Development And Primary Application Of Monoclonal Antibodies Against Salmonella SpiC Protien

Salmonella spp. has been over 2600 serotypes up to date. It has a great influence on livestock and poultry industry and even human health. Its detection and diagnosis is important to prevent and control of Salmonella.SpiC protein is proposed to be an effector protein of T3SS encoded within Salmonella pathogenicity island 2 (SPI-2). It is required for the survival of Salmonella within phagocytic cells. SpiC protein may be exported by T3SS-2 into the host cell cytosol to interact with host proteins to interfer the trafficking of host cell and inhibit the fusion between phagosome and lysosome in Salmonella.. In the previous study, we found that spiC gene is specific gene, only in Salmonella spp.. and SpiC protein could induce its antibody in the infected host. So we thought that it might used as a kind of specific biomarker with ELISA method.Monoclonal antibody is important in detection and diagnosis to pathogen in addation to the study of the structure and function of protein. And so, we would make the specific monocional antibodies against SpiC protein to specifically detect and diagnose Salmonella.In this study, six-week-old BALB/c mice were immunized with the purified rHis-SpiC protien. After cell fusions being performed through the lymphocyte hybridoma technique,7 hybridoma cell lines which could steadily secrete SpiC monoclonal antibodies (MAbs) screened by indirect ELISA with rGST-SpiC were obtained after subclones for three times, and they were named as 3G9,3B2,4D9,4F7,5F8,6H2,6B9, respectively.Isotype analysis revealed MAbs of 6B9,3B2,4D9,5F8 and 3G9 were IgG1, MAbs of 4F7 and 6H2 were IgG2. The indirect ELISA results showed that the titers of all MAbs were very high up to 1×105-1×107 in primary ascetic fluids. Western blotting analysis clarified that these seven Mabs could specifically react with rHis-SpiC at 20 KDa and GST-SpiC at 40 KDa, this result was consistant with His Mab and rGST Mab, respectively. It suggested that the seven MAbs had good reactivity with recombinant SpiC protien.Dot-ELISA was established to detect the Salmonella, seven MAbs only reacted with Salmonella specifically.189 isolates were detected by this Dot-ELISA, positive isolates were 173(91.53%.) and negative were 16(8.47%). This result was consisitant with PCR detecting of stn gene.Using purified fusion protein rGST-SpiC as the coating antigen, and the MAbs 5F8 antibody labelled by HRP as detecting antibody, a competitive ELISA (cELISA) was developed for determing SpiC antibodies. The conditions including serum dilution, blocking reagents, competitive time of cELISA were optimized,149 chicken serum samples were detected,47% (70/149) was posistive and 19%(28/149) was negative with slide agglutination test and cELISA. 34%(51/149) was positive with cELISA but was negative with slide agglutination test. All results showed that cELISA developed in our study was high sensitive and specific, and could be widely used for detecting chicken serum antibodies against Salmonella.In summary, seven hybridoma cell lines secreted SpiC MAbs were developed. These Mabs would be vaulable for studying mechanisms of Salmonella infection, diagnosis of Salmonellosis, and development of vaccines.