Establishment Andapplication Of Doubleantibody Sandwich ELISA For Chicken Shigella Boydii

Shigellosis of chicken is a new infectious disease, which found in recent years. The disease is an acute infectious disease caused by Shigella, which can give rise to different species, different age of chicken diarrhea and death of chickling. The current study results show that Shigellosis of chicken was widespread and harmful. Because of the spread of Shigellosis, the poultry industry of China has tremendous economic losses. Shigella may cause cross-infection between human and poultry, which led to serious security risks for human public health. Therefore, the study of Shigellosis of chicken not only has important veterinary health significance, but also has great medical public health significance.Chicken S.boydii as research material of the project was isolated, identified and conserved by the laboratory. The study of double antibody sandwich ELISA was carried, and that was based on the indirect ELISA for quantitative detecting S.boydii vaccine immune antibody .The results are as follows:Firstly, S.boydii inactivated aluminum hydroxide vaccine was used to mmunized adult rooster. The method that indirect ELISA for quantitative detecting S.boydii vaccine immune antibody was used during the process of immunization, and obtained high titer immune serum. S.boydii IgG was extracted by saturated ammonium sulfate and purified by ion exchange chromatography by using DEAE-52.Secondly, horseradish peroxidase (HRP) was used to mark purified S.boydii IgG by two-step Glutaraldehyde method and sodium periodate method. The IgG-HRP labelled by sodium periodate method was selected and purified after product testing.Then, double antibody sandwich ELISA for chicken S.boydii was established successfully. And chicken S.boydii IgG were used as coated antibody and chicken S.boydii IgG-HRP were used as enzyme-labeled antibody. The best working conditions were optimized for double-antibody sandwich ELISA. The results were: the concentration of coated antibody were 5μg/mL and the best conditions was 4℃ ?12h; the best conditions for each hole was closed 100μL 1%BSA, 37℃1h; the optimum conditions tested antigen and antibody coated was 37℃1.5 h; the best dilution of IgG-HRP was1:200 and the optimum conditions was 37℃1h; chromogenic substrate time was 37℃20min.Specificity tests,sensitive tests and replicate tests show that the method has good reproducibility, great specificity, high sensitivity. The Coated S.boydii IgG and other pathogens that cause diarrhea had no cross reaction. The coefficient of variation of one-plate was 0.31%～0.53% , and 0.50%～0.84% of inter-plates; The detection limit of the method was 105cfu/mL.The method was initially applied, it's a new technical means for Chicken S.boydii pathogen detection, and is easy to expand and apply in the production.