Optimization of apple allergen preparation for in vivo and in vitro diagnostics

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.