Immunocheraical investigation of allergens from Rhizopus nigricans

The allergenic proteins of mould, Rhizopus nigricans extract (RNE) were identified and characterized by crossed immunoelectrophoresis (CIE), thin-layer isoelectrofocusing (TLIEF) and RAST inhibition. CIE revealed that the extract contained at least 31 distinct antigens. On TLIEF the extract resolved into 22 bands in pI 3.5-6.8. Two important allergens, Rhiz IIIb and VIb were purified by a combination of ammonium sulfate precipitation, anion exchange chromatography on DEAE-Sephadex column and gel filtration. Twenty and 12 micrograms of Rhiz IIIb and Rhiz VIb were sufficient to give 50% RAST inhibition as against 43 micrograms of crude RNE. Rhiz IIIb and Rhiz VIb were found to be glycoproteins with molecular weights of 12,400 daltons and 14,200 daltons, respectively. Rhiz IIIb was found to be homogeneous on polyacrylamide gel electrophoresis (PAGE) and TLIEF with a pI of 4.8, while Rhiz VIb gave a single band on PAGE and resolved into two Coomassie blue stained bands with pI 3.6 and 3.8. It was possible to separate the components of RNE on fast protein liquid chromatography (FPLC) using an anion exchanger Mono Q column. The identification and characterization of the antigenic and allergenic proteins in the extract will be useful in standardization of RNE and in preparation of an in-house reference standard.