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. 2021 Jul 22;11:15000. doi: 10.1038/s41598-021-94603-9

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© The Author(s) 2021

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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Comparison of the plate-based PSMA-EV sandwich ELISA with and without purification of EVs from serum. (a) Schematic diagram of the preparation of “purified EVs” by Tim4 and the measurement of the PSMA levels on EVs recaptured by Tim4 immobilized on the microplate. (b) Serum containing increasing doses of C4-2B EVs was prepared for each normal donor (n = 5). The serum containing C4-2B EVs was incubated with Tim4-bound magnetic beads and then EVs were released from Tim4 by adding EDTA. Serum containing increasing doses of C4-2B EVs (50 µL, filled circle) or EVs purified from the same amount of the serum (open circle) were transferred to a well of 96-well plate where they were recaptured by immobilized Tim4, followed by detection of PSMA on EVs. When PSMA-EVs in serum containing C4-2B EVs was measured, EV binding to Tim4 was conducted at its final concentration of 50%. Inset: Enlarged plot at low concentrations. Data are shown by mean ± SD and those for each donor is presented in Supplementary Fig. S4.