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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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Determination of the optimal conditions for the plate-based PSMA-EV sandwich ELISA. EVs isolated by sequential centrifugation from culture medium of C4-2B cells (C4-2B EVs) were used as a positive control for PSMA-expressing EVs. Samples were applied to a well of 96-well plate coated with Tim4. After washing, ALP-labeled anti-PSMA antibody was added to the well, followed by detection of chemiluminescence in the presence of ALP substrate. (a) Antibody selection. Commercially-available anti-PSMA antibodies (A: Miltenyi Biotec, REA408; B: BioLegend, LNI-17; C: MBL, 107-1A4; D: Abcam, GCP-05, E: R&D, sheep-polyclonal; F: Millipore, 3/A12) were labeled with ALP. The PSMA-EV levels in samples containing increasing doses of C4-2B EVs (0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, and 25 ng/well in 50 µL) were determined. (b) Matrix effect on sandwich ELISA. Measurement of the PSMA-EVs in C4-2B EVs was performed in the presence of increasing concentration of pooled serum from 6 normal donors (0, 10, 25, and 50%). (c) Competitive sandwich ELISA. Measurement of the PSMA-EVs in pooled serum containing increasing doses of C4-2B EVs (50 µL) was performed in the presence and absence of an excess amount of non-labeled anti-PSMA antibody (fivefold).