Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/22763
Title: Establishing Improved Platforms for Dengue Diagnosis and Hybridoma Development Using Dengue Virus Envelope Domain III Antigen
Authors: MELVIN TAN LIK CHERN
Keywords: Dengue, Diagnosis, Hybridoma, Envelope, Antigen, ELISA
Issue Date: 24-Nov-2010
Source: MELVIN TAN LIK CHERN (2010-11-24). Establishing Improved Platforms for Dengue Diagnosis and Hybridoma Development Using Dengue Virus Envelope Domain III Antigen. ScholarBank@NUS Repository.
Abstract: Dengue is a mosquito-transmitted viral disease of global importance. By exploiting the immunogenic properties of dengue virus (DENV) envelope recombinant Domain III (rDIII) proteins, several innovative platforms were established for research and diagnosis of dengue infection. In this study, a metal affinity membrane chromatography method for rapid purification of DENV rDIII proteins (serotypes 1 - 4) was established for the first time for flavivirus research. This method of purification is superior over traditional packed-bed chromatography because it is faster, more consistent and more cost-effective to perform. Purified DENV rDIII proteins were subsequently characterized and used for functional studies. Biotin-streptavidin enhanced indirect ELISA platforms [iELISA-(BS)] for detection of DIII-specific antibodies in mice and human sera were also established. When compared against the normal indirect ELISA, the former demonstrated three times improvement in signal-to-noise ratio and at least one logarithmic improvement in the limit of detection. Further to having improved sensitivity, these monovalent DENV rDIII iELISA-(BS) platforms (serotypes 1 - 4) exhibited excellent high-throughput screening potential (z-factor > 0.75). These assays were used for detection of seroconversion in mice after DENV rDIII immunization, as well as characterization of monoclonal antibodies. Additionally, the monovalent DENV rDIII iELISA-(BS) platforms were modified to detect for relative avidity rDIII protein-specific antibodies to its corresponding proteins. Significantly, human IgM and IgG tetravalent DENV rDIII protein-based ELISAs developed in this study were able to effectively detect DIII protein-specific antibodies in DENV patient sera. The assays were able to assist in differentiation between primary and secondary infection in patient sera. These results supported that DIII protein-based ELISAs are potential alternatives for commercially available diagnostic kits. A further study was performed to establish an integrated platform (using state-of-the-art technologies such as sortagging, microengraving and micromanipulation) to dengue research. The sortagging protein labelling method allowed for site-directed labelling of DENV rDIII proteins (serotypes 1 - 4) without masking its epitopes. Microengraving method was improved for examination of individual primary B cells (obtained from mice immunized with DENV rDIII protein) for its antibody secreting capability. It was also used for rapid screening of hybridomas which secreted DIII protein-specific monoclonal antibodies. Each hybridoma clone was identified and retrieved using a specialized robot, via the process of micromanipulation. Monoclonal antibodies produced by these clones were subsequently characterized via monovalent DENV rDIII-protein iELISA-(BS) and plaque reduction neutralization test. Through this process, four commercially viable clones of hybridomas secreting mono-specific neutralizing monoclonal antibodies (1B2, 1B3, 3B4 and 3B22) were developed.
URI: http://scholarbank.nus.edu.sg/handle/10635/22763
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