Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Detection of Finfish Tropomyosin

• Other Authors: Zhao, Yaqi (author), Rao, Qinchun, 1974- (professor directing thesis), Singh, Prashant (committee member), Chase, P. Bryant (committee member), Florida State University (degree granting institution), College of Health and Human Sciences (degree granting college), Department of Nutrition and Integrative Physiology (degree granting department)
• Format: Master Thesis
• Language: English
• Published: Florida State University 2021
• Subjects: Food; Gadus morhua
• Online Access: https://diginole.lib.fsu.edu/islandora/object/fsu%3A797721/datastream/TN/view/Monoclonal%20Antibody-Based%20Enzyme-Linked%20Immunosorbent%20Assay%20for%20the%20Detection%20of%20Finfish%20Tropomyosin.jpg

Introduction: Finfish is one of the Big Eight allergenic foods in the United States (U.S.). Economic-driven finfish substitution or adulteration and unintentional cross-contamination not only violate food regulations (e.g., labeling) but also cause food safety concerns (e.g., finfish allergy). The composition and distribution of finfish allergens differ in species, resulting in various levels of allergenicity. These issues underline the essentiality for the detection of undeclared allergenic finfish ingredients. Tropomyosin, a myofibrillar muscle regulatory protein, has been recognized as a finfish allergen. The objective of this study was to establish a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) for the detection of finfish tropomyosin. Methods: Finfish species were verified using the standard DNA barcoding method. Western blot was performed to characterize anti-tropomyosin mAbs (species selectivity and antigenicity). The immunoaffinity was determined using indirect non-competitive ELISA (inELISA). Cod (Gadus morhua) tropomyosin was purified using anion-exchange chromatography (AEC). The purity of cod tropomyosin was verified by non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stain-free gel. Tropomyosin monomer isoforms were studied using non-reducing SDS-PAGE, two-dimensional gel electrophoresis (2D-PAGE), and amino acid (AA) sequencing. The molecular structure of tropomyosin was determined under reducing, non-reducing, and native conditions. The indirect competitive ELISA (icELISA) and sandwich ELISA (sELISA) were developed and validated. Results and discussion: Seven mAbs were purified and characterized. From Western blot, three laboratory-developed mAbs were immunoreactive to tropomyosin. As for species selectivity, two mAbs (1G11 and 7B8) could bind with tropomyosin from finfish, shellfish, mammalian, and poultry species, while mAb8F5 was specific to finfish tropomyosin. The Immunoaffinity of mAb7B8 was higher than that of mAb8F5. The ...