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 an Ames test‐negative, alternative substrate for HRP. The quality of several commercially available anti‐IgE antibodies was examined. For some of these reagents very high non‐specific binding to food and pollen proteins occurred. The application of our method to apple and birch pollen allergen characterization is described. Results of IgE‐ and IgG‐immunoblotting and immuno‐detection of allergens following two‐dimensional‐ electrophoresis, as well as cross‐reactivity studies by means of immunoblot inhibition, are presented.