Studies of immunological and molecular biological techniques with infectious laryngotracheitis virus of chickens Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/pv63g289p

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  • Monoclonal antibodies (MCA) produced against infectious laryngotracheitis virus (ILTV) of chickens reacted in western blotting experiments with several different ILTV protein bands in the absence of tunicamycin which inhibits carbohydrate synthesis. Most of the MCA lost their reactivity in western blotting experiments when extracts of tunicamycin-treated ILTV CELC were used, suggesting their specificity for carbohydrate-based epitopes. In an indirect immunofluorescence test most of the MCA bound primarily to cytoplasmic antigens except some MCA which bound primarily to nuclear antigens. Additivity ELISA was also performed to study whether MCA are against the same epitope or different epitopes. The polymerase chain reaction (PCR) was developed as a diagnostic technique for detection of ILTV using primers made from a portion of the ILTV thymidine kinase gene. The 647-basepair amplified ILTV PCR product was labeled to create a non-radioactive, biotinylated DNA probe. Hybridization was performed using the probe to detect ILTV. Both PCR and hybridization detected ILTV, and neither hybridization nor PCR gave positive results with any other pathogen. Hybridization was specific for ILTV, However, slight hybridization occurred with CELC DNA when relatively relaxed conditions were used. In another experiment, diagnostic tests to detect ILTV in tracheas of experimentally-infected chickens, including the indirect fluorescent antibody test (IFAT), immunoperoxidase (IP), virus isolation (VI), histopathology, PCR, and hybridization, were performed and compared. Using virus isolation as a reference, the sensitivity and specificity of the tests were calculated. The IP test and IFAT performed better than any other test used in this study.
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