The Establishment Of Hybridoma Cell Lines Secreting Monoclonal Antibodies To Chicken Siga

Mucosal imnunity of poultry is very important as the first defense barrier against infectious pathogens. The Secretory Immunoglubulin A(SIgA) is the effective factor of mucosal immunity in poultry, which has many functional attributes, serving to prevent adhering and multiplication of pathogens, priming biological activity in immunology to clean infective agents and preventing poultry from infection.The hybridoma technology invented in the 1960-1970's is a high-tech of biology. Monoclonal antibodies are produced by cell fusion technology. In contrast to polyclonal antibodies, monoclonal antibodies have following virtues: identical structure, uniform component, high specificity, animate bio-activity and fewer crossing reactivity.In this study, used the material of the fresh galls of chicken, the improved method of ammonium sulfate was employed to extract chicken SIgA from the galls of chicken for the first time. Sodium sulphate polyacrylamide gel electrophoresis (SDS-PAGE) indicated and purified the heavy chain of SIgA, The depurated SIgA emulsified in freund's complete adjuvant were chosen to immunize the 8-week-old female Bal/c mice with 50μg at the first time. Two weeks later, the same antigen emulsified in freund's incomplete adjuvant were intraperitoneally, and three days after final immunizad with 100μg by abdominal cavity and vena caudalis. Three days after, the spleen cells of immunized Balb/C mice were fused with NS0 mouse plasmacytoma cells using PEG-1500. When cells have paved the whole cell, the positive hybridoma cell secreting McAb against SIgA were screened by indirect ELISA, subclone the positive cells by limiting dilution for three times to get identical cell. One hybridoma cell lines named 5H9 against chicken SIgA, the result indicated by using indirect ELISA, the McAb ELISA titers from cell culture supernatant and ascites were 1:400-1:800 and 1:1.6×10~4, respectively. The McAb showed no cross-reactivity and high affinity to chicken SIgA. During six months, the hybirdoma cell can stably secrete McAb against chicken SIgA after ten times serial passages and three times freezing and revival. It provided technical support for deeply researching the mucosal imnunity of poultry and diagnosing the chicken infectious disease.