Fig. 3: Effects of the Pert-AF647 concentration and the balance between qAb and HER2 expression levels on evaluation sensitivity.

a Evaluation of epitope similarity between qAb and various concentrations of Pert-AF647. K562/HER2/scFv(Dac) and K562/HER2/scFv(Per) cells were incubated with 0.1–100 nM Pert-AF647 prior to FCM analysis. Representative contour plots are shown. Percentage of Pert-AF647-non-binding cells calculated using FCM plots from three independent experiments is also shown as the mean ± SEM. **p < 0.0001 (Tukey’s multiple comparisons test). n = 3. b Expression levels of scFv(Per) and HER2 in Pert-AF647-non-binding [rAb(−)] and Pert-AF647-binding [rAb(+)] cell populations. K562/HER2/scFv(Per) cells were incubated with 1 nM Pert-AF647 prior to FCM analysis and the expression levels of scFv(Per) and HER2 were estimated by the fluorescent intensity of sfGFP and mCherry, respectively. Representative contour plots are shown. Mean fluorescence intensity (MFI) values of sfGFP and mCherry in the rAb(−) and rAb(+) cell populations calculated using FCM plots from three independent experiments are also shown as the mean ± SEM. **p < 0.0001 (t-test). n = 3. c Expression level of HER2 on K562/HER2^Lo and K562/HER2^Hi cells, as estimated by the fluorescent intensity of mCherry. Representative histograms are shown. d Balance between scFv(Per) and HER2 expression levels in K562/HER2^Lo/scFv(Per) (Lo) and K562/HER2^Hi/scFv(Per) (Hi) cells. Expression levels of scFv(Per) and HER2 were estimated by the fluorescent intensity of sfGFP and mCherry on FCM analysis, respectively, and the relative expression ratio was calculated using the MFI values of sfGFP and mCherry from three independent experiments. The mean ± SEM is shown. **p < 0.0001 (t-test). n = 3. e Epitope similarity evaluation between qAb and Pert-AF647 using cells with different HER2 expression levels. K562/HER2^Lo/scFv(Per) (Lo) and K562/HER2^Hi/scFv(Per) (Hi) cells were incubated with 1 nM Pert-AF647 prior to FCM analysis. Representative contour plots are shown. Percentage of Pert-AF647-non-binding cells calculated using FCM plots from three independent experiments is also shown as the mean ± SEM. ** p < 0.0001 (t-test). n = 3.