Immunodetection of Allergens from Mullet (Mugil Cephalus) and Salmon (Salmo Salar)

The presence of misbranding and undeclared allergenic residues was the number one cause of food recalls in the United States, between 2012 to 2016. In this study, parvalbumin was used as a model to study the matrix effect on thermostability of this protein. In general, parvalbumin is thermostable; however, its thermostability varies among different fish species. Identification of new fish allergens is also another important factor for the development of immunoassays to determine allergen-specific IgE antibodies. The specific objectives of this study were to: 1) study the matrix effect on thermostability of parvalbumin from mullet and salmon using three sample models, and 2) identify new potential fish allergen(s) and evaluate the in vitro pepsin digestion stability of suspect allergen(s). To fulfill objective 1, three sample systems were studied: soluble protein extracts from hot smoked samples, protein extracts (PE), and purified parvalbumin (PP). The PE and PP samples were heated for 0, 2, 5 and 8 min at 100 °C, respectively. BCA assay, SDS-PAGE, indirect non-competitive ELISA (inELISA), and Western blot (WB) were used to study the relative protein solubility (RPS), molecular integrity, relative immunoreactivity (RI), and antigenicity of parvalbumin in PE and PP samples., respectively. The amino acid (AA) sequence of mullet parvalbumin was determined using LC-MS/MS. Overall, RPS of PE samples heated for 8 min, compared with unheated samples, were significantly decreased (P < 0.05) in both species; however, no significant decreases (P < 0.05) were observed in RPS of PP samples. From SDS-PAGE, parvalbumin color intensity in PP model did not change over the heating time. Whereas, it was decreased in the salmon smoked and heated PE. From ELISA, in mullet PE, RI of heated samples did not significantly change (P > 0.05). However, RI of salmon PE significantly decreased (P < 0.05) as a function of heating time. The RI of mullet PP was not significantly different over the heating time (P > 0.05), while RI in 8-min heated salmon PP was significantly increased. From the WB, antigenicity of parvalbumin was decreased in the salmon heated PE samples. No changes were observed in the parvalbumin antigenicity in hot smoked mullet samples as well as mullet PE and PP samples. Amino acid sequence comparison also revealed a greater number of cysteine amino acid in salmon parvalbumin compared with mullet parvalbumin. This indicates that salmon parvalbumin is more engaged in heat-induced protein interactions and aggregation. In summary, PP was thermostable in both species. Mainly due to heat-induced protein interactions, the matrix effect on the thermostability of salmon parvalbumin was greater than that of mullet parvalbumin. This study demonstrates that the sample matrix effect can significantly affect in vitro studies related to food allergenic proteins. To fulfill objective 2, identification of new potential allergens in crude proteins extracts of heated mullet and salmon mullet and salmon was performed using seven fish allergic human sera. Pepsin digestion stability of identified potential allergen was evaluated and antigenicity of resulted peptides was tested using pooled human IgE and anti-TM polyclonal antibody in WB. From WB, parvalbumin and TM were identified as major IgE-binding proteins. TM-bound IgE was identified in five of seven tested sera (about 71%). The presence of TM was confirmed using MS analysis. Pepsin digestion stability also showed that pepsin digestion destroyed both IgE and IgG epitopes of TM after 15 min of digestion. No antigenic peptides were detected after TM digestion. These findings show that pepsin digestion can effectively reduce potential allergenicity of TM from mullet and salmon. Clinical studies are needed to further assess allergenicity of TM.