1a. Objectives (from AD-416):
1. Develop simple model food systems to evaluate processing effects. 2. Assess the effects of various types of processing on allergenic foods, and the specific allergens contained therein, and methods for their detection using the model food systems.
1b. Approach (from AD-416):
1. Simple pastry squares (solid matrix) and liquid extracts of foods will be prepared with known amounts of commonly allergenic foods (milk, peanut). 2. We will apply various processing methods to the solid and liquid foods as appropriate, including boiling, baking, deep-fat frying, retorting, ultra-high temperature processing, microwaving, high pressure processing, radiofrequency processing, ultrasonic processing, pulsed electric field processing, acidification, proteolysis, and selected fermentations (specifically cheese as an example of fermentation of milk). 3. The effects of processing will be assessed using commercial immunoassay kits that detect either a mix of proteins from the allergenic source (e.g., total milk, total peanut) or a specific allergen (e.g., casein, ß-lactoglobulin, Ara h 1, Ara h 2, Ara h 3). Comparisons will also be made to human IgE-based immunoassays. 4. New approaches will be developed and comparatively evaluated to address assay deficiencies. New immunoassay approaches will be designed either (a) to detect pairs of epitopes on short peptides from specific allergens or (b) to use antisera prepared to process-modified antigens, or (c) to employ various alternative extraction techniques intended to resolubilize aggregated proteins, or (d) combinations of these approaches.
3. Progress Report:
This work was in-part performed by a post-doctoral from University of Nebraska Lincoln, who traveled to the ARS scientist's laboratory for training in allergen-specific enzyme-linked immunosrobent assay (ELISA) and other assays. The post doctoral was successfully trained and learned how to perform anti-peanut and anti-peanut allergen-specific (anti-Ara h 1, 2, and 3) ELISA with roasted peanut extracts and model samples. Also, the post doctoral was trained to perform fluorescent labeling of Ara h 1, a major peanut allergen, and to perform fluorescent polarization assays to measure protein-protein interactions.