Résumé
Objective:To develop an indirect enzyme-linked immunosorbent assay (ELISA) for semi-quantification of major allergen parvalbumin in fish.Methods:The soluble proteins were prepared from both white and dark muscles of seven species of freshwater fish and five species of marine fish.Tricine-SDS-PAGE and Western blot were performed to examine the protein patterns of fish muscle extracts.Natural parvalbumin being used to make calibration curve was purified from silver carp (Hypophthalmichthy molitrix) by ammonium sulphate fractionation,followed by ion exchange and gel filtration chromatography.The molecular mass of purified protein was estimated by Tricine-SDS-PAGE and identified by Western blot with anti-frog parvalbumin monoclonal antibody PARV-19.ELISA using PARV-19 was carried out to evaluate parvalbumin contents in white and dark muscles.Results:Tricine-SDS-PAGE revealed species-specific differences in proteins of heated extracts.Western blot confirmed that the major bands were showed in Tricine-SDS-PAGE with the molecular masses of 10-14 kD corresponded to parvalbumins recognized by PARV-19 and various numbers of isoforms of parvalbumin existed in different species of fish.There might be some differences in the parvalbumin contents and the epitope region was recognized by PARV-19 based on the differences in relative intensities of protein immunodetection.The ELISA showed that the contents of parvalbumin were 4 to 33 folds higher in the white muscle than in the dark muscle and varied greatly in different species of fish.Conclusion:These results validate that the dark muscle might be less allergenic than the white muscle due to the lower content of parvalbumins,and it is suggested that the commercial anti-parvalbumin antibody PARV-19 can be used to detect parvalbumins from the commercially important species of fish tested in this study and the method we develope succeeds to detect the major allergen in various species of fish.