Joseph L. Baumert
Date of this Version
Schmidt, M. K. (2020). Development of a Sandwich ELISA Targeting Cashew Ana o 2 and Ana o 3. [Unpublished Master's Thesis]. University of Nebraska-Lincoln.
Cashew nut is the second leading tree nut allergy in the US. Cross-contact of cashew nut poses potential food safety risks for individuals with cashew allergies. Highly-processed foods, such as HTST/UHT cashew milks may lead to problems in cashew protein detection by current allergen detection methods. Therefore, the aim of this study was to develop a robust sandwich ELISA for detection of highly processed cashew proteins. Commercial cashew ELISAs were evaluated for their robustness and sensitivity in detecting cashew milk cashew protein. After unreliable results were determined, cashew Ana o 2 (11S) and Ana o 3 (2S) were semi-purified using established methods. Cashew Ana o 2 was reduced (11S R/A) for improved extraction and both the 2S and 11S R/A were used for rabbit immunization. Cashew specific IgG antibodies were monitored by determining their titer values. A 1:1 pool of the rabbit sera (11S R/A:2S) was used as the capture reagent while sheep anti-roasted cashew sera was used as the detector reagent in the optimized sandwich ELISA (LOQ; 0.3 ppm cashew protein). Potential matrix interference and cross-reactivity were evaluated in 58 food matrices including plant milks, tree nuts, spices, baking ingredients, and seeds. No matrix interference was found with any tested plant milk, with matrix interference found from 9 select seeds, spices and tree nuts. Certain foods in the Anacardiaceae family (pink peppercorn, pistachio, mango seed) were found to be cross-reactive. The sensitivity of the developed ELISA was evaluated further with cashew protein incurred in pre- and post-processed almond milk and cookies. The high percentage recovery of cashew protein in almond milk, above 10 ppm cashew protein, before and after processing indicates that the developed ELISA can reliably detect heat-processed cashew nut proteins in foods. With cookies, high percentage recovery was obtained with incurred baked cookie while incurred cookie dough showed overestimation. More validation work is needed to ensure that the developed ELISA will support allergen detection for various food matrices and processes.
Advisor: Joseph L. Baumert