Preparation,stability and in vitro activity of egg yolk immunoglobulin Y against human Sucrase / 中国免疫学杂志

ABSTRACT

Objective:To prepare the egg yolk immunoglobulin Y ( IgY) against human Sucrase and study its stability,in vitro activity. Methods:Hy-line laying hens were immunized with human Sucrase protein,IgY was isolated and purified from egg yolks of im-munized hens using water dilution and salting out method. Indirect ELISA was used to evaluate the titer and stability of IgY. The purity and specificity of IgY were analysed by SDS-PAGE and Western blot respectively. The inhibitory effects of IgY on α-glucosidase was studied by PNPG method. Results:Indirect ELISA results showed IgY could be detected on the tenth day after the first immunization, and the peak titer of IgY was 112 800 after the 40th day of immunization. SDS-PAGE showed that the heavy chain and light chain of IgY were 65 kD and 25 kD respectively, and the IgY against human Sucrase could specifically recognize the protein of human Sucrase. The IgY maintained primary titer when it was kept between 29-69℃ for 15 min,and pH 4-7,37℃,4 h. The titer of IgY was maintained 50% after digestion by pepsin and trypsin respectively for 2 hours. IgY had a higher resistence to pepsin than trypsin after longer digestion time. IgY showed an inhibitory effect on α-glucosidase in concentration dependent manner. The half inhibitory concentration (IC50) was 0. 540 mg/ml. Conclusion:The IgY against human Sucrase has been successfully obtained,which established foundations for its study of Type 2 diabetes mellitus rat models in vivo.