Preparation Of Monoclonal Antibodies Against GB And GD To Establish A Double Antibodies Sandwich ELISA For Dtection Of Infectious Laryngotracheitis Virus

Infectious laryngotracheitis is a respiratory disease of chickens caused by infectious laryngotracheitis virus, it is highly contagious. This disease can cause the infected chicken’s difficulty breathing, cough with blood-tinged sptum, laying rate decreased or even death. Therefore, it is important to develop a simple, rapid and accurate diagnosis to control ILT. This study is preparation of the monoclonal antibody of ILTV gB and gD protein, established a sandwich ELISA antigen detection method, provided an effective means for the rapid detection and diagnosis of ILT.In this study, prokaryotic expression ILTV gB and gD protein was used as the immunogen to immunized the BALB/c mice aged of 6-8 weeks, then the spleen cells from immunized mouse were fused with SP2/0 cells after the antibody titer of the mice serum were greater than 1:104; After that, an indirect ELISA method was established to screening and subcloning the hybridomas. Ultimately, two hybridoma cell lines secreting monoclonal antibodies against ILTV gB and the other two hybridoma cell lines secreting monoclonal antibodies against ILTV gD were obtained, named as 5A8,1C1,4D6,7D9 separately. After preparation of ascites, they are purified by protein G column; then the antibody titer was determined by indirect ELISA method established before. The results show that antibody titer of both four ascites induced by hybridoma cell lines greater than1:105; Western Blot results show that four monoclonal antibodies can specifically identify its corresponding protein; indirect immunofluorescence results show that four monoclonal antibodies can be used to test ILTV infected cells with specific fluorescence reaction.In this study, a double antibodies sandwich ELISA method for detection of ILTV was established by using monoclonal antibody 7D9 as the coating antibody and 7D9-HRP labeled antibody as a detection antibody. Optimum conditions were as follows:monoclonal antibody coated at a concentration of 5μg/ml,4℃ coated overnight; with 1% BSA blocking solution, 37℃ incubated for 120min; antigen to be detected at 37℃ for 60min; HRP labeled antibody working at a concentration of 2.5μg/ml,37℃ incubated for 60min. Specific tests show that the sandwich ELISA method has no cross-reaction with the avian influenza virus (AIV), Newcastle disease virus(NDV),infectious bronchitis virus (IBV); sensitivity test show that the sandwich ELISA method can detect ILTV at the lowest amount of 102TCID50/ml; repeatability tests show that coefficient of variation is within 8% both intra and inter batch. Fourty samples were tested by the sandwich ELISA method, the coincidence rate was 89.2% comparing with polymerase chain reaction amplification results. All the above results show that the sandwich ELISA method for detection of antigen has great specificity, high sensitivity and good reproducibility, establish of sandwich ELISA method can be helpful with ILT epidemiological investigation and diagnosis.