Development And Application Of Indirect Elisa Antibody Test Kit For Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia is caused by Actinobacillus pleuropneumoniae, which is a highly contagious and occasionally fatal respiratory disease. The virulence of A.pleuropneumoniae in not understood,but it seems clear that more than one virulence factors is involved. Factors responsible for virulence include toxins, outer membrane protein,capsule polysaccharides, lipopolysaccharides and so on.Toxins are not only the main virulence factors,and also the immunogen. A. pleuropneumoniae generated four kinds of toxins.ApxⅡ was secreted by all serotypes of A. pleuropneumoniae except serotype10.ApxⅣ was expressed all serotypes of A.pleuropneumoniae only after infection of host,but not in vitro.1Optimization and development of ApxⅡ-ELISA antibody test kit for Actinobacillus pleuropneumoniaeThe induction conditions of the recombinant protein ApxⅡ had been optimized.The optimal temperature was37℃and the optimal concentration was0.8mM. The ApxⅡ-ELISA has been established for detecting antibodies of Actinobacillus pleuropneumoniae.Some factors of the ApxⅡ-ELISA was optimized.The coating concentration of rApxⅡ protein was2.23μg/mL,and incubated for1hour at37℃and then overnight at4℃.The plate were subsequently blocked1hour with1%BSA-PBST.The work concentration of HRP-antibody was1:20000.Based on the indirect ELISA,the ELISA test kit was developed,whose stability, sensitivity,specificity and repeatability were evaluated. The variant coefficient of ApxⅡ-ELISA test kit in a batch and between batches varied from2.52%to7.41%and2.96%to7.16%,respectively. The ApxⅡ-ELISA test kit was compared with IHA kit, the coincidence was86.7%.The ApxⅡ-ELISA test kit was specific and sensitive, and rapidly detected porcine contagious pleuropneumonia.2Development of ApxⅣ-ELISA antibody test kit for Actinobacillus pleuropneumoniaeThe induction conditions of the recombinant protein ApxⅣ had been optimized.The optimal temperature was30℃and the optimal concentration was0.6mM.The ApxIV-ELISA was established for detecting antibodies of Actinobacillus pleuropneumoniae previously.Based on the ApxⅣ-ELISA, the coating condition of the antigen, the storage conditions of the plates,diluted HRP-antibody,the control positive/negative serum and the test kit were explored,and the ApxⅣ-ELISA test kit was developed. The variant coefficient of ApxⅣ-ELISA test kit in a batch and between batches varied from2.65%to8.40%and2.69%to11.3%,respectively. the ApxⅣ-ELISA test kit was stable at-20℃for at least9months and4℃for3months. The test kit was compared with ApxⅣ-ELISA kit produced by Keqian Company, the coincidence was67.5%. The test kit distinguished antibodies of infection and vaccination.3Application of ApxⅡ-ELISA and ApxIV-ELISA test kitsThe ApxⅡ-ELISA and ApxⅣ-ELISA kit detected a large number of clinical serum samples.The positive rates of the two toxins antibody showed the trend of rising after declining in serum samples collected from pigs of different days.The positive rate deteced by declined to the minimum ApxⅡ-ELISA kit in50～60days old,while the positive rate deteced by the ApxⅣ-ELISA was the minimum in90-110days old.The ApxⅡ-ELISA kit deteced288postive serum from clinical samples and the positive rate was34.3%.191positive serum samples were detected by ApxIV-ELISA kit and the positive rate was22.8%.