Purification Of Chicken IgG And Characterization Of Monoclonal Antibody Against Chicken IgG

IgG is the highest content in serum immunoglobulin (Ig), accounting for 75%~80% of the total serum Ig, which is the primary antibodies in the blood. Determination of serum IgG levels is the common indicators evaluating vaccine effectiveness and humoral immunity. Monoclonal antibody has the specificity of antigen binding compared with conventional serum antibody. It is the most important tool of the immunology and serology and also has been used in the veterinary diagnosis and treatment of diseases widely. In the poultry industry, the use of these monoclonal antibodies to detect disease-specific antibodies has important significance in comprehensive and systematic understanding of the poultry pathogen infection in the animal immune response and taking appropriate control measures. In addition, it is the basis for the development of effective kit to detecting poultry disease and the levels of vaccination.The following work done in this paper mainly focuses on purification of chicken Ig and characterization of monoclonal antibody against chicken IgG.First,IgG was isolated from chicken serum by a series of precipitation, such as purification with saturated ammonium sulfate and chromatography with Sephadex G-200. After SDS-PAGE electrophoreisis, IgG with a high degree of purity was obtained and the concentration of 2.4mg/mL was determined by Folin-phenol method.Secondly, the 6-8 week-old female BALB/c mice were immunized three times with purified IgG, the spleen cells from the immunized mouse and SP2/0 cells were fused in proper proportion. Then, indirect ELISA was established to screen the positive hybridoma cell strains. Four hybridoma cell strains producing monoclonal antibodies, named C44, C45, C67, C68, were obtained by twice subcloning. Four monoclonal hybridoma could be able steadily to secrete antibody anti IgG after five generations.Finally, the properties of these monoclonal antibodies were identified such as the titers, Ig subclasses, antibody specificity and cross-reaction. These four monoclonal hybridoma cell strains had the titers 1:400, 1:1600, 1:3200, 1:400 and 1:640000, 1:320000, 1:640000, 1:80000 in the culture supernatant and in the ascites respectively by indirect ELISA test; and belonged to IgG1 isotype by an identification with Mouse Monoclonal Antibody Isotyping Kit.We used the methods of Western Blot to analyze the specificity of four monoclonal antibodies and found that C44, C45 could recognize chicken IgG light chain and C67, C68 recognize chicken IgG heavy chain. These monoclonal antibodies could not bind Ig of pig duck and rabbit Western Blot as well as in indirect ELISA. These results showed that monoclonal antibody C44, C45, C67, C68 could only specifically recognize chicken IgG, and had no cross reaction to the Ig from other animals.