Virus Assays on Microplate Readers

Martin Mangold discusses improving efficiency and decreasing costs

Viruses, nowadays more than ever, pose growing threats to our health, society and economy. Detection systems capable of analysing the mechanisms of viral infections as well as quantifying related diagnostic parameters are important tools to understand virus-host interactions and develop new treatments or diagnostic tests.

In the past, these applications mainly revolved around microscopy-related techniques. Today more and more experiments are performed with microplate readers, as these offer several advantages in comparison to more traditional methods. On one hand, the microplate platform drastically reduces sample volume and accordingly assay cost. On the other, the higher number of tested samples per run reduces overall measurement times and enables a higher throughput. In addition, microplate readers provide a quantitative output that is less prone to interpretation or human error, compared to more qualitative, image-based readouts. Many microplate readers come also equipped with a variety of detection modes, providing the possibility to measure a huge variety of different assays on one device.

Accordingly, microplate readers have become an indispensable tool in virology research, covering a multitude of assays from basic to drug research. Roughly speaking, these assays can be categorised in three groups: functional virus assays, diagnostic assays, and anti-viral drug screening, which span the whole spectrum of detection modes from fluorescence intensity to AlphaScreen.

Functional Virus Assays

Functional virus assays deal with the analysis of host-virus interactions. They are employed to clarify the cellular and molecular mechanisms behind infection and the role of the involved virus and host proteins. Generally, the influence of these interaction partners is investigated by gene knockout or inhibition and virus replication and viral gene expression are used as readouts. Furthermore, plate readers with atmospheric and temperature control enable real-time kinetic data acquisition of infection, replication and neutralisation assays monitored by fluorescent or luminescent reporter proteins in living cells. Together with the measurement of cell viability, cytopathic effect (CPE) and TCID50, which can easily be performed on a microplate reader, this allows to study virus infectivity and replication directly in host cells.

Diagnostic Assays

Diagnostic assays are employed to efficiently assess the presence of a virus and its spread in a population. For this purpose, assay sensitivity and speed are essential to allow for the fast and accurate evaluation of infection rates. These quantitative detection methods rely mainly on the detection of viral antigens or antibodies (ELISA) or of the viral genome (PCR, LAMP) in patient samples. One example is the loop-mediated isothermal amplification (LAMP) assay which amplifies viral gene material in a single 65°C incubation step (Fig. 1). LAMP products can be easily detected on a plate reader and the assay can be directly run and measured in real-time on plate readers with 65°C heating. While PCR and LAMP products are detected by absorbance or fluorescence measurement, ELISAs for the quantification of antibodies and viral antigens or cytokines in serological samples rely on a colorimetric, fluorescent, or luminescent output. As all these detection methods can be incorporated in a single device, microplate readers are a viable option for performing large quantities of different diagnostic tests due to their high throughput and sensitivity.

Screening for Anti-Virals

Anti-viral screenings are used to discover drug-based treatment options for viral infections. Once druggable targets are identified, assays for the detection of their activity need to be scaled up to efficiently screen for anti-viral molecules. For this purpose, labelled substrates are employed to allow plate reader-based detection of enzymatic assays such as neuraminidase or RNA-dependent RNA Polymerase activity and inhibition. FRET, BRET, TR-FRET, fluorescence polarisation and AlphaScreen are extremely useful for the analysis of virus-host protein interactions in high-throughput and of utmost importance for the identification of inhibitors of these interactions. Furthermore, the detection of viral replication in host cells can be used as readout to assess the potency of anti-virals (Fig. 2). Irrespective of the method, microplate readers with highest sensitivity and speed are essential for high-throughput screening approaches, delivering robust and reproducible results for thousands of samples. To achieve even higher throughput, many microplate readers are robot compatible and can easily be integrated into automated systems.

The BMG Labtech Clariostar Plus plate reader with LVF Monochromators, temperature and atmospheric control capabilities is an ideal tool for virology research. For high-throughput screening applications, the Pherastar FSX provides excellent sensitivity and detection times.

Martin Mangold is with BMG Labtech


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