An Improved Assay for Virus Research and Therapeutic Development
The PRMNT assay (3,4) is a quantitative immunofluorescence assay that demonstrates the specificity of a Western blot alongside the repeatability and sampling rate of an ELISA-based test.
PRMNT is based upon the In Cell Western Assay™ used widely elsewhere (1, 2) and, as the name implies, measurement is made of the total expression of a protein (native or from infection) with a directly or indirectly fluorescently-tagged antibody, across all cells within a single well, under a given treatment or stress. The signal in that well is correlated to relevant controls and corrected for the variance in number of cells per well. This correction is achieved using a second fluorescence signal reporting the number of cells. This is commonly achieved with DRAQ5™ due to its rapid and stoichiometric labelling of nuclear DNA which is therefore proportional to the number of cells. Typically, this high throughput assay is performed in 96- or 384-well microplate formats and the integrated fluorescence signal captured for each well. However, it is possible, depending on the optical characteristics of the plate-reading device, to more deeply investigate the protein expression and nuclear counterstain signals on a cell-by-cell basis which may give an indication of biological variance and cell and nuclear morphometrics (e.g. indicating cell death or unwanted toxicity). Likewise, it may be possible to quantify more than one protein target using non-overlapping fluorescence tags.
In brief, after treatment(s), cells are fixed and permeabilized and probed intracellularly with target antigen-specific primary antibodies. After washing off excess unbound antibody, fluorescently-tagged secondary antibody and nuclear counterstain are added and incubated. Excess reagents are washed off and plates stored appropriately prior to reading on a suitable plate reader, plate-based cytometer or imager.
In the specific context of antiviral research, PRMNT represents a significant improvement over the conventional plaque reduction neutralisation test (PRNT). It has already been applied to the screening and evaluation of convalescent plasma, the efficacy of candidate anti-virals and as a means of detecting population-level virus exposure by the presence of neutralising antibodies all in the context of SARS-CoV-2.
Importantly, PRMNT can detect and quantify protein expression or the reduction of viral infection or replication in situ in a relevant cellular context, while limiting operator bias.
Experimental design
When working with potentially hazardous, infectious material one should assess the containment and safety precautions required before proceeding.
It is necessary to pre-determine i) the optimal number of cells to seed per well and ii) the multiplicity of infection (MOI) of viral particles to the cells.
Create a standard curve for DRAQ5™ signal versus a suitable range of calculated cell numbers per well.
One should run the assay to determine an acceptable robustness (i.e. Z’-factor) based on negative, positive and background controls. Procedures for this are available elsewhere.
Outline protocol
1. Seed cells at a pre-determined number (typically 4-5,000) per well of a 96-well microtiter plate allowing the cells to adhere (6-24 h depending on cell type). Prepare sufficient wells to perform the assay in triplicate (or as statistically required).
Note: the assay may be scaled to 384-well microtiter plate depending upon the statistical robustness of lower numbers of cells.
2. Add virus inoculum to each well, according to a dilution series. And, as required:
a. Virus inoculum may be pre-treated with neutralising antibody (e.g. convalescent serum, monoclonal antibody).
b. Virus-infected cells may be subsequently treated with candidate anti-infective drugs or neutralising antibody.
3. Following the required period(s) of incubation for the assay, wells are fixed and inactivated by addition of 10% formaldehyde. Incubate for 24 hours at 4 °C.
4. Wash the cells with PBS. Repeat twice. Aspirate.
5. Permeabilize the cells with PBS/0.1% Triton X-100; incubate for 2 minutes. Repeat twice. Aspirate.
6. Apply the blocking solution and incubate for 2 hours at room temperature.
7. Aspirate. Apply the unlabelled primary antibody, in blocking solution. Incubate for 2 hours.
8. Aspirate. Wash with PBS/0.1% Tween-20. Repeat twice.
9. Aspirate. Dilute labelled primary antibody as required and combine with DRAQ5™ at a final concentration of 5 μM in PBS/0.5% Tween-20. Incubate for 1 hour at room temperature.
10. Aspirate. Wash with PBS/0.1% Tween-20. Repeat.
11. Aspirate. Replace PBS and read wells.
12. The antibody signal for each well is corrected according to the corresponding DRAQ5™ signal.
Notes:
i) The streptavidin-biotin system can be an alternative to fluorescently-labelled secondary antibody
ii) The primary antigen-detecting antibody may be fluorescently-tagged to further simplify the protocol.
iii) The number and volume of washes required at each step should be determined and may further vary between microplate manufacturers, cell type, and the reagents used.
References:
- Hannoush, R.N. (2008). Kinetics of Wnt-Driven b-Catenin Stabilization Revealed by Quantitative and Temporal Imaging. PLoS ONE 3(10): e3498.doi:10.1371/journal.pone.0003498
- Cox, Kara S., et al. "Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures." mAbs. 8.1. (2016): 129-140
- Park, Jun-Gyu, et al. "Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2." Journal of Virological Methods 287 (2020): 113995.
- Focosi, Daniele, et al. "Viral infection neutralization tests: A focus on severe acute respiratory syndrome‐coronavirus‐2 with implications for convalescent plasma therapy." Reviews in Medical Virology (2020): e2170
In-Cell Western Assay™ is a trademark of LICOR Biosciences.
