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Fish provides a valuable source of many essential nutrients. However, it is also one of the "Big Eight" allergenic foods that account for more than 90% of food allergic reactions, so reliable detection of its presence is crucial. The addition of acid ingredients such as vinegar, lemon juice, and tomato sauce has shown to markedly reduce the detectability of fish in an immunoassay using a previously developed fish-specific monoclonal antibody (MAb 8F5), whose antigenic protein is fish tropomyosin-a 36 kDa myofibrilar protein, and acid ingredients may also reduce fish's allergenicity. This study, therefore, focused on studying the effects of vinegar on the detectability (assay immunoreactivity) and allergenicity of three commonly consumed fish species (whiting, cod, and red grouper). MAb 8F5 [Immunoglobulin G (IgG)] and human plasma [Immunoglobulin E (IgE)] from three fish allergic patients were individually used to investigate the effects of vinegar on the detectability and allergenicity of each fish sample, using indirect enzyme-linked immunosorbent assay (iELISA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot were used to reveal changes in the overall and antigenic protein banding patterns in vinegar-treated samples. The results of iELISA with MAb 8F5 demonstrated that vinegar dramatically reduced the detectability of fish samples (up to 90% of the OD reading) when compared with water-treated and non-treated control samples. SDS-PAGE results showed that the intensity of bands of vinegar-treated samples became lighter than those of controls in all three fish species. The vinegar-treated samples in western blot showed little or no band at 36 kDa, which agreed with the results of the MAb-8F5 based iELISA. Considerable reductions of the OD readings were also apparent in all the fish samples cooked (100 °C) with vinegar for 60 min when tested by IgE-based iELISA. However, there were variations among species and subjects: of the three fish species tested, red grouper was more resistant to vinegar treatment and the subject with a higher IgE concentration in plasma was less affected by vinegar-induced alterations in the fish allergens. Moreover, the chemical reactions that attribute to the vinegar's effects on antigenic tropomyosin-IgG binding and fish allergen-IgE binding are distinctively different. These results indicate that vinegar treatment of fish decreases x the detectability of finfish using MAb 8F5-based iELISA via acidic precipitation of the antigenic protein-tropomyosin, while the decreased allergenicity caused by vinegar was due to the acid denaturation of the allergen.
A Thesis submitted to the Department of Nutrition, Food and Exercise Sciences in partial fulfillment of the requirements for the degree of Master of Science.
Includes bibliographical references.
Yun-Hwa Peggy Hsieh, Professor Directing Thesis; Shridhar Sathe, Committee Member; Ming Cui, Committee Member.
Florida State University
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