Separation And Purification Of Chicken Immunoglobulin And Establishment Of ELISA For Their Detection

Chicken infectious diseases are popular. With the development of the poultry industry, early diagnoses and early response to poultry diseases are important. The measure of chicken serum IgG is a common index to clinic immunology inspection and humour immunology research. Compared to the general immunological sera, monoclonal antibody against chicken immunoglobulin has several advantages, such as high specificity, steady-going titre. The development of the monoclonal antibody against chicken immunoglobulin, which can provide high quality, standardized reagent for diagnosing to various poultry disease, is essential for the further research to chicken communicable diseases.This study is composes of two parts. In the first part, we aimed to separate and purify IgG from chicken sera. And indirect ELISA was established in the latter one.The chicken serum immunoglobulin was primarily purified by multistep deposition of (NH₄)₂SO₄ and ethanol deposition. SDS-PAGE shows that the sera immunoglobulin was purified by the two methods and the former method was better. Then, the primarily purified serum immunoglobulin was filtered by SephadexG-200 to further separate IgG. Immune electrophoresis and SDS-PAGE electrophoreisis showed that the electrophoretic purity was reached. The purified IgG has two bands in SDS-PAGE electrophoresis, and one band in immune electrophoresis. furthermore, the molecular weight(MW) of chicken IgG heavy chain is 65KD, and the molecular weight of light chain is 25KD.And the concentration of IgG was 1mg/mL by Lowry method, which were enough to fulfill the continuous experiment.The purified chicken IgG was used to immunize the animals as antigen to preparate rabbit and mouse sera against chicken serum IgG. Then, the polyclonal antibody of the immunized animals was measured by ELISA and AGP: the titre of rabbit serum to be 1:8 by AGP, 1:512000 by indirect-ELISA, and the titre of mouse sera to be 1:2 by AGP, 1:128000 by indirect-ELISA.Indirect ELISA was established to screen the positive hybridoma cell lines. By optimization, optimal reactive conditions of indirect-ELISA for screening the McAb coated with chicken IgG was determined as follows, the optimal peridium concentration of the IgG was 0.5μg/mL, the dilution of positive and negative sera were both 1:2000, the dilution of IgG-HRP was 1:10000, the optimal time of substrate solution was 20min.