Allergen Extraction: Factors Influencing Immunogenicity and Sensitivity of Immunoassays

Abstract

Because of the high social impact of Food allergy, it is of great importance to correctly diagnose this disease using reliable tests. Knowledge of the allergenicity properties of proteins, how they react 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 (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 C57/BL6) with solution containing 100μg of the extracted proteins and 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 to 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.

Identification of linear epitopes and their major role in the immunoglobulin E-binding capacity of tropomyosin from Alectryonella plicatula

“…That might be due to the difference in the extraction buffer, especially the use of β-Me which formed a monomer by destroying the disulfide bonds. 33 More critically, the obtained sequence could be used to analyze the epitopes of ATM.…”