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Immunochemical investigation of allergens from Rhizopus nigricans
S Sridhara1, S V Gangal, A P Joshi
1CSIR Centre for Biochemicals, Delhi, India.
Abstract:
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.
Insights
Researchers identified and characterized allergenic proteins in Rhizopus nigricans extract (RNE). Two key allergens, Rhiz IIIb and VIb, were purified and found to be potent inhibitors of RAST, aiding RNE standardization.
Area of Science:
- * Immunology
- * Mycology
- * Protein Chemistry
Background:
- * Moulds like Rhizopus nigricans are common allergens.
- * Understanding mould allergens is crucial for diagnosis and treatment.
- * Rhizopus nigricans extract (RNE) is used in allergy testing.
Purpose of the Study:
- * To identify and characterize allergenic proteins in RNE.
- * To purify major allergens for potential use as standards.
- * To investigate the properties of purified allergens.
Main Methods:
- * Crossed immunoelectrophoresis (CIE) for antigen profiling.
- * Thin-layer isoelectrofocusing (TLIEF) for charge heterogeneity analysis.
- * Radioallergosorbent test (RAST) inhibition for allergen potency.
- * Gel filtration and ion-exchange chromatography for purification.
- * Polyacrylamide gel electrophoresis (PAGE) and Fast Protein Liquid Chromatography (FPLC) for homogeneity assessment.
Main Results:
- * RNE contained at least 31 antigens, resolving into 22 bands on TLIEF.
- * Two major allergens, Rhiz IIIb and VIb, were purified.
- * Rhiz IIIb and VIb demonstrated significantly higher RAST inhibition potency than crude RNE.
- * Both allergens are glycoproteins with distinct molecular weights and isoelectric points.
- * FPLC enabled further separation and analysis of RNE components.
Conclusions:
- * Identification and characterization of RNE allergens are essential for diagnostic accuracy.
- * Purified allergens Rhiz IIIb and VIb can serve as valuable reference standards.
- * This work facilitates the standardization of RNE for clinical applications.