Stefan Vieths

Apple allergy: the IgE‐binding potency of apple strains is related to the occurrence of the 18‐kDa allergen

Low‐temperature, acetone powder extracts were prepared from mature fruit of 16 apple strains. SDS‐PAGE and immunoblot analysis revealed great variation in the relative amounts of the 18‐kDa apple allergen in these extracts. EAST (RAST) scores, measured with individual and pool sera from patients allergic to birch pollen and apples, ranged from 0.2 to 4.0 and were related to the relative amount of the 18‐kDa protein. These findings were confirmed by ELISA‐inhibition assays, dose‐related histamine release, semiquantitative evaluation of immunoblots by absorption/reflection densitometry, and skin prick tests with extracts of Golden Delicious, Boskoop, and Jamba apples (corresponding to a high, low, and very low 18‐kDa allergen content). Additional open oral challenge tests were performed with two apple‐allergic patients and 15 and 16 apple strains. With all methods, the deduced allergenic potency decreased in the following order: Golden Delicious>Boskoop>Jamba. Therefore, we concluded that the IgE‐binding potency of apple strains depends on the occurrence of the 18‐kDa allergen.

Characterization of the 18-kDa Apple Allergen by Two-Dimensional Immunoblotting and Microsequencing

A low-temperature extract taken from Golden Delicious apples was separated by two-dimensional polyacrylamide gel electrophoresis. By means of two-dimensional immunoblotting with patients serum containing IgE specific to Bet v I, a rabbit polyclonal antiserum raised against Bet v I, and two Bet v I specific monoclonal antibodies, epitopes cross-reactive to Bet v I were identified on an apple allergen with a molecular mass of 18 kDa and pI 5.5. Furthermore, certain antibody reactivities with 4 isoproteins of a molecular mass of 16 kDa and pIs ranging from 4.9 to 5.5 were observed, which may indicate the presence of Bet v I related epitopes on these proteins. Based on 26 amino acid residues, N-terminal sequencing of the 18-kDa apple allergen revealed 62% sequence identity between Bet v I from birch pollen and the apple allergen. Our results therefore support the view that both proteins express common as well as non-related IgE-reactive epitopes.

Isolation and characterization of the 18-kDa major apple allergen and comparison with the major birch pollen allergen (Bet v I).

The major allergen from birch pollen, Bet v I, and the cross‐reacting 18‐kDa major allergen from Golden Delicious and Granny Smith apples were isolated by micropreparative SDS‐PAGE followed by electroelution. In the case of apples, highly active, low‐temperature extracts were used. The purity of the allergens was checked by analytic SDS‐PAGE and immunoblotting with allergic patients’ sera, as well as by N‐terminal amino acid microsequencing, and the allergens were found to be very pure. The strong immunologic activity of the isolates was determined by the enzyme allergosorbent test (EAST) and EAST inhibition assays; this activity was, in the case of Bet v I, similar to that of a preparation obtained by monoclonal antibody affinity chromatography. The allergenic potency of Bet v I and of the cross‐reactive apple allergen was determined by EAST inhibition and dose‐related histamine release. With both assay systems, the allergenic reactivity of Bet v I was considerably higher than that of the major apple allergen. Fürthermore, skin prick tests with the purified allergens and with whole allergenic extracts were performed on a group of 33 patients suffering from birch‐pollen and apple hypersensitivity, and on a control group of 10 patients. The frequency of positive prick test results in the allergic patient group ranged from 73% for the major allergen from Golden Delicious apples to 97% with Bet v I and whole birch pollen extract, respectively. In contrast to our low‐temperature extracts, commercial prick test solutions of four different manufacturers were found to be unreliable for the diagnosis of apple allergy. The skin test results again indicated the strong immunologic activity of the allergen isolates and the predominance of the major allergens in context with birch‐pollen and apple hypersensitivity. Taken together, the results support the view that the 18‐kDa major allergen represents most of the allergenicity of the the apple fruit, and that all allergenic epitopes of the apple proteins are present on Bet v I.

Allergy to fruits and vegetables in pollen‐sensitive patients: Allergen characterization by IgE immunoblotting and peroxidase staining

Allergies to several plant foods, i.e. apples, nuts, stone fruits, celery and spices, are highly associated with pollen allergies. The observed crossreactivities are due to structural similarities between food and pollen allergens. Using horseradish peroxidase (HRP) as the marker enzyme we have further developed and optimized a sensitive and specific immunoblotting procedure for the detection of human IgE antibodies specific to food and pollen proteins separated by SDS‐PAGE and immobilized on nitrocellulose blots. Our optimization studies involved four colour substrates and six buffer systems. The best results were obtained when 0.3% Tween 20 and no protein was used for membrane blocking, whereas blocking with proteins yielded high backgrounds. Immuno‐ detection amplification was accomplished by the following incubation steps: (a) sample (human serum); (b) secondary antibody; (c) biotinylated tertiary antibody; and (d) streptavidin‐HRP. Staining was performed with 3,3,5,5‐tetramethylbenzidine which is a...

Investigation of the stability of apple allergen extracts

To determine optimal conditions for allergen preservation, we investigated the influence of different stabilizing additives and of storage temperature on the allergen activity of apple protein preparations, obtained by extraction in phosphate buffer or by precipitation in diacetone alcohol and resolubilization in phosphate buffer in the presence or absence of enzyme inhibitors. For this purpose, the extracts were stored for 6 months either in frozen state at −20° C or in lyophilized state at −20° C, 4° C, or room temperature and were characterized by SDS‐PAGE, immunoblot, ELISA inhibition, and prick test. The highest stability revealed the extracts that were prepared by precipitation in the organic solvent in the presence of enzyme inhibitors, lyophilized, and stored at −20° C. For storage of extract solutions at 4° C, PBS/glycerol and cysteine/sodium citrate/glycerol were found to be the most effective stabilizing additives.

Occurrence of IgE binding allergens during ripening of apple fruits

Apple allergy, as well as allergies to nuts, stone fruits and other fruits and vegetables, is very common in patients with hay fever caused by birch pollen. The observed clustering of hypersensitivities is due to cross‐reactions of allergen‐specific IgE antibodies. In the case of apple allergy, patients frequently report that the symptoms are more severe after ingestion of mature fruits. We therefore investigated whether changes of IgE binding properties arose during the development of Boskoop and Golden Delicious apples. Using one tree for each strain, extracts were prepared from fruits at different states of maturity. Sera of patients allergic to birch pollen and apples were studied by means of non‐competitive and competitive ELISA (EAST and EAST inhibition) as well as by immunoblotting followed by absorption/reflection densitometry. The results of the three methods agreed closely and revealed that the allergenic potency increased strongly during maturation of Golden Delicious apples and to a lesser deg...

Food allergy: Specific binding of IgE antibodies from plant food sensitized individuals to carbohydrate epitopes

The presence of anti‐carbohydrate immunoglobulin E (IgE) antibodies was investigated in a group of 54 patients who were sensitive to at least two pollen species and at least five vegetable foods. Enzyme‐linked immunosorbent assays (ELISAs) and ELISA inhibition assays were performed with immobilized food‐related oligosaccharides (e.g. stachyose from legumes) and extracts of plant gums (e.g. gum arabic), as well as with allergen extracts from apples, celery and other fruits and vegetables. Furthermore, a combined procedure of immunoblotting and periodate treatment was applied to identify the epitope nature of glycans from vegetable food glycoproteins. The results suggested that 12/54 of the sera contained IgE antibodies against carbohydrate structures. The antibodies were also found to be highly cross‐reactive. For six patients, galactose may be an important epitope. The clinical relevance of the phenomenon requires further investigation.