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. 2022 Aug 27;12:14651. doi: 10.1038/s41598-022-18681-z

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© The Author(s) 2022

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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Evaluation of the incorporation of the Spike S, N and M in fluorescent SARS-CoV-2 VLPs using immuno-spotting coupled to TIRF-Microscopy. (A) TIRF images of M(GFP)NES VLPs, M(GFP)NE VLPs and control. An anti-S antibody coupled to a AF555 secondary antibody was used for S immunolabeling. Yellow arrows show the anti-S antibody-AF555 bound to VLPs-(GFP). Green arrows show VLPs-(GFP). Red arrows show antibody-labeled VLPs. The scale bar is 5 µm. (B) Percentage of S incorporation in M(GFP)NES versus M(GFP)NE and the control (no VLP). For each condition, 125 fluorescent particles were counted over 10 images (frames). One dot in the graph represents the % of S incorporation in the fluorescent VLP(M-GFP), i.e. % of colocalization in each frame. The t-test was calculated and the p-value between VLP M(GFP)NES and VLP M(GFP)NE is 0.0001 and p-value between VLP M(GFP)NES and No VLP is < 0.0001. (C) Images of SARS-CoV-2 M(GFP)NES and MN(GFP)ES VLPs as revealed by immuno-spotting coupled to TIRF-M, showing N or M incorporation in the MN(GFP)ES or M(GFP)NES VLP respectively with an anti-N or anti-M antibody coupled with an Alexa555 labeled secondary antibody, as indicated. The scale bar is 10 µm. (D) Quantification of the incorporation (%) of S, M or N on respective GFP-VLPs. N = 2, 11 images per sample and the number of particles counted was between 500 and 650 for each condition.