Over the years CSIR-CDRI has developed a large number of Biological Assays and Screening Protocols to carry out biological activity studies of compounds against various diseases. Given below is the list of assays that are available at CSIR-CDRI. As a policy, CSIR-CDRI prefers to carry out these tests for outside samples on mutual collaboration basis after signing appropriate undertaking by the concerned institutes/universities to enable a scope for further development of any active molecule towards new drug discovery. In addition, the running costs for such screenings need to be paid. Researchers willing to get any of the tests carried out may please contact
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The Flow cytometry-based growth inhibition assay is a pivotal method for screening of potential inhibitors against dengue virus (DENV) and Japanese Encephalitis Virus (JEV). It is a live virus, cell-based assay used to evaluate test compounds' ability to either directly inhibit the virus (Type I assay) or hinder viral proliferation by targeting its life cycle (Type II assay). Flow cytometry operates by detecting the scattering of light and the emission of fluorescence from cells labelled with specific fluorescent probes as they pass through a laser beam.
In the Type I assay, varying concentrations of test compounds are mixed with the virus and incubated at 4ºC overnight prior to infecting Vero cells, green monkey kidney cell line. After infecting Vero cells, the mixture is incubated for 24 hours followed by fluorescent staining. This assay directly quantifies the ability of compounds to inhibit the virus attachment to the host cell. Effective compounds will result in reduced infectivity compared to untreated virus controls. The Type II assay involves infecting Vero cells with the virus first followed by 2 hours incubation at 37ºC humidified incubator with 5% CO2. Subsequently, the cells are treated with compound dilutions and further incubated for 48 hours. After incubation, fluorescent staining (virus specific Abs tagged with fluorophore) is performed. This assay evaluates how well compounds can disrupt the virus's life cycle once it's already inside the cells. To detect infected cells, a monoclonal antibody called 2H2 labelled with Alexa flour 488, specific to the Flavivirus prM, is used. Flow cytometry, followed by analysis using FlowJo software, is employed to quantify the stained cells. The concentration of the compound that results in a 50% reduction in virus compared to the untreated control is termed the IC50 (Inhibitory Concentration 50%) and is calculated using GraphPad prism. This method provides a precise assessment of compound efficacy against the virus.
Sample can be provided in either salt or solution. For Salt: ≥1/10th amount in mg of molar weight (Ex. If MW of compound is 300, 30mg of sample is required) For solution: 100-500ul of 100-500mM in DMSO. For Extracts: ≥50mg (Solid); ≥1ml (Liquid) Required details of the sample: Molecular weight, Solubility (Organic/Aqueous), Nature, MSDS, Storage conditions. Samples should be provided in leak proof containers with printed labels. Exact weight or volume, name/code, date of packing should be printed on every individual container. All samples should be free from contaminants (Microbial).
The NS1 test is vital for diagnosing DENV infection due to its release into the bloodstream, showing a direct link with virus replication in mammals. Moreover, in vertebrate cells, NS1 secretion into the extracellular space is closely tied to immune response modulation, including the inhibition of complement system function. The introduction of NS1 detection has significantly enhanced DENV diagnosis capabilities, underscoring the importance of NS1 antigen detection as a valuable confirmation method for DENV infection. The purpose of the assay is to measure the amount of NS1 produced when various dosages of the test compound are applied to Vero cells infected with DENV.
The aim of this assay is to quantify NS1 production when different doses of the test compound were applied to DENV-infected Vero cells. Vero cells were initially seeded in a 96-well plate at a density of 2.5 X 104 cells. Twenty-four hours post-seeding, viral infection was initiated, and cells were incubated for 2 hours at 37°C in a humidified incubator with 5% CO2. Subsequently, compound treatment was administered at varying concentrations, and cells were further incubated under the same conditions. Culture supernatant aliquots were collected every 12 hours over a five-day period post-infection. In the experiment, cells were divided into different groups for treatment. Cells treated with varying concentrations of the compound were designated as the test samples. Cells treated solely with the virus served as the virus control group, showcasing an elevated level of NS1 release, as indicated in the figure. Meanwhile, cells not exposed to either the virus or the compound were assigned as the cell control group, demonstrating minimal NS1 release levels, as depicted in the figure. NS1 antigen levels were quantified using a commercially available dengue NS1 antigen sandwich ELISA kit.
The NS1 assay serves as a valuable tool for testing the efficacy of compounds against viral infections, particularly for viruses like Dengue virus (DENV) where NS1 plays a crucial role. It can use for assessing anti-viral activity and testing various concentrations of the compound allows to identify the minimum concentration required to significantly reduce NS1 release, providing insight into the compound's potency.
PRNT (Plaque based viral reduction/neutralisation test) is an efficient cell-based assay for directly quantifying infectious virions and evaluating antiviral substances by counting discrete plaques, which represent infectious units and areas of cellular death, in a well-organized cell culture setup. This method involves diluting the virus sample, infecting a monolayer of susceptible cells, and allowing the virus to replicate. Overlaid with a semi-solid medium, the cells form plaques where the virus has lysed or killed them. By staining and counting these plaques, we can determine virus titres and assess the effectiveness of antiviral agents.
The viral infection in Vero cells is quantified by the development of discrete plaques using virus specific HRP labelled antibodies. The viral prM specific antibodies (2H2) are labelled with HRP and are allowed to bind to the cells and the presence of bound antibodies is visualised using TMB solution. Vero cells are infected with 0.5 MOI PFU and were incubated with different concentrations of drug in carboxy methyl cellulose containing DMEM to localise the viral infection. After 48 hrs, the media is removed and cell is washed and incubated with HRP labelled 2H2 antibodies. Post incubation the plate is treated with TMB solution and incubated for 30 minutes in dark. The developed blue colored plaque is counted. The reduction is plaque numbers will be considered as functional activity against viral infection. The reduction of >50% plaques is considered as active compound.
Plaque assays remain indispensable in virology for their direct visualization of viral infectivity and their ability to provide valuable insights into antiviral drug efficacy within a biological context.
User should ensure the proper transport of the sample, if the sample is stable only in 4oC the temperature should be taken care of while transportation to avoid degradation and hence the false result. The compound received with contaminants will not be proceeded for the assay. Half-life of the compound, if known, should be shared so accordingly it would be considered.
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