Ultrasensitive Point-of-Care Dengue Diagnostics and Vaccine Applications

• Author / Creator

  Ganguly, Advaita

• Dengue virus infections can result in a range of clinical manifestations from asymptomatic infection to dengue fever and the severe disease dengue haemorrhagic fever/dengue shock syndrome. The disease is now endemic in more than 100 countries in Africa, the Americas, the eastern Mediterranean, Southeast Asia, and the Western Pacific, threatening more than 2.5 billion people. The World Health Organization estimates that there may be 50 million to 100 million cases of dengue virus infections worldwide every year, which result in 250,000 to 500,000 cases of Dengue Hemorrhagic fever and 24,000 deaths each year. The dengue virus non-structural NS1 protein is a 46–50 kDa glycoprotein when expressed in infected mammalian cells. A high circulating level of NS1 was demonstrated in the acute phase of dengue infection by antigen capture ELISAs. The precise function of dengue NS1 protein remains unclear. However, antigen detection of non-structural dengue antigens may be of benefit for an early stage rapid diagnosis of infection due to its long half-life in the blood. Five high affinity monoclonal antibodies were developed and characterized against the recombinant dengue NS1 protein using hybridoma technology. Anti-NS1 and anti-HRPO hybridomas were fused and sorted to develop a series of bi-specific antibodies. The recombinant NS1 protein was also used to immunize chickens for the development of Anti-NS1 chicken IgY polyclonal antibody. The different combinations of anti-NS1 mAbs, bi-specific antibodies and chicken IgY were used in the development of simple, rapid, inexpensive, highly sensitive, specific and easy to perform assays for the detection of dengue virus infection.
  The dengue envelope protein was also expressed using recombinant techniques for the evaluation of its role as a vaccine candidate. The envelope protein is known to induce neutralizing antibodies against dengue virus. The recombinant dengue envelope protein was used to immunize mice as a vaccine candidate. Serum antibody analysis confirmed virus neutralization characteristics. We also raised monoclonal antibodies against the envelope protein to better understand dengue pathogenesis as well as potential reagents in dengue diagnostic development.

• Subjects / Keywords

  • Monoclonal Antibody
  • Bispecific Antibody
  • Dengue
  • Chicken IgY
  • Immunoswab

• Graduation date

  Fall 2013

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R38G8FV0S

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.