Tapping allergen repertoires by advanced cloning technologies.
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.
Crameri
R
R
Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland. crameri@siaf.unizh.ch
Kodzius
R
R
Konthur
Z
Z
Lehrach
H
H
Blaser
K
K
Walter
G
G
D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012371:Robotics / Q000379:methods*
D000485:Allergens; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007073:Immunoglobulin E