The majority of monoclonal antibodies against dengue virus had been generated during the 1980s. During that time period, little was known about the structure and function of the ten proteins (C, prM, E, NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) encoded in dengue virus genome. In the past decade, several studies has provided detailed information on these proteins, including the three dimensional structure of E protein of tick-borne encephalitis virus (closely related to dengue) and dengue protease-helicase (NS3) protein, and also reveal the complexity of their posttranslational changes, intermolecular interactions and functions. It is now clear that currently available monoclonal antibodies against dengue proteins are inadequate with regards to the range of the target protein (ie. no monoclonal antibody is available for NS2-5 proteins) and the choice of specific epitopes which relate to functional site/domain within each protein. The lack of such important tool has hampered research in all areas of dengue study. To alleviate this problem, we propose to combine the expertise, experience and facilities of three collaborating laboratories (Molecular Biotechnology Unit, Siriraj Medical School/BIOTEC; Department of Clinical Immunology, Faculty of Associated Medical Sciences and Department of Microbiology, Faculty of Medicine, Chiang Mai University) to produce a bank of monoclonal antibodies specific to various dengue proteins by employing two immunization approaches (DNA and conventional antigen immunization) in conjunction with new screening strategies. For DNA immunization, gene segments encoding the C, prM, E, NS1, NS3 and NS5 proteins of dengue type 2 virus will be amplified from a full-length cDNA clone and inserted into a plasmid vector specially designed for surface expression on mammalian cells and used directly for immunization of mice. In conventional immunization, concentrated virus suspension, highly enriched preparation of selected viral proteins, or different functional forms of E and NS1 will be employed. Novel screening strategies will be utilized to allow the identification of monoclonals specific not only for each viral protein but also novel epitopes of prM, E and NS1 which can be related to their posttranslational changes and intra/intermolecular interaction. These strategies include indirect immunofluorescence assay against distinct gene-transfected cells, ELISA using dengue-infected cells as target, and ELISA or western blot analysis using different functional forms of E and NS1 as targets, etc. Selected monoclonals will then be characterized with regards to their isotype and functional characteristics. By employing mammalian cell expression, DNA immunization and rapid screening using gene-transfected cells, it should be possible to efficiently generate monoclonals to dengue proteins without having to laboriously purify them from infected cells. Screening with various forms of proteins may result in monoclonals with wider ranges of target epitope and functional property, which might be used in the development of new assay systems for diagnosis and for research and commercial purposes.