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. 2020 Jan 29;14(1):e0008039. doi: 10.1371/journal.pntd.0008039

Fig 2. Validation of rNS1 antigens by ELISA in immunized mice.

Fig 2

A) Expression and secretion of V5-tagged recombinant NS1 proteins of TBEV, WNV, ZIKV, and DENV1-4 in cell extract and supernatant of transfected HEK293T cells. Actin blots were included as a loading control for cell extracts and of clean supernatants. SDS PAGE was conducted in denaturing/reducing conditions. B) Schematic representation of the protocol followed for the immunization of BALB/c mice by gene gun technology. Numbers indicate the day when the prime-boost immunization was performed and the day of collection. C and D) TBEV rNS1 IgM/IgG ELISA with sera from four different mice immunized with TBEV-NS1 (mice were differentiated with a mark in the right ear (Rx), left (Lx), both ears (RxLx) or without the mark (unmarked)). Detection of IgM/IgG antibodies was performed at day 0, 19, 26, 33 and 46. E and F) TBEV, WNV, ZIKV, and DENV1-4 IgM/IgG rNS1 ELISA with sera from TBEV-NS1 immunized mice. Sera from day 46 post-immunization were diluted in blocking solution (2% milk in PBS) as indicated. G and H) Heatmaps of the IgM/IgG cross-reactivity. Several dilutions of each serum sample were analyzed in plates coated with homologous and heterologous rNS1 6xHis-tagged antigens. * each ELISA result includes the average of two biological replicates.