Preparation and Immunological Reactions of a Purified Egg Allergen Ovotransferrin

Abstract

Ovotransferrin is one of the major allergens in egg white. To define the properties of ovotransferrin, a simple protocol for ovotransferrin purification was developed and a broad physicochemical and immunological assay were used for the characterization. The prepared ovotransferrin revealed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and had an estimated purity of more than 97%. The identity of the prepared protein identified by mass spectrometry analysis demonstrated that it was iron-free ovotransferrin with a molecular weight of 77,726 Da and pI value of 6.85, respectively, and its nature folded structure was kept well as defined by circular dichroism spectroscopy. Western blotting and enzyme-linked immunosorbent assays showed strong immune reactions between the prepared ovotransferrin and rabbit polyclonal antibody against ovotransferrin/egg-allergic patients' sera, suggesting that the prepared ovotransferrin bound to IgG and IgE strongly. In addition, 24 of the potential linear IgE binding epitopes on the prepared ovotransferrin were predicted using a web-based allergen database for food safety. The preparation method of ovotransferrin in the present work might provide scientific support to the authenticity of ovotransferrin for developing certified reference materials.