Allergen extraction: Factors influencing immunogenicity and sensitivity of immunoassays

Food allergy prevalence is increasing worldwide, therefore there is a high demand for reliable tests to correctly diagnose this disease. Knowledge of proteins allergenicity and how they react both in the body and in diagnostic tests is necessary to adequately assess the potential immunogenicity of both natural foods and those produced through biotechnological processes. Thus, our aim was to analyze the factors that influence the protein extraction of foods in terms of, immunogenicity and immunoassays sensitivity. Peanut proteins were extracted using four distinct extraction buffers with different pH values (physiological saline, tris buffer, borate buffer with and without β-mercaptoethanol), the protein concentration was determined by the Lowry method and polyacrylamide electrophoresis (SDS-PAGE) was used to compare the protein profile of each extract. The immunogenicity of each extract was verified by sensitizing two mouse strains (Balb/c and C57Bl/6) with a solution containing 100 μg of the extracted proteins and was determined by ELISA. Results show that extraction with the distinct buffers resulted in protein solutions with different yields and profiles. The immunogenicity of the different extracts also demonstrated distinct patterns that varied depending on the extraction methods, mouse strain and in vitro test. Immunoreactivity varied in accordance with the protein extract used to coat the microtitration plates. In conclusion, the protein 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.