Honors Program
Honors Program: Embargoed Theses
First Advisor
Joseph Baumert
Second Advisor
Shyamali Jayasena
Third Advisor
Jennifer Auchtung
Committee Members
Joseph Baumert, Shyamali Jayasena, Jennifer Auchtung
Date of this Version
2-24-2025
Document Type
Thesis
Citation
Merkle, J. L., Baumert, J. & Jayasena, S. 2025. Comparison of Six Commercial Sesame ELISA Kits on Their Reliability of Detecting and Quantifying Sesame Residue from Buffer & Cracker Matrices, Kit Extracted Protein Profiles, & Kit Antibody Specificity to Extracted Sesame Protein. Undergraduate Honors Thesis. University of Nebraska-Lincoln
Abstract
With sesame being recently added to the allergens requiring labeling by the FDA, there is little information on sesame detection and quantification on which producers can create food safety plans. In this study, commercial sesame ELISAs were evaluated to determine allergen recovery from sesame flour spiked into buffer at known concentrations and sesame flour, extract, and seeds spiked into crackers. Additionally, the protein profiles of kit extractions were analyzed and antibody specificity was observed.
10, 50, and 100 ppm sesame flour spikes were prepared in buffer and were evaluated using Veratox (Neogen), R-Biopharm, Romer, Morinaga, BioFront, and 3M (Neogen) sesame ELISAs. 10, 50 and 100 ppm sesame flour spikes were spiked into crackers. Additionally, 10, 50, and 100 ppm sesame extract spikes were prepared similar to the buffer samples prior to spiking into crackers. The sesame seed spikes were prepared by adding a sesame seed to a 15 g cracker sample (corresponding to 20 ppm sesame protein) before or after homogenization. The samples were homogenized using either a benchtop blender or a freezer mill. The R-Biopharm and BioFront Sesame ELISAs were used to evaluate the sesame flour, extract, and seed spikes in crackers. After conducting 2D Quant Protein Analysis on the Veratox and R-Biopharm sesame protein extracts, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to analyze the protein profiles of the extracts. Western Blots were conducted using kit conjugate antibodies to understand antibody specificity.
Recovery varied among the six kits and buffer spike concentrations. The R-Biopharm and BioFront kits were the most consistent in recovering sesame from the buffer spikes as both recovered 90%. For the sesame extract and flour samples, R-Biopharm had an average recovery of 102% and 72%, respectively while BioFront had an average recovery of 81% and 146%, respectively. Spiking a sesame seed into homogenized crackers resulted in more consistent recovery than spiking into cracker pieces. With the exception of the 3M kit, the extracted protein profiles only differ in the amount of soluble protein extracted and the banding patterns are consistent with current literature. The conjugated antibodies of the six kits differ in targets, as some are monoclonal and other may be polyclonal or mixtures of monoclonal. Some are able to detect protein in all six extracts while others (specifically Romer) was not able to detect protein in two extracts from other kits.
Sesame ELISA kits differ in recovery and detection capacity across different spiking materials and type of sesame residue used for spiking. As the protein extraction and antibody specificity differs between the kits, this was to be expected. These results are useful for understanding the limits of ELISA testing methods. Further evaluation of allergen recovery from different matrices incurred with sesame is warranted.
Comments
Copyright Janine L. Merkle, Joseph Baumert, and Shyamali Jayasena 2025.