Tapping allergen repertoires by advanced cloning technologies

Background: Complex allergenic sources such as moulds, foods and mites cont ain complex panels of IgE-binding molecules which need to be cloned, produc ed and characterized in order to mimic the entire allergenicity of whole ex tracts reconstituted by mixing single standardized recombinant allergens. M ethods: Phage surface display of cDNA libraries selectively enriched for al lergen-expressing clones using IgE from allergic patients allows rapid isol ation of large panels of allergens. For the characterization of all differe nt clones present in enriched cDNA libraries in a fast and cost-effective w ay, high-throughput screening technology is required. Results: The combinat ion of selective enrichment of cDNA libraries based on biopanning against s erum IgE from sensitized patients and automated robot technology for pickin g and high-density gridding of clones onto filter membranes, followed by hy bridization, enables fast identification of all the different clones presen t in an enriched library. The consequent application of selective enrichmen t and robotic-based screening allows, within weeks, cloning and characteriz ation of the whole allergenic repertoire of any organisms. Conclusions: Rob otic-based high-throughput screening of clones selected for IgE-binding cap acity from phage surface-displayed cDNA libraries of Aspergillus fumigatus, Cladosporium herbarum, Coprinus comatus, Malassezia furfur, peanut and hum an lung tissue allowed rapid characterization of 81, 28, 37, 27, 8 and 151 different sequences, respectively. All these cDNAs bear a high probability to encode allergens derived from the respective allergenic source. Copyrigh t (C) 2001 S. Karger AG,Basel.