Author:

Keywords:

Aptamer, CE-SELEX, lysozyme

Abstract:

Food allergy is a major health concern in both children and adults. Hen egg is one of the food components mostly reported to cause adverse reactions in infants and young children. Lysozyme is one of major allergens in egg – white protein appearing not only in foods but also in many health care products. It may cause mild to severe allergic reactions in small amounts. In this study we present a novel approach for selecting aptamers with high affinity and selectivity using Capillary Electrophoresis - Systematic Evolution of Ligands by Exponential enrichment (CE-SELEX). T o perform CE-SELEX, the random DNA library was incubated with lysozyme and the bound sequences were isolated from unbound sequences u sing capillary electrophoresis and detected with laser-induced fluorescence (LIF) detection. Subsequently, the bound sequences were PCR amplified and purified to obtain an enriched ssDNA pool for further selection rounds. During the selection process the dissociation constant of the ssDNA pool decreased from the micromolar to the low nanomolar range within five rounds of selection. The affinity and the specificity of the selected ssDNA aptamers to lysozyme were evaluated using fluorescence anisotropy, surface plasmon resonance and affinity capillary electrophoresis. The selected aptamer had a dissociation constant of 3 nM as determined by fluorescence anisotropy. The aptamers were successfully challenged for specificity against other egg white proteins. The high affinity aptamer opens up possibilities for the development of an aptamer based bio-assay for the detection and quantification of lysozyme in food and pharmaceutical products.