Development of Monoclonal Antibody Based Immunoassays for Tree Nut Detection and Quantification

NON-TECHNICAL SUMMARY: Currently there are no methods available that: 1) can help validate available assays for tree nut detection and quantification and 2) can adequately address the issue of antibody (Ab) cross-reactivity that may lead to false positive identification of the offending food. We propose to develop monoclonal antibody (MAb)-based immunoassays to 1) detect and quantify trace amounts of almonds and cashew nuts, in various foods, as both are often implicated in tree nut-induced allergies in humans; and 2) assess effects of various processing methods on the stability of select epitopes on targeted almond and cashew nut proteins The selection of nut seed proteins will be based on their demonstrated stability against several processing methods commonly encountered in processing the tree nuts, either alone or as an ingredient, in a variety of food uses. Results of experiments directed at assessing effects of food processing on epitope stability will, for the first time, define the stability of epitopes on specific allergenic protein(s) in almonds and cashew nuts leading to improved fundamental understanding of epitope stability thereby helping in the design of specific processing methods to inactivate defined epitopes, thus improving the safety of consumers specifically sensitive to those epitopes. The information gained will provide critical information to help improve food labeling and consumer food safety.

<P>

APPROACH: The overall approach is to use molecularly defined reagents such as mouse monoclonal antibodies (MAbs) and, where possible, cloned target antigens. Two formats of enzyme-linked immunosorbent assays (ELISAs), inhibition and capture, will be assessed. For assay development, MAbs will be selected that are devoid of cross-reactivity and are directed against stable epitopes that are not compromised by food processing. The proteins used for MAb production will contain 1:2 (w/w) mixture of proteins extracted from unprocessed and processed nut seeds. The processed component will include equal amounts of roasted, blanched, fried, autoclaved, and microwaved tree nuts. For assessment of the effects of processing and storage, the stable epitopes will be compared to MAb-epitope combinations that are labile. Where possible recombinant indicator proteins, rather than their heterogeneous and less well-defined native nut seed-derived counterparts, will be used as defined components of the assays. To assess the effects of food matrices, selected high affinity MAbs will be tested for interference by variety of foods that have been spiked with known quantities of the target nut protein. This testing will be done in three phases. In the first, ground foods will be spiked with known amounts of target nut extracts and tested. In the second, ground foods will be spiked with target nut flours and co-extracted, and in the third, foods will be processed (e.g. baked, cooked, stir fried) to include known amounts of target nut ingredients and their extracts tested by ELISA.