Development And Preliminary Application Of Competitive ELISA For Detection Of Salmonella Enteritidis Based On Monoclonal Antibody Against H:g Antigen

Salmonella is a common zoonotic pathogen,which has brought huge losses to the breeding industry.Epidemiological studies have shown that the predominant serotypes of Salmonella in large-scale chicken farms in China are Salmonella enteritidis(SE)and Salmonella pullorum(SP).Although the competitive ELISA based on monoclonal antibody against O9 antigen(O9-cELISA)for detection of group D Salmonella antibody established in our laboratory can detect SE and SP infection rapidly and efficiently,it is still unable to distinguish them.Reports have suggested that the detection method based on specific antibody against H antigen can solve this problem.In this study,we developed a competitive ELISA based on monoclonal antibody against H:g antigen(H:g-cELISA)of SE.After optimization,the H:g-cELISA kit was assembled for rapid identification of SE infection.Further analysis indicated that the combination of H:g-cELISA and O9-cELISA can distinguish SP from SE infection,which provides technical support for the eradication of SP.1.Development of H:g-cELISAThe hybridoma cell 12E3 was used to prepare previously to produce monoclonal antibody against SE H:g antigen in our laboratory.After peritoneal injection with 12E3,the mouse peritoneal fluid was purified and then labeled with horseradish peroxidase(HRP).The titer of HRP-labeled antibody was up to 32000.The SE flagellin extracted after acidolysis was used as coating antigen.The H:g-cELISA determined by checkerboard method showed that the optimum concentration of coating antigen and HRP-labeled antibody was 5.8 ?g/ml and 0.5 ?g/ml,respectively.On this basis,the optimum dilution of serum was determined to be 1:8.After optimization,PBS was selected to dilute coating antigen.Then,the antigen-coated plate was incubated at 4? for 20 h.After wash,the PBS containing 5%skimmed milk was added as blocking buffer and incubated at 37? for 3 h.After wash,the HRP-labeled antibody was added and incubated at 37? for 0.5 h.Finally,the coloring condition was at 37? for 10 min.The cut-off value was determined by the ROC curve analysis and the serum was determined as positive when the inhibition rate was over 35%.With this standard,it was determined that the detection limit is 0.56 ?g/ml.No cross-reaction was found in chicken serums between SE and other pathogens infection using the H:g-cELISA.By detecting 132 SE-infected chicken serums and 136 SPF chicken serums,the specificity and sensitivity of H:g-cELISA were found to be 96.97%and 97.79%,respectively.In conclusion,the method has good stability,specificity and sensitivity,which can be used for the detection of SE antibodies.2.Assembly and application of H:g-cELISA kitThrough comparative analysis,it was found that the kit can be stored at 37? for 25 days with Liquid Plate Sealer liquid and protective agent(Candor).The kit showed better stability after being stored at 4? for 300 days.Compared with commercial kit(IDEXX)and PAT,the coincidence rate of H:g-cELISA kit was 97%and 97.8%,respectively.894 serums from two different farms in jiangsu were detected using the H:g-cELISA kit and the results showed that the positive rate of layer farm was 4%and that of broiler farms was 1.16%.In addition,77 SE-infected chicken serums and 65 SP-infected chicken serums were tested using the H:g-cELISA kit combined with the competitive ELISA kit based on O9-cELISA.The results showed that 98.5%of SP infected serums could be identified.These indicated that the combination of H:g-cELISA kit and O9-cELISA kit can distinguish SP from SE infection.In summary,the kit can provide technical support for the prevention and control of SE and the eradication of SP.