Allergy in the 21st Century: New Answers to Old Questions
23rd Symposium of the Collegium Internationale Allergologicum
May 18-23, 2000, Hakone, Japan
Editors: Takeru Ishikawa, Kumamoto; Terumasa Miyamoto, Tokyo; Hirokazu Okudaira, Tokyo; Hisao Tomioka, Chiba; Rudolf Valenta, Vienna; Dietrich Kraft, Vienna

Allergens

Tapping Allergen Repertoires by Advanced Cloning Technologies
Reto Crameri^a, Rimantas Kodzius^a,b, Zoltan Konthur^b, Hans Lehrach^b, Kurt Blaser^a, Gerald Walter<sup>b

a</sup>Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland;
^bMax Planck Institute of Molecular Genetics, Berlin, Germany

Address of Corresponding Author

Int Arch Allergy Immunol 2001;124:43-47 (DOI: 10.1159/000053664)

  Key Words

• Allergens
• Cloning
• Phage surface display
• Robotics
• DNA array
• Hybridization

  Abstract

Background: Complex allergenic sources such as moulds, foods and mites contain complex panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Methods: Phage surface display of cDNA libraries selectively enriched for allergen-expressing clones using IgE from allergic patients allows rapid isolation of large panels of allergens. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, high-throughput screening technology is required. Results: The combination of selective enrichment of cDNA libraries based on biopanning against serum IgE from sensitized patients and automated robot technology for picking and high-density gridding of clones onto filter membranes, followed by hybridization, enables fast identification of all the different clones present in an enriched library. The consequent application of selective enrichment and robotic-based screening allows, within weeks, cloning and characterization of the whole allergenic repertoire of any organisms. Conclusions: Robotic-based high-throughput screening of clones selected for IgE-binding capacity from phage surface-displayed cDNA libraries of Aspergillus fumigatus, Cladosporium herbarum, Coprinus comatus, Malassezia furfur, peanut and human 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.

Copyright © 2001 S. Karger AG, Basel

  Author Contacts

Correspondence to: Prof. Reto Crameri
Swiss Institute of Allergy and Asthma Research (SIAF)
Obere Strasse 22
CH-7270 Davos (Switzerland)
Tel. +41 81 410 08 48, Fax +41 81 410 08 40, E-Mail crameri@siaf.unizh.ch

  Article Information

Number of Print Pages : 5
Number of Figures : 1, Number of Tables : 1, Number of References : 30