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. 2023 Sep 5;13:14632. doi: 10.1038/s41598-023-41092-7

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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In vitro characterization of mRNA-encoded VNAs. (a) Design of VNA targeting C. difficile toxins (Tcd) A and/or B, C. botulinum toxin (BoNT) A, including a signal peptide (SP), V_HH domains specific for the toxins, O-tag epitopes (O-tag), and an albumin binding peptide (ABP). (b) Western blot analysis of VNA-TcdA, VNA-TcdB, VNA-TcdA/B and VNA-BoNTA from cell lysates (CL) and supernatants (SN) of transfected BHK cells harvested 48 h after transfection. Cells were transfected in triplicates, and for each VNA, a representative sample was loaded on denaturing SDS-PAGE. Signals correspond to individual VNAs (upper blot) and α/β tubulin (lower blot) as a loading control. An uncropped version can be found in Supplement Fig. 11. (c) Detection of VNA-TcdA and VNA-TcdA/B from BHK supernatants (conditioned medium) by O-tag detection ELISA (Supplement Table 2). Control cells were transfected by VNA-TcdB. TcdA toxin was used as capture agent. Supernatants from (b) were assayed in 96 well-plates, starting at a 1:5 dilution (5⁻¹) and serially diluted in a fivefold dilution series. A450 values (y-axis) are plotted against the dilution (x-axis). Error bars represent the standard deviation (SD) from triplicates. (d) Detection of VNA-TcdB and VNA-TcdA/B from BHK supernatants (conditioned medium) or medium-only (control) by O-tag detection ELISA (Supplement Table 2). Control cells were transfected by VNA-TcdA. TcdB toxin was used as capture agent. Supernatants from (b) were assayed in 96 well-plates, starting at a 1:5 dilution (5⁻¹) and serially diluted in a fivefold dilution series. A450 values (y-axis) are plotted against the dilution (x-axis). Error bars represent the SD from triplicates. (e) Neutralization assay to analyze VNA-TcdA and VNA-TcdA/B functionality. Serial dilutions from c) were subjected to a neutralization assay in Vero cells. Error bars represent the SD from triplicates. (f) Neutralization assay to analyze VNA-TcdB and VNA-TcdA/B functionality. Serial dilutions from d) were subjected to a neutralization assay in Vero cells. Error bars represent the SD from triplicates.