Application Of A Monoclonal Antibody-based Capture ELISA To Detect Pseudorabies Virus And Development Of A Pichia Pastoris Expressing Apxâ…¢A-based ELISA To Detect Antibody Against Actinobacillus Pleuropneumonia

This thesis consisted of two parts.1 Development of monoclonal antibody-based capture ELISA for detection of pseudorabies virus (PrV)Pseudorabies virus (PrV), a member of Alpha Herpesvirus, is the causative agent of Pseudorabies (Aujeszky's disease), one of the most severe and economically important infectious diseases of domestic and wild animals. Swine was the natural host and reservoir of PrV.1.1 Balb/C mice were immunized with purified PrV. After fusion of SP2/0 myeloma cells with the stimulated splenocytes, two hybridoma secreting monoclonal antibody against PrV, designated as 3G7E3 and 4A6F5, were identified by ELISA. The ELISA titers of 3G7E3 and 4A6F5 in ascites were both 1:100×2¹². No cross reactions were found when Japanese encephalitis virus (JEV), Porcine respiratory and reproduce syndrome virus (PRRSV), porcine parvavirus (PPV), hog cholera virus (HCV) and swine influenza virus (SIV) were tested. The specific reactivity of Mabs with PrV was confirmed by IFA and Mab-based sandwich ELISA. These results indicated that specific hybridomas secreting Mab against PrV were developed and possessed the potential of usage in development for detection of PrV antigen .1.2 The Pig anti-PrV serum was produced through immunization of animals with purified PrV, followed by isoaltion of IgG. Using 3G7E3 as capture antibody and purified IgG as detector antibody, a sandwich ELISA was developed to detect pseudorabies virus (PrV). After optimization, the microplates were coated with 100μl monoclonal antibody at concentration of 12.2 μg/ml and the working concentration of IgG was determined as 16.0 μg/ml. No cross reaction was found when JEV, PRRSV, PPV, HCV and SIV were tested by this technique. The detection limit of the test was 0.312μg/ml of PrV. The varition indexes between or within batches were 4.87% and 1.07%, respectively. The tissue samples collected from experimentally and clinically infected pigs were used to validate the sandwich ELISA. The highest positive rates were obtained from detection of tonsils, brains and lungs. Up to 77.4% agreement between sandwich ELISA and PCR was obtained from detection of 159 clinical samples and new ELISA was more sensitive. These data indicated that a sensitive and specific sandwich ELISA for detection of PrV was developed, providing a useful tool for diagnosis of PrV infection.1.3 By using sandwich ELISA and PCR, the presence of PrV in the pork, mutton and beef samples, which were collected from markets in Wuhan, were investigated. Theresults showed that 19 positive samples were detected in 97 pork samples by DAS-ELISA while 25 positive samples were picked up by PCR. Only two positive samples were detected from 50 beef samples by PCR. All of 50 mutton samples were proved to be PrV free by both PCR and ELISA. Up to 892 % agreement between PCR and DAS-ELJSA was achieved. PrV could be isolated from the ELJSA-positive samples. These data indicated that the latent infection of PrV in pigs might play an important role in present prevalence of Aujeszky's disease.2. Expresion of actinobacillus pleuropneumoniae ApxIII in yeast system and development of recombiant ApxEQ-ELISAActinobacillus pleuropneumoniae is the etiological agent of porcine contagious pleuropneumonia, a respiratory disease that continues to have a worldwide economic significance. RTXs determine the bacterial pathogenicity, so, detection of antibodies against RTXs is helpful in diagnosis of APP and evaluation of vaccine efficacy. Due to the difficulty in purification of native toxins, the easily-purified recombinant proteins have been used in development of diagnostic methods with high specificty. Comapred to other protein expression system, pichia pastoris system has its unique advantages. This paper describes the expression of ApxIII in the novel system and development of recombinant ApxlII-based ELISA.The full length of gene encoding ApxIIIA of Actinabacillus Pleuropneumoniae was amplified by polymerse chain reaction (PCR) and ligated to pMD-18T for purpose of sequence analysis. The amplicon was further cloned to pPIC9K expressing vector and transformed to GS115 competent cells. The protein with the size of approximate 110 kd was expressed by addition of methanol. The products were used to coat microtiter plates for develoment of antibody-detection ELISA that was optimized through checkerboard assay. The recombinant apxIHA-based ELISA possessd specificity, no false positive results occurred when Hps, PrV and PRRSV were tested. The variation indexes ranged from 8.24% to 2.46%. 82.4 % of agreement between the novel ELISA and IHA was observed after detection of 284 clinical samples, while novel ELISA was more sensitive. These results revealed that our new ELISA was of sensitivity, specificity and reproducibility, suitable for clinical diagnosis of infection caused by ApxIII-producing actinobacillus pleuropneumoniae.