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. 2020 Dec 10;10:21738. doi: 10.1038/s41598-020-78895-x

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© The Author(s) 2020

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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Overview of the BLI-ISA experiment. To begin, a tray of fiber optic biosensors and a 96- or 384-well plate of samples are placed into the Octet BLI instrument (Supplementary Fig. S2), and the assay program is run. Throughout the experiment, real-time measurements are recorded as the change in the wavelength of reflected light returning from the biosensor surface. First, biosensors are equilibrated by dipping into wells containing BLI assay buffer. In the antigen loading step, biosensors are dipped into wells containing tagged antigen (e.g. streptavidin SA biosensors dipped into biotinylated antigen). After a wash, antigen-loaded biosensors are placed into diluted plasma, and a Total Antibody Binding signal is measured. After another wash, the antigen–antibody-coated biosensors are dipped into wells containing isotype-specific binding reagents (e.g. colloidal gold-conjugated anti-human IgG), and a Detection signal is measured. Created in BioRender.com.