Development And Application Of Monoclonal Antibodies Against Infectious Bronchitis Virus By Isolates From Shanxi

Infectious bronchitis (IB) is an acute, highly contagious respiratory and urogenital tract disease that caused by Infectious bronchitis virus, which belonged to Coronaviridae family. Many features of IB V, for example, antigen complexity, high incidence of point mutation and gene recombination, many of serotypes and weak cross protection between the strains of different serotypes, cause IB continuous outbreaks and epidemics, and lead to differ in the virus serotype due to different regions. Although the vaccine has been widely used in disease prevention, still can not avoid the non-vaccine serotypes IB prevalent, and has brought serious economic losses of the poultry industry. So that, accurate identification of local epidemic serotypes and formulate reasonable and targeted immunization program have a vital significance.It has important significance to study the monoclonal antibody against fundamental research, diagnosis and prevention of IB, because of its relative stabilities, high purity, sustainable acquired character and identify specificity characteristics. Monoclonal antibody against IBV namely SX2009isolated from Shanxi Province was prepared in this study. This study not only laid the foundation for establishing the rapid diagnosis kit of IB, also has important significance for researching on IBV antigenic variation, pathogenic mechanisms, diagnosis and treatment of IB.The6-8week-old (12-18g)female BALB/c mice immunized with purified virus after IBV (SX9) proliferation cultured by egg inoculation method, differential centrifugation, sucrose density gradient centrifugation. The cells were fused by PEG fusion method after detected titer more than104to mice post-inoculation. Indirect ELISA assays the first step prepared was used to detect of serum antibody titers of mice. The hybridoma cells were screened after cell fusion by indirect ELISA method. Strong positive holes were subcloned by limiting dilution method, and got2hybridoma cell lines that capable of stably secreted the monoclonal antibody anti-IBV were named2D6and11C10respectively. A lot of monoclonal antibody was prepared by the preparation method of the ascites. Ascites antibody titer of2 strains of monoclonal antibodies was measured by indirect ELISA method that has been established in the first step. The titers of2D6and11C10were105and106respectively.2D6and11C10were IgG1by subtype identification. The experimental results showed that two hybridoma cell lines had well passaging stability and specificity. Two strains monoclonal antibodies specifically reacted with IBV, and not reacted with Newcastle disease virus (NDV), avian influenza virus (AIV), infectious bursal disease virus (IBDV) and poultry egg drop syndrome (EDS76) virus.The2strains of monoclonal antibodies against IBV were prepared successfully in this experiment, and laid the foundation for the further development of diagnostic reagents.