| Summary: | 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 ...
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