Effect of freeze-thaw cycles on the immunogenicity of Litopenaeus vannamei allergens

In order to study the activity of shrimp allergens in freeze-thaw cycles, Penaeus vannamei were used to extract allergen,respectively stored at -3 ℃,-20 ℃ and -80 ℃ for one to five cycles.For the purpose of exploring the best activated condition of the allergen extract,protein concentration was determined by BCA;SDS-PAGE electrophoresis was used to evaluate protein composition;indirect ELISA and Western-blotting were used to survey allergens activity.The results showed that when shrimp allergen extract was in the cycle of freezing and thawing for five cycless at -80 ℃,-20 ℃ and -3 ℃,the protein concentration decreased by 33.1%,30.0% and 24.2% respectively.The major allergen bands were degraded ,when the shrimp protein extract was freezed and thawed for four cycles at -80 ℃ and -20 ℃,on the other hand the major allergen bands didn’t change at -3 ℃ for five cycles of freezing and thawing.The allergenic activity of shrimp protein extract had reduced when freezed and thawed for five cycles at -80 ℃ and -20 ℃ and three cycles at -3 ℃.The major allergen Western-blotting bands disappeared when five cycles of freezing and thawing were advanced at -80 ℃ and -20 ℃,while the major Western-blotting bands didn’t change at -3 ℃.Therefore,when the allergens protein was storaged,the cycles of freeze-thawing should be reduced to ensure the allergenic activity of shrimp didn’t change,then at -80 ℃ and -20 ℃ within four freeze thaw cycles and at -3 ℃ less than two freeze-thaw cycles were allowed.