Christian Radauer,a Farzaneh Adhami,a Irene Fürtler,a Stefan Wagner,a Dorothee Allwardt,a Enrico Scala,b Christof Ebner,c Christine Hafner,d Wolfgang Hemmer,e Adriano Mari,b and Heimo Breitenedera,⁎
Christian Radauer
aDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
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Farzaneh Adhami
aDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
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Irene Fürtler
aDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
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Stefan Wagner
aDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
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Dorothee Allwardt
aDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
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Enrico Scala
bCenter for Molecular Allergology, IDI-IRCCS, Rome, Italy
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Christof Ebner
cAmbulatorium für Allergie und Immunologie, Vienna, Austria
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Christine Hafner
dKarl Landsteiner Institute for Dermatological Research, St. Pölten, Austria
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Wolfgang Hemmer
eFloridsdorfer Allergiezentrum, Vienna, Austria
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Adriano Mari
bCenter for Molecular Allergology, IDI-IRCCS, Rome, Italy
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Heimo Breiteneder
aDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
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Author information Article notes Copyright and License information Disclaimer
aDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
bCenter for Molecular Allergology, IDI-IRCCS, Rome, Italy
cAmbulatorium für Allergie und Immunologie, Vienna, Austria
dKarl Landsteiner Institute for Dermatological Research, St. Pölten, Austria
eFloridsdorfer Allergiezentrum, Vienna, Austria
Heimo Breiteneder: ta.ca.neiwinudem@redenetierb.omieh
⁎Corresponding author. Tel.: +43 1 40400 5102; fax: +43 1 40400 5130. ta.ca.neiwinudem@redenetierb.omieh
Received 2010 Aug 3; Revised 2010 Oct 21; Accepted 2010 Oct 25.
Copyright © 2011 Elsevier Ltd.
Open Access under CC BY-NC-ND 3.0 license
Abstract
Allergies to certain fruits such as banana, avocado, chestnut and kiwi are described in 30–70% of latex-allergic patients. This association is attributed to the cross-reactivity between the major latex allergen hevein and hevein-like domains [HLDs] from fruit class I chitinases. We aimed to assess the extent of cross-reactivity between hevein and HLDs using sera from latex-allergic patients with and without plant food allergy. Hevein and HLDs of latex, banana, and avocado chitinases were expressed in Escherichia coli as fusion proteins with the maltose-binding protein and purified by affinity chromatography. IgE binding to these proteins was studied in sera from 59 latex-allergic patients and 20 banana-allergic patients without latex allergy by ELISA and ELISA inhibition. Additionally, 16,408 allergic patients’ sera were tested for IgE binding to hevein, latex chitinase, and wheat germ agglutinin using an allergen microarray. Hevein-specific IgE was detected in 34/59 [58%] latex-allergic patients’ sera. HLDs of latex, banana, and avocado chitinases were recognized by 21 [36%], 20 [34%], and 9 [15%] sera, respectively. In contrast, only one of 20 banana-allergic patients without latex allergy was sensitized to chitinase HLDs. In most tested latex-allergic patients’ sera, IgE binding to hevein was only partially reduced by preincubation with HLDs. Among hevein-sensitized, latex-allergic patients, the percentage of plant food allergy [15/34 = 44%] was equal to latex-allergic patients without hevein sensitization [11/25 = 44%]. In the general allergic population, 230 of 16,408 sera [1.4%] reacted to hevein and/or a hevein-like allergen. Of these, 128 sera showed an isolated sensitization to hevein, whereas only 17 bound to latex chitinase or wheat germ agglutinin without hevein sensitization. In conclusion, the IgE response to HLDs is elicited by hevein as sensitizing allergen in most cases. Despite considerable cross-reactivity between these allergens, no correlation between latex-associated plant food allergy and sensitization to hevein or HLDs was found.
Abbreviations: HCW, health care worker; HLD, hevein-like domain; ISAC, immuno solid-phase allergen chip; MBP, maltose binding protein; OD, optical density
Keywords: Latex allergy, Latex-fruit syndrome, Hevein, Class I chitinase, Wheat germ agglutinin, IgE cross-reactivity
1. Introduction
Allergy to natural rubber latex became a major health concern in the 1980s due to the frequent use of natural rubber latex gloves by healthcare workers [HCWs] as a consequence of the HIV pandemic [Bousquet et al., 2006]. In the last decade, the incidence of latex allergy among HCWs decreased in industrialized countries due to a switch to powder-free gloves with low protein content [Nienhaus et al., 2008]. However, other population groups remain at risk: the prevalence of latex allergy is especially high among children with spina bifida and other individuals that undergo frequent surgical treatments early in life [Cremer et al., 2007]. In addition, the use of latex gloves is still increasing among occupational groups outside the healthcare system [such as food handling personnel and greenhouse workers] and in newly industrializing countries such as India and China [reviewed in Rolland and O’Hehir, 2008].
About 30–50% of latex-allergic patients show allergic symptoms to plant-derived foods, especially fresh fruits. This association was named latex-fruit syndrome [reviewed in Blanco, 2003; Wagner and Breiteneder, 2002]. The fruits most commonly involved are banana, avocado, chestnut, and kiwi. Several latex allergens were discussed as mediators of the latex-fruit cross-reactivity, such as Hev b 2 [endo-β1,3-glucanase] [Barre et al., 2009; Palomares et al., 2005; Wagner et al., 2004], Hev b 6.02 [hevein] [Chen et al., 1997], Hev b 7 [patatin-like protein] [Schmidt et al., 2002], Hev b 8 [profilin] [Ganglberger et al., 2001], and Hev b 12 [non-specific lipid-transfer protein] [Beezhold et al., 2003].
The allergen whose contribution to the latex-fruit syndrome was studied in greatest detail is the major latex allergen hevein [Hev b 6.02]. Hevein is the N-terminal domain of its precursor prohevein [Hev b 6.01], which is cleaved in vivo upon latex coagulation [Lee et al., 1991]. Plant-derived class I chitinases contain N-terminal domains with high sequence similarities to hevein, thus designated hevein-like domains [HLDs]. Several class I chitinases were cloned and characterized as allergens, such as Hev b 11 from latex [O’Riordain et al., 2002], Pers a 1 from avocado [Sowka et al., 1998a] [originally designated Prs a 1], and Cas s 5 from chestnut [Diaz-Perales et al., 2002]. It was shown that the major part of their IgE reactivities resided in their HLDs, even though the C-terminal domains of class I chitinases as well as homologous class II chitinases, which lack an HLD, contain low-affinity, denaturation sensitive IgE binding sites [Blanco et al., 1999; Diaz-Perales et al., 1998, 2002]. Another hevein-like allergen is wheat germ agglutinin [Tri a 18], a minor allergen for patients with bakers’ asthma, which contains four hevein-like domains [Sutton et al., 1984].
It is currently believed that hevein is the sensitizing agent in the hevein-HLD cross-reactivity. In inhibition assays, IgE binding to class I chitinases or isolated HLD from banana [Mikkola et al., 1998] and avocado [Chen et al., 1998] was completely blocked by preincubation of the sera with hevein, but not vice versa. However, most studies that examined hevein-HLD cross-reactivity exclusively used sera of hevein-sensitized patients. Thus, the question if HLDs could act as sensitizers without prior hevein sensitization remains unanswered. Data on the clinical relevance of this cross-reactivity are scarce. No comparison of allergen sensitization profiles and presence of latex-associated food allergy has been published.
In this work, we examined the sensitization patterns to and the cross-reactivity between hevein and HLDs from latex, banana and avocado chitinases in a latex-allergic population using IgE ELISA and ELISA inhibition assays. In contrast to previous studies, our patient sample was not preselected for hevein sensitization or the presence of plant food allergy. Additionally, we analyzed the sensitization patterns to hevein and HLDs in a large number of sera from the general allergic population tested by the immuno solid-phase allergen chip [ISAC] technology. While confirming the previously assumed predominant role of hevein as sensitizing agent, we found sensitization to HLDs without IgE binding to hevein in a minority of patients. Despite the high cross-reactivity between these allergens, the clinical relevance of sensitization to hevein and HLDs for latex-associated plant food allergy appears to be limited.
2. Materials and methods
2.1. Isolation of total RNA from Hevea latex, avocado and banana
Latex RNA was isolated from fresh latex obtained from regularly tapped rubber trees [Hevea brasiliensis, clone RRIM 600] at the Rubber Research Institute of Malaysia's Experimental Station, Sungai Buloh, Selangor as described [Sowka et al., 1998b]. Total RNA of banana and avocado was prepared following previously described protocols [Clendennen and May, 1997; Starrett and Laties, 1993].
2.2. Cloning of cDNAs encoding hevein and HLDs of class I chitinases from latex, banana and avocado
Reverse transcription was carried out with 1 μg total RNA using Moloney Murine Leukemia Virus reverse transcriptase [Fermentas, St. Leon-Rot, Germany] and the primer T25NN [5′-GGAGAAGGA[T]25[AGC]N-3′].
The coding regions for hevein [Hev b 6.02] and HLDs of class I chitinases from latex [Hev b 11-HLD], banana [Mus a 2-HLD] and avocado [Pers a 1-HLD] were amplified by PCR using primers designed according to the published cDNA sequences [Table 1]. The cloned PCR products were sequenced using the Thermo Sequenase fluorescent labeled primer cycle sequencing kit [GE Healthcare, Little Chalfont, UK] and a DNA sequencing system using infrared fluorophore labeled primers [LI-COR 4000L, LI-COR Biosciences, Lincoln, NE, USA].
Table 1
PCR primers used for cloning of hevein and hevein-like domains from class I chitinases.
AllergenSourceAcc. no.aPCR primersbHev b 6.02Hevea brasiliensis [Para rubber tree]{"type":"entrez-nucleotide","attrs":{"text":"M36986","term_id":"168208","term_text":"M36986"}}M36986Forward: 5′-GGAATTCGAGCAATGTGGTCGGCAAGC-3′Reverse: 5′-CAAGCTTTTAGTCTTTGCAATTGCTTTGGC-3′Hev b 11-HLDHevea brasiliensis{"type":"entrez-nucleotide","attrs":{"text":"AJ238579","term_id":"14575524","term_text":"AJ238579"}}AJ238579Forward: 5′-GGAATTCGAGCAATGTGGAAGGCAA-3′Reverse: 5′-CAAGCTTTTAACCTCCACCGTCACATTG-3′Mus a 2-HLDMusa acuminata [banana]{"type":"entrez-nucleotide","attrs":{"text":"AJ277278","term_id":"17932709","term_text":"AJ277278"}}AJ277278Forward: 5′-GGAATTCGAGCAATGCGGAAGGCAAG-3′Reverse: 5′-CAAGCTTTTAGCCGCTACCGCCGCATT-3′Pers a 1-HLDPersea americana [avocado]{"type":"entrez-nucleotide","attrs":{"text":"Z78202","term_id":"3201546","term_text":"Z78202"}}Z78202Forward: 5′-GGAATTCGAACAATGTGGTAGACAAGC-3′Reverse: 5′-CAAGCTTTTAGGTGACCCCACCGCATT-3′
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aNucleotide sequence accession numbers from the European Molecular Biology Library/GenBank/DNA Data Bank of Japan sequence databases.
bRestriction endonuclease cleavage sites for Eco RI [forward] and Hind III [reverse] are underlined.
2.3. Expression and purification of recombinant proteins
The coding sequences were inserted into the Escherichia coli expression vector pMAL-p2 according to the manufacturer's directions [New England Biolabs, Beverly, MA, USA]. The recombinant proteins Hev b 6.02, Hev b 11-HLD, and Mus a 2-HLD fused to maltose binding protein [MBP] were expressed in E. coli XL1 blue cells and Pers a 1-HLD in E. coli BL21 cells. For the expression of fusion proteins, 400 mL super broth medium [25 g/L tryptone, 15 g/L yeast extract, 0.5 g/L NaCl] containing 100 mg/L ampicillin were inoculated with 1 mL of an overnight culture and grown to an optical density [OD] at 600 nm of 0.5. Expression was induced by adding isopropyl β-d-thiogalactoside to a final concentration of 0.3 mM. After growing overnight, cells were harvested, resuspended in column loading buffer [20 mM Tris–HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4] and extracted using a French Pressure Cell [SLM Instruments, Rochester, NY, USA]. The recombinant fusion proteins were purified by affinity chromatography on an amylose resin column according to the manufacturer's protocols [New England Biolabs]. The isolated fusion proteins were analyzed by SDS-PAGE.
2.4. Purification of natural hevein
An extract of the lutoid fraction [B-serum] of freshly tapped latex [H. brasiliensis clone RRIM 600] was prepared as described previously [Wagner et al., 2004]. Twenty-five milligrams of freeze-dried latex B-extract were dissolved in 2 mL extraction buffer [25 mM Tris–HCl, 10 mM EDTA, pH 7.5] supplemented with protease inhibitors [Complete EDTA-free Protease Inhibitor Cocktail Tablets, Roche Applied Science, Vienna, Austria] and centrifuged through a Centricon 10 ultrafiltration device [Millipore, Billerica, MA, USA]. The flow-through was purified from low molecular weight contaminants by using a PD-10 desalting column [GE Healthcare, Vienna, Austria]. Purity and identity of hevein were confirmed by SDS-PAGE and matrix-assisted laser desorption–ionization mass spectrometry [data not shown].
2.5. Patients and controls
This study was performed using sera of 59 latex-allergic patients from Austria and Italy, who were admitted to an allergy outpatient clinic for diagnosis of their allergy. Sera were drawn during routine clinical examinations. Allergy to latex and plant inhalant allergens [birch, grass, ragweed and mugwort pollen, Ficus benjamina] was established based on typical case histories and positive skin prick tests with commercial extracts [ALK-Abello, Hørsholm, Denmark]. Food allergy was diagnosed based on the symptoms reported by the patients. As controls, sera of 20 banana-allergic patients without latex allergy were included. Diagnosis of banana allergy was based on reported symptoms of type I allergy following the ingestion of bananas. Criteria for diagnosing the absence of latex allergy were a lack of typical symptoms and a negative skin prick test with latex extract [ALK-Abello].
For a retrospective epidemiological study of the sensitization to hevein and HLDs, patients were enrolled at the Center for Molecular Allergology, IDI-IRCCS [Rome, Italy]. Serum sample storage and use of routine data has been approved by the institutional ethical committee. The study comprised 16,408 subjects complaining about any suspected IgE-mediated allergic symptom.
As controls for ELISA experiments, five sera of individuals without clinical histories of allergic reactions were drawn.
2.6. IgE ELISA and ELISA inhibition
Microtiter plates [Maxisorp F96, Nunc-Nalge, Roskilde, Denmark] were coated with 10 μg/ml of a monoclonal anti-MBP antibody [Serotec, Oxford, UK] in sodium carbonate buffer, pH 9.5 overnight at 4 °C. After blocking non-specific binding sites with 2% non-fat dry milk in TBST [50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% Tween 20], recombinant allergens and MBP [New England Biolabs] were applied to the plates at 10 μg/ml in TBST, 0.5% BSA and incubated for 2 h at room temperature. For detection of IgE binding to natural hevein, CovaLink NH plates [Nunc-Nalge] were activated with 1.25% glutaraldehyde in 50 mM Na-phosphate, pH 8.2 overnight at 37 °C. Hevein was covalently coupled to the plates at 3 μg/ml for 3 h at 37 °C. Non-specific binding sites were blocked as described above.
Sera were diluted 1:5 in TBST, 0.5% BSA and applied to the allergen-coated plates overnight at 4 °C. Bound IgE was detected using an alkaline phosphatase-labeled monoclonal anti-human IgE antibody [BD PharminGen, Heidelberg, Germany] and a p-nitrophenyl phosphate substrate [Sigma–Aldrich, St. Louis, MO, USA]. All samples were assayed in duplicates. Five sera of non-allergic individuals were used as negative controls. The mean OD of the negative controls plus three standard deviations was subtracted from all sample OD values. For statistical analyses, OD values were counted positive if they exceeded the OD of the MBP controls.
For ELISA inhibition assays, patients’ sera were preincubated with 50 μg/ml of recombinant allergens or MBP before proceeding with the assay as described above. This inhibitor concentration was found to be greater than the saturating value in a preliminary concentration-dependent inhibition experiment with two sera. Percent inhibition values were calculated relative to the OD value of the serum preincubated with buffer [0% inhibition] and the mean OD value of the sera of non-allergic patients [100% inhibition].
2.7. ISAC testing
IgE to recombinant Hev b 6.02 and Hev b 11-HLD MBP fusion proteins and to natural Tri a 18 [wheat germ agglutinin] was measured by the ISAC technology [Phadia Multiplexing Diagnostics, Vienna, Austria] in sera of 16,408 subjects. The ISAC technology allows the detection of specific IgE to microarrayed single allergenic molecules, either natural or recombinant, spotted in triplicates on a solid surface [Harwanegg and Hiller, 2005]. A microarray carrying 89 different allergens was used. Briefly, 20 μl of serum were incubated on the reaction site of the microarray in a humid chamber for 2 h. Slides were washed, and fluorescein-labeled anti-IgE antibody was added and incubated for 1 h. Fluorescence was detected by a laser scanner. Images were processed and raw fluorescence values were analyzed by the ISAC software [Phadia Multiplexing Diagnostics]. Values are expressed as kUA/L. Values above 0.1 kUA/L were counted positive.
2.8. Statistical analyses
The amounts of IgE binding to hevein and HLDs were compared using the Wilcoxon test for paired samples. Pearson linear correlation coefficients were calculated for the correlations of the IgE ELISA OD values of different allergens. Both analyses were performed using only the results of those patients who were sensitized to at least one of the tested allergens. The correlation between sensitization to hevein or HLDs and presence of food allergy symptoms was evaluated using the chi-squared test. Statistical analyses were performed with SPSS 14.0 [SPSS, Chicago, IL, USA].
For the analysis of ISAC data, the one-way ANOVA test was used to compare IgE value distributions. The Spearman rank correlation test was used to evaluate correlations between double positive values.
3. Results
3.1. Sequence and structure comparison of hevein and HLDs
Fig. 1A shows a sequence alignment of hevein, HLDs from latex, banana, and avocado chitinases, and all four domains of wheat germ agglutinin. Identities between hevein and chitinase HLDs were at least 68% with identities among chitinase HLD sequences being higher than between hevein and chitinase HLDs. The sequences of the domains of Tri a 18 diverged to a greater extent both from hevein and chitinase HLDs with the exception of domain 3, which shows 66% identity to hevein.
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Fig. 1
Sequence and structure comparison of hevein and HLDs. [A] Multiple sequence alignment; grey: residues identical to corresponding ones in hevein, bold: conserved cysteine residues, dashes: gaps introduced during the alignment. [B] Mapping of conserved residues onto the molecular surface of hevein [PDB ID: 1hev]. Black: residues conserved between hevein and HLDs of class I chitinases [left] or Tri a 18 [right], respectively. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco [supported by NIH P41 RR-01081] [Pettersen et al., 2004].
A comparison of the NMR-structure of hevein [PDB ID: 1hev] with the crystal structure of Tri a 18 [PDB ID: 2uwg] and homology models of Mus a 1-HLD and Pers 1-HLD based on cereal class I chitinases [downloaded from the Swiss-Model Repository [Kiefer et al., 2009]] revealed that the backbone conformations of hevein and HLDs were nearly identical [data not shown]. Thus, it is justified to assess the extent of surface conservation by only taking into account the surface contributions of conserved and variable side chains. The mapping of the conserved residues onto the molecular surface of hevein [Fig. 1B] showed that, despite the high extent of sequence conservation, many of the conserved residues did not contribute to the molecular surface of hevein, resulting in a much larger proportion of variable surface than expected from the sequence comparison. In addition, residues conserved between hevein and class I chitinases on the one hand and hevein and Tri a 18 on the other hand were located on only partially overlapping regions of the surface indicating that the cross-reactivity between hevein and different hevein-like allergens might be mediated by different epitopes.
3.2. Patients
Fifty-nine latex-allergic patients admitted to allergy outpatient clinics were included in this study [Table 2]. Most of them suffered from mild, local symptoms [urticaria and rhinoconjunctivitis] upon contact with latex products. Adverse reactions to plant foods were reported by 26 [44%] of the patients. In eight patients, reported food allergy was confirmed by positive skin tests, in 4 patients by food allergen-specific IgE and in four patients by both methods. Thirteen patients reported mild, local reactions [oral allergy syndrome] as their sole adverse reactions to plant foods [Table 2]. Other reported symptoms were rhinoconjunctivitis [1 patient], atopic dermatitis [3 patients], urticaria [1 patient], angioedema [1 patient], gastrointestinal symptoms [1 patient], and anaphylaxis [6 patients].
Table 2
Clinical characteristics and ELISA results of the latex-allergic and banana-allergic patients included in this study.
Latex-allergic patientsBanana-allergic patientsNumber [female, male]59 [46, 13]20 [13, 7]Mean age [range]38 [9–67]36 [5–70]Occupational latex exposure29 [49%]n.d.Latex CAP > 0.35 kUA/L54 [92%]2/7 [29%]
Latex allergy symptoms Urticaria37 [63%]0 Rhinoconjunctivitis20 [34%]0 Angioedema15 [25%]0 Asthma10 [17%]0 Eczema7 [12%]0 Anaphylaxis1 [2%]0
Allergy to plant inhalants Pollen33 [56%]16 [80%] Ficus benjamina5 [8%]5 [25%]
Plant food allergy Any26 [44%]20 [100%] Banana12 [20%]20 [100%] Avocado6 [10%]3 [15%]
Plant food allergy symptoms Oral allergy syndrome14 [24%]17 [85%] Rhinoconjunctivitis1 [2%]0 Atopic dermatitis3 [5%]0 Urticaria1 [2%]0 Angioedema2 [3%]1 [5%] Gastrointestinal symptoms1 [2%]1 [5%] Anaphylaxis6 [10%]1 [5%]
IgE ELISA: positive results Hev b 6.0234 [58%]0 Hev b 11-HLD21 [36%]1 [5%] Mus a 2-HLD20 [34%]1 [5%] Pers a 1-HLD9 [15%]0
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n.d.: not determined.
The foods most frequently eliciting allergic reactions were banana [12 patients, 20%], avocado [6 patients, 10%] and apple [6 patients, 10%; Table 2]. Other foods reported to cause allergic reactions were kiwi fruit, chestnut, hazelnut, walnut [5 patients each], mango and peach [3 patients each], and melon, tomato, citrus fruit and soybean [2 patients each], In addition, adverse reactions to fig, almond, eggplant, apricot, peanut, pea, bean, lentil, caper, passion fruit, lettuce, zucchini, pear, pistachio and hot spices were reported by a single patient each.
As a control group, 20 banana-allergic patients without latex allergy were included [Table 2]. In four individuals, banana allergy was confirmed by a positive prick-to-prick test with fresh banana. Reported symptoms were oral allergy syndrome [17 patients], angioedema [1], abdominal pain [1], and anaphylaxis [1].
3.3. Comparison of natural and recombinant hevein
Natural hevein was purified from latex B-serum to a purity of at least 95% as verified by SDS-PAGE [data not shown]. MALDI-MS analysis yielded two peaks with masses of 4703 Da and 4721 Da matching the calculated masses of hevein with an N-terminal pyroglutamic acid [M = 4702 Da] and with an unmodified N-terminus [M = 4720 Da].
In order to check the suitability of MBP fusion proteins for the analysis of IgE binding, natural and recombinant hevein were tested in an ELISA with 31 latex-allergic patients’ sera. IgE binding to both allergen preparations showed a good linear correlation with a Pearson correlation coefficient of 0.94 [data not shown].
3.4. IgE binding to hevein and HLDs among latex and banana-allergic patients
All sera of latex and banana-allergic patients were tested for IgE-binding to recombinant hevein and HLDs by a sandwich ELISA with an anti-MBP antibody coated to the solid phase [Table 2]. Thirty-four sera of latex-allergic patients [58%] contained IgE specific for hevein. The number of sera recognizing HLDs of class I chitinases was lower with 21 [36%], 20 [34%], and 9 [15%] for Hev b 11, Mus a 2, and Pers a 1, respectively. In contrast, only one of 20 sera from banana-allergic patients displayed IgE binding to HLDs from Hev b 11 and Mus a 2. Seven latex-allergic patients’ sera also bound to MBP without a fused allergen. However, all but one OD value were just above the threshold.
Of the 42 sera [71%] from latex-allergic patients recognizing at least one of the tested allergens, 14 [24%] bound exclusively to hevein [Fig. 2A]. Twenty sera [34%] bound to hevein and to at least one HLD. The amounts of IgE binding to hevein were significantly [p