D3.71 - A research tool for development of in-house specific IgE immunoassays - comparison between different methods

The possibility to quantitatively measure allergen-specific IgE antibodies is a valuable tool in allergy research to identify and characterize new allergens, as well as help elucidate novel cross-reactivity patterns; however, the allergen of interest may not be commercially available. The aim of this study was to evaluate the potential use of the ImmunoCAP Streptavidin test for developing in-house assays and quantifying specific IgE (sIgE) to novel allergens, and to compare an automated and a manual method for coupling.

Allergens (whole allergen extracts and recombinant allergen components) were biotinylated and coupled to the ImmunoCAP Streptavidin test. Different biotinylation conditions and allergen-coupling methods and concentrations were evaluated. In addition, automated and manual coupling of biotinylated allergen sources were compared.

Two allergen sources (rAra h 1 and horse dander extract), biotinylated using 5-fold and 20-fold molar excesses of biotin, were coupled to the ImmunoCAP Streptavidin test and analyzed on the Phadia 200 instrument. Biotin molar excess had little or no impact on assay performance in this study. Three allergen coupling concentrations were tested, yielding responses of 71–106% relative to conventional tests. At the optimal coupling concentration, response levels were 98% for the whole allergen extract and 89%–100% for the allergen components, and no total IgE interference was detected above the LoQ. Overall, optimization of coupling concentration appeared more critical than the biotinylation step in these experiments. Agreement between an automated method for coupling biotinylated allergens and a manual method was evaluated (Figure 1). The manual method had a variation of 3.61% (CV) between replicates and is acceptable for use when the automated method (Phadia 200) is not available.

The ImmunoCAP Streptavidin test is a research tool that enables development of in-house assays and quantification of sIgE to novel allergens. In this study, coupling concentration appeared more important than biotinylation conditions. A manual coupling method can be used when a Phadia 200 instrument is not available. This approach may facilitate the discovery of novel allergens and investigations of sensitization patterns in allergy research.