Effect Of Glycation On Conformation, Antigenicity And Allergenicity Of Egg Ovalbumin

Hen egg was officially recognized as one of the eight big allergic foods by WHO and FAO. With the improving of our living standard, hen egg consumption increases. So egg allergy has become a public health issue and food safety problem in food industry with an increasing attention. Ovalbumin (OVA) is one of the four major allergens in hen egg white, comprising 54% of total egg white protein. Currently, there are reports on various treating methods on functional properties and nutritional properties of OVA, but report on effect of glycation on antigenicity and IgE binding ability of OVA is not available. Thus, it is necessary to investigate the effect of glycation on antigenicity and IgE binding ability of OVA.Hen egg OVA was purified by column chromatography from fresh hen egg white and was detected by SDS- PAGE with 5% stacking gel and 15% running gel. It was shown that the OVA purity was over 98 percent, and the recovery rate was about 87.66%. A simple, reproducible and suitable approach to purify egg OVA for laboratory was developed.The preparation of rabbit polyclonal antibody against OVA was performed according a routine way, including multiple subcutaneous sites injection with complet Freund's adjuvant at the first time and incomplete Freund's adjuvant for the bostering, respectively. We optimized the operating condition of the indirect ELISA by chessboard titration firstly, and the optimal concentration of coating antigen was 5p.g/ml, and the optimal dilution of HRP-IgG was 1:10,000, followed by detecting the variety of titre of the specific rabbit serum..To investigate the effect of glycation on the antigenicity, IgE binding ability and conformation of OVA, OVA was treated by glycation. The secondary and tertiary structure of OVA was defined by circular dichroism, fluorescence and UV absorption spectrum, respectively. Analysis of conformation indicated that after treating, an increase in UV absorption intensity and surface hydrophobicity of OVA was found, and the secondary structure, mainlyβ-sheet andp-turn, was also changed.Analysis of antigenicity and IgE binding ability showed that the antigenicity of OVA had a gradual increase after glycation, and the IgE binding ability of OVA decreased as treating time increases. It can be concluded that OVA antigenicity and IgE binding ability changes oppositely after glycation.Since five distinct regions in OVA were found to contain dominant allergic IgE epitopes, and these epitopes were composed primarily ofβ-sheet and (3-turn structures, it is possibly theseβ-sheet andβ-turn changes destroyed or buried some IgE epitopes, causing OVA IgE reactivity reduction.