BACKGROUND: A series of studies was performed to identify optimal elution conditions for production of desired Alternaria allergens with simultaneous reduction of undesired ones. METHODS: Proteins and allergens from four strains of Alternaria extracted for differing time intervals and in different buffers were analyzed by one- and two-dimensional electrophoresis, as well as by immunoglobulin E enzyme-linked immunosorbent assay inhibition and immunoblotting. RESULTS: The amount of protein and carbohydrate released varied with each time interval but was consistent between buffers. Extracts from longer time intervals tended to contain more carbohydrate. Electrophoresis of the four strains demonstrated many similar proteins; however, the concentrations of these proteins showed considerable interstrain difference. Comparison of extraction times for single strains by immunoblotting showed that certain allergens are preferentially released during specific time intervals. Some allergens were seen to be most prevalent in a 24-hour extract, whereas others were most prevalent in a 1-hour extract. Two-dimensional electrophoresis resolved bands into discrete spots. The major shared elements of the four strains could be easily identified. The appearance and disappearance of individual protein elements with time was seen. CONCLUSIONS: Elution conditions have a significant impact on quantities of specific glycoproteins contained in extracts of Alternaria and must be controlled and optimized when such extracts are produced for allergen purification.
BACKGROUND: A series of studies was performed to identify optimal elution conditions for production of desired Alternaria allergens with simultaneous reduction of undesired ones. METHODS: Proteins and allergens from four strains of Alternaria extracted for differing time intervals and in different buffers were analyzed by one- and two-dimensional electrophoresis, as well as by immunoglobulin E enzyme-linked immunosorbent assay inhibition and immunoblotting. RESULTS: The amount of protein and carbohydrate released varied with each time interval but was consistent between buffers. Extracts from longer time intervals tended to contain more carbohydrate. Electrophoresis of the four strains demonstrated many similar proteins; however, the concentrations of these proteins showed considerable interstrain difference. Comparison of extraction times for single strains by immunoblotting showed that certain allergens are preferentially released during specific time intervals. Some allergens were seen to be most prevalent in a 24-hour extract, whereas others were most prevalent in a 1-hour extract. Two-dimensional electrophoresis resolved bands into discrete spots. The major shared elements of the four strains could be easily identified. The appearance and disappearance of individual protein elements with time was seen. CONCLUSIONS: Elution conditions have a significant impact on quantities of specific glycoproteins contained in extracts of Alternaria and must be controlled and optimized when such extracts are produced for allergen purification.
Authors: Charles Barnes; Jay Portnoy; Michelle Sever; Samuel Arbes; Ben Vaughn; Darryl C Zeldin Journal: Ann Allergy Asthma Immunol Date: 2006-09 Impact factor: 6.347