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.
Abstract: BACKGROUND: Food allergies are usually managed by food avoidance. Hidden allergens in food, due to cross-contamination and/or allergenic additives added during production, place an important concern in today’s increasing food allergy cases worldwide. Previous studies showed that introduction of new food components, in an inflamed intestine, results in sensitization to this food. Thus, our aim was to evaluate the kinetics of multiple food allergy induction. METHODS: Adult male C57BL/6 mice were divided into five groups, four of which were submitted to an intestinal inflammation induction protocol to peanuts. Egg white (OVA) diluted 1:5 v/v in distilled water was instilled by gavage 6h-before (EXP-1), concomitant (EXP-2) and 6h-after (EXP-3) the onset of the peanut challenge diet. Positive control (POS CONT) and NEG CONT received saline per gavage. Finally, animals were challenged with subcutaneous injections of OVA. RESULTS: No changes in diet intake were observed. Anti-OVA total IgG antibody titers significantly increased in EXP-2. Flow cytometry revealed significant decrease in CD4+CD25+Foxp3+ and significant increase in TCD8+ in EXP-2. Histomorphometrically, EXP-2 and EXP-3 were classified as Infiltrative and Partial Destruction stages. EXP-1 was classified as Infiltrative, while POS CONT was classified as Partial Destruction. NEG CONT was classified as Normal. CONCLUSION: The introduction of a new food only a few hours before the initiation of a gut inflammation is able to induce oral tolerance, however the introduction of a new dietary protein concomitant to the onset or during an ongoing gut inflammation may induce multiple allergies.
BACKGROUND: Food allergies are usually managed by food avoidance. Hidden allergens in food, due to cross-contamination and/or allergenic additives added during production, place an important concern in today’s increasing food allergy cases worldwide. Previous studies showed that introduction of new food components, in an inflamed intestine, results in sensitization to this food. Thus, our aim was to evaluate the kinetics of multiple food allergy induction. METHODS: Adult male C57BL/6 mice were divided into five groups, four of which were submitted to an intestinal inflammation induction protocol to peanuts. Egg white (OVA) diluted 1:5 v/v in distilled water was instilled by gavage 6h-before (EXP-1), concomitant (EXP-2) and 6h-after (EXP-3) the onset of the peanut challenge diet. Positive control (POS CONT) and NEG CONT received saline per gavage. Finally, animals were challenged with subcutaneous injections of OVA. RESULTS: No changes in diet intake were observed. Anti-OVA total IgG antibody titers significantly increased in EXP-2. Flow cytometry revealed significant decrease in CD4+CD25+Foxp3+ and significant increase in TCD8+ in EXP-2. Histomorphometrically, EXP-2 and EXP-3 were classified as Infiltrative and Partial Destruction stages. EXP-1 was classified as Infiltrative, while POS CONT was classified as Partial Destruction. NEG CONT was classified as Normal. CONCLUSION: The introduction of a new food only a few hours before the initiation of a gut inflammation is able to induce oral tolerance, however the introduction of a new dietary protein concomitant to the onset or during an ongoing gut inflammation may induce multiple allergies.