Culture filtrate antigens and allergens of Epicoccum nigrum cultivated in modified semi-synthetic medium.

Abstract

Epicoccum nigrum (EN) is an important fungal allergen for nasobronchial allergy. Fungal extracts should contain all the relevant allergen components from spores, mycelium and culture medium for the purpose of allergy diagnosis and therapy. EN extract from spore-mycelial mass has been standardized, but the culture filtrate (CF) allergens of EN have not been studied as EN grows poorly in synthetic medium. The objective of the present study was to obtain a standard CF extract of EN by cultivating the source material in a modified semi-synthetic medium and to compare this with the EN cellular extract. Sabouraud's medium containing yeast extract (50 mg/l) was filtered using 10-kDa cut-off membrane and the lower molecular mass media components were used to cultivate EN. The CF obtained after removing the spore-mycelia was dialyzed to remove media components. The CF extract was characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot. It was compared with EN spore-mycelial extract by enzyme-linked immunosorbent assay (ELISA), ELISA inhibition and by intradermal testing on allergy patients. The CF extract of EN resolved into 30 protein bands on SDS-PAGE. About 27 IgG bands were detected using anti-EN rabbit antibodies and 12 IgE bands by EN-sensitive pooled patients' sera. Periodate modification of CF proteins showed that the carbohydrate moieties are not important for IgE binding. Protein components of 26, 34 and 43 kDa were recognized as the major CF allergens. Three different batches of CF extract required 7.5-9 ng of self protein for 50% inhibition of binding to anti-EN rabbit antibodies in ELISA. Intradermal testing with CF extract showed comparable allergenic potency to standardized EN spore-mycelial extract, although it contained some allergenic proteins in higher amounts as compared to the spore-mycelial extract. In summary, the semi-synthetic medium has been suitably modified for obtaining EN CF antigens. This medium can be an important substitute for producing potent CF allergens of fungi that grow poorly in synthetic medium. The EN CF extract elicited good allergenic reactivity and may be used for allergy diagnosis along with spore-mycelial extract.