RESUMO
Dengue is a mosquito borne infection, which in recent years has become a major international public health concern. Annually, 100 million dengue virus infections are reported worldwide. The nonstructural protein 1 (NS1) of dengue virus is a useful target for diagnostics of dengue infection since the protein is abundantly circulating in the blood during acute phase of the disease, in both primary and secondary infections. This research paper highlights the development of a panel of Mab and bsMab for dengue NS1 detection. The P148 series of Mabs showed high specificity for recombinant dengue NS1 antigen. These antibodies showed no cross reactivity with recombinant dengue envelope protein and other viral proteins. The hybrid-hybridoma approach to generate the P156.1 and P156.2 bsMabs from the P148 monoclonal antibody method was used during this study. Furthermore, the affinity purification provided good yields of quadromas associated with HRPO in two steps. Direct detection method involved coating of plates with different concentrations of recombinant antigen and detecting with bsMab. Sensitive sandwich assay with Mabs and bsMabs was also done. Detection of nonstructural dengue antigens may be of benefit for early and rapid diagnosis of dengue infection due to their long half-life in the blood.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Vírus da Dengue/imunologia , Proteínas não Estruturais Virais/sangue , Animais , Anticorpos Biespecíficos/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/genética , Antígenos Virais/sangue , Antígenos Virais/imunologia , Dengue/diagnóstico , Feminino , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas não Estruturais Virais/imunologiaRESUMO
Dengue and the more severe dengue hemorrhagic fever have been a very critical public health problem globally. Millions of people especially in the tropical areas get infected with dengue. An efficient diagnostic is very important for early screening of dengue infection. In dengue-infected patients, the nonstructural protein NS1 is present on the surface of infected cells and secreted in plasma. The NS1 antigen is an important target for developing a quick diagnostic largely due to its long presence in the blood. We have developed a simple-to-use immunoswab-based diagnostic procedure employing monoclonal antibodies and the second-generation quadromas. The detection limit for NS1 has been established to be in the subnanogram range. The assay is very sensitive, has a visual end point, and also being extremely inexpensive. With this assay, screening time for a dengue-infected person would be very rapid.