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.
Parvalbumins have long been identified as major allergens in fish, but our research has found that parvalbumins are not the main cause of allergic reactions to tilapia. After homogenization, proteins were extracted from freshwater tilapia to react with a pooled sera sample taken from patients (n = 20) tested to be allergic to tilapia. Enzyme-linked immunosorbent assay revealed immunoglobulin E antibody activity, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, Western blot, 2D electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to carry out protein separation and analysis. Protein identification against different databases yielded three known high molecular weight proteins as tilapia allergens: chromosome undetermined SCAF7145, fructose-bisphosphate aldolase A and enolase 3 (beta muscle). A fourth, new, unidentified protein with two T-cell receptors was discovered.
The prevalence of fish allergies has become a serious health problem and has increased alarmingly over the past few years. To contain this problem, we would need to identify all commonly consumed fish allergens. To date, however, no report has identified largemouth bass allergens. This study attempted to identify and purify the major allergen implicated in the allergic response to largemouth bass (Micropterus salmoides), a freshwater fish widely consumed in China. Fifteen patients with a positive history of type I allergy to fish were recruited from skinprick test and the allergy screen. Total protein extracts and purified allergenic protein from bass were tested for their immunoglobulin EÁbinding properties. Immunoblot assay resulted in strong reactivity with the 17-kDa protein in all patients. In summary, nucleoside diphosphate kinase B was identified as a novel fish allergen in largemouth bass. This finding is important for allergy diagnoses and the treatment of freshwater fishÁallergic disorders.
The aim of this paper is to study the influence of high pressure treatment on the structural changes and allergenicity of largemouth bass. We treated the allergens at 100, 200, 300 and 400 MPa for 15 min and at 300 MPa for 5, 10, 15, 20 and 30 min at 20 • C. The treated samples from largemouth bass were tested for their IgE-binding properties by combining Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) with western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). Circular dichroism analysis was performed to characterize the structural change. In summary, we can determine that the greatest structure changes were found for samples treated by 400 MPa for 15 min. High pressure treatment did change the structure, subunit composition and molecular weight of largemouth bass allergens, but it did not change the allergenicity of the allergens.
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