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Partial purification and characterization of mold antigens commonly found in foods
1Department of Food Science, Purdue University, West Lafayette, Indiana 47907-1160.
Abstract:
Rapid methods are needed for detection of molds in foods; therefore, an enzyme-linked immunosorbent assay was developed. The extracellular and mycelial antigens for Mucor, Aspergillus, Cladosporium, and Geotrichum species were partially purified and characterized. The molecular masses of the mycelial and extracellular antigens, as determined by size exclusion chromatography, ranged from 4.5 x 10(5) to 6.7 x 10(5) Da. There was only one main antigenic peak separated by Sepharose CL-4B and concanavalin A-Sepharose columns for Mucor, Cladosporium, and Geotrichum mycelial and extracellular antigens, but there were two for Aspergillus mycelial antigens and three for Aspergillus extracellular antigens. These antigens contained 10 to 50% protein which was part of the active site since protease digestion significantly decreased antigenic activity. Neutral sugars, ranging from 13 to 75%, made up the rest of the active site, and < 1% phosphate was detected in mycelial antigens. Geotrichum, Cladosporium, and Aspergillus antigens contained mainly glucose, galactose, and mannose. Mucor antigens contained these sugars plus fucose. The percentage of sugars differed between the mycelia and extracellular antigens. Enzymatic digestion and competitive inhibition tests using different sugar derivatives showed that galactosyl residues with beta linkages were immunodominant for Aspergillus, Geotrichum, and Cladosporium antigens and mannosyl residues with alpha linkages were immunodominant for Mucor antigens.
Insights
Rapid detection of food molds like Aspergillus and Mucor is crucial. An enzyme-linked immunosorbent assay (ELISA) was developed using partially purified fungal antigens, identifying specific sugar residues for improved mold detection.
Area of Science:
- Food microbiology
- Immunochemistry
- Analytical chemistry
Background:
- Rapid detection methods for molds in food are essential for safety and quality control.
- Existing methods can be time-consuming, necessitating the development of faster diagnostic tools.
- Molds like Aspergillus, Mucor, Cladosporium, and Geotrichum are common food contaminants.
Purpose of the Study:
- To develop a rapid enzyme-linked immunosorbent assay (ELISA) for detecting common foodborne molds.
- To characterize the extracellular and mycelial antigens of selected mold species.
- To identify immunodominant epitopes within these antigens for assay development.
Main Methods:
- Partial purification and characterization of extracellular and mycelial antigens from Mucor, Aspergillus, Cladosporium, and Geotrichum species.
- Size exclusion chromatography to determine molecular masses of antigens.
- Sepharose CL-4B and Concanavalin A-Sepharose chromatography for antigen separation.
- Protease digestion and enzymatic analysis to identify protein and sugar components of antigenic sites.
- Competitive inhibition assays with sugar derivatives to determine immunodominant residues.
Main Results:
- Antigen molecular masses ranged from 4.5 x 10^5 to 6.7 x 10^5 Da.
- Aspergillus antigens showed more complex separation profiles than other mold antigens.
- Antigens comprised 10-50% protein and 13-75% neutral sugars; phosphate content was <1%.
- Galactosyl residues with beta linkages were immunodominant for Aspergillus, Geotrichum, and Cladosporium.
- Mannosyl residues with alpha linkages were immunodominant for Mucor.
Conclusions:
- Partially purified fungal antigens possess distinct molecular and compositional characteristics.
- Specific sugar residues (galactose or mannose) are immunodominant in different mold species.
- These findings support the development of an ELISA for rapid and specific mold detection in foods.