Allergen Extraction: Factors Influencing the Yield, Allergenicity and Sensitivity of Immunoassays

Abstract

AbstractFood allergy is an IgE and or IgG immune-mediated reaction to food antigens. Knowledge of the allergenicity properties of proteins, how they react in the body and in diagnostic tests is necessary to adequately assess the allergenic potential of both natural foods and those produced through biotechnological processes. Thus, our aim was analyze the factors those influence the protein extraction of foods in terms of yield, allergenicity and sensitivity in immunoassays. Peanut proteins were extracted using 4 distinct extraction buffers (physiological saline, tris buffer, borate buffer with and without β-mercaptoethanol). The protein concentration of the obtained extracts was determined by the Lowry method. Polyacrylamide electrophoresis (SDS-PAGE) was used to compare the protein profile of each extract. Immunogenicity of each extract was verified by sensitizing two mouse strains (Balb/c and C57/BL6) with 100μg of the proteins, and the immunoreactivity was determined by ELISA. Our results show that extraction with the distinct buffers resulted in protein solutions with different yields and profiles. The antigenicity of the different extracts in a murine model of systemic immunization also demonstrated distinct patterns that varied depending on the extraction methods and mouse strain. The immunoreactivity varied in accordance to the protein extract used to coat the microtitration plates. In conclusion,the protein yield and profile in the extracts is critically influenced by the salt composition and pH of the extraction buffers. This in turn influences both in vivo immunogenicity and in vitro immunoreactivity.