Separation, Purification And Analys Of The Main Allergen Components In Milk

AIM:Separation,purification and standardization of the allergen components in milk were the key for the diagnosis and treatment of milk allergy and preparation of the low/no allergens formula milk.To provide the standardization vaccin for the Chinese people,the main allergens in milk were separated,purified,and then identified by analysis of the relationship between the histamine release and the immune activity.METHODS:The allergen proteins of milk were purified by the techniques of isoelectric precipitation,(NH₄)₂SO₄ salting-out,sephadex G-50 gel-filtration chromatography and DEAE-sepharose anion-exchange chromatography.The C57/BL6 mice were respectively injected intraperitoneally with the extracts of skim milk,whey,casein andβ-lactoglobulin mixed with the aluminum hydroxide adjuvant.The allergic components of the milk were analized by western-blotting.The titer of sIgE and sIgG were tested by indirect ELISA.The peritoneal mast cells degranulation was induced by different allergens components in vitro and the histamine release rate was detected by fluorescence analysis.RESULTS:The milk allergenic components casein,β-lactoglobulin andα-lactalbumin were isolated and purified and the purity was more than 90%.The main allergen component of milk was casein by western-blotting.The titer of sIgE and sIgG of immune serum from C57/BL6 mice were tested by indirect ELISA.The sIgG titer was significantly increased and the sIgE titer was low.The histamine release rate was consistent with the level of sIgG and no obvious association with sIgE.Casein was confirmed as the main allergic components in skim milk andβ-lactoglobulin in whey.The results of histamine release showed that there was similar epitopes between casein andβ-lactoglobulin.CONCLUSION:Histamine fluorescence detection and sIgG levels can be used for the identification and evaluation of allergen components in milk and possible be a new way of food allergens standardized.