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. 2017 Dec 5;7:17011. doi: 10.1038/s41598-017-17274-5

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© The Author(s) 2017

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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Optimising the detection of antibody mediated inhibition of sporozoite invasion. (a) 10000 PbGFP sporozoites were incubated with 1 μg/mL mAb-PbCSP (3D11) on ice for 0, 15 or 30 minutes prior to addition to 30000 Huh7 cells. Cells were harvested the following day and the percentage of GFP⁺ cells determined by flow cytometry. The graphs represent the percentage of GFP⁺ cells (left) or percentage of inhibition (right) when compared to the sporozoite only control well. (b) 20000 PbGFP sporozoites together with 1 μg/ml mAb-PbCSP were added to 30000 Huh7 cells and incubated at 21 ^oC or 37 ^oC for 1 hour, prior to overnight incubation at 37 ^oC. Cells were harvested the following day and the percentage of GFP⁺ cells determined by flow cytometry. Graphs represent the percentage of GFP⁺ cells (left) or percentage of inhibition (right) when mAb-PbCSP wells were compared to sporozoite only wells incubated under the same conditions. (c) 30000 (black) or 15000 (grey) sporozoites (PbGFP-Luc) were added to 30000 Huh7 cells in the presence of a range of mAb-PbCSP concentrations (0–5 μg/mL) and percentage of infection measured by flow cytometry the following day. Graphs represent the percentage of GFP⁺ (left) or percentage of inhibition (right) when addition of mAb-PbCSP was compared to sporozoite only control wells. Data was analysed with a two-way ANOVA which showed a significant effect of antibody concentration but no significant effect of sporozoite number. (d) 15000 sporozoites (PbGFP-Luc) were added to 30000 Huh7 cells per well in the presence of mAb-PbCSP (0.25 or 0.5 μg/mL) and harvested at different times after infection. Graphs represent the percentage of GFP⁺ cells over time (left), percentage of inhibition (middle) or log GFP MFI of infected cells over time (right). The percentage of inhibition data was analysed with a two-way ANOVA to test for the effect of antibody concentration over time, no significant effect of time was observed. Log (MFI GFP⁺) data was analysed with a two-way ANOVA and post-hoc Dunn’s multiple comparisons test, but no difference between groups at each timepoint was observed.