Characterization of allergens isolated from the freshwater fish blunt snout bream (Megalobrama amblycephala)

Fish are an important source of dietary protein for humans throughout the world. However, they are recognized as one of the most common food allergens and pose a serious health problem in countries where fish consumption is high. Many marine fish allergens have been extensively studied, but relatively little is known about freshwater fish allergens. This study identified two main allergens from blunt snout bream (Megalobrama amblycephala), a freshwater fish widely consumed in China. Sera from 11 patients with convincing clinical history of blunt snout bream allergy were utilized in IgE immunoblot analysis to identify prominent allergens. Several blunt snout bream proteins revealed specific binding to serum IgE, with the 47 and 41 kDa proteins being the most immunodominant among them. Two-dimensional gel electrophoresis (2D SDS-PAGE) enabled resolution of the 47 and 41 kDa proteins into several protein spots with distinct isoelectric points. 2D SDS-PAGE along with IgE immunoblot analysis further confirmed the strong reactivity of these protein spots with the pooled sera from blunt snout bream-sensitive patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of the peptides generated by trypsin digestion of the different spots corresponding to the 47 and 41 kDa proteins indicated that these spots were isoforms of enolase and muscle creatine kinase, respectively. The potential allergenicity of these proteins was further verified by an bioinformatics approach using the full-length and 80 amino acid sliding window FASTA searches, which revealed a significant amino acid sequence homology between blunt snout bream allergens and several known inhaled and crustacean allergens.