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. 2020 Jun 1;10:8869. doi: 10.1038/s41598-020-65860-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 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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Antibody Streptavidin linked to Biotin-Saporin as payload can be used to investigate antibody internalization (a) Schematic of Antibody-SB-Saporin conjugate generation. Streptavidin is conjugated randomly to lysine residues of the antibody with an average of two molecules of streptavidin per antibody. Streptavidin-linked antibody was conjugated to Monobiotin-Saporin at a molar ratio of 1:6. (b) Binding assay of trastuzumab, Trastuzumab-SB-Saporin and Trastuzumab-S to Her2-high SKBR-3 breast cancer cells and their concentrations at 50% of the maximum mean fluorescence intensity. (c–f) Top panels, target antigen recognition by naked antibodies, anti-Her2 trastuzumab (c), anti-CSPG4 (d), anti-EGFR Cetuximab (e), anti-FRα MOv18 (f) on cancer cell lines expressing different target antigens expression (Her2 (c), CSPG4 (d), EGFR (e), FRα (f)). Bottom panels (c–f), investigation of cell viability upon treatment with the naked antibody (black), antibody-Streptavidin-Biotin-Saporin conjugate (orange) or Saporin alone (grey). The ribosome inhibitor Saporin, unable to enter the cell alone, can be used to investigate antibody internalization by measuring viability (MTS) of antibody-SB-Saporin conjugate-treated cells. N = 1 for all binding assays and N = 3-4 independent experiments for MTS studies. All experiments were performed in triplicate, error bars represent Standard Deviation (SD).