High-throughput isolation of recombinant antibodies against recombinant allergens.

BENCHMARKS Immunoglobulin E (IgE)-mediated allergic diseases have become a serious health problem in industrialized countries (1,2). Routine diagnosis of allergic conditions is based on clinical history, skin test reactivity, and serum IgE determinations against the offending agents (3). Both skin test reactivity and determinations of serum IgE levels reflect directly on the quality of the al-lergen extracts used (4). Depending on the exact content of a single allergen in the extract and on the sensitization pattern of the patient, discrepant diagnostic results can be obtained using commercial allergen preparations. Unfortunately , most of the extracts used for diagnosis are not standardized (5,6). The biochemical nature and content of the allergenic components are unknown; therefore, the allergenicity of different extracts cannot be compared. The application of modern molecular biology techniques in allergology allowed for the cloning and molecular characterization of major and minor allergens (7) shown to be powerful reagents for the in vitro and in vivo diagnosis of allergic conditions (4). However, in the majority of cases, reagents allowing for the quantification of individual allergens in commercial allergenic products are still lacking. Monospecific antibodies raised against allergens would substantially facilitate improved quality control and standardization of allergen extracts and lead to the development of more consistent products for clinical use (6,8). Recombinant immunoglobulin gene libraries cloned in phage or phagemid vectors are an in vitro simulation of antibody repertoires, allowing for the production of antibodies in prokaryotic hosts without immunization or the use of animals (9). The fast-increasing number of recombinant allergens and the need for sensitive reagents for their detection prompted us to implement high-through-put technologies (10) for the rapid selection of allergen-specific antibodies. Starting from " single pot " naive human antibody libraries that have been previously described (11), we performed parallel selections of phage carrying functional, binding single-chain fragment variables (scFvs) against different recombinant allergens. Phage antibody libraries were processed and enriched using a prototype pin-based magnetic particle processor (ThermoLabsystems, Helsinki, Finland) combined with liquid-handling robots (KingFisher ® ; ThermoLabsystems) (10). The magnetic particle processor is an automated device that enables the parallel handling of up to 96 individual screenings using 96 magnetic pins that correspond to the position of a 96-well microplate. The magnetic particle processor has a head that consists of two slide-in holders in standard microplate format that are equipped with up to 96 pin-based permanent magnets in rows of eight and the corresponding …