Although the physicochemical characteristics of isolated Cav p 1 are very similar to those for other rodent allergens and furthermore partial sequence identity with Mus m 1 was found, it is clearly shown here to be an immunologically independent major allergen.
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 degrees C or in lyophilized state at -20 degrees C, 4 degrees 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 degrees C. For storage of extract solutions at 4 degrees C, PBS/glycerol and cysteine/sodium citrate/glycerol were found to be the most effective stabilizing additives.
Chronic urticaria, recurrent angioedema and non-allergic asthma have all been associated with pseudoallergic reactions to food ingredients. For atopic dermatitis and diseases of the gastrointestinal tract, this association is controversial. Pseudoallergic reactions can be elicited by additives as well as by natural food ingredients. An altered histamine metabolism may be associated with pseudoallergy. Acute urticaria or a short episode of angioedema is not an indication for exhaustive evaluation. If basic diagnostic screening is negative in chronic urticaria, a low-pseudoallergen diet can be considered. Skin and serological tests are not objective diagnostic parameters for pseudoallergic reactions. The severity of symptoms should be documented while the patient is on a low-pseudoaller-gen diet. Oral provocation with additives leads to reproducible symptoms only in a few cases. Therefore, if a low-pseudoallergen diet brings improvement, the patient is then exposed to a pseudoallergen-rich "super meal". After a positive reaction to the "super meal" the challenge with additives takes place in the form of collective group exposition. When the patient has asthma or a history of anaphylac-toid reactions, testing with individual substances in carefully increasing dosages is required. The suspicion of adverse reactions against histamine can be confirmed by a challenge with histamine dihydrochloride. In the case of respiratory symptoms, provocation by inhalation should be considered. Objectifying symptoms especially in gastrointestinal diseases is mandatory and should include double-blind placebo-controlled food challenge, if possible.
The aim of our investigation was to obtain a well-characterized active apple extract suitable for both in vivo and in vitro diagnostics by a technically simple method. For this purpose, apple extracts were prepared by homogenization in potassium phosphate buffer or by precipitation in organic solvents and resolubilization in potassium phosphate buffer in the presence or in the absence of enzyme inhibitors. These extracts were comparatively investigated by means of SDS-PAGE, two-dimensional electrophoresis, immunoblotting, RAST inhibition, and prick test. The in vitro investigations indicated that extracts prepared by precipitation in organic solvents (diacetone alcohol) at -20 degrees C have a higher allergen activity than those prepared by extraction in aqueous solutions. From the in vivo tests (prick test), it was concluded that application of inhibitors of cytoplasmic enzymes (phenol oxidases, peroxidases, proteases) already during extraction is an essential precondition for active prick test solutions. Correspondingly, the extract obtained by solvent precipitation in the presence of enzyme inhibitors appeared to be most suitable for clinical application.
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