Establishment And Application Of The Kit For Detecting The Antbodies Against Actinobacillus Pleuropneumoiae

1 Optimization, standardization and Development of rApxâ…£-ELISA Kit For Detecting Antibodies against Actinobacillus pleuropneumoiaeThe recombination protein(rApxâ…£) was induced with IPTG by 220g. The rApxâ…£-ELISA has been established for detecting antibodies against Actinobacillus pleurapneumoiae. Some factors of the rApxâ…£-ELISA have been optimized. The ELISA board closed by 1%BSA at 37℃for 2h. The work concentration of H₂O₂ was reduced from 0.025‰to 0.020‰and the time ofcolour reaction was 8～10min. According to the rApxâ…£-ELISA, the kit was developed for detecting antibodies against Actinobacillus pleuropneumoiae. The reagents were stable well when storing at -20℃for 12 months. The rApxâ…£-ELISA test k(?) has repeat(?)ili(?), all right. The coeilicient of variations are 0.1～6.4%of the different sets and 3.2～6.9% of the same set. The kit was specific, sensitive and rapid for monitoring antibodies against Apxâ…£of APP and diagnosing improvisely.2 Establishment of rApxâ…£-IHA for the Antibodies against Actinobacillus pleurapneumoiaeAn indirect hemagglutination assay to detect the antibodies to active Actinobacillus pleuropneumoiae (APP) was established by using recombinant Apxâ…£(rApxⅣ—IHA).The purified protein of 15μg /mL from recombinant E.coli. was coated with 1% (v/v) pyruvaldehyde- formaldehyde sheep red blood cells in TAP buffer solution with pH4 at 37℃for 1h . The absorbed red blood cells were incubated with the swine serum of 30 microliters at 37℃for 45mins.The samples were determined as positive when they showed entire agglutination at 1:8 or above. rApxⅣ—IHA are linearly related with coefficient of 0.974 to rApxⅣ—ELISA established formerly.3 Development and Application of rApxâ…£- IHA Kit for Detecting the Antibodies against Actinobacillus pleuropneumoiae According to established rApxâ…£-IHA, the kit of rApxâ…£-IHA was developed for detecting de antibodies against Actinobacillus pleuropneumoniae. The coefficient of variations are 0～6.4% of the different sets and 0～6.8% of the same set. The reagents were stable well when storing at -4℃for 6 months. The positive rate of antibodies against was Apxâ…£of APP of the swine sera from different sources was 32.0% (176/550) and the swine sera of infected artificially was 92.2% by rApxâ…£IHA. The result displays that the low infected rate was in the first culturist according to 6.1% positive rates and the positive rates of the other four culturists were between 27.6%. to 45.0%. The results demonstrated that rApxⅣ—IHA for detecting Apxâ…£for detecting Apxâ…£antibodies of APP is rapid, simple and specific and can be used widely.