Purification of natural Mal d 1 and Mal d 2 allergens and monitoring of their expression levels during ripening in Golden Delicious apple

Food Research International 44 (2011) 2674–2678

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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Purification of natural Mal d 1 and Mal d 2 allergens and monitoring of their expression levels during ripening in Golden Delicious apple J. Szamos a,⁎, K. Takács a, E.E. Szabó a, E. Kovács b, É. Gelencsér a a b

Central Food Research Institute, Department of Food Safety, Unit of Biology, H-1022 Budapest, Herman Ottó út 15., Hungary Corvinus University of Budapest, Faculty of Food Science, Department of Postharvest Science and Technology, H-1118 Budapest, Ménesi út 45/F., Hungary

a r t i c l e

i n f o

Article history: Received 2 November 2010 Accepted 20 May 2011 Keywords: Apple allergens Purification Chromatography ELISA PCR

a b s t r a c t Apple (Malus domestica) is the most widely cultivated fruit crop in Europe. Consumption of fresh apples can cause allergic reaction with a variable degree of severity in susceptible individuals. Previous studies have indicated that expression pattern of Mal d allergens is affected by growing and storage circumstances. At the same time only a few data are available on purification and expression levels of the heat labile Mal d 1 apple allergen localised in peel and pulp of the fruit because this protein can be extracted in an active form only if reactions with phenolic compounds present in apple are inhibited. Our aim was to concurrently purify and monitor the expression levels of the heat labile Mal d 1 (18 kDa), a Bet v 1 related allergen and the heat resistant Mal d 2 (32 kDa), a thaumatin-like allergenic protein from Golden Delicious apple cultivar with high potential to express Mal d 1 allergens. This assumption was proven by amplification of a polymorphic pattern of PCR products with 154–174 bp fragments by using Mal d 1.06A SSR primers. The apple proteins were extracted from frozen apple pulp under acidic circumstances at pH 3.0 to prevent reaction of the allergens with phenolic compounds of apple. Under such circumstances protein-binding brown pigments can be avoided and its deleterious effect on analysis of Mal d allergens can be eliminated. The separation of Mal d allergens was performed by cation-exchange chromatography and gel filtration. Mal d allergens were quantified during ripening in Golden Delicious apple extract. Mal d 1 and Mal d 2 specific ELISAs were developed to be used for quantification of the immune reactive Mal d allergens. It was found that during the ripening period, the contents of Mal d 1 and Mal d 2 allergens were continuously, but a bit differentially increased. According to the chromatographic results the content of Mal d 1 (0.1 –0.25 mg/100 g apple) increased intensively from 144th until 152th day, then it did not change (152th–164th days), while the content of Mal d 2 (0.18–0.75 mg/100 g apple) started to increase from 144th until 158th day. ELISA results showed the same tendency. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction The consumption of fresh apple belongs to healthy and balanced diet in Europe, however it can cause allergic reaction with a variable degree of severity in susceptible individuals. So far, four major allergens Mal d 1, Mal d 2, Mal d 3 and Mal d 4 have been identified in apple (Malus domestica). Mal d 1 and Mal d 4 allergens are heat labile and sensitive to proteolytic degradation, while Mal d 2 and Mal d 3 allergens are resistant to heat and stable to proteolytic degradation. Mal d 1 and the Mal d 2 proteins are the most significant apple allergens inducing IgE-mediated hypersensitivity reaction (Hoffmann-Sommergruber & Radauer, 2004). Mal d 1 proteins belong to pathogenesis-related proteins (PRs), PR10 protein family. Mal d 1 are about 18 kDa molecular weight proteins (Hoffmann-Sommergruber et al., 1997; Puehringer, Zeinoecker,

⁎ Corresponding author. Tel: + 36 1 355 8244 190; fax: + 36 1 225 3342. E-mail address: [email protected] (J. Szamos). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.05.033

Marzban, Katinger, & Laimer, 2003). Mal d 1 allergens have a high homologue sequence identity with another member of PR-10 proteins and birch Bet v 1 pollen allergen (Calkoven et al., 1987; Fritsch et al., 1998). According to Gao et al. (2005), the Mal d 1 allergens are coded by several gene families and the expression of these allergenic proteins is generated by several biotic (pathogens, environmental stress) and abiotic (injury) factors (Atkinson, Perry, Matsui, Ross, & Macrae, 1996; Puehringer et al., 2000). Mal d 1 allergens are very labile proteins and can be easily degraded even at room temperature, and by different proteolytic enzymes such as pepsin and trypsin (Jensen-Jarolis et al., 1999). Mal d 2 are 31 kDa molecular weight proteins, members of PR-5 stress-protein family, which have sequence homology with thaumatin-like proteins (TLPs). Mal d 2 proteins are fungicides, and have homology with Bet v 2 birch pollen allergen. The Mal d 2 proteins are exceptionally stable molecules, due to the 8 disulfide bridges fixing their structure. These stable molecules have significant roles in protection against denaturation caused by heat and/or protease enzymes (Breiteneder & Mills, 2005).

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The preparation of functionally active apple allergen extracts by isolation of fruit proteins is not a simple task in general, because fractionation of proteins present in low amount makes the isolation procedure to a struggle in an environment influenced steadily by reaction components of enzymatic browning. Several efforts have been made to eliminate the adverse binding of apple allergens to end-products of enzymatic browning, using different extraction buffers and isolation techniques. The first apple allergen extract with good immune reactivity was prepared by Björkstén, Halmepuro, Hannuksela, and Lahti (1980). They studied the effects of several additives as EDTA (ethylene diamine tetra acetic acid), DIECA (diethyldithiocarbamic acid), and PVP (polyvinylpyrrolidone). Another approach, elaborated by Vieths, Schöning, Brockmann, and Aulepp (1992; 1995), used acetone and dry ice for preparation of allergenic proteins from apple. The extract had a good allergenic activity. Successful isolation of a small amount of apple allergens was achieved by a micro preparative SDS-electrophoresis (Vieths, Janek, Aulepp, & Petersen, 1995), while an improved yield of extracted allergenic proteins was attained with anion-exchange chromatography (Martínez, Fernández-Rivas, Martínez, & Palacios, 1997). For chromatographic separation of Mal d 2 and Mal d 3, anion or cation exchange chromatography and gel filtration were applied (Marzban et al., 2009; Menu-Bouaouiche et al., 2003; Pastorello et al., 1999; Pastorello and Trambaioli, 2001). Previous studies have confirmed that the allergen expression profile in apple is affected by several factors as growing, storage conditions, and apple cultivars. Several publications have indicated that the Golden Delicious is an apple cultivar having one of the highest potential to express allergenic proteins (Hsieh, Moos, & Lin, 1995; Marzban et al., 2005; Vieths, Jankiewicz, Schoning, & Aulepp, 1994). At the same time, data on the purification and expression levels of the heat labile Mal d 1 apple allergen localised in peel and pulp of the fruit are very limited. Presumably, the reason why such efforts have failed is these proteins can be extracted in an active form if only reactions with phenolic compounds present in apple are inhibited (Björkstén et al., 1980). Contrary to Mal d 1 proteins, more results are available on other apple allergens, including Mal d 2. Considering the differences in stability between the two proteins, our aim was to develop a simple liquid chromatographic method to concurrently purify the heat labile Mal d 1 (18 kDa) and the heat resistant Mal d 2 (31 kDa) allergenic proteins from Golden Delicious apple cultivar having high potential to express Mal d 1 allergens and monitor their expression levels during ripening. 2. Materials and methods 2.1. Materials 2.1.1. Apple samples Golden Delicious apple cultivar harvested in Érd, Hungary was used in our experiments. The apple samples were harvested on 130th, 137th, 144th, 158th, and 164th days after apple tree blooming time. A batch of about 20 kg apples was picked in the year of 2008 at different times in autumn. One representative sample was prepared from randomly taken 5 apples for analyses. 2.2. Methods 2.2.1. Extraction of allergenic apple proteins Apples (slices without peel) were cut into small pieces as fast as possible to avoid sudden browning, and then stored at −25 °C. The frozen apple pieces were ground to pulp, then 100 g of apple pulp was extracted with 200 ml cold, 40 mM citrate-phosphate buffer (pH 3.0) containing 2% PVPP (polyvynilpolypyrrolidon, Sigma) at 4 °C for 30 min. Thereafter the homogenate was filtered through a nylon

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cloth, and then centrifuged at 10,000 ×g for 10 min. The collected extracts were divided into two equal parts. One part of supernatant was diluted with distilled water at a ratio of 1:1, and used directly for CarboxyMethyl Cellulose (CM)-chromatography as well as gel filtration to purify Mal d allergens used as antigens for the immunisation. The other part of supernatant was tested by ELISA and cation exchange membrane assays. To 50 ml of extract made by 40 mM citrate-phosphate buffer (pH 3.0) 50 ml of distilled water was added and the solution was loaded on a strong acidic cation exchanger, (SF5 membrane, Sartorius) equilibrated with 20 mM citrate-phosphate start buffer (pH 3.0) beforehand, at a flow rate of 18 ml min − 1. The membrane was washed with an additional 10 ml start buffer, and then the proteins bound to the membrane were eluted with 0.2 M NaCl in start buffer at a flow rate of 0.5 ml min − 1. The typical elution volume ranged from 3 ml to 5 ml, to which an additional 1/5 volume of saturated Na2HPO4 (disodium-hydrogen-phosphate) was added to adjust the pH of the eluted fraction to 7.0. 2.2.2. Cation exchange (IEX) and size exclusion (SEC) chromatographic purification of Mal d 1 and Mal d 2 antigens for immunisation To 1000 ml of apple extract (pH 3.0) an equal volume of distilled water was added and this solution was loaded on a CM-cellulose IEX column (100 cm 3 CM 52 SERVACEL (SERVA) in an Econo column 5 × 20 cm (BIO-RAD), flow rate: 10 ml min − 1), equilibrated with 20 mM citrate-phosphate start buffer (pH 3.0) beforehand. The protein fraction bound on the column was eluted with 0.2 M NaCl in start buffer, and then lyophilized. The powder was redissolved in a small volume of start buffer and 200 μl aliquots was loaded on a Superose 12 10/30 FPLC column (Pharmacia). In phosphate-buffered saline (PBS, pH 7.4) eluent at a flow rate of 0.5 ml min− 1 the peaks emerging at tr = 30 min, and tr =35 min were collected as Mal d 2 and Mal d 1, respectively. The purity and the molecular masses of apple allergens were checked by SDS–polyacrylamide electrophoresis (Laemmli, 1970). The purified Mal d 1 and Mal d 2 were used also as chromatographic controls. Before loading on the size exclusion chromatographic column, the protein content of the purified controls was determined with Qubit™ Fluorometer (Invitrogen). 2.2.3. Chromatographic quantification of membrane bound Mal d 1 and Mal d 2 allergens in different apple samples To determine Mal d 1 and d 2 expression levels in apple sample by size exclusion chromatography, 200 μl aliquots of the purified extracts was loaded on a Superose 12 10/30 FPLC column. The chromatographic separation was followed by an EM-1 Econo UV monitor (BioRad), and the Mal d 1 and Mal d 2 contents were evaluated according to the height of the chromatographic peaks. For the evaluation of the allergen content of the apple samples, purified Mal d 1 (49 μg) and Mal d 2 (20 μg) controls were loaded on the column. The results were expressed in mg kg − 1. 2.2.4. Development of polyclonal antibodies specific to natural Mal d 1 and Mal d 2 apple allergens The antibody was developed according to Harboe & Inglid (1973) as published before (Hajós et al, 1995). Briefly, two out-bred Hungarian Vadas rabbits weighing 2–2.5 kg were used for the immunisation with highly purified Mal d 1 or Mal d 2 antigens. For the primary injection (s.c.) of rabbits 25 μg antigen/kg body weight emulsified in Complete Freund's Adjuvant (FCA) (1:1) was used followed by 4 booster injections (s.c.) in 14 day intervals (days: 14, 28, 42, 56) with 25 μg antigen/kg body weight emulsified in incomplete Freund's Adjuvant ICA (1:1). Rabbit antibodies (RbIgG) specific to Mal d 1 or Mal d 2 derived from hyperimmune sera were obtained by ammonium-sulphate precipitation, dialysed, freeze-dried and characterised for efficacy and used for the development of Mal d 1 and Mal d 2 specific ELISAs.

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2.2.5. Competitive indirect ELISA to quantify Mal d allergens in apple extracts Microtiter plates were coated with 100 μl 5 μg ml − 1 Mal d 1 or Mal d 2 proteins. The purified Mal d proteins were lyophilized before using for coating the microtiter plates. Free binding sites were blocked with 250 μl 0.01 M phosphate-buffered saline containing 0.5% gelatine (pH 7.4 PBS). The apple-extract (50 μl), containing the competitive Mal d 1 or Mal d 2 or the competitive Mal d 1/Mal d 2 standards (2000– 0.002 μg ml − 1) was added in PBS together with 50 μl Mal d 1 or Mal d 2 specific rabbit IgG diluted (1:160 or 1:20) in PBS. For the detection of the formed immune-complex, 100 μl HRPO labelled goat anti-rabbit IgG diluted (1:20,000; Jackson) in PBS was applied. H2O2/OPD (Sigma Fast) was used for the colour development, before the reaction was stopped with 50 μl 4N H2SO4. The absorbance was measured at 490 nm, against 630 nm reference filter. For evaluation the results a half-linear logarithmic regression has been used. The results obtained by competitive ELISA method were expressed in mg kg − 1. 2.2.6. PCR amplification of Mal d 1 gene The first step of identification of apple allergens by PCR analysis is the DNA isolation from apple leaves. In this study the gDNA was extracted according to Wizard DNA Clean-up System protocol (Promega, USA). After the isolation procedure the concentration and the purity of DNA solution were determined by UV-spectroscopy (UV1601, Shimadzu) relating to the absorbance measured at 260 and 280 nm, respectively. The amplifications were carried out in a total volume of 25 μl reaction mixture containing 25 μM appropriate primers, 0.75 U JumpStart Ready Mix REDTaq DNA Polymerase (Sigma). The optimal annealing temperature, the number of cycles and the sequences of Cy5 fluorescence labelled primer pairs, adapted by Gao et al. (2005), are listed in Table 1. The use of agarose electrophoresis is not suitable to determine 1–2 base pairs differences, so Pharmacia Biotech ALF express Genetic analyzer was used to establish the exact size of the amplificated products. In this equipment 10 μl PCR products was separated on 10% acrylamid gel in TBE buffer (pH 8.0) and a laser detected DNA fragments. Grape DNA sample containing well-known sequences was used as control. 3. Results and discussion 3.1. Identification of polymorphic Mal d 1 gene in Golden Delicious apple cultivar Mal d 1.06A SSR primer pairs developed by Gao et al. (2005) were used to characterise the Mal d 1 gene specific PCR products. According to the authors this primer-pair is able to discriminate between apple cultivars with varying Mal d 1 allergen content as their results showed polymorphic pattern of different 154–174 bp fragments. The apple cultivar which holds homozygote 154 bp allele may contain low amount of Mal d 1 allergens. According to our results the Golden Delicious apple cultivar showed polymorphic character holding 156 and 164 bp alleles. As this apple cultivar did not contain 154 bp allele

Table 1 Reaction condition of PCR amplification. Primer pairs

Forward: 5′-GGTGAAGGTTAGTTTAATTTCCACA-3′ Reverse: 5′-TTCACATAGCTGTATTCACTCCCT-3′

Amplified fragment lengths Denaturation Annealing Extension Final extension Number of cycles

154–174 bp 94 °C, 90 s — hot start + 94 °C, 60 s 60 °C, 30 s 72 °C, 120 s 72 °C, 5 min 38

either homozygotic or heterozygotic forms we suspected that the Golden Delicious apple cultivar would contain high level of Mal d 1 allergens therefore it would be a good test material for purification of Mal d 1 allergens and monitoring of the allergen expression profile during ripening. 3.2. Purification of natural Mal d 1 and Mal d 2 allergens and monitoring of their expression levels during ripening in Golden Delicious apple In our study we focused on the development of a new liquid chromatographic method for quantification of Mal d 1 and Mal d 2 proteins in Golden Delicious apple cultivars. The apple samples were harvested on 130th, 137th, 144th, 158th, and 164th days after apple tree blooming time. During the method development, the first step was to elaborate an efficient extraction procedure to isolate the target Mal d 1 and Mal d 2 proteins. Since the purification of Mal d 1 from natural source is not a simple task, a reliable preparative procedure is missing, while there are some published methods on purification of Mal d 2. The cause of this is the instability of Mal d 1 at room temperature, while Mal d 2 is a stable molecule. As it was outlined above, the purification of apple proteins is greatly hindered by the enzymatic browning at pH-s of 7.0–8.0, because the pigments (phenolics) produced finally in this reaction bind proteins to various extents. The extraction of small amounts of proteins from fruits is influenced by the presence of plant phenols and polyphenol-oxidase enzyme. Homogenization releases plant phenols that will be oxidised into quinones enzymatically, and then the quinones will form brown pigments that partially bind proteins. Finally, this leads to the loss of allergenicity. This unintended reaction can be minimised by decreasing of the extraction-time, adjustment of low temperature and acidity of fluid, application of a solution with inhibitors as organic polymers (PVPP), and chelating agents (EDTA, DIECA) for extraction of apple proteins. The latter creates complexes with the copper atom in the active centre of the polyphenol-oxidase (PPO) (Björkstén et al., 1980). Vieths et al. (1992; 1995) used acetone and dry ice for the extraction, which resulted in an extract with a good allergen activity. Otherwise, it is important to mention, that salting-out or acetone precipitation proved not to be successful for concentration of allergenic apple proteins Martínez et al. (1997)). Taking into account these results, a simple system was elaborated to isolate apple proteins with citrate-phosphate buffer pH 3.0 in the presence of PVPP, at room temperature, within a short time after peeling (20 min extraction, 20 min centrifugation). Conditions of acidic and neutral extraction methods are illustrated in Table 2. Compared to Björkstén's et al. (1980) procedure, the acidic extraction method has several advantages, as almost complete inhibition of PPO catalysed browning reaction and the favourable fact that CM-cellulose binds Mal d 1 and Mal d 2 exclusively under the applied conditions.

Table 2 Comparison of neutral and acidic extractions.

Extraction at Raw material Buffer

I. Neutral pH

II. Acidic pH

pH 7.0

pH 3.0

Lyophilized apple

Frozen apple pulp

10 mM K-phosphate (pH 7.0), 20 mM citrate-phosphate (pH 3.0), 2% PVP 2 mM EDTA, 2% PVP 1:20 1:2

Extraction ratio Enzymic Partly inhibited browning

Practically inhibited

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apple allergen conte (mg/100 g apple)

a. chromatographic method 1,0 0,8 0,6 0,4 0,2 0,0 130

137

144

152

158

164

Days after bloom time Mal d 1

Mal d 2

b. ELISA method apple allergen conte (mg/100 g apple)

According to other authors (Vieths et al., 1992; 1995), Mal d 1 apple allergen was previously separated by micro-preparative electrophoresis or by anion-exchange chromatography, RP-HPLC, or gel filtration (Hsieh et al., 1995; Sanchez-Monge et al., 1999). In our procedure, the extracts containing Mal d 1 and Mal d 2 allergens were processed by IEX and size exclusion chromatography as it is illustrated in Fig. 1. The quantities of Mal d 1 and Mal d 2 allergens were calculated from the ratio of the heights of chromatographic peaks of the controls and the samples and expressed in mg/100 g apple. The protein extracts containing Mal d 1 and Mal d 2 allergens were suitable for both the chromatographic analysis and the allergen specific antibody based ELISAs which was intended to be used for confirming the chromatographic data. The chromatographic results can be seen in Fig. 2a, which were confirmed by ELISA measurements (Fig. 2b). Our aim was to monitor the allergenic protein composition of apple extract made of 5 apples per sample, instead of measuring statistically significant differences using expensive, uncertain and time-consuming repetitions (e.g. the usage of SF5 membranes). It must be stated that apple proteins Mal d 1 and Mal d 2 represent extremely different analytical signals, because Mal d 1 is an especially labile protein, while Mal d 2 has very good stability. Both the chromatographic and ELISA results showed that during the ripening period, the content of Mal d 1 and Mal d 2 allergens continuously increased. While the Mal d 1 content slowly increased day-by-day, the Mal d 2 protein level increased slowly at the beginning (until 152th day after bloom time), then it quickly raised when the growing was finished, and the ripening period has started (152–164th days) . These results confirmed the previous

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1,0 0,8 0,6 0,4 0,2 0,0 130

137

144

152

158

164

Days after bloom time Mal d 2

Mal d 1

Fig. 2. Changes in Mal d 1 and Mal d 2 allergen protein content of Golden Delicious apple during the ripening period, measured by the developed liquid chromatographic and ELISA method. a, chromatographic method. b, ELISA method.

findings (Oh, Song, Shin, & Chung, 2000), that Mal d 2 content of apple is related to the ripening state. To explain the practical consequence of the results that the Mal d 1 and Mal d 2 allergens develop differentially during the ripening process of Golden Delicious apples is not easy. The role of Mal d 1 during maturation is not clear; probably it has a function in regulation of physiological stress processes. Mal d 2 has a function in plant defence against fungal pathogens. That function is probably connected with the change in tissue structure (softening), and cell wall disassembly. The purification of Mal d allergens and the development of methods for quantifying Mal d allergens were acceptable approaches. Comparing the applied methods, it was found that the allergen contents measured by antibody based ELISA and size exclusion chromatography followed similar tendency. It seems that the chromatographic method was comparable in quantifying the Mal d 1 and Mal d 2 allergen contents in Golden Delicious apple cultivar with ELISA. 4. Conclusions

Fig. 1. Separation of Mal d 1 and d 2 by size exclusion chromatography. Apple proteins were extracted from pulp with acidic buffer, then processed by CM-cellulose chromatography, and finally the eluted proteins were separated on Superose column.

Our aim was to develop a liquid chromatographic method to concurrently purify the heat labile Mal d 1 from Golden Delicious apple cultivar having high potential to express Mal d 1 allergens and monitor their expression levels during ripening. Golden Delicious apple cultivar was a good source of the Mal d 1 allergens which was confirmed previously by PCR techniques. In respect to differences in stability between the heat labile Mal d 1 (18 kDa) and the heat resistant Mal d 2 (31 kDa) allergenic proteins the IEX/SEC chromatography proved to be a reliable analytical technique for separation and simultaneous quantitative determination of target apple allergens. The main advantage of the acidic extraction of apple proteins is the inhibition of enzymatic browning in the extract. As it is known, lowering of the pH to less than 4.0 inhibits the activity of polyphenoloxidase, which is responsible for enzymatic browning. The inhibition of this reaction is important, inasmuch as the

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end-product brown pigment binds the extracted proteins to an indefinite extent. Additional advantage of the acidic extraction is that it supports the processing and analyses of apple extract with chromatography and ELISA as well. The acidic extract could be easily applied to ionic exchange chromatography and ELISA. One of the most developed methods published in the literature is that of Vieths et al. (1992). However the use of low temperatures and the large volume of cold acetone make this procedure less feasible. It was established that the acidic extraction can successfully protect the active allergens from the reactions with phenolic compounds present in apple. The results were comparable to competitive ELISA during ripening of Golden Delicious apple cultivar. Acknowledgements We wish to thank Mrs. Katalin Háder-Sólyom and Mrs. Éva KissValentin in assisting in the practical laboratory work. This research was supported by 67809 Hungarian Research Grant OTKA. References Atkinson, R. G., Perry, J., Matsui, T., Ross, G. S., & Macrae, E. A. (1996). A stress-, pathogenesis-, and allergen-related cDNA in apple fruit is also ripening related. New Zealand Journal of Crop and Horticultural Science, 24, 103–107. Björkstén, F., Halmepuro, L., Hannuksela, M., & Lahti, A. (1980). Extraction and properties of apple allergens. Allergy, 35, 671–677. Breiteneder, H., & Mills, E. N. C. (2005). Molecular properties of food allergens. The Journal of Allergy and Clinical Immunology, 115, 14–23. Calkoven, P. G., Aalbers, M., Koshte, V. L., Pos, O., Oei, H. D., & Aalberse, R. C. (1987). Cross-reactivity among birch pollen, vegetables and fruits as detected by IgE antibodies is due to at least three distinct cross-reactive structures. Allergy, 42, 382–390. Fritsch, R., Bole, B., Vollmann, U., Wiedemann, U., Jahn-Schmid, B., Krebitz, M., Breiteneder, H., Kraft, D., & Ebner, C. (1998). Bet v 1, the major birch pollen allergen, and the Mal d 1, the major apple allergen, cross-react at the level of allergenspecific T helper cells. The Journal of Allergy and Clinical Immunology, 102, 679–686. Gao, Z. S., Van de Weg, W. E., Matos, C., Van der Meer, I. M., Li, Y. H., Bolhaar, S. T. H. P., Knulst, A. C., Zuidmeer, L., van Ree, R., Hoffmann-Sommergruber, K., & Gilissen, L. J. W. J. (2005). Allelic constitution of Mal d 1 genes on linkage group 16 is related to the differences in allergenicity among apple cultivars. Theoretical and Applied Genetics, 110, 769–783. Hajós, G., Gelencsér, É., Pusztai, Á., Grant, G., Sakhri, M., & Bardócz, Zs. (1995). Biological effects and survival of trypsin-inhibitors and the agglutinin from soybean in the small-intestine of the rat. Journal of Agricultural and Food Chemistry, 43(1), 165–170. Harboe, N., & Inglid, A. (1975). A manual of quantitative immunelectrophoresis, methods and applications. In N. H. Axelsen, J. Kroll, & B. Weeke (Eds.), Scan. J. Immunology, vol. 2 Suppl. No.1. (pp. 161–164). (2) Marchall, J. J., & Lauda, C. M. (1975). Purification and properties of phaseolamin, an inhibitor of a-amylase, from kidney bean, Phesolus vulgaris. The Journal of Biological Chemistry, 250, 8030–8037.

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