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. 2020 Jul 9;10:11344. doi: 10.1038/s41598-020-68226-5

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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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Optimization and improvement of ex vivo ELISpot assay. (A) Triplicates of 2 × 10⁵ vaccinated donor PBMCs (n = 10) were stimulated at 5 µg/mL/peptide with respective OLP pools. (B) 1–5 × 10⁶ vaccinated donor PBMCs (n = 13) were plated and simultaneously pulsed at 10 µg/mL/peptide with all OLP pools. Signal-to-noise ratios were calculated. (C) 2 × 10⁶ vaccinated donor PBMCs (n = 7) were stimulated with 10, 5, and 1 µg/mL/OLP. (D) 2 × 10⁶ vaccinated donor PBMCs (n = 10) were pulsed at 5 µg/mL/peptide with OLP pools (8) for each HBV antigen. Dotted lines indicate respective positive thresholds.