Description
The Viral ToxGlo™ Assay is a simple, quantifiable method of determining viral-induced cytopathic effects (CPE) in host cells caused by lytic virions. The assay measures cellular ATP as a surrogate measure of host cell viability. When CPE occurs due to viral infection, ATP depletion can be measured and correlated with viral burden. The amount of ATP detected is directly proportional to the number of viable host cells in culture and can be used as a simple method to quantify viral-induced CPE. The homogeneous "add-mix-measure" assay procedure involves adding the single reagent (ATP Detection Reagent) directly to host cells following viral treatment. A “glow-type” luminescent signal is generated that is proportional to the amount of ATP present. Cell washing, multiple pipetting steps and visual assessment are not required to assess CPE. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after reagent addition and mixing and is designed for use in multiwell formats, making it ideal for automated high-throughput screening (HTS).
Applications include the determination of viral infectivity and the corresponding tissue culture infective dose (TCID ) and potential antiviral potency or off-target toxicity of test compounds.
Features and Benefits
Objectively Quantify CPE: The assay provides quantifiable data by luminescence detection, which obviates subjective operator error associated with visual scoring methods.
Decrease Time to Results: Data can be recorded and analysis begun 10 minutes after reagent addition.
Simplify Assessment of CPE: The homogeneous “add-mix-measure” protocol dramatically reduces the manual steps required for CPE assessment.
Choose Your Format: The reagent is scalable from 96- to 1536-well plate formats.
Amenable to High-Throughput Screening: Luminescent signal is very stable with a half-life generally >5 hours dependent on cell type and medium used, allowing batch or consecutive processing. No fluorescence interference results in high signal to background and delivers excellent Z′ values in screening applications.
This method is only useful for viruses that produce cytotoxicity and CPE.
Quantifiable Measure of CPE
Increased luminescence indicates less infective virus and less CPE. Panel A. Dilutions of Influenza virus H1N1 were applied to MDCK monolayers. Panel B. Dilutions of VEEV applied to Vero E6 monolayers. Panel C. Dilutions of dengue virus applied to BHK-21 monolayers. Panel D. Dilutions of RSV applied to A549 monolayers. After incubation, ATP Detection Reagent was added directly to cells and luminescence measured.
Accurately Calculate Toxicity of Test Compounds
Panel A. Test compound reduces viral CPE with no off-target cytotoxicity. MDCK cells with 100 TCID 50 of H1N1 (on-target antiviral) or MDCK cells only (off-target cytotoxicity). Panel B. Test compound reduces viral CPE and also causes off-target cytotoxicity. BHK-21 cell monolayer with 100 TCID 50 of Dengue virus (on-target antiviral) or BHK-1 cells only (off-target cytotoxicity). After incubation, ATP Detection Reagent was added directly to cells and luminescence measured.
