SARS-CoV-2 Protease Sensor Kit

For measuring Papain-like protease (PLpro) Activity in unprocessed cellular lysates or in live cells

Catalog No: MP-PLpro-96
Size: 96 reactions
Price: $599

Figure 1. Schematic of SARS-CoV-2 PLpro turn on assay.
A peptide with a proprietary sequence is quenched by the electron withdrawing properties of the amide bond and fluoresces when the bond between the terminal amino acid and rhodamine 110 is cleaved by PLpro.
Tests can be performed in multi-well plates using cellular lysates or in live, whole cells. Examples are shown in Figure 2.

Figure 2. Application of SARS-CoV-2 PLpro sensor.
Left. In lysates from VeroE6 cells (Demonstrated by S. Bradfute, University of New Mexico, USA.
Right. In live Vero-CCL81 cells (Demonstrated by Edmarcia Elisa de Souza and Carsten Wrenger University of São Paulo, Brazil and Christian Betzel, University of Hamburg, Germany).
Specifications:
  • Detects SARS-CoV-2 activity in ~ 10 hours after infection
  • Lysis buffer immediately inactivates SARS-CoV-2 activity
  • Detects wildtype, delta, and omicron variants (others not yet tested)
  • Adaptable to robotic screening of therapeutic antibodies and PLpro inhibitors in multi-well plates
  • Works in cellular lysates or whole, live cells
  • Validated in 3 independent BSL-3 laboratories
  • Further validated as a rapid Point-of-care test in saliva from patients

Background.

SARS-CoV-2 Papain-Like Protease (PLpro) plays an essential role in polypeptide processing during viral replication. In addition to processing the viral polyprotein, PLpro strongly binds to ubiquitin and ISG15 and removes these signaling proteins to counter the host cell innate response. As a result, PLpro is an important potential target for antiviral drugs that may inhibit viral replication and restore the host cell immune response.

Current biochemical drug screening approaches use recombinant, truncated PLpro that does not reflect PLpro as it is produced by an infected cell. Current cell-based assays that determine the effect of inhibitors measure cytopathic effects (CPE), plaque reduction, or viral RNA reduction. These assays are complex, involve several handling steps and take days to complete.

Mesa Photonics SARS-CoV-2 Protease Sensor.

The sensor allows for simple and direct measurement of PLpro inhibition in less than 10 hours after infection. The test is homogeneous and does not require any processing steps, making it ideally suited for screening applications in multi-well plates.

The cell-based assay uses a patented reporter peptide that fluoresces when cleaved by SARS-CoV-2 PLpro in lysates from cells infected with SARS-CoV-2 and/or in infected whole cells (Figure 1.). Unlike biochemical assays, the assay kit measures the activity of full-length, endogenous papain-like protease as it is produced by the cell.

Performance has been demonstrated in lysates from VeroE6 cells for detection of anti-SARS-CoV-2 neutralizing antibodies and PLpro inhibitors. Real time monitoring of PLpro cleavage activity was further demonstrated in whole, live Vero-CCL81 cells.

Patent protected by US11851698B2, US11603552B2
Reference: Anahi G. Jimenez-Campos et al., A cell-based Papain-like Protease (PLpro) activity assay for rapid detection of active SARS-CoV-2 infections and antivirals. PLOS One, December 26, 2024
https://doi.org/10.1371/journal.pone.0309305.