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Keywords:

  • food allergy;
  • lipid transfer protein;
  • peach;
  • polyclonal antibodies;
  • Rosaceae

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

Background: Lipid transfer proteins are molecules widely distributed in fruits. Sensitization to LTP is frequent in fruit sensitive patients. The aims of this study were to purify LTP and to assess the content of LTP in ripe peach peel and pulp extracts by ELISA inhibition using polyclonal antibodies.

Methods: LTP was purified from ripe yellow peach peel by two different column chromatography methods. A polyclonal antibody was produced by injecting purified LTP into two New Zealand white rabbits. ELISA inhibition and rabbit monospecific polyclonal antibody were used to calculate the LTP content in Springcrest and Miraflores varieties of peach peel and pulp extracts. Purified LTP (2.5 mg/ml) was used to skin test 24 peach-sensitive patients.

Results: The purified LTP showed a single band at approximately 9 kDa. The polyclonal antibody raised anti LTP recognized only the LTP molecule in the peach extracts. LTP content, expressed in µg/mg of freeze-dried extract in four extracts were: yellow peach peel, 15.48; yellow peach pulp 2.25; red peach peel 14.67 and red peach pulp 1.84. Twenty patients (83.3%) had a positive skin test with purified LTP.

Conclusions: We have developed a system to determine the concentration of LTP in peach extracts. LTP in peel extracts is approximately seven times greater than in pulp.

The severity of the adverse reactions induced by the ingestion of fruits varies, and depends on the type of fruit and if the fruit is ingested with or without peel (1, 2). Martínez et al. (3) attributed this phenomenon to the fact that the peel may have a greater protein content than the pulp. Many members of the botanical family Rosaceae are implicated in allergic reactions, especially fruits belonging to the subfamilies Prunoideae (peach, apricot, plum, cherry, etc.) and Pomoideae (apple, pear, medlar, etc.). Peach (Prunus persica) is one of the most consumed Rosaceae fruits in Mediterranean countries and is responsible for an important number of food allergies (4, 5). In 1999 Pastorello et al. (6) identified and sequenced the major allergen of peach (Pru p 3) as a molecule with a molecular weight of 9 kDa. This allergen was subsequently included in the group of lipid transfer proteins (LTP) (7). Asero et al. (8) suggested that LTP could be considered as a group of panallergens, with a high degree of cross-reactivity, not only with other non-related fruits but also with pollens and nuts (9).

Some fruit allergens are degraded rapidly upon manipulation of the fruit, and therefore, the appropriate measures should be taken to avoid this deleterious process. This fact makes the standardization of food extracts particularly difficult, and, therefore, the prick test with natural fruits is the method of choice.

Whereas major allergens can be measured in extracts of several mite species, epithelia, moulds, insects and pollens, major allergen content in foods is rarely assessed. Standardization of food allergens still remains an important issue, especially for fruits. The measurement of LTP in peach, and other fruit extracts, including apples, may improve the overall quality of peach extracts and enhance the diagnostic capabilities of these extracts. It also enhances the overall knowledge of the allergenic composition in these extracts.

The aims of this study were to purify LTP and to assess the content of LTP in ripe peach peel and pulp extracts by ELISA inhibition using polyclonal antibodies.

Preparation of extracts

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

Ripe yellow (var. Miraflores) and red (var. Springcrest) peaches were bought at a local market. Peel and pulp were carefully separated, obtaining approximately 460 and 130 g of pulp and peel, respectively, from each kind of peach. The extracts were prepared according to the method of Björksten (10) with minor modifications. A total of four extracts were prepared, two of pulp (Springcrest and Miraflores peaches) and two of peel (Springcrest and Miraflores peaches). The raw materials were homogenized and extracted (1 : 2 w/v) in PBS 0.01 m, pH 7.4, 20% polyvinylpolypyrrolidone (PVPP) (Sigma, Madrid, Spain), ethylenediaminetetracetic acid disodium salt dihydrate (EDTA) 2 mm (Sigma) and diethyldithiocarbamic acid sodium salt trihydrate (DIECA) 10 mm (Sigma). After an extraction of 30 min, the extracts were centrifuged at 12,000 g for 30 min at 4°C. The supernatants were sterile filtered, dialyzed overnight in 3.5 kDa membranes (Spectra/Por, Rancho Domínguez, USA) and freeze-dried.

Purification of LTP

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

LTP was purified from a Miraflores peach peel extract by gel filtration and hydrophobic interaction chromatography using a FPLC system (Pharmacia, Sweden). Gel filtration (Sephadex G-75) was conducted in a 36 × 1.6 cm column at a constant flow of 1.5 ml/min. The column was equilibrated and eluted with PBS 0.01 m, pH 7.4. Fractions were collected every 3.5 min and absorbance monitored at 280 nm. Fractions where LTP were present (as identified by SDS-PAGE) were pooled, dialyzed, filtered and freeze-dried. Hydrophobic interaction chromatography was conducted in a 20 × 1 cm column, using a phenyl Sepharose matrix. The column was equilibrated with (NH4)2CO3 and the elution of the allergen was done using decreasing concentration of (NH4)2CO3 at a constant flow of 1.5 ml/min. Fractions were collected every 3 min. Fractions containing LTP, as indicated by SDS-PAGE gels, were dialyzed in membranes with a nominal cut-off of 3.5 kDa against bidistilled water, filtered and freeze-dried.

Patient population

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

Twenty-four individuals residing in Madrid and Alicante, Spain, with a clinical diagnosis of IgE mediated food allergy (11) (oral allergy syndrome after ingestion of peach) were skin prick tested with a peel extract from the peach variety Miraflores (5 mg/ml) and with purified LTP (2.5 mg/ml). Patients with a wheal diameter greater than 7 mm2 after 20 min were considered positive according to the guidelines of European Academy of Allergology and Clinical Immunology (12). Skin test areas were measured using technical software mac draft v. 4.3.

SDS-PAGE

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

SDS-PAGE analysis was used to determinate the protein profile of the extracts evaluated in this study. Samples were run in SDS-PAGE gels with 2.67% C, 15% acrylamide as described by Laemmli (13). Five different samples were separated (yellow peach peel, LTP, yellow peach pulp, red peach peel and red peach pulp). Twelve µg of protein of each pulp extract, 7 µg of protein of each peel extract and 1 µg of LTP were loaded on the gels. Reference markers with known molecular weights (Bio-Rad Laboratories. Hercules, CA. USA) were run in the same gel. After electrophoresis, the gels were fixed for 45 min and silver-stained. The gels were studied with the Scanner Sharp JX-330 and analysed with Image Master 1-D Elite v. 2.01 Pharmacia Biotech.

Rabbit antiserum production

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

Two New Zealand white rabbits were immunized with purified LTP following a methodology previously described (14). Briefly, four injections were administrated to each rabbit, with an interval of 2 weeks. The first two injections contained 150 µg of protein, dissolved in 0.7 ml of Freund's complete adjuvant and 0.3 ml of bidistilled water. The third and the fourth injections contained 150 µg of protein, dissolved in 0.7 ml of Freund's incomplete adjuvant and 0.3 ml of bidistilled water. Every injection was administered subcutaneously in the armpit and groin area in four different places in each rabbit. Serum samples were obtained from the marginal vein of the ear before and after the immunization process to evaluate antibody production.

Antibody titres were measured using direct ELISA. Briefly, 10 µg of LTP/ml were dissolved in carbonate/bicarbonate buffer, pH 9.6, and coated onto a plastic microtitre plates (Immulon IV, Dynex Technologies, Chantilly, USA) and blocked with 100 µl per well of 1% HSA dissolved in PBS 0.01 m pH 7.4 for 1 h. One hundred µl of rabbit serum was added to the wells in serial dilutions (from 1 : 2 to 1 : 20 000 dilution and incubated for 2 h. Specific IgG was detected with peroxidase-conjugated monoclonal anti-rabbit IgG (Sigma, Madrid. Spain). IgG was expressed in OD units.

Immunoblot

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

Separated bands were electrophoretically transferred to P-Immobilon membrane (PVDF) (Millipore, Bedford, USA). After transfer, the membrane was dried at room temperature for 4 h. The membrane was incubated overnight with polyclonal rabbit serum diluted 1 : 5000 in PBS 0.01 m Tween 2%. Specific IgG binding was detected with peroxidase-conjugated monoclonal anti-rabbit IgG for 2 h diluted 1 : 10 000 in PBS 0.01 m, foetal calf serum 15%, heparin 1%. Afterwards, the membrane was dried overnight at room temperature.

Quantity assessment of LTP in peach extracts by ELISA inhibition

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

Plastic microtitre plates (Immulon IV, Dynex Technologies, Chantilly, USA) were coated with LTP at a concentration of 10 µg protein/ml in carbonate/bicarbonate buffer pH 9.6 and incubated overnight in a humidified chamber at room temperature. Several dilutions of LTP and of the four peach extracts were made in a low absorbance microwell plate (Nunc, Roskilde, Denmark) using PBS 0.01 m. Equal amounts of the rabbit polyclonal serum pool (diluted 1 : 5000 in PBS 0.01 m) were added to the same wells and allowed to stand for 2 h. After this inhibition period, the dilutions were placed into the wells previously coated with LTP and incubated overnight. After washing, 100 µl of peroxidase-conjugated monoclonal anti-rabbit IgG, dilution 1 : 15 000 were added to the wells for 2 h. After the final wash, 100 µl of peroxidase substrate was added to each well for 30 min and the reaction was stopped with 100 µl/well of sulphuric acid 1 n. The results were read at 450 nm. The regression curve of LTP dilutions (from 1 to 0.125 µg of protein/ml) was used as a standard curve to calculate total concentrations of LTP in the samples; the 50% inhibition point of LTP was calculated.

LTP purification and protein content

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

At the end of the purification process, 19.8 mg of purified LTP were obtained. Peel extracts revealed a higher concentration of protein than the pulp extracts. Protein content of each extract were: yellow peach peel protein contained 60.2 µg protein/mg of freeze-dried material (6%); yellow peach pulp, 28.8 µg (2.8%); red peach peel 56.3 µg (5.6%); and red peach pulp 26.8 µg (2.68%).

Skin tests

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

All 24 positive patients (100%) had a positive skin test reaction with the peel extract; 20 of these patients (83.3%) were also positive to the purified protein. The geometric mean of the wheal sizes were 69 mm2 (18–202) for the peel extract and 35.5 mm2 (11–150) for the purified protein. The statistic analysis (Wilcoxon signed rank test) showed significant differences (P < 0.01) in the wheal sizes induced by both skin test reagents.

SDS-PAGE

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

Protein profile of four peach extracts is shown in Fig. 1. Several bands were detected in every extract in a molecular weight range of 9–70 kDa. The most prominent band in all extracts was seen at 9 kDa. The purified LTP showed only a band of 9 kDa.

image

Figure 1. SDS-PAGE. Lane 1, molecular markers; lane 2, peach peel (Miraflores variety); lane 3, purified LTP; lane 4, peach pulp (Miraflores variety); lane 5, peach peel (Springcrest variety); lane 6, peach pulp (Springcrest variety).

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Immunoblot

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

The rabbit polyclonal IgG antibodies only recognized LTP in the four analysed extracts and in the LTP lane. The serum of both rabbits collected prior to the immunizations was used as a negative control. No bands were detected using this control. Results are shown in Fig. 2.

image

Figure 2. Immunoblot using rabbit polyclonal antibodies. Lane 1, peach peel (Miraflores variety); lane 2, purified LTP; lane 3, peach pulp (Miraflores variety); lane 4, peach peel (Springcrest variety); lane 5, peach pulp (Springcrest variety).

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Quantity assessment of LTP in peach extracts by ELISA inhibition

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

A standard curve was obtained with several dilutions of LTP inhibition (r = 0.993). The amount of LTP needed to produce 50% inhibition was 0.005 µg of protein. A curve was obtained with each extract. Results were extrapolated from the LTP standard curve. The concentrations of protein to produce 50% inhibition were calculated for each extract: yellow peach peel 0.0194 µg; yellow peach pulp: 0.064 µg; red peach peel 0.0191 µg and red peach pulp 0.073 µg. All curves of all the samples had correlation coefficients, r > 0.95. There were no significant differences in the curves in respect to the standard curve (Fig. 3 A and B), confirming the parallelism and the consistency of the experiment.

image

Figure 3. ELISA inhibition assay using extracts of peel and pulp of the two varieties of peaches. LTP was used in the solid phase.

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LTP content, expressed in µg of LTP/mg of freeze-dried material, in the four extracts was: yellow peach peel 15.48; yellow peach pulp 2.25; red peach peel 14.67 and red peach pulp 1.84. LTP represented 25.72% of the total protein in yellow peach peel, 7.81% in yellow peach pulp, 26.06% in red peach peel and 6.85% in red peach pulp.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References

In this paper we describe the purification process of peach LTP and the production of rabbit antibodies to assess the quantity of LTP in different peach extracts. We purified LTP from peach peel using different chromatography methods and prepared polyclonal rabbit antibodies. The specificity of the serum was demonstrated using immunoblots. These experiments showed that the antiserum only recognized a single band, which corresponded to LTP in each one of the extracts. The activity of the purified protein was evaluated by skin prick test. The results confirm the allergenic activity of LTP on skin test. A great proportion of the patients (83.3%) recognized the allergen, demonstrating that LTP from peach induces positive skin test reactions.

We confirmed that peach peel has a greater protein and LTP content than the pulp. On an average, peel contained approximately twice as much protein as the pulp and approximately seven times more LTP per mg of freeze-dried material. This fact could explain why some individuals experience symptoms after skin contact with the peel and why some individuals may ingest the pulp but not the peel, as suggested by some authors. The results are consistent for the two types of peaches analysed. Both peel extracts have approximately 25% of LTP and pulp peach extracts 7% of LTP.

In order to assess the quantity of LTP ingested by an individual, we made the following calculations. Five peaches weigh approximately 1 kg, of which 160 g are peels, 450 g pulp and 390 g seeds. After extracting, dialysing and freeze-drying these peels, we obtained 1.87 g of freeze-dried material (yield: 1.17%) and 4.41 g (yield: 0.92%) of the pulp. The total protein content in these materials was 112.57 mg for the peel and 127 mg for the pulp. Since the LTP content was 25% of the total protein in the peel and 7% in the pulp extracts, we can assume that 28.14 mg of LTP and 8.89 mg were recovered from the peel and pulp of the 5 peaches, respectively. Therefore, we can speculate that the entire peel of a single peach contains, approximately, 5.62 mg of LTP and the pulp, 1.78 mg. One single peach would therefore contain a total of 7.4 mg of LTP. Considering that a bite may represent a tenth of a peach, one bite would contain approximately 9 g of pulp and 3.2 g of peel. Therefore, if the whole bite is ingested, the individual would ingest a total of 750 µg of LTP, of which 572 originate from the peel and 178 from the pulp. These calculations were based on the general assumption that 1 kg of peaches (five peaches) contains 450 g of pulp and 160 g of peel. The differences between LTP content in peel and pulp could explain why some patients with oral allergy syndrome may resist the intake of peeled peaches. Therefore, we suggest that concentrations of 750 µg of LTP can be associated with the induction of clinical symptoms in peach-sensitive individuals. Similar quantities of allergen have been associated with allergic symptoms (anaphylaxis) after the ingestion of mite-contaminated foods (15).

We have developed a quantitative method to determine the total concentration of an important allergen in fruit extracts. The quantification of Pru p 3, an allergen that is recognized in vitro and in vivo by the majority of peach-allergic patients, may be suitable for establishing selection criteria for raw materials, while also establishing the concentration of this allergen in peach extracts.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Preparation of extracts
  5. Purification of LTP
  6. Protein content
  7. Patient population
  8. SDS-PAGE
  9. Rabbit antiserum production
  10. Immunoblot
  11. Quantity assessment of LTP in peach extracts by ELISA inhibition
  12. Results
  13. LTP purification and protein content
  14. Skin tests
  15. SDS-PAGE
  16. Immunoblot
  17. Quantity assessment of LTP in peach extracts by ELISA inhibition
  18. Discussion
  19. References