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Doctoral Thesis, Stefan van Hoof, Maastricht University 2019

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Quantifying virus-mediated cytopathic effect with xCELLigence platform

Jul 20, 2016

Virus infection of a host cell typically includes the selective suppression of host cell functions and redirection of resources towards viral replication and assembly, ultimately leading to host cell lysis and dissemination of new virus. While host cell rounding, detachment from the plate surface and/or lysis are readily detected by real-time impedance monitoring, more subtle changes in host cell morphology occurring during earlier phases of viral infection can also be monitored. This sensitivity to virus-induced changes in host cell morphology and behavior makes the xCELLigence technology very well suited for a wide array of virology applications, including: differentiating between virus strains/isolates based on the kinetics of replication and cytopathic effect, determining viral titers, determining neutralizing antibody titers, and studying virus-host cell interactions using physiologically relevant cell types that cannot typically be used because they aren’t compatible with traditional assay techniques.

Evaluating the relative fitness of different virus strains/isolates, and determining the identity of a virus isolate can involve a large number of techniques, including: ELISA, PCR, RT-PCR, Western blotting, plaque assays, immunofluorescence, etc. Owing to its ability to kinetically characterize a virus-induced cytopathic effect, xCELLigence real-time cell analysis (RTCA) can be used in place of, or in addition to, some of these traditional assays for characterizing virus fitness and/or identity.

RTCA-based kinetic comparison can be used for assessing the relative fitness/virulence of different virus isolates/strains, or to help identify a virus using RTCA traces from known standards.

Key Benefits:

- Quantify virus titer: An automated, simple, reduced workload alternative to plaque assays.
- Evaluate the fitness of different strains/isolates: The relative fitness of different viruses (natural isolates, engineered mutants, etc.) are readily evaluated using the onset and kinetics of virus-mediated cytopathic effects.
- Determine/confirm virus identity: Real-time kinetic traces of virus-mediated cytopathic effects can be compared to those of characterized viruses to help determine/confirm the identity of a virus.
- Quantify neutralizing antibody titer: Because the time of cytopathic effect onset correlates with neutralizing antibody concentration, standard curves are easy to generate. These can be used for quantifying neutralizing antibody in samples of unknown concentration.
- Rapid assay optimization: Quickly identify the optimal viral titer and assay time point for subsequent screening of inhibitory compounds, neutralizing antibodies and neutralizing serums.

Application
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Related technologies: Real-time, label free cell analysis

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Agilent technologies

Agilent provides xCELLigence impedance-based, label-free, real time cell analysis system and NovoCyte flow cytometers.

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Located inside a tissue culture incubator, the RTCA DP Analyzer has three integrated stations for E-Plates 16 or CIM-Plates 16

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Located inside a tissue culture incubator, it is capable of switching any one of the wells on the E-Plate 96 to the RTCA Analyzer for impedance measurement

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Located inside a tissue culture incubator, it is capable of switching any one of the wells on any of six E-Plates to the RTCA Analyzer for impedance measurement

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