Abstract
BackgroundCurrent diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens.Methodology/Principal FindingsWe sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences.Conclusions/SignificanceThese results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy.
Highlights
Allergy affects 10–40% of the population [1] and results in elevated IgE [2] - a condition that is often diagnosed with skin prick tests (SPT) that can cause discomfort, risk anaphylaxis [3] and can increase patient sensitivity to allergens [4]
Statistical Analyses Coefficient of variances (CV) were calculated as the standard deviation divided by the mean of replicates
Here, we developed a protein microarray containing 80 different allergen fractions from 31 different species of allergen sources, and have demonstrated that it can reproducibly measure allergen-specific IgE in small amounts of sera
Summary
Allergy affects 10–40% of the population [1] and results in elevated IgE [2] - a condition that is often diagnosed with skin prick tests (SPT) that can cause discomfort, risk anaphylaxis [3] and can increase patient sensitivity to allergens [4]. According to the International Union of Immunological Societies Allergen Nomenclature Sub-Committee, over 600 allergens have been identified to date (http://www.allergen.org). Most of these were identified by immunoblotting soluble allergen extracts separated by electrophoresis with patient sera or monoclonal antibodies [18]. Allergens identified in these studies are typically 10–70 kD cytoplasmic proteins and have diverse biological functions [2,18,19]. Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens
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