RESUMO
A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study were theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use.
Assuntos
Actinidia/química , Alérgenos/análise , Artemisia/química , Frutas/química , Extratos Vegetais/química , Pólen/química , Alérgenos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Troca Iônica , Peso Molecular , Proteínas de Plantas/análise , Proteínas de Plantas/isolamento & purificação , Espectrofotometria UltravioletaRESUMO
Kiwifruit has become a frequent cause of fruit allergy in the recent years. The molecular basis of type I hypersensitivity to kiwifruit is attributed to 11 IUIS allergens, with Act d 1, Act d 2 and Act d 5 characterized in extenso. Evaluation of the allergenic properties of Act d 4, a cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa) was performed in this study. Identity of the purified glycoprotein was determined by Edman degradation and by mass fingerprint whereby more than 90% of the primary structure of the mature kiwifruit cystatin was confirmed. Using MALDI TOF analysis, molecular masses of 10902.5 and 11055.2 Da were detected for Act d 4, respectively. Positive skin prick reactivity with Act d 4 was induced in three kiwifruit allergic patients, as well as the upregulation of CD63 and CD203c molecules in the basophile activation assay. The IgE reactivity was detected in dot blot analysis while Western blot analysis was negative using sera from six kiwifruit patients, suggesting the presence of conformational IgE epitopes on the Act d 4 molecule. As activator of effector cells in type I hypersensitivity Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy.