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

Abstract

19Food allergy is an IgE and or IgG immune-mediated reaction to food antigens.20 Knowledge of the allergenicity properties of proteins, how they react in the body and 21 in diagnostic tests is necessary to adequately assess the allergenic potential of both 22 natural foods and those produced through biotechnological processes. Thus, our aim 23 was analyze the factors those influence the protein extraction of foods in terms of 24 yield, allergenicity and sensitivity in immunoassays. Peanut proteins were extracted 25 using 4 distinct extraction buffers (physiological saline, tris buffer, borate buffer with 26 and without β-mercaptoethanol). The protein concentration of the obtained extracts 27 was determined by the Lowry method. Polyacrylamide electrophoresis (SDS-PAGE) 28 was used to compare the protein profile of each extract. Immunogenicity of each 29 extract was verified by sensitizing two mouse strains (Balb/c and C57/BL6) with 30 100μg of the proteins, and the immunoreactivity was determined by ELISA. Our 31 results show that extraction with the distinct buffers resulted in protein solutions with 32 different yields and profiles. The antigenicity of the different extracts in a murine 33 model of systemic immunization also demonstrated distinct patterns that varied 34 depending on the extraction methods and mouse strain. The immunoreactivity varied 35 in accordance to the protein extract used to coat the microtitration plates. In 36 conclusion, the protein yield and profile in the extracts is critically influenced by the 37 salt composition and pH of the extraction buffers. This in turn influences both in vivo 38 immunogenicity and in vitro immunoreactivity. 39 Introduction 40 Considering that 4% to 8% of children present allergies and that a significant 41 portion of these present symptoms that prevent school attendance which in turn takes, 42 at least one of their caregivers away from work we can say that allergies have a high 43 social impact [1]. In addition to absence at work, altering meal preparation routine 44 and other social activities of the family negatively impacts stress levels [2]. Thus, 45 adequate diagnosis is fundamental. 46 The best-known measure to prevent food allergy symptomatology in allergic 47 people is the adoption of exclusion diets. To ensure clearer food labeling many 48 countries have food allergen labeling laws. For example, the Food Allergen Labeling 49 and Consumer Protection Act (FALCPA/USA-2004) requires that foods are labeled to 3 50 identify the eight major food allergens: milk, egg, fish, crustacean-shellfish, tree nuts, 51 wheat, peanuts and soybeans which account for over 90% of all documented food 52 allergies in the U.S. [3]. Along with these 8 the Australian law requires the labeling of 53 two other foods -sesame and lupin [4]. In the European Union 14 foods are on the 54 required labeling list: the ten previously cited plus celery, mustard, Sulphur dioxide, 55 and mollusks. 56 Regardless of whether ingested food is derived from animals or plants, the 57 food matrix i...