Covid-19 assays and studies

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covid-19 ASSAYS and studies

IITRI can perform the following COVID-19 assays and studies under BSL-3 using SARS-CoV-2.

Multiple relevant strains available upon request (wildtype and variants as available).

Efficacy assay (Immunostain)

Looks at 50% inhibitory concentration (IC50) of the drug or serum against SARS-CoV-2
Briefly, cells are plated in a 96 well plate. Serial dilutions (i.e. 1:2, 1:3 or 1:10) of TA or sera are mixed with equal volumes of the virus at a low MOI (i.e. 0.005 TCID50/cell). The virus:TA or sera mixture is added to the cells. The cells are washed and overlaid with media with or without the test article.
Other variations are possible to sponsor’s specific requests (TA addition prior to virus inoculation (pretreatment) or after virus inoculation (post treatment).
This assay looks at the extent of viral replication measured and expressed as a percentage of virus controls (set to 100% virus replication) and cell controls (set to 0% or background). Virus Controls and Cell Controls are loaded in each plate.
48 hours after virus inoculation, cells are fixed and detected with coronavirus specific monoclonal antibody.

SARS-CoV-2 efficacy assay immunostain

Neutralization assays (PRNT, ELISA)

SARS-CoV-2 48hr neutralization assays for antibodies to determine reduction of plaques and viral measurements.

PRNT

– Plaques are visualized and counted using an ELISPOT instrument

– Plaque counts are fitted to a dose-response curve and plaque reduction neutralization test titers (PRNT) expressed in terms of conventional 50% PRNT end-point titers.

ELISA

– For each well, the inhibition of virus by neutralizing antibodies is calculated as the percentage of reduction of the absorbance value in respect of the virus control

– The neutralizing antibody titer is defined as the reciprocal dilution that caused 50% reduction of the absorbance value of the virus control (50% A450 reduction)

In vivo studies

Syrian Hamster Model

– General clinical observations

– Body weight loss

– Viral load in nasal washes (TCID50 for live virus, SARS-CoV-2-specific RTqPCR)

– Tissue viral burden by TCID50 and/or RT-qPCR

– Histopathology of tissues, to be evaluated with standard H.E. staining

– Neutralizing antibody titer

– Cytokine expression by RT-qPCR

More about this model: IITRI COVID-19 hamster model

K18- hACE2 Transgenic Mouse Model

– General clinical observations

– Body weight loss, mortality

– Tissue viral burden by TCID50 and/or RT-qPCR neutralizing antibody titer

– Histopathology of tissues, to be evaluated with standard H.E. staining

– Cytokine expression by RT-qPCR and/or ELISA

Dose range-finding data for the humanized ACE2 – transgenic mouse model

More about this model: IITRI COVID-19 mouse model

Ferret Model

– Favorable model for looking at effects solely in upper respiratory tract

– Infection model only, no clinical signs

– Viral load and shedding in nasal washes

– Viral load in nasal washes (TCID50 for live virus, SARS-CoV-2-specific RTqPCR)

– Tissue viral burden by TCID50 and/or RT-qPCR

– Histopathology of tissues, to be evaluated with standard H.E. staining

– Neutralizing antibody titer

– Cytokine expression by RT-qPCR (ELISA reagents for ferrets are very limited)

Read more about our ferret models: IITRI influenza ferret model

IITRI has established itself as a leading COVID-19 CRO and due to the importance for this testing during the pandemic, has made provisions to turn these assays and studies around rapidly. Other related capabilities include a BSL-3/ABSL-3 aerobiology environment for inhaled drug challenge studies.

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