The Characterization Of Quantitative And Qualitative Relationship Of Shrimp Allergen Extracts

This paper based on the characterization of quantitative and qualitativerelationship of shrimp allergen extracts. Firstly, the procedure of shrimp allergenextraction is established and the standardization reference of shrimp allergen extract isselected. Secondly, the immunogenicity of shrimp allergen extracts in the storageprocess and freeze-thaw cycles are researched, the effect of antifreeze in allergenextracts is compared. At last, the method of allergen extracts immunogenicitycharacterization is investigated. The main results are as follows:1. The selection of shrimp allergen extraction solutionThe acetone powder of Penaeus vannamei is extracted with seven existing ormodified extracting solutions. The means of protein concentration, SDS-PAGE,indirect ELISA and immunoblotting are compared. It shows that the shrimp proteinextracted by1M KCl with DTT has high concentration and immunogenicity,including major and minor allergens.2. The immunogenicity of different crustacean allergen extractsSeven crustaceans which are extensively consumed in Qingdao are chose. Thecomponents of seven crustaceans are analyzed by SDS-PAGE and their allergenactivity is compared by indirect ELISA and Western blotting. The results show thatPenaeus vannamei allergen extracts have comparatively large immunogenicity. It canbe used as the reference of shrimp allergen extracts. 3. The immunogenicity of shrimp allergen extracts in the storage processFor the purpose of explore the best storage conditions of the allergen extracts,protein concentration is determined by BCA; protein composition is evaluated bySDS-PAGE electrophoresis; allergens immunogenicity is compared by indirectELISA. The results show that when shrimp allergen extracts are stored at-20℃and-80℃, the major allergen bands don’t degraded in less than five weeks, and theallergy immunogenicity of the extract remained unchanged.4. The immunogenicity of shrimp allergen extracts in the freeze-thaw cyclesprocessThe freeze-thaw cycles effect on immunogenicity of Penaeus vannamei allergenextracts are studied. The results show that the protein concentration decreased by33.1%,30.0%and24.2%respectively when shrimp allergen extract in the cycle offreezing and thawing for five times at-80℃,-20℃and-3℃. Therefore, when theallergens protein are stored, the cycles of freeze-thawing should be reduced to ensurethe immunogenicity of shrimp didn’t change, then at-80℃and-20℃within fourfreeze-thaw cycles and-3℃less than two freeze-thaw cycles are allowed.5. The effect of store endurance to allergen extracts with antifreezeThe usage of antifreeze can retain the concentration, composition andimmunogenicity of proteins. The results demonstrate that the use of sucrose andtrehalose are better than the glycerine. The sucrose and trehalose concentration of10%and20%could maintain anti-freezing effect. With the temperature of-20℃、-40℃and-80℃, the use of antifreeze is much more better than the room temperatureand0℃.6. The anti-enzymolysis of allergen extracts immunogenicityThe stability of different crustacean allergen extracts and Penaeus vannameiallergen extracts with different extracting solutions in vitro simulated gastric fluid （SGF） were studied. With the comparison of the immunogenicity and the enzymolysistime of allergen extracts, the conclusion is that the stability of allergen extracts invitro simulated gastric fluid （SGF） used to be the characterization of allergen extractsimmunogenicity is unfeasible.7、The characterization of shrimp allergen extraction activity by the RBL-2H3released β-hexosaminidaseThe result show that10ng/mL is the best allergen extracts concentration toprovoke cells releasing β-hexosaminidase. Different crustacean allergen extracts withthe same concentration could make different β-hexosaminidase release. The dilutionreaching the half maximal release was calculated (ED₅₀). The reciprocal value of theED50was defined as the biological potency of Penaeus vannamei allergen extracts.