Figure 1.
Dynamic monitoring of Vero cell proliferation.
Serial dilutions of Vero cells were seeded at indicated densities of 50 000 to 390 cells/well in a 96-well E-plate. The attachment phase, lag-phase and proliferation phase were dynamically monitored every 15 min for 22 h, as indicated in the text. Data shown are representative of three independent experiments showing similar results.
Figure 2.
Real-time measurement of cytotoxicity and time-dependent standard curves for ricin and agglutinin.
Vero cells were seeded in a 96-well E-plate (12 500 cells/well). Immediately after seeding, cells were exposed to the indicated concentrations of ricin (A, C), agglutinin (B, D), or medium (control). Cell proliferation was dynamically monitored every 15 min for 24 h. Figures A and B show the time-dependent alteration of the CI for different ricin or agglutinin concentrations. Figure C and D display the percentage of viable cells plotted against toxin concentrations at selected time points (conversion of the data from figure [A, B] to [C, D] is described in material and methods). Data shown are representative of five (A, C) or three (B, D) independent experiments with similar results.
Table 1.
IC50 values for ricin and agglutinin at different time points of the real-time cytotoxicity assay.
Figure 3.
Comparison of ricin and agglutinin cytotoxicity in RT-CES system and MTT assay.
Vero cells (12 500 cell/well RT-CES system, 10 000 cells/well MTT assay) were seeded in a 96-well E-plate (RT-CES system) or 96-well cell culture plate (MTT assay), respectively. In the RT-CES system (filled symbols), serial dilutions of ricin (grey) and agglutinin (black) were incubated immediately after cell seeding, and cell proliferation was monitored online for 24 h. For the MTT assay (open symbols), cells were cultivated for 18 h and incubated afterwards with ricin or agglutinin. After 2 h cells were washed and cultured for a further 20 h, before the MTT reagent was used to determine cell viability. Data shown are representative of two independent experiments showing similar results.
Figure 4.
Specificity of the cytotoxicity test.
Vero cells (12 500 cells/well) were seeded in a 96-well E-plate. (A) Serial dilutions of ricin were preincubated with polyclonal anti-ricin IgY for 1.5 h at 37°C, and then added to the cells. Cell proliferation was dynamically monitored every 15 min for 23 h. Data shown are representative of five independent experiments showing similar results. (B) Vero cells were exposed to 10 µg/mL ricin, agglutinin, abrin, DBA or TVA, respectively (white columns). In order to show the specificity of the assay, the different lectins were preincubated with anti-ricin IgY as above (black columns). The viability of the cells after 21 h is depicted as percentage of the viability of untreated control cells (100%). Data shown are exemplary data out of two independent experiments showing similar results. (C) Ricin (20 000 ng/mL) was heated in PBS for 30 min at 95°C (denatured, white circles), or was left untreated (native, black circles) and then added to the cells. Cell proliferation was dynamically monitored every 15 min for 23 h. In parallel, cell growth was monitored in medium only (negative control, black diamond). Data shown are representative of two independent experiments showing similar results.
Figure 5.
Detection of functionally active ricin in complex matrices.
(A) Vero cells (12 500 cells/well) were treated immediately after seeding into E-plates with 1∶14-diluted food extracts from milk (dotted grey line), carrot juice (dotted black line), baby food (grey line) or medium (black line). Characteristic growth phases of the cells were dynamically monitored every 15 min for 43 h. (B) Vero cells were exposed to ricin spiked into milk, carrot juice, baby food or medium, respectively. The indicated toxin concentrations are post-dilution concentrations. The viability of the cells is depicted as percentage of viable cells plotted against toxin concentrations in the different food matrices, measured after 24 h. (C) Vero cells were treated as described in (B). The viability of the cells is depicted as percentage of viable cells plotted against toxin concentrations in the different food matrices, measured after 42 h. (D) Vero cells were incubated with different dilutions of Ricinus communis-containing fertilizer extract (1∶3.5, black; 1∶14 dark grey; 1∶56 light grey; 1∶224, hatched) either without treatment (native), preincubated with 6 µg polyclonal anti-ricin IgY for 1.5 h at 37°C (+Ab) or heated for 30 min at 95°C (heated). For guidance, Vero cells were treated in parallel with different concentrations of purified ricin (white bars, 230 ng/mL, 2.3 ng/mL, 0.23 ng/mL). The viability of the cells after 21 h is depicted as percentage of the viability of the untreated control cells (100%). Data shown are exemplary data out of two independent experiments showing similar results.