Novel anti-NS1 human single domain antibody from dengue infected NS1 positive patients.

Viruses of the flavivirus family significantly affect human health worldwide. Dengue virus (DENV), a member of the flavivirus family, causes pathogenic symptoms like high fever, severe headache and body pain upon infection. In some cases, it develops into severe dengue that is fatal. As a crucial pillar of immune response antibodies are produced against different epitopes of the dengue proteins and a collective response is generated by targeting critical proteins. A vaccine against dengue is still to be developed and new antibodies are the need of the hour. In our work we have isolated novel full length heavy chain variable region genes from peripheral blood mononuclear cell (PMBC) of NS1 positive DENV infected patients. The sequencing suggests that the isolated heavy chains to be novel and not yet reported. The sequence verified clones were sub cloned into expression vector pET-28a(+) for protein expression in E. coli. Selected heavy chains were overexpressed and purified to near homogeneity and further specific binding of purified heavy chain variable region with rNS1 was confirmed through competitive inhibition ELISA, dose dependent competitive inhibition ELISA and western blotting. ITC further confirmed the binding of rNS1 and SdAb3 with favourable free energy (ΔG = -9.22 kcal/mol). Identity of purified protein was confirmed by western blotting through anti-His antibody. It was found that a large amount of the purified protein was going in insoluble fraction and a protein refolding strategy was optimized with different buffer conditions. A buffer containing arginine, histidine and sucrose was found to be most efficient in solubilization of protein from insoluble fraction.