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Fig. 5 | Virology Journal

Fig. 5

From: Investigation of the immunogenicity of Zika glycan loop

Fig. 5

The immunogenicity of an unglycosylated glycan loop construct in humans and mice. a Dot blot analysis of human sera to detect IgM and IgG response to TMV-GL and TMV. Purified antigen (either TMV or TMV-GL) was spotted onto each square of nitrocellulose, dried, blocked, and then incubated with a 1:500 dilution of the indicated human serum in blocking buffer. After washing, the antibody bound to the membrane was detected using horseradish-peroxidase conjugated secondary antibodies and Clarity Western Substrate. b Digital quantification of the magnitude of dot blot assays using ImageJ software. The y-axis represents the mean pixel count ratio of the TMV-GL signal divided by the TMV signal. c Dot blot analysis of human sera to detect IgM and IgG responses to rE. Pooled sera from each of the 5 groups of immunized mice (TMV, TMV-GL, TMV-E, rE, and adjuvant only) were dotted onto nitrocellulose, blocked, and then incubated with a goat anti-mouse IgM HRP-conjugated secondary antibody and detection using Clarity Western Substrate. d Plant-expressed rE was run on a 4–20% SDS-PAGE gel and stained with Coomassie. rE was run either reduced or non-reduced, where reducing conditions break the disulfide bonds. Non-reducing conditions indicate that rE is not aggregated through inter-subunit disulfide bond, and rE runs as a monomer. e Endpoint ELISA titers against TMV-GL using sera from TMV-GL immunized mice, TMV immunized mice, TMV-E immunized mice, and rE immunized mice. Each point represents the endpoint titer of the sera from one mouse, and the bars indicate the average endpoint titer for each group. The difference between ELISA values generated by TMV-GL and TMV, TMV-E, or rE are statistically significant (P < 0.0001, F test)

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