Preparation Of Monoclonal Antibodies Against Salmonella Flagellin And Establishment Of Blocking ELISA

Salmonella,an important zoonotic pathogen,is widely distributed in the link of livestock and poultry production.Salmonella infection not only affects the healthy development of animal husbandry but also can infect human beings through breeding,processing environment,meat and egg products,causing a variety of diseases and seriously threatening public health security.The traditional method to identify the Salmonella through biochemical tests and PAT,but takes a long time and can not meet the market demand for rapid detection.The molecular detection technology based on nucleic acid consumes less time and own higher sensitivity,but has higher requirements about equipment.Both of the methods need the bacteria to be extracted from the samples,which is time-consuming.Immunological detection methods have been widely studied due to their advantages of realizing serum detection,high specificity and high throughput.However,most of the current Salmonella detection methods are for one or several Salmonella serotypes,and the diversity of Salmonella serotypes makes the detection prone to false-negative results.Therefore,it is necessary to establish a detection method that can detect different Salmonella serotypes simultaneously.Salmonella flagellin exists in all kinds of Salmonella serotypes except S.Pullorum and S.Gallinarum.The common epitopes of Salmonella flagellin have been proved to be an ideal diagnostic target with broad-spectrum screening.In this study,Salmonella flagellin was selected as the immunogen to prepare monoclonal antibodies(MAbs)for recognizing Salmonella flagellin;and a blocking ELISA method was established to realize the rapid and efficient detection to different serotypes of Salmonella.1.Preparation and identification of MAbs against Salmonella flagellinIn this study,S.Enteritidis flagellin was extracted by acid hydrolysis method,and the protein band was identified as 57kDa by SDS-PAGE,with high purity.The BALB/c mice were immunized with the purified flagellin.Cell fusion was conducted after three times of immunizations at intervals.Screening hybridoma cells that secretion MAbs for recognizing Salmonella flagellin by an indirect ELISA.Six hybridoma cells were obtained,which were stably secreted of MAbs after repeatedly sub-cloned.Their subtypes and titers were determined by indirect ELISA,results showed 1F6 MAb subtype was IgG2a,7A10 MAb subtype was IgG2b,and the other four MAbs subtypes were IgG1.The results of indirect ELISA showed that all of the cell culture supernatant titers were more than 2.0×105.MAbs 1F6 and 7A10 were selected to prepare ascites,and both of them had a titer of more than 3.0×107.MAb 1F6 was anti-Salmonella flagellin,and its reactivity,affinity,purity and specificity were furtherly identified by Western blot and ELISA.The results revealed that the MAb 1F6 could recognize Salmonella flagellin and the affinity constants were 1.11×1010 M-1,and 1F6 could stably bind with antigen.Identification of the reactive specificity of MAb 1F6 by Western blot and ELISA revealed that MAb 1F6 reacted with all Salmonella containing flagellum and definite negative reacted with S.Pullorum(without flagellin)and non-Salmonella.The MAb with high affinity and high specificity was successfully obtained,which can be used as a candidate to establish an immunological detection method for the detection of Salmonella.2.Establishment and application of blocking ELISA based on Salmonella flagellin MAbThe purified and HRP-labeled MAb 1F6 was used for the establishment of a MAb blocking ELISA method for common epitopes of Salmonella flagellin.It was determined that the best working concentration of the coating antigen and the MAb were 1.0 ?g/mL and 0.534 ?g/mL,respectively,in the blocking ELISA assay by checkerboard method.After optimizing various experimental conditions,the optimal detection conditions were determined as follows:the optimal incubation time of serum was 1 h;PBS solution containing 5%skimmed milk was used as blocking buffer and incubated at 37? for 2 h;The optimum dilution of serum was determined to 1:10;HRP-labeled MAb was added and incubated at 37? for 0.5 h.Finally,substrate action time development at 37? for 4 min.The detection threshold was determined by ROC curve analysis,and when the cut-off was 36.40%,to reveal the positive result,otherwise,it was negative;meanwhile,the Youden index was the highest,0.9756;the method showed the highest sensitivity and specificity were 97.56%and 100%,respectively.The inter-batch and intra-batch tests showed that the blocking ELISA method had good reproducibility.For 10 chicken serum of different pathogenic microorganisms infection tested by the blocking ELISA,results show that no cross-reaction was found between Salmonella infection serum and other pathogens infection serum.In addition,227 chicken serums were detected by this method with the test results of 221 samples were consistent with the background,and an overall coincidence rate was 97.4%.In conclusion,this method was well stable,with high specificity and high sensitivity,and it can be used for broad-spectrum screening of poultry Salmonellosis.