Fractionation and identification of the allergic proteins in species.

BACKGROUND AND PURPOSE

Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of strains including , , and could be mentioned. In this study, we aimed to separate allergic proteins from strains and determine their identity.

MATERIALS AND METHODS

Standard species of strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freeze-thawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods.

RESULTS

Among the studied species, showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE-reactive proteins was confirmed to be 45 kDa mycelia catalase.

CONCLUSION

Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase.