or 2 ■ or 3 ■ or 4 ■ or ≥ 5 ■ culprit plant foods. b) Highest Mueller grade for reaction(s) in patients with suspected Pru p 7 allergy.</figcaption></figure>
<figure><img src="image_5.png" /></figure>

434 a)

<figure><img src="image_6.png" /></figure>

435 b)

**Figure 3.** a) Basophil activation test in patients with suspected Pru p 7 allergy. Percentages of basophils expressing CD63 for each individual of group 1 (n=23) are reported, in response to cypress pollen extract (1 µg/ml, blue columns), peach extract (1 µg/ml, red columns), Pru p 7 (1 µg/ml, orange columns).

436
437
438
439
440
441
442

b) Percentage of patients with positive BAT results to cypress pollen extract, peach extract and rPru p 7 among individuals with suspected Pru p 7 allergy (group 1), control individuals with LTP-mediated food allergy (group 2) and subjects with respiratory allergy sensitized to cypress pollen (group 3).

442
<figure><img src="image_7.png" /></figure>

a)

443

444

<figure><img src="image_8.png" /></figure>

b)

445

446

**Figure 4.** **a)** IgE immunoreactivity of 37 patients tested by western blots on cypress pollen proteins separated by SDS-PAGE. Patients are classified in 3 groups according to figure 1. Molecular weights are indicated in kDa and the lane "Total proteins" corresponds to the total proteins in the extract stained with silver nitrate. Double-headed arrows indicate reactivity to Cup s 7, the cypress pollen GRP. **b)** IgE immunoreactivity of 5 patients from group 1 and 2 patients from each of the groups 2 and 3. Patients were tested against the total cypress extract (Cups) and 3 recombinant GRPs: Pru p 7 from peach, Cry j 7 from Japanese cedar pollen and snakin-1 from potato. Proteins are separated by SDS-PAGE and transferred to nitrocellulose membranes. Molecular weights are indicated in kDa. Reactivities to GRPs are around 14kDa.

447

448

449

450

451

452

453

454
1 **eSupplement**

2

3 **Supplementary eFigure 1. Dose-response of basophil activation with tested allergens.**

4 Cypress extract (A), peach extract (B) and Pru p 7 (C) were initially tested at 1000, 100, 10, and 1 ng/mL on

5 5 selected patients, to define the optimal allergen concentration for BAT. Percentage of CD63+ basophils at

6 each dose for each patient is reported. Black lines correspond to mean values.

7

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

8

9
10 Supplementary eFigure 2. Gating strategy for the identification of resting and activated basophils.

11

12 Total basophils were identified for CD45 positivity and low SSC-A, then by FSC-A and SSC-A parameters
13 and high IgE positivity. CD63 expression differentiates resting and activated basophils. Resting basophils are
14 identified as IgE+CD45+CD63- cells. Activated basophils are identified as IgE+CD45+CD63+ cells.
15 Histograms represents percentage of CD63+ basophils in medium condition and after incubation with Pru p 7
16 in a representative patient of group 1.

17

<figure><img src="image_12.png" /></figure>

18

19
20 *Agreement among in vitro test results*

21 WB results do not correlate at 100% with Pru p 7 sensitization as revealed by ALEX
22 multiarray assay, ImmunoCAP and BAT. Pt014 and Pt017 are Pru p 7supos but Cup s 7neg; conversely,
23 Pt026 is Cup s 7pos while Pru p7neg in both ALEX and BAT assay and Pru p7pos in ImmunoCAP.
24 Pt027 is Cup s 7pos while Pru p 7neg in ALEX but positive in BAT and ImmunoCAP. In three other
25 patients (Pt006, Pt011, Pt016), despite basophil activation by Pru p7 and IgE to Pru p7 detection by
26 ImmunoCAP, Pru p 7 specific IgE are negative in ALEX multiarray. In Pt032 IgE immunoreactivity
27 is confirmed by both singleplex and multiplex, while basophil activation test gives a negative result.
28 Pt036 cannot be confirmed allergic to Pru p 7, because he was found negative in Pru p 7 and Cup s
29 7 immunoassays as well as Pru p 7 BAT despite a positive SPT with cypress pollen and peach and
30 no Pru p 3 sIgE.

31 As a result, 91.3% concordance is observed between Pru p 7 determination in either in vitro
32 test and Cup s 7 determination in WB. The same concordance is found between BAT and
33 ImmunoCAP (Supplementary Table 1). These results might be explained by differences in the relative
34 concentration of FcεRI-bound IgE and circulating serum IgE, and also by the fact that the allergens
35 molecules used in each method are not exactly identical or similarly exposed. The latter might be
36 related to a differential exposure of the different epitopes on the GRP molecular surface. Moreover,
37 ImmunoCAP, ALEX and BAT assays differ in their sensitivity.

38
Supplementary eTable 1. Discordant results between ALEX multiplex assay, ImmunoCAP, Basophil Activation Test and Cup s 7 WB in a selection of patients in group 1. Pink boxes highlight positive results.

Pru p 7 BAT +++: more than 60% CD63 expressing basophiles. Cup s 7 WB semi quantitation based on signal intensity: +: low, ++: medium, +++: high.

<table><thead><tr><th></th><th>Pru p 7<br>ALEX<br>kUA/L</th><th>Pru p 7<br>ImmunoCAP<br>kUA/L</th><th>Pru p 7<br>BAT</th><th>Cup s 7<br>WB</th></tr></thead><tbody><tr><td>Pt 006</td><td>0.19</td><td>1.54</td><td>+++</td><td>+</td></tr><tr><td>Pt 011</td><td>&lt;0.1</td><td>0.83</td><td>+++</td><td>-</td></tr><tr><td>Pt 014</td><td>1.69</td><td>1.67</td><td>+++</td><td>-</td></tr><tr><td>Pt 016</td><td>&lt;0.1</td><td>1.57</td><td>+++</td><td>-</td></tr><tr><td>Pt 017</td><td>1.8</td><td>1.81</td><td>+++</td><td>-</td></tr><tr><td>Pt 026</td><td>&lt;0.1</td><td>0.93</td><td>-</td><td>+++</td></tr><tr><td>Pt 027</td><td>&lt;0.1</td><td>3.01</td><td>+++</td><td>+++</td></tr><tr><td>Pt 032</td><td>3.57</td><td>22.4</td><td>-</td><td>++</td></tr></tbody></table>