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. 2020 Jan 24;10:1163. doi: 10.1038/s41598-020-58305-y

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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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Confirmation of putative receptor-binding sites on the ligands using synthetic analogues by semi-quantitative ELISA. Framed reagents were coated into microtiter wells. Data present means of triplicates with ± S.D. CB – coating buffer; hBMECs – protein extract of human brain microvascular endothelial cells; DIII-1 – GTTYGVCSK-biotin; DIII-2 – VLIELEPPFGDSYIVVGRK-biotin; NadA-1 – AATVAIVAAYNNGQEINGFKAGETIYDIGEDGTITQK-biotin; NadA-2 – LADTDAALADTDAALDETTNALNKLGENITTFAEETK-biotin. Coating of DIII-2 synthetic peptide was very low on the Nunc™ ELISA plate, that is why detection of coated DIII-2 on the plate (input control) with streptavidin-HRP conjugate was weak. Please note that all wells were blocked with blocking buffer after overnight coating. Interaction was detected with streptavidin-HRP conjugate.