Preparation And Preliminary Application Of Monoclonal Antibody Against Salmonella O:9 Lipopolysaccharide

Salmonella spp.is an important zoonotic pathogen,which can infect many livestock and poultry around the world,which poses a threat to the breeding industry of livestock and poultry and animal-origin food safety.In the modern large-scale poultry farming industry,O:9 serotype,Salmonella Pullorum and Salmonella Enteritidis,were main serotype among avian Salmonella infection,they easily cause high morbidity and mortality to young broilers.Adult chickens show asymptomatic infection,transmiting vertically Salmonella to eggs,and egg production will be significantly decreased.All these will bring to huge economic losses in the poultry industry.Poultry infected with Salmonella in large-scale group is one of the main sources and also a key control point for the prevention and control of Salmonella.And so,rapid,accurate and effective Salmonella detection is conducive to early find Salmonella infections in poultry as soon,and Salmonella-infected chickens as pathogen sources were eliminated to ensure food hygiene and human health.The classification of Salmonella serotypes is based on their difference in O-antigens.O-antigens as an O-specific side chain on the surface of Gram-negative bacteria,are composed of different repeating oligosaccharide units and located in the lipopolysaccharide(LPS)on the cell wall,which can induce its host to produce specific antibodies as diagnostic target for Salmonella infection.At present,glass plate agglutination test(PAT)was widely used for avian Salmonella infection based on the interaction between O-antigen and its antibodies.However,in clinical use,the PAT test results are not stable,which lead to the difficulty in the diagnosis of salmonellosis in the poultry industry,because PAT testing only relies on eye observation and the stained antigens vary from batch to batch.Being compared with PAT,enzyme-linked immunosorbent assay(ELISA)has better sensitivity and specificity and the detection result can be digitized,which make the results clearer.And so,monoclonal antibodies against Salmonella 0:9 antigen were successfully prepared,and a sandwich ELISA method with anti-O:9 antibody were initially established.1.Preparation of monoclonal antibody against Salmonella 0:9 antigenThe LPS of Salmonella Enteritidis C50041 was extracted by hot phenol water method and its concentration was 3.641 mg/mL as determined by the anthrone colorimetric method.O-specific side chains were further purified from LPS mixtures after lipid A and core polysaccharide were degraded by acetic acid and coupled with KLH protein to obtain complete thymus-dependent antigens.Using inactivated Salmonella,Salmonella LPS and O-antigen-KLH as immunogens,5 different immunization programs were designed to immunize 6-week-old female BALB/c mice.After cell fusion between B cells from mice with high expression of anti-O:9 antibody and SP2/0,and the combined screening of indirect ELISA and PAT method,7 hybridoma cell lines which were capable to stably secrete monoclonal anti-O:9 antibody were obtained and named 1D3,1H3,2A4,5C11,5E1,5F11,5F12,respectively.Identification of antibody subtypes showed that only 5F11 was IgG3 type and other 6 monoclonal anti-O:9 antibodies were IgM type.The antibody titer in the culture supernatant of the hybridoma cells of 7 monoclonal antibodies is at least 2.0×105,and the antibody titer in ascites of mice is up to 3.0×106.After analysis by PAT,indirect ELISA,and Western blotting,all 7 monoclonal anti-O:9 antibodies can specifically react with the O:9 antigen,without cross-reaction with other bacterial,which revealed that they had good specificity.2.Preliminary establishment of sandwich ELISA detection method with anti-O:9 monoclonal antibodiesAfter anti-O:9 monoclonal antibodies were prepared,a sandwich ELISA for detecting the level of O:9 antigen for Salmonella infection were initially established.5F11 was selected as the detection antibody in this ELISA method.After 5F11 being labeled by HRP,its titer was over 51,200.During establish sandwich ELISA,monoclonal antibody 1D3 was identified as the capture antibody by sandwich ELISA.After optimation,the concentration of the capture antibody was 320 ng/mL,and the optimal concentration of detection antibody was 320 ng/mL,Following bacteria preenrichment at least 24 h and bacteria pretreated in boiling water for 10 min,and the lowest detection load of bacteria is 1.0×106 CFU/well.Simulated bacterial samples could be specifically detected Salmonella with O:9 antigen by sandwich ELISA method,and other bacteria are negative,there was no cross-reaction,it specificity is good.This method could also detect out the corresponding Salmonella from the fece of chicken artificially infected with Salmonella Pullorum.The sandwich ELISA method could use Salmonella as detected target,avoiding traditional process to isolate Salmonella and greatly improving detection speed.