CONICET | Buscador de Institutos y Recursos Humanos

Dengue is an important viral disease transmitted by mosquitoes, and in recent years it has become endemic in Argentina. High levels of the Dengue virus protein NS1 has been detected in the sera of infected patients, and for this reason this protein has been used for the development of diagnostic kits. Nanoantibodies (NAbs), which are fragments of heavy chain antibodies obtained from camelids, are a potentially useful tool for Dengue detection. These small molecules typically exhibit strong specificity and affinity for different antigens, are thermostable and can be easily produced in microorganisms at a low cost. Most importantly they can be straightforwardly modified by standard molecular biology techniques. Given the need for innovative and affordable dengue detection systems, here we report the production of NAbs with ability to recognize the NS1 protein of Dengue virus.For this purpose, two llamas were immunized with purified and inactivated supernatant of Dengue infected cells containing high levels of NS1 protein. Total RNA was isolated from peripheral blood. After cDNA synthesis, part of the genes encoding antibody heavy chains were amplified by PCR and cloned into the pHEN4 phage display vector. This plasmid was transformed into TG1 E. coli cells, which were later infected with the M13K07 helper phage. Selection of NS1 specific binders was carried out by phage display. NAbs were separated from TG1 periplasm by osmotic shock, and specific binding to purified NS1 was tested by ELISA.Two libraries of about 2x108 transformants with more than 88% of inserts of the right size were obtained. After 3 rounds of panning, 192 candidate clones were isolated and amplified in TG1 cells. Periplasmic extraction of NAbs allowed us to determine that isolated candidates specifically recognize NS1 protein of Dengue virus. Selected clones will be modified and produced at a large scale to generate a Dengue NS1 detection kit.