| ObjectiveSalmonella is Gram-negative bacteria parasitized in human and animal intestines, and act as main pathogen of human diarrhea. Chicken egg yolk antibody (Immunoglobulin Yolk, IgY), differs from mammalian Immunoglobulin G (IgG) in terms of structural and immunological properties. It also has advantageous compared with mammalian IgG, including stability under various physicochemical conditions and its suitability as an immunological tool. IgY can be produced in large quantities from the yolk of chicken eggs by simple methods, and without distress to the animals. Therefore, IgY has received much attention due to its potential as an alternative to conventional mammalian antibodies.This research aim at separating and purifying IgY against-Salmonella, as well as analysing and authenticating its basic physicochemical characteristic, it will provide us ideas and strategies for immunotherapy of Salmonella. Methods1. Immunogen was attenuated Salmonella Ty21a of K antigens sorts.2. The hens we used were named Jing-Bai Hen, aged around 6 months, weighing about 1300±100g, in good health and laying five to six eggs per week. The hens were ready for immunization after a week's normal feeding.3. Preparing the single cell suspension, 109 cells were innoculated on both wing skin and both back skin of the hens (with the same quantity of complete Freund's adjuvant at first injection). After the primary inoculation, we immunized the animal 3 times at 14, 28, 42 days respectively with the same dose of Ty21a without adjuvant. Intensive immunization was given once a month after the above mentioned treatment, so as to maintain a high antibody titer in animal. Eggs were collected and marked immediately before and during the course of immunization. The eggs were stored at 4℃before used to prepare IgY.4. The eggs before and after ( at 15, 20, 25, 30, 35, 40, 45 days) immunization with Ty21a were used in our study. In order to get pure IgY antibody, the yolk liquid was collected from the eggs,extracted through distilled water, salted out in 50% saturated ammonium sulfate, dialyzed, concentrated with PEG.5. The obtained antibodies were identified through SDS-PAGE, Western blotting and ELISA.6. Testing activity of inhibiting Salmonella growth in vitro.ResultsAbout 10 days after immunization, a special antibody of IgY could be detected from the yolk. The level of the antibody got to a high peak on 30th day. 100~200 mg of antibody could be harvested from each egg. It was strongly heat-proof and acid-proof. And its effect could last 2~6 months. Two straps were shown while detecting the purified IgY with SDS-PAGE, with the molecular weight reaching approximate 70KD and 20KD respectively. The results of ELISA shown that no cross reaction was observed between IgY and other antigens, such as Streptococcus, Colibacillus, Staphylococcus aureus. It could particularly inhibit Salmonella growth in vitro.ConclusionIn this study, the AS-IgY were isolated and purified successfully. Through studying on it's physicochemical property, SDS-PAGE, Western blotting and ELISA, IgY was indicated to be a new kind of antibody which had strong resistance of acid and heat. The method of IgY preparation introduced here was feasible and high-yield. It was demonstrated to inhibit Salmonella growth in vitro, with better particularity but no cross reaction with other irrelevant antigen. Therefore, this research would develop a new method for the prevention and therapy of human Salmonellosis.
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