Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

BACKGROUND: In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. METHODOLOGY/FINDINGS: This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. CONCLUSIONS/SIGNIFICANCE: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices

Detection of functionally active ricin in complex matrices.

<p>(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 <i>Ricinus communis</i>-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.</p

Comparison of ricin and agglutinin cytotoxicity in RT-CES system and MTT assay.

<p>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.</p

Dynamic monitoring of Vero cell proliferation.

<p>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.</p

IC50 values for ricin and agglutinin at different time points of the real-time cytotoxicity assay.

<p>IC50 values for ricin and agglutinin at different time points of the real-time cytotoxicity assay.</p