Chemically modified allergens (allergoids) have found use in both traditional and novel forms of immunotherapy of allergic disorders. Novel forms of immunotherapy include local allergen delivery, via the gastrointestinal tract. This study conveys the gastrointestinal stability of three types of mugwort pollen allergoids under simulated conditions of the gut. Allergoids of the pollen extract of Artemisia vulgaris were obtained by means of potassium cyanate, succinic and maleic anhydride. Gastrointestinal tract conditions (saliva, and gastric fluid) were simulated in accordance with the EU Pharmacopoeia. The biochemical and immunochemical properties of the derivatives following exposure to different conditions were monitored by determining the number of residual amino groups with 2,4,6-trinitrobenzenesulfonic acid, SDS PAGE, immunoblotting and inhibition of mugwort-specific IgE. Exposure to saliva fluid for 2 min did not influence the biochemical and immunochemical properties of the derivatives. In the very acidic conditions of the simulated gastric fluid, the degree of demaleylation and desuccinylation, even after 4 h exposure, was low, ranging from 10 to 30 %. The digestion patterns with pepsin proceeded rapidly in both the unmodified and modified samples. In all four cases, a highly resistant IgE-binding protein the Mwof which was about 28-35 kD, was present. Within the physiological conditions, no new IgE binding epitopes were revealed, as demonstrated by immunoblot and CAP inhibition of the mugwort specific IgE binding. An important conclusion of this study is the stability of the modified derivatives in the gastrointestinal tract of patients, within physiological conditions. The means that they are suitable for use in much higher concentrations in local forms of immunotherapy than unmodified ones.

The characterization of an allergen is a troublesome and difficult process, as it requires both the precise biochemical characterization of a (glyco)protein molecule and the establishment of its susceptibility to IgE antibodies, as they are the main link to histamine release in some hypersensitivity states (type I allergies). As the characterization of an allergen includes molecular weight determination of the allergenic molecule, its structure determination, physicochemical properties, IgE binding properties of the allergen molecule, and its allergenicity, an overall review of which biochemical and immunochemical methods are used in achieving this goal are presented in this paper. The information on the molecular level on the structures of allergens indicates that allergens are considerably heterogeneous protein structures, and that there is no particular aminoacid sequence which is responsible for the allergenicity. Therefore, information gained from detailed structural, functional and immunochemical studies of these intriguing molecules, which nowadays modulate a variety of pathophysiological conditions, would greatly improve our understanding of the underlying disease mechanisms, and the way to handle them.

An acid phosphatase from an extract of mugwort (Artemisia vulgaris) pollen was purified by a factor of 48 by a combination of ion exchange and gel-chromatography. The molecular weights of the enzyme were 76 kDa and 73 kDa, determined by gel filtration on a Sephadex G-100 sf column and by SDS PAGE(under reducing and non-reducing conditions), respectively. In analytical isoelectrofocusing, the enzyme appears as two very close bands pI at about 4.2. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.16mM. The purified enzyme has broad specificity, and hydrolyses p-nitrophenyl phosphate and ?-naphthyl phosphate. Pyrophosphate and O-phospho-L-tyrosine were estimated to be the best substrates for this enzyme as potential in vivo substrates. The enzyme is inhibited competitively by phosphate (Ki = 1.25 mM), molybdate (Ki = 0.055 mM) and pyrophosphate (Ki = 6.7 mM) and non-competitively by fluoride (Ki = 9.8 mM). Metal ions such as Hg2+, Cu2+ and Zn2+ express an inhibitory effect on the enzyme, while the enzyme is slightly activated by non-ionic detergents, Tween 20 and Triton X-100. There is no change in the enzyme activity in the presence of tartrate, citrate, EDTA, 1,10-phenanthroline and sulfhydryl-group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.