Characterization and Detection of Parvalbumin from Four Finfish Species
Jiang, Xingyi (author)
Rao, Qinchun, 1974- (professor directing dissertation)
Logan, Timothy M., 1961- (university representative)
Salazar, Gloria (committee member)
Singh, Prashant (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)
Introduction: As the major allergen in finfish, parvalbumin is responsible for more than 90% of fish allergy. To reduce the occurrence of finfish allergy, on the one hand, food allergen characterization could assist in protein modifications to decrease allergenicity. One way to evaluate parvalbumin antigenicity under different conditions is to utilize monoclonal antibody (mAb). On the other hand, it is critical to develop assays for allergen residues quantification. Currently, many antibody-based immunoassays are available for finfish parvalbumin detection, while there is limited research on aptamer development against finfish parvalbumin.Objectives: (1) to purify and characterize parvalbumin from Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar), striped mullet (Mugil cephalus), and tilapia (Oreochromis niloticus); (2) to characterize monoclonal antibody (mAb) specific to finfish parvalbumin; and (3) to develop and characterize ssDNA aptamers. Methodology: For objectives 1 and 2, parvalbumin from each finfish species was purified using column chromatography. The purity and amino acid sequence were obtained using gel electrophoresis and LC-MS/MS, respectively. Effect of chelators, reducing chemical and oxidizing agent on antibody-antigen interaction was studied using immunoblot. For objectives 3 and 4, ssDNA aptamers were developed using systematic evolution of ligands by exponential enrichment (SELEX). The obtained sequences and the published anti-parvalbumin aptamer sequences were commercially synthesized. Their target analyte, selectivity, structure and affinity were analyzed. Results: Parvalbumin was successfully purified from four fish species with more than 97% purity. The yield of parvalbumin from cod, salmon, mullet, and tilapia was 1.1 mg/g, 0.4 mg/g, 3.4 mg/g, and 1.2 mg/g of wet muscle, respectively. It suggested that parvalbumin content and distribution were species-dependent. All purified parvalbumin was identified with a sequence coverage of at least 89%. PAS results showed that parvalbumin was not glycosylated. Parvalbumin from different fish species showed different electrophoretic mobility. Native parvalbumin contained both monomers and oligomers. In the presence of reducing agent (dithiothreitol) and detergent (sodium dodecyl sulfate), parvalbumin oligomers dissociated into monomers. As for the immunochemical properties, immunoreactivity of all four fish parvalbumin decreased when its calcium was chelated. By using the microplate SELEX, a total of 20 candidate aptamer sequences were obtained. All aptamers had a hair-pin structure, which occurred when two regions of the same strand are complementary to each other and form the Watson-Crick base pairs. It is noted that at different temperatures, the same candidate sequence may have different structures. The developed and published aptamers all showed reaction with several finfish proteins. Conclusion: This study (1) investigated the purity, conformation, and immunoreactivity of parvalbumin from four finfish species; (2) characterized the mAbs specific to finfish parvalbumin; and (3) developed and characterized the ssDNA aptamers. In summary, parvalbumin is a mixture of monomers and oligomers due to covalent and non-covalent interaction under the native condition. Its immunoreactivity is affected by calcium chelation. The purified parvalbumin has the potential to be used in the development of capturing agents such as antibodies and aptamers, which can be further utilized in the assay development for the detection of undeclared fish allergenic residues in foods.
November 5, 2021.
A Dissertation submitted to the Department of Nutrition and Integrative Physiology in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Qinchun Rao, Professor Directing Dissertation; Timothy M. Logan, University Representative; Gloria Salazar, Committee Member; Prashant Singh, Committee Member.
Florida State University