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. 2019 Mar 5;9:3492. doi: 10.1038/s41598-019-40175-8

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© The Author(s) 2019

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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Phospholipid reactivity of HA stem-reactive mAbs. (a) Reactivity of CR6261 mAb with the human HEp-2 epithelial cells. To indicate CR6261 (gray scale) reactivity with the plasma membrane maximum intensity Z-projection of 20 stacks was taken with 60x N.A. = 1.2 objective. The cells were co-stained with DAPI (blue). The scale bar is 50 μm. (b–h) Phospholipid reactivity in enzyme-linked immunosorbent assay (ELISA). Microtiter plates were coated with lipids 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cardiolipin (CL) or sphingomyelin (SM), blocked with phosphate-buffered saline (PBS) supplemented with 2% bovine serum albumin (BSA). All mAbs were diluted either in PBS (empty bars, −) or PBS+ 2% BSA (shaded bars, +). Binding was detected with horseradish peroxidase-conjugated secondary Ab (Invitrogen), incubated with ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) and absorbance read at 405 nm. mAb 2F5 was used as control. Reactivity at 0.5 μM is shown as mean ± SD.