© 1990 S. Karger AG. Basel 0020-5915/90/0922-0168 $ 2.75/0
Int Arch Allergy Appl Immunol 1990;92:168-174
IgE-Mediated Allergic Reactions to Potatoes R. Wahla , Susanne Laub, H.J. Maasch a , U. Wahnh a Allergopharma Joachim Ganzer KG, Research and Development of Allergen Extract Preparations, Reinbek bei Hamburg, FRG; b Universitätsklinikum Rudolf Virchow, Charlottenburg, Berlin
I
Abstract. We investigated sera of 12 patients with IgE-mediated hypersensitivity reactions to potato, using different in vitro methods. Radioallergosorbent test classes 2-4 (7.6-46.5% binding) were measured with potato allergen disks. Immunoblot detected allergen bands with an isoelectric point range of approximately 4.5-5.2 and mainly in a molecular weight range between 16.00 and 30.00 kD. But also at molecular weights of 43.00 and 65.00 kD allergen bands (IgE specificities) could be detected. Histamine release assay performed with isolated fractions of the potato extract showed a great individual variation and positive results of fractions of molecular weights between 10.00 and 80.00 kD. From our data we conclude that raw potato is a ‘multiallergenic’ vegetable.
Adverse reactions to foods are most common dur ing infancy and childhood. IgE antibodies frequently occur in atopic children and at low titers also in some healthy children [1]. Symptoms may occur if food al lergens penetrate the gut barrier and reach their corre sponding IgE antibodies on the mast cells in gut wall, skin, or respiratory tract. The most important allergens in food allergies are egg, milk, nuts, and wheat [2-7], There are only few cases recorded in the literature of allergic reactions to potatoes. Pearson [8] described 29 female subjects with asthma who noted sneezing and/or wheezing while they were scraping unboiled potatoes. Nater and Zwartz [9] described a 24-year-old woman who suffered from allergic reactions after inhalation of finely dispersed particles of raw potatoes. Castells et al. [10] described an 11-year-old girl who developed anaphylactic symptoms to potatoes when she ate po tatoes for the first time at 5 months of age. Recently Quirce et al. [11] described a housewife who devel oped bronchial asthma after peeling potatoes. We investigated sera of patients who suffered from potato allergy. For our investigations we used several in vivo a n d in vitro m ethods such as skin prick test,
histamine release assay (HRA), radioallergosorbent test (RAST), RAST inhibition, and immunoblots. By testing single isolated fractions from a potato extract using HRA, we attempted to determine which frac tions caused the strongest reactions. By immunoblot we characterized the immunologic proteins of the potato extract. Materials and Methods Patients 12 children (age range 1-16 years, mean age 6.5 years) with immediate hypersensitivity reactions (urticaria, gastrointestinal symptoms) and elevated serum IgE concentrations (RAST classes 2-4) were selected for the study. The parents of the patients were in formed about the aim of the study and gave consent. Leukocytes and serum were collected from each patient on the same day. Allergen Extract For the extract preparation potatoes were peeled and dejuiced at room temperature, centrifuged for 15 min, and filtered. One part was lyophilized, the other part was used for the fractionation. Frac tionation was done using a Sephadex G-100 column and 0.004 M NH4 H C 0 3 eluent. Seven fractions in the following molecular weight ranges were isolated: fraction I: 10.00-20.00 kD; fraction II: 20.00- 30.00; fraction III: 30.00-40.00; fraction IV: 40.00-50.00; fraction V: 50.00-60.00; fraction VI: 60.00-70.00, and fraction VII: 70.00- 80.00 kD. The fractions were adjusted to the same protein content and stored freeze-dried until use for HRA. The column was Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:18:13 PM
Introduction
Potato Allergy
169
calibrated with a protein mixture of molecular weights between 12.50 (cytochrome c) and 67.00 kD (bovine-serum albumin). For all investigations the same lot of the potato extract was used.
Table 1. RAST class measurements performed with sera from potato-allergic patients and with the nonallergic control pool serum
Histamine Release Assay HRA was done as described elsewhere [12, 13]. Briefly, blood was collected in 50-ml plastic tubes containing 0.01 M ethylenediaminetetraacetic acid and sedimented for 90 min at room tempera ture with 2.5 ml dextran (Braun Melsungen, FRG; molecular weight 60,000) and 3% glucose/10 ml blood. The upper layer was transferred to another plastic tube and centrifuged at 150 g for 8 min at 4°C. The pelleted cells were suspended in Pipes A contain ing 4 mAf ethylenediaminetetraacetate and centrifuged at 150 g for 8 min at 4°C. The cells were then washed in Pipes A. After centrifu gation, the cells were resuspended in Pipes ACM buffer (2 mM CaCI2, 0.5 m M MgCU) and incubated with serial dilutions of po tato extracts (fractions I—VII) at concentrations ranging from 10"4 to 1 pg/ml. After 40 min the tubes were centrifuged at 400 g, and the supernatants were analyzed for histamine contents. Histamine release reactions were assayed in duplicate using an automated fluorometric technique. The results were expressed as released hista mine as a percentage of total histamine. Basophil sensitivity (Ag30) [s the antigen concentration in micrograms per milliliter required to release 30% cellular histamine; reactivity is the maximum histamine release. Histamine release of 20% was considered to be significant. F]RA was only done with the isolated fractions and not with the crude extract.
Sera
RAST classes
% binding
Bie Br Sa Po Pom Pf Ja Jü K.Ö Sch Kr Sz Nonallergic control pool serum
2 4 3 3 2 3 3 3 3 4 3 2
9.2a 46.5a 13.8a 23.4a 7.6b 21.0” 24.0” 21.5* 17.6e 42.3a 28.7e 8.0b
0
1.6
Radioallergosorbent Test The sera of 12 potato-allergic patients and a nonallergic control pool serum (n = 112) were measured by RAST (Pharmacia, Frei burg, FRG) using potato allergen disks produced according to the prescription of Ceska et al. [14] and Maasch et al. [15]. For the pre paration of the allergen disks 1 pg protein [16] of the nonfractionated extract was used for coupling to cyanogen bromide activated paper disks. After coupling the amount of protein coupled to each disk was measured. For nonspecific binding the allergen disks were measured with a nonallergic control pool serum.
a Histamine release positive with single fractions. b Negative HRA. c HRA not performed.
A g 3o
(pg Protein/ml)
R A ST Inhibition The allergenic activity of potato extract, potato flour, and potato starch was measured by RAST inhibition [17, 18] using the potato allergen disks and a pool serum prepared from four sera of potato-allergic patients (RAST class 3, 1:5 diluted). As tracer for bound specific IgE, l25I-antihuman IgE (Pharmacia) was used. Isoelectric Focusing (IEF) and Sodium Dodecyl Sulfate-Polyacrylamide Cel Electrophoresis (SDS-PAGE) Agarose IEF and SDS-PAGE (gradient 3-22%) of the raw po tato extract were done in accordance with the prescription of the manufacturer (LKB, Munich, FRG). For IEF 50 pg protein and for SDS-PAGE 25 pg protein were applied onto the gel. Marker pro teins were obtained from Pharmacia. The proteins were stained with Coomassie brilliant blue R 250 (Merck, Darmstadt, FRG). (Kd) 10-20 20-30 30-40 40-50 50-60 60-70 70-80
Fig. 1. Basophil sensitivity (Ag30) in 7 children with positive his tamine release to at least 1 of 7 potato fractions of different molecu lar weight.
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Immunoprint Immunoprint was carried out as described by Peltre et al. [19]. The proteins were transferred from the gel onto nitrocellulose membranes (Schleicher and Schiill, Dassel, FRG) by capillary transfer. The membranes were incubated overnight with sera of po-
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170
Table 2. Percentage of histamine release to seven potato frac tions of different molecular weights in 8 patients with positive his tamine release to at least I of the 7 potato fractions
Antigen, pl/ml
io-J Fraction 1 Sa Bie Po Br JÜ Ja Pf Sch
Patients
3 0 ND 2 1 0 0 0
io-J 7 0 72 2 1 0 0 0
10‘2
3 3 66 0 2 0 4 I
io-' 9 3 43 2 5 0 8 2
10°
59 12 42 6 39 2 4 16
Fraction II Sa Bie Po Br Jü Ja Pf Sch
4 3 ND 0 3 0 0 0
4 3 56 1 6 1 0 0
3 5 69 3 23 0 2 0
5 5 50 4 59 0 5 26
42 14 36 4 39 0 47 30
Fraction III Sa Bie Po Br Jü Ja pf Sch
0 3 ND 1 3 0 0 0
1 5 67 2 8 0 0 0
0 7 74 4 38 0 6 0
3 9 69 4 61 0 14 3
16 32 42 5 36 0 73 4
Fraction IV Sa Bie Po Br Jü Ja Pf Sch
1 0 ND 1 4 1 0 0
2 0 67 8 10 1 0 0
5 3 71 4 54 0 5 0
4 8 68 10 ND 0 39 0
28 48 47 21 43 7 82 4
Fraction V Sa Bie Po Br Jü Ja Pf Sch
3 2 ND 0 2 1 0 0
5 3 52 0 4 0 0 0
5 49 72 2 6 2 0 0
7 ND 79 4 43 23 64 14
43 55 61 13 58 23 86 18
Fraction VI Sa Bie Po Br Jü Ja pf Sch
Antigen, gl/ml
io-4
io-3
io-2
io-'
10°
7 5 ND 0 2 0 0 0
5 3 58 0 0 1 0 0
4 3 69 0 1 1 5 2
5 5 74 1 11 0 24 15
31 11 59 2 57 11 84 32 I
Fraction VII Sa Bie Po Br Jü Ja pf Sch
5 3 ND 0 1 4 0 0
5 7 48 0 1 4 0 0
4 7 59 0 3 4 0 12
6 11 69 1 11 2 46 22
24 19 49 6 54 2 84 39
ND = Not determined.
tato-allergic patients (1:5 diluted), washed, incubated with l2SI-antihuman IgE (3 kBq/ml) overnight, and washed once more. Then autoradiography was performed at -2 0 °C over I and 2 weeks us ing Kodak X Omat AR films (Eastman Kodak, Rochester, N.Y., USA). Western Blotting Utilizing western blot analysis [20], the molecular weight of the allergen bands of the potato extract and the IgE specificities of the single patient sera were determined. In order to perform western blotting, 25 pg of protein of the potato extract was separated via SDS-PAGE according to the molecular weight. The proteins were transferred from the gel onto nitrocellulose membranes (Schleicher and Schiill) using electrotransfer. The following procedure was done as described for immunoprint.
Results Histamine Release Assay Histamine release was performed in 10 patients (Sa, Bie, Br, Po, Jü, Ja, Pf, Sch, Sz, and Pom). Two patients (Sz and Pom) did not release histamine from mixed leukocytes to the seven potato fractions at the concentrations used in the assay. Eight patients showed a positive histamine release to at least one fraction. Two patients released hista-
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Patients
Table 2 (continued)
Potato Allergy
171
Histamine Release (%)
Fig. 2. Patient Sch. Dose-response curves of histamine release to seven potato fractions.
\
Radioallergosorbent Test By preparation of the potato allergen disks, 0.300 (xg protein was bound on each cyanogen bro mide activated paper disk. Table 1 shows that the po tato allergen disks were of good quality. With all sera RAST classes 2-4 (7.6-46.5% binding) and with the nonallergic control pool serum RAST class 0 (1.6% binding) were measured.
Fig. 3. RAST inhibition curves of potato extract (O), potato flour (□), and potato starch (A).
R A ST Inhibition Allergenic activity could only be found with the potato extract by RAST inhibition. In total 75% inhi bition was obtained. In contrast, the extracts prepared from potato flour and starch showed no allergenic activity (fig. 3). IEFand SDS-PAGE By IEF most of the protein bands could be de tected in the isoelectric point range of approximately 4.3-7.2 (fig. 4) and by SDS-PAGE in a molecular weight range between 14.40 and 43.00 kD. Further-
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mine to only one fraction (patient Ja: fraction V; pa tient Br: fraction IV), 2 patients to three fractions (pa tient Bie: Fractions III, IV, V; patient Sch: fractions II, VI, VII), 1 patient to four fractions (patient Sa: fractions I, II, V, VI), 1 patient to six fractions (pa tient Pf: fractions II-VII), and 2 patients to all 7 frac tions (patients Jii and Po). Median values of Ag30: pa tient Br released only 21% to fraction IV, patient Ja only 23% to fraction V, and patient Sa only 28% to fraction IV and 24% to fraction VII (table 1). Therefore, these positive results were not included in figure 1. The results of the percentage of histamine release to the different fractions are shown in table 2 for each of the 7 patients. There was no significant difference for basophil reactivity and sensitivity (fig. 1) between the different fractions, and the patients demonstrated considerable variations in their spectra of sensitiza tion to potato allergens. Figure 2 shows the dose-re sponse curves of HRA from patient Sch. Patient Sch released histamine to fractions II, VI, and VII.
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172
-
94.000 67.000 43.000 30.000
=?&88
Sch Kr Ko
more, weak protein bands could be seen at molecular weights of 67.00 and 95.00 kD (fig. 5). Immunoprint and Western Blotting Figures 4 and 5 show besides IEF and SDS-PAGE immunoprint and western blot performed with the potato extract using sera of potato-allergic patients. By immunoprint most allergen bands (IgE specifici ties) could be detected in an isoelectric point range of 4.5-5.2. By western blot the radiostaining intensity of the allergen bands in the range between 16.00 and 30.00 kD was more pronounced in the blots of pa tients Kr and Sch than in the blot of patient Ko. The blot of patient Sch showed also allergen bands with molecular weights of 43.00 and 65.00 kD. In the blots of patients Kr and Ko, only a further allergen band could be detected at molecular weights of 43.00/ 44.00 kD. At a molecular weight of 65.00 kD no al lergen band could be seen.
M
D
Fig. 5. SDS-PAGE (right) and western blot (left) of the potato extract. Western blotting was performed with sera of 3 potato-aller gic patients. M = Marker proteins; D = daltons. Time of auto radiography: 2 weeks.
Discussion Atopic infants frequently show allergic reactions to food. Milk, egg, nuts, and wheat are the most com mon allergens [2-7], Although potatoes are a very im portant vegetable, especially in Europe and North America, and, therefore, exposure is high, potatoes are rarely known to cause food allergy [8-11], In the present study sera were investigated from patients who showed allergic reactions to potatoes. By RAST using potato allergen disks, in all sera IgE specificities could be found, expressed in percent bindings. With the nonallergic control pool serum only very low per cent bindings were measured, so that the positive RAST results with the sera of the potato-allergic pa tients could not be attributed to nonspecific binding. By RAST inhibition, we showed that only the raw po tato extract and not potato flour or starch contained allergenic activity. By western blot of sera of potato-
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Fig. 4. IEF (right) and immunoprint (left) of the potato extract. Immunoprint was performed with sera of 5 potato-allergic patients. M = Marker proteins; Ip = isoelectric point. Time of autoradiogra phy: 2 weeks.
SDS
Potato Allergy
so the allergens seem to be proteins or glycoproteins. Characterization of major allergens still has to be worked out on a larger number of patients. Acknowledgements We thank Mrs. M. Bormann, Mrs. M. Morawietz, Mrs. A. Zieren, C. Schwerter, and I. Werthman for the technical assistance; for typing the manuscript we thank Mrs. L. Witt.
References 1 Foucard T, Johansson SGO: A following study of children with asthmatoid bronchitis. I. Skin test reactions and IgE-antibodies to common allergens. Acta Paediatr Scand 1973;62:633-644. 2 Edwards HJ, McConnockie H, Trotman DM, et al: Allergy to inhaled egg material. Clin Allergy 1983;13:427-432. 3 Langeland T: A clinical and immunological study of allergy to hen’s egg white. Clin Allergy 1983; 13:371 -382. 4 Bahna SL, Gandni MD: Milk hypersensitivity. II. Practical aspects of diagnosis, treatment and prevention. Ann Allergy 1983;50:295-301. 5 Bock A, Atkins FH: The natural history of peanut allergy. J Allergy Clin Immunol 1989;83:900-904. 6 Viencci A, Novembre E, Martino M, et al: Reliability of tests for specific IgE to food in atopic dermatitis. Allergy 1989;44 (suppl 9):90-96. 7 Renz H, Rieger CHL: Kriterien zur Erfassung kindlicher Atopiker mit Sensibilisierungen gegen Nahrungsmittel. Allergologie 1989;12:366-370. 8 Pearson RS: Potato sensitivity on occupational allergy in housewives. Acta Allergol 1966:21:507. 9 Nater JP, Zwartz JA: Atopic allergic reactions due to raw pota toes. J Allergy 1967;40:202-206. 10 Castells MC, Pascual C, Esteban M, et al: Allergy to white pota toes. J Allergy Clin Immunol 1986:78:1110-1114. 11 Quirce S, Diez Gomez ML, Hinogosa M, et al: Housewives with raw potato-induced bronchial asthma. Allergy 1989;44: 532-536. 12 Siraganian RP: An automatic continuous flow system for the extraction and genometric analysis of histamine. Anal Biochem 1974;57:383-394. 13 Maasch HJ, Fischer B, Wahl R, et al: Comparison of histamine release assay and RAST inhibition as tools for allergen extract standardization. Int Arch Allergy Appl Immunol 1984:73: 314-320. 14 Ceska M, Eriksson R, Varga JM: Radioimmunosorbent assay of allergens. J Allergy Clin Immunol 1972;49:1-9. 15 Maasch HJ, Wihl JA, Schultze-Werninghaus G, et al: A manu facturer’s criteria for in-house reference preparations for RAST inhibition. Ann Allergy 1987;59:29-33. 16 Wahl R, Geissler W, Maasch HJ: Comparison of classical Lowry, modified Lowry and a dye-binding assay for the estima tion of protein in allergen extracts and influence of different parameters on the modified Lowry assay. Biol Chem Hoppe Seyler 1985;366:979-984.
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allergic patients, often allergens of molecular weights between 16.00 and 40.000 kD could be detected, whereas histamine release showed comparable posi tive results to a broader range of molecular weights between 10.00 and 30.00 kD. Patient Sch released his tamine to fractions II, VI, and VII, i.e., to allergens of a molecular weight between 20.00 and 30.00 and 60.00 and 80.00 kD which is in rather good accordance with the western blot results where bands in the range between 16.00 and 65.00 kD could be detected. Histamine release was negative for fraction IV (40.00-50.00 kD), and the results for cellular IgE and serum IgE do not correspond. Between cell-bound IgE and serum IgE no consistent results have been re ported [21], An explanation for the discrepancy be tween HRA and western blot might be that the separ ation according to molecular weight by chromatogra phy is not as exact as by SDS-PAGE. Furthermore, the proteins are denatured and change their tertiary structure by SDS-PAGE and, therefore, might lose epitopes or form new epitopes. For the molecular weight determination by size exclusion chromatogra phy nondenatured extracts were used. Unfortunately, we could investigate only 3 patients using western blot and 5 patients with immunoprint. This was due to the small volume of blood we could take from some young infants. So the comparison be tween the different assays was difficult. Quirce et al. [11] assumed that potato allergens > 10 kD caused immediate reactions to raw potatoes in housewives. Castells et al. [10] demonstrated spe cific IgE antibodies directed against several proteins of raw potato extract in the same range, as we did us ing western blotting. The patients described by these authors like our patients, showed allergic reactions to boiled potato. Although raw potato extract for immu noprint, western blot, and HRA was used, one can as sume that those allergens detected by laboratory tests might also be present in boiled potato. The IgE speci ficities in the sera detected by immunoblot were not induced by eating unboiled, but boiled or fried potatoes. Nevertheless, we cannot exclude that the reaction patterns might have been slightly different if we had used boiled potato extract. Similar studies on boiled potato extracts should be performed. Our results show that potatoes contain several al lergens of different isoelectric points and molecular weights. Potato allergy should be kept in mind, if the patients suffer from allergic reactions to food. Neither flour nor starch are the reason for allergic reactions,
173
17 Yman L, Ponterius B, Brandt R: RAST-based allergen assay methods. Dev Biol Stand 1975;20:151-165. 18 Wahl R, Maasch HJ, Geissler W: Comparison of three varia tions of the radioallergosorbent test-inhibition assay for mea suring allergenic activity of grass pollen extracts. Anal Biochem 1983;134:189-194. 19 Peltre G, Lapeyre J, David B: Heterogeneity of grass pollen allergens (Daclylis glomerata) recognized by IgE antibodies in human patient sera by a new nitrocellulose immunoprint technique. Immunol Lett 1982;5:127-131. 20 Burnette WN: ‘Western blotting’: Electrophoretic transfer of proteins from sodium dodecyl-polyacrylamide gels to unmodi fied nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981; 112:195-203.
W ahl/Lau/M aasch/W ahn
21 Lau S, Biemeier M, Urbanek R, et al: Hypersensitivity to oval bumin in children with hen’s egg white allergy. Eur J Pediatr 1988;147:606-608.
Received: March 15, 1990 Accepted after revision: May 31, 1990 Correspondence to: Dr. Rüdiger Wahl Allergopharma Joachim Ganzer KG Research and Development of Allergen Extract Preparations Hermann-Körner-Strasse 52 D-2057 Reinbek bei Hamburg(FRG)
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174
Int Arch Allergy Appl Immunol 1990;92:168-174
IgE-Mediated Allergic Reactions to Potatoes R. Wahla , Susanne Laub, H.J. Maasch a , U. Wahnh a Allergopharma Joachim Ganzer KG, Research and Development of Allergen Extract Preparations, Reinbek bei Hamburg, FRG; b Universitätsklinikum Rudolf Virchow, Charlottenburg, Berlin
I
Abstract. We investigated sera of 12 patients with IgE-mediated hypersensitivity reactions to potato, using different in vitro methods. Radioallergosorbent test classes 2-4 (7.6-46.5% binding) were measured with potato allergen disks. Immunoblot detected allergen bands with an isoelectric point range of approximately 4.5-5.2 and mainly in a molecular weight range between 16.00 and 30.00 kD. But also at molecular weights of 43.00 and 65.00 kD allergen bands (IgE specificities) could be detected. Histamine release assay performed with isolated fractions of the potato extract showed a great individual variation and positive results of fractions of molecular weights between 10.00 and 80.00 kD. From our data we conclude that raw potato is a ‘multiallergenic’ vegetable.
Adverse reactions to foods are most common dur ing infancy and childhood. IgE antibodies frequently occur in atopic children and at low titers also in some healthy children [1]. Symptoms may occur if food al lergens penetrate the gut barrier and reach their corre sponding IgE antibodies on the mast cells in gut wall, skin, or respiratory tract. The most important allergens in food allergies are egg, milk, nuts, and wheat [2-7], There are only few cases recorded in the literature of allergic reactions to potatoes. Pearson [8] described 29 female subjects with asthma who noted sneezing and/or wheezing while they were scraping unboiled potatoes. Nater and Zwartz [9] described a 24-year-old woman who suffered from allergic reactions after inhalation of finely dispersed particles of raw potatoes. Castells et al. [10] described an 11-year-old girl who developed anaphylactic symptoms to potatoes when she ate po tatoes for the first time at 5 months of age. Recently Quirce et al. [11] described a housewife who devel oped bronchial asthma after peeling potatoes. We investigated sera of patients who suffered from potato allergy. For our investigations we used several in vivo a n d in vitro m ethods such as skin prick test,
histamine release assay (HRA), radioallergosorbent test (RAST), RAST inhibition, and immunoblots. By testing single isolated fractions from a potato extract using HRA, we attempted to determine which frac tions caused the strongest reactions. By immunoblot we characterized the immunologic proteins of the potato extract. Materials and Methods Patients 12 children (age range 1-16 years, mean age 6.5 years) with immediate hypersensitivity reactions (urticaria, gastrointestinal symptoms) and elevated serum IgE concentrations (RAST classes 2-4) were selected for the study. The parents of the patients were in formed about the aim of the study and gave consent. Leukocytes and serum were collected from each patient on the same day. Allergen Extract For the extract preparation potatoes were peeled and dejuiced at room temperature, centrifuged for 15 min, and filtered. One part was lyophilized, the other part was used for the fractionation. Frac tionation was done using a Sephadex G-100 column and 0.004 M NH4 H C 0 3 eluent. Seven fractions in the following molecular weight ranges were isolated: fraction I: 10.00-20.00 kD; fraction II: 20.00- 30.00; fraction III: 30.00-40.00; fraction IV: 40.00-50.00; fraction V: 50.00-60.00; fraction VI: 60.00-70.00, and fraction VII: 70.00- 80.00 kD. The fractions were adjusted to the same protein content and stored freeze-dried until use for HRA. The column was Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:18:13 PM
Introduction
Potato Allergy
169
calibrated with a protein mixture of molecular weights between 12.50 (cytochrome c) and 67.00 kD (bovine-serum albumin). For all investigations the same lot of the potato extract was used.
Table 1. RAST class measurements performed with sera from potato-allergic patients and with the nonallergic control pool serum
Histamine Release Assay HRA was done as described elsewhere [12, 13]. Briefly, blood was collected in 50-ml plastic tubes containing 0.01 M ethylenediaminetetraacetic acid and sedimented for 90 min at room tempera ture with 2.5 ml dextran (Braun Melsungen, FRG; molecular weight 60,000) and 3% glucose/10 ml blood. The upper layer was transferred to another plastic tube and centrifuged at 150 g for 8 min at 4°C. The pelleted cells were suspended in Pipes A contain ing 4 mAf ethylenediaminetetraacetate and centrifuged at 150 g for 8 min at 4°C. The cells were then washed in Pipes A. After centrifu gation, the cells were resuspended in Pipes ACM buffer (2 mM CaCI2, 0.5 m M MgCU) and incubated with serial dilutions of po tato extracts (fractions I—VII) at concentrations ranging from 10"4 to 1 pg/ml. After 40 min the tubes were centrifuged at 400 g, and the supernatants were analyzed for histamine contents. Histamine release reactions were assayed in duplicate using an automated fluorometric technique. The results were expressed as released hista mine as a percentage of total histamine. Basophil sensitivity (Ag30) [s the antigen concentration in micrograms per milliliter required to release 30% cellular histamine; reactivity is the maximum histamine release. Histamine release of 20% was considered to be significant. F]RA was only done with the isolated fractions and not with the crude extract.
Sera
RAST classes
% binding
Bie Br Sa Po Pom Pf Ja Jü K.Ö Sch Kr Sz Nonallergic control pool serum
2 4 3 3 2 3 3 3 3 4 3 2
9.2a 46.5a 13.8a 23.4a 7.6b 21.0” 24.0” 21.5* 17.6e 42.3a 28.7e 8.0b
0
1.6
Radioallergosorbent Test The sera of 12 potato-allergic patients and a nonallergic control pool serum (n = 112) were measured by RAST (Pharmacia, Frei burg, FRG) using potato allergen disks produced according to the prescription of Ceska et al. [14] and Maasch et al. [15]. For the pre paration of the allergen disks 1 pg protein [16] of the nonfractionated extract was used for coupling to cyanogen bromide activated paper disks. After coupling the amount of protein coupled to each disk was measured. For nonspecific binding the allergen disks were measured with a nonallergic control pool serum.
a Histamine release positive with single fractions. b Negative HRA. c HRA not performed.
A g 3o
(pg Protein/ml)
R A ST Inhibition The allergenic activity of potato extract, potato flour, and potato starch was measured by RAST inhibition [17, 18] using the potato allergen disks and a pool serum prepared from four sera of potato-allergic patients (RAST class 3, 1:5 diluted). As tracer for bound specific IgE, l25I-antihuman IgE (Pharmacia) was used. Isoelectric Focusing (IEF) and Sodium Dodecyl Sulfate-Polyacrylamide Cel Electrophoresis (SDS-PAGE) Agarose IEF and SDS-PAGE (gradient 3-22%) of the raw po tato extract were done in accordance with the prescription of the manufacturer (LKB, Munich, FRG). For IEF 50 pg protein and for SDS-PAGE 25 pg protein were applied onto the gel. Marker pro teins were obtained from Pharmacia. The proteins were stained with Coomassie brilliant blue R 250 (Merck, Darmstadt, FRG). (Kd) 10-20 20-30 30-40 40-50 50-60 60-70 70-80
Fig. 1. Basophil sensitivity (Ag30) in 7 children with positive his tamine release to at least 1 of 7 potato fractions of different molecu lar weight.
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Immunoprint Immunoprint was carried out as described by Peltre et al. [19]. The proteins were transferred from the gel onto nitrocellulose membranes (Schleicher and Schiill, Dassel, FRG) by capillary transfer. The membranes were incubated overnight with sera of po-
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Table 2. Percentage of histamine release to seven potato frac tions of different molecular weights in 8 patients with positive his tamine release to at least I of the 7 potato fractions
Antigen, pl/ml
io-J Fraction 1 Sa Bie Po Br JÜ Ja Pf Sch
Patients
3 0 ND 2 1 0 0 0
io-J 7 0 72 2 1 0 0 0
10‘2
3 3 66 0 2 0 4 I
io-' 9 3 43 2 5 0 8 2
10°
59 12 42 6 39 2 4 16
Fraction II Sa Bie Po Br Jü Ja Pf Sch
4 3 ND 0 3 0 0 0
4 3 56 1 6 1 0 0
3 5 69 3 23 0 2 0
5 5 50 4 59 0 5 26
42 14 36 4 39 0 47 30
Fraction III Sa Bie Po Br Jü Ja pf Sch
0 3 ND 1 3 0 0 0
1 5 67 2 8 0 0 0
0 7 74 4 38 0 6 0
3 9 69 4 61 0 14 3
16 32 42 5 36 0 73 4
Fraction IV Sa Bie Po Br Jü Ja Pf Sch
1 0 ND 1 4 1 0 0
2 0 67 8 10 1 0 0
5 3 71 4 54 0 5 0
4 8 68 10 ND 0 39 0
28 48 47 21 43 7 82 4
Fraction V Sa Bie Po Br Jü Ja Pf Sch
3 2 ND 0 2 1 0 0
5 3 52 0 4 0 0 0
5 49 72 2 6 2 0 0
7 ND 79 4 43 23 64 14
43 55 61 13 58 23 86 18
Fraction VI Sa Bie Po Br Jü Ja pf Sch
Antigen, gl/ml
io-4
io-3
io-2
io-'
10°
7 5 ND 0 2 0 0 0
5 3 58 0 0 1 0 0
4 3 69 0 1 1 5 2
5 5 74 1 11 0 24 15
31 11 59 2 57 11 84 32 I
Fraction VII Sa Bie Po Br Jü Ja pf Sch
5 3 ND 0 1 4 0 0
5 7 48 0 1 4 0 0
4 7 59 0 3 4 0 12
6 11 69 1 11 2 46 22
24 19 49 6 54 2 84 39
ND = Not determined.
tato-allergic patients (1:5 diluted), washed, incubated with l2SI-antihuman IgE (3 kBq/ml) overnight, and washed once more. Then autoradiography was performed at -2 0 °C over I and 2 weeks us ing Kodak X Omat AR films (Eastman Kodak, Rochester, N.Y., USA). Western Blotting Utilizing western blot analysis [20], the molecular weight of the allergen bands of the potato extract and the IgE specificities of the single patient sera were determined. In order to perform western blotting, 25 pg of protein of the potato extract was separated via SDS-PAGE according to the molecular weight. The proteins were transferred from the gel onto nitrocellulose membranes (Schleicher and Schiill) using electrotransfer. The following procedure was done as described for immunoprint.
Results Histamine Release Assay Histamine release was performed in 10 patients (Sa, Bie, Br, Po, Jü, Ja, Pf, Sch, Sz, and Pom). Two patients (Sz and Pom) did not release histamine from mixed leukocytes to the seven potato fractions at the concentrations used in the assay. Eight patients showed a positive histamine release to at least one fraction. Two patients released hista-
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Patients
Table 2 (continued)
Potato Allergy
171
Histamine Release (%)
Fig. 2. Patient Sch. Dose-response curves of histamine release to seven potato fractions.
\
Radioallergosorbent Test By preparation of the potato allergen disks, 0.300 (xg protein was bound on each cyanogen bro mide activated paper disk. Table 1 shows that the po tato allergen disks were of good quality. With all sera RAST classes 2-4 (7.6-46.5% binding) and with the nonallergic control pool serum RAST class 0 (1.6% binding) were measured.
Fig. 3. RAST inhibition curves of potato extract (O), potato flour (□), and potato starch (A).
R A ST Inhibition Allergenic activity could only be found with the potato extract by RAST inhibition. In total 75% inhi bition was obtained. In contrast, the extracts prepared from potato flour and starch showed no allergenic activity (fig. 3). IEFand SDS-PAGE By IEF most of the protein bands could be de tected in the isoelectric point range of approximately 4.3-7.2 (fig. 4) and by SDS-PAGE in a molecular weight range between 14.40 and 43.00 kD. Further-
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mine to only one fraction (patient Ja: fraction V; pa tient Br: fraction IV), 2 patients to three fractions (pa tient Bie: Fractions III, IV, V; patient Sch: fractions II, VI, VII), 1 patient to four fractions (patient Sa: fractions I, II, V, VI), 1 patient to six fractions (pa tient Pf: fractions II-VII), and 2 patients to all 7 frac tions (patients Jii and Po). Median values of Ag30: pa tient Br released only 21% to fraction IV, patient Ja only 23% to fraction V, and patient Sa only 28% to fraction IV and 24% to fraction VII (table 1). Therefore, these positive results were not included in figure 1. The results of the percentage of histamine release to the different fractions are shown in table 2 for each of the 7 patients. There was no significant difference for basophil reactivity and sensitivity (fig. 1) between the different fractions, and the patients demonstrated considerable variations in their spectra of sensitiza tion to potato allergens. Figure 2 shows the dose-re sponse curves of HRA from patient Sch. Patient Sch released histamine to fractions II, VI, and VII.
W ahl/Lau/M aasch/W ahn
172
-
94.000 67.000 43.000 30.000
=?&88
Sch Kr Ko
more, weak protein bands could be seen at molecular weights of 67.00 and 95.00 kD (fig. 5). Immunoprint and Western Blotting Figures 4 and 5 show besides IEF and SDS-PAGE immunoprint and western blot performed with the potato extract using sera of potato-allergic patients. By immunoprint most allergen bands (IgE specifici ties) could be detected in an isoelectric point range of 4.5-5.2. By western blot the radiostaining intensity of the allergen bands in the range between 16.00 and 30.00 kD was more pronounced in the blots of pa tients Kr and Sch than in the blot of patient Ko. The blot of patient Sch showed also allergen bands with molecular weights of 43.00 and 65.00 kD. In the blots of patients Kr and Ko, only a further allergen band could be detected at molecular weights of 43.00/ 44.00 kD. At a molecular weight of 65.00 kD no al lergen band could be seen.
M
D
Fig. 5. SDS-PAGE (right) and western blot (left) of the potato extract. Western blotting was performed with sera of 3 potato-aller gic patients. M = Marker proteins; D = daltons. Time of auto radiography: 2 weeks.
Discussion Atopic infants frequently show allergic reactions to food. Milk, egg, nuts, and wheat are the most com mon allergens [2-7], Although potatoes are a very im portant vegetable, especially in Europe and North America, and, therefore, exposure is high, potatoes are rarely known to cause food allergy [8-11], In the present study sera were investigated from patients who showed allergic reactions to potatoes. By RAST using potato allergen disks, in all sera IgE specificities could be found, expressed in percent bindings. With the nonallergic control pool serum only very low per cent bindings were measured, so that the positive RAST results with the sera of the potato-allergic pa tients could not be attributed to nonspecific binding. By RAST inhibition, we showed that only the raw po tato extract and not potato flour or starch contained allergenic activity. By western blot of sera of potato-
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Fig. 4. IEF (right) and immunoprint (left) of the potato extract. Immunoprint was performed with sera of 5 potato-allergic patients. M = Marker proteins; Ip = isoelectric point. Time of autoradiogra phy: 2 weeks.
SDS
Potato Allergy
so the allergens seem to be proteins or glycoproteins. Characterization of major allergens still has to be worked out on a larger number of patients. Acknowledgements We thank Mrs. M. Bormann, Mrs. M. Morawietz, Mrs. A. Zieren, C. Schwerter, and I. Werthman for the technical assistance; for typing the manuscript we thank Mrs. L. Witt.
References 1 Foucard T, Johansson SGO: A following study of children with asthmatoid bronchitis. I. Skin test reactions and IgE-antibodies to common allergens. Acta Paediatr Scand 1973;62:633-644. 2 Edwards HJ, McConnockie H, Trotman DM, et al: Allergy to inhaled egg material. Clin Allergy 1983;13:427-432. 3 Langeland T: A clinical and immunological study of allergy to hen’s egg white. Clin Allergy 1983; 13:371 -382. 4 Bahna SL, Gandni MD: Milk hypersensitivity. II. Practical aspects of diagnosis, treatment and prevention. Ann Allergy 1983;50:295-301. 5 Bock A, Atkins FH: The natural history of peanut allergy. J Allergy Clin Immunol 1989;83:900-904. 6 Viencci A, Novembre E, Martino M, et al: Reliability of tests for specific IgE to food in atopic dermatitis. Allergy 1989;44 (suppl 9):90-96. 7 Renz H, Rieger CHL: Kriterien zur Erfassung kindlicher Atopiker mit Sensibilisierungen gegen Nahrungsmittel. Allergologie 1989;12:366-370. 8 Pearson RS: Potato sensitivity on occupational allergy in housewives. Acta Allergol 1966:21:507. 9 Nater JP, Zwartz JA: Atopic allergic reactions due to raw pota toes. J Allergy 1967;40:202-206. 10 Castells MC, Pascual C, Esteban M, et al: Allergy to white pota toes. J Allergy Clin Immunol 1986:78:1110-1114. 11 Quirce S, Diez Gomez ML, Hinogosa M, et al: Housewives with raw potato-induced bronchial asthma. Allergy 1989;44: 532-536. 12 Siraganian RP: An automatic continuous flow system for the extraction and genometric analysis of histamine. Anal Biochem 1974;57:383-394. 13 Maasch HJ, Fischer B, Wahl R, et al: Comparison of histamine release assay and RAST inhibition as tools for allergen extract standardization. Int Arch Allergy Appl Immunol 1984:73: 314-320. 14 Ceska M, Eriksson R, Varga JM: Radioimmunosorbent assay of allergens. J Allergy Clin Immunol 1972;49:1-9. 15 Maasch HJ, Wihl JA, Schultze-Werninghaus G, et al: A manu facturer’s criteria for in-house reference preparations for RAST inhibition. Ann Allergy 1987;59:29-33. 16 Wahl R, Geissler W, Maasch HJ: Comparison of classical Lowry, modified Lowry and a dye-binding assay for the estima tion of protein in allergen extracts and influence of different parameters on the modified Lowry assay. Biol Chem Hoppe Seyler 1985;366:979-984.
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allergic patients, often allergens of molecular weights between 16.00 and 40.000 kD could be detected, whereas histamine release showed comparable posi tive results to a broader range of molecular weights between 10.00 and 30.00 kD. Patient Sch released his tamine to fractions II, VI, and VII, i.e., to allergens of a molecular weight between 20.00 and 30.00 and 60.00 and 80.00 kD which is in rather good accordance with the western blot results where bands in the range between 16.00 and 65.00 kD could be detected. Histamine release was negative for fraction IV (40.00-50.00 kD), and the results for cellular IgE and serum IgE do not correspond. Between cell-bound IgE and serum IgE no consistent results have been re ported [21], An explanation for the discrepancy be tween HRA and western blot might be that the separ ation according to molecular weight by chromatogra phy is not as exact as by SDS-PAGE. Furthermore, the proteins are denatured and change their tertiary structure by SDS-PAGE and, therefore, might lose epitopes or form new epitopes. For the molecular weight determination by size exclusion chromatogra phy nondenatured extracts were used. Unfortunately, we could investigate only 3 patients using western blot and 5 patients with immunoprint. This was due to the small volume of blood we could take from some young infants. So the comparison be tween the different assays was difficult. Quirce et al. [11] assumed that potato allergens > 10 kD caused immediate reactions to raw potatoes in housewives. Castells et al. [10] demonstrated spe cific IgE antibodies directed against several proteins of raw potato extract in the same range, as we did us ing western blotting. The patients described by these authors like our patients, showed allergic reactions to boiled potato. Although raw potato extract for immu noprint, western blot, and HRA was used, one can as sume that those allergens detected by laboratory tests might also be present in boiled potato. The IgE speci ficities in the sera detected by immunoblot were not induced by eating unboiled, but boiled or fried potatoes. Nevertheless, we cannot exclude that the reaction patterns might have been slightly different if we had used boiled potato extract. Similar studies on boiled potato extracts should be performed. Our results show that potatoes contain several al lergens of different isoelectric points and molecular weights. Potato allergy should be kept in mind, if the patients suffer from allergic reactions to food. Neither flour nor starch are the reason for allergic reactions,
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17 Yman L, Ponterius B, Brandt R: RAST-based allergen assay methods. Dev Biol Stand 1975;20:151-165. 18 Wahl R, Maasch HJ, Geissler W: Comparison of three varia tions of the radioallergosorbent test-inhibition assay for mea suring allergenic activity of grass pollen extracts. Anal Biochem 1983;134:189-194. 19 Peltre G, Lapeyre J, David B: Heterogeneity of grass pollen allergens (Daclylis glomerata) recognized by IgE antibodies in human patient sera by a new nitrocellulose immunoprint technique. Immunol Lett 1982;5:127-131. 20 Burnette WN: ‘Western blotting’: Electrophoretic transfer of proteins from sodium dodecyl-polyacrylamide gels to unmodi fied nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981; 112:195-203.
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21 Lau S, Biemeier M, Urbanek R, et al: Hypersensitivity to oval bumin in children with hen’s egg white allergy. Eur J Pediatr 1988;147:606-608.
Received: March 15, 1990 Accepted after revision: May 31, 1990 Correspondence to: Dr. Rüdiger Wahl Allergopharma Joachim Ganzer KG Research and Development of Allergen Extract Preparations Hermann-Körner-Strasse 52 D-2057 Reinbek bei Hamburg(FRG)
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174
