Preparation And Application Of Monoclonal Antibodies Against Pasteurella Multocida Toxin

Toxigenic Pasteurella multocida(T~+Pm) is a major pathogen of progressive atrophic rhinitis(PAR).T~+Pm secretes Pasteurella multocida toxin(PMT),a 146 kDa dermonecrotic toxin,which is encoded by toxA gene.PMT is considered to be a main pathogenic factor of T~+Pm.Injection of little purified PMT into SPF pigs can cause PAR.Considering that PMT plays a important role in PAR,the detection of PMT and antibodies against PMT become the key of PAR diagnosis.Detection of PMT antibodies can provide an important method for the evaluation of the quality of vaccine and PMT antibodies detection.It can also provide evidence for the diagnosis of PAR.In this study,monoclonal antibodies against PMT were prepared,and a competitive ELISA based on HRP-labled monoclonal antibody was established.The ELISA can be used to detect PMT antibodies and provide a useful tool for the eradication of PAR.The results are as follow:1.Generation of monoclonal antibodies against Pasteurella multocida toxinUsing two purified Escherichia coli expressed recombinant PMT proteins as antigen to immune Balb/c mice,then spleen cells and myeloma cells SP2/0 were fused.The indirect ELISA method and Western-blot assay were employed to screen target hybidoma cells.The positive cells were cloned three times by Limited dilution.After 3 times of fusion,eleven strains of Hybridoma cell which could stablely secret antibodies againt PMT were obtained.Six of the monoclonal antibodies recognized PMT-C and five ones recognized PMT-N.The titers of ascites were above 100×2~9.Relative affinity of the monoclonal antibodies tested by ELISA were above 10~4.All the monoclonal antibodies were proved to be specific.No cross-reaction with nontarget proteins was found.2.Establishment of the competitive ELISA and the prime:applicationMonoclonal antibodies was purified by the method of Caprylic Acid/Ammonium Sulfate Precipitation.Then,purified monoclonal antibodies was labled by HRP using heptaiodic acid oxidation method.The competitive ELISA method used to detect the antibody against PMT was established by using PMT-C protein as antigen and the HRP-labled monoclonal antibody AH12 as detection antibody.All the items which may influence the reaction were perfected.Chess-board assay showed that the concentration of recombinant protein for coating ELISA plates is 223ng/mL and the working titer for HRP-labelled monoclonal antibody was 1:3200.＞40%of inhibition rate was the positive judging standard.A total of 50 known sera were detected by both the competitive ELISA and in vitro neutralization assay.The result indicated the positive coincidence rate and the negative coincidence rate were 90%and 100%respectively.82 unknow sera were detected in parallel by both the competitive ELISA and in vitro neutralization assay.The total coincidence rate of the two assays is 91.5%.1054 serum samples from pig fields of Hubei province were tested using the competitive ELISA and the positive rate was 26.7%.