54I-5 |
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L. CHEN, R. E. Goodman, and S. L. Hefle. Food Allergy Research and Resource Program, Univ. of Nebraska, Dept. of Food Science and Technology, 143 Food Industry Building, Lincoln, NE 68583-0919 Fish is a common allergenic food source. Parvalbumin is a calcium-binding muscle protein that is highly conserved across fish species and amphibians, and is the major cross-reactive allergen associated with fish allergy. With the increasing consumption of fish products and the apparent increase in prevalence of food allergies, food manufacturers and regulatory agencies are seeking methods to detect major allergens. Our objective was to test the efficacy of using commercially available anti-parvalbumin antibodies to develop a quantitative immunoassay method to detect fish contamination in foods. Frog (Sigma®, USA), rat, and carp (Swant®, Switzerland) anti-parvalbumin antibodies were evaluated by 1D western blots of extracts of carp, catfish, cod, tilapia, and tuna. The anti-frog parvalbumin was further evaluated with western blots of 2D electrophoretically separated fish extracts to identify potential isoforms of parvalbumin in each fish species. An indirect ELISA was developed and optimized to measure the relative parvalbumin content in fish protein extracts. Of the three antibodies tested, the mouse monoclonal anti-frog parvalbumin IgG from Sigma® exhibited the highest specificity against fish parvalbumins. No parvalbumin band was present in tuna fish. The 2D electrophoresis and western blots revealed species-specific differences in proteins that appear to represent isoforms of parvalbumin in carp (5), catfish (3), cod (1), and tilapia (2). Based on differences in protein staining and immunodetection, parvalbumin isoforms may present slightly different epitopes that are recognized by the anti-frog IgG (Sigma). An indirect inhibition ELISA was developed by using the anti-frog IgG. Extracts of fish were tested as inhibitors and the detection limit, defined as the protein content needed for 25% inhibition, ranged from 0.03 (catfish) to 7.6 (cod) ìg total protein/ml. These results suggest that the indirect ELISA we developed using the frog anti-parvalbumin antibodies is a valuable method to detect fish parvalbumin in foods.
Session 54I, Toxicology & Safety Evaluation: General
2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana |