The plaque reduction neutralization test (PRNT), pseudovirus neutralization assay (PsVNA), and enzyme-linked immunosorbent assay (ELISA) are commonly used methods to detect and measure the neutralizing activity of antibodies against viruses. Due to differences in their methodologies and application, the choice of assay can depend on factors such as experimental requirements, resources, and the specific virus being studied. We examine their property and why NAB-Sure™, an ultrasensitive SARS-CoV-2 Neutralizing Antibody (sVNT) has definitive advantages over other methods.

Plaque Reduction Neutralization Test (PRNT)

PRNT is a widely accepted standard for measuring neutralizing antibodies. In this serological test, cells are incubated with live virus and serial dilutions of the sample (typically serum or plasma). The virus infects the cells, resulting in the formation of plaques, which are regions of dead/dying cells caused by cytopathic effects of viral infection. Neutralizing antibodies, if present in the test sample, block viral infection, leading to a reduction in plaque formation. By comparing the number of plaques in the presence and absence of the test samples, the neutralizing antibody titer (NT50) can be determined.

Advantages:

• Uses live cells and virus to reflect the functional ability of antibodies to neutralize the virus.
• Can provide quantitative data on neutralizing antibody titers.
• Can be used to identify the specific virus strain.
• Can assess neutralization of NAbs that bind epitopes outside the RBD.

Disadvantages:

• Requires handling live virus cultures and use of BSL-3 facilities.
• Labor-intensive and time consuming, can take 5 days to complete.
• High lab-to-lab variability due to the use of live cells and viruses
• Lower throughput and higher costs compared to other methods.

Pseudovirus Neutralization Assay (PsVNA)

The pseudovirus neutralization assay is an alternative to PRNT that avoids the need to handle live viruses. A genetically engineered, non-replicating pseudovirus is used. This pseudovirus carries surface proteins of the target virus, enabling it to enter host cells but not replicate. The neutralizing antibodies present in the test samples can inhibit the entry of the pseudovirus into the cells. The inhibition is typically quantified by measuring reporter gene expression (e.g., luciferase or GFP) in the host cells.

Advantages:

• Safer than working with live viruses, BSL-2 facilities can be used.
• Can be adapted to high-throughput screening.
• Can be read after 24 hours incubation.

Disadvantages:

• Requires generation of pseudoviruses.
• Does not measure impact on replication.
• High lab-to-lab variability due to the use of live cells.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is a versatile and widely used immunoassay technique. For neutralizing antibody detection, typically a competitive ELISA is used. In this assay, samples with increasing amounts of neutralizing antibodies lead to decreasing ELISA signal. The degree of antibody binding is typically quantified using a colorimetric or fluorescent readout.

Advantages:

• Cost effective, easy to perform and typically completed within 8 hours.
• Amenable to high-throughput screening through automation.
• High precision, low lab-to-lab variability.
• Requires BSL-1 or BSL-2 facilities.

Disadvantages:

• The correlation between ELISA results and virus neutralization may vary unless concordance has been previously established.
• Typically less sensitive than other methods.

If you need to quantify NAb’s from micro samples like dried blood spots using a cell-free sVNT assay that does not require proprietary equipment, the above solutions likely won’t provide the required sensitivity.

NAB-Sure™ will.

NAB-Sure is an ultrasensitive SARS-CoV-2 Neutralizing Antibody Test. Using a competitive binding assay based on SPEAR Bio’s SPEAR immunoassay platform, NAB-Sure quantifies neutralizing antibodies from serum, plasma, and even dried blood spots by measuring their ability to block the SARS-CoV-2 spike protein from binding to the ACE2 receptor of a human cell.

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Advantages:

• Cost-effective and system-agnostic – Only standard real-time qPCR and thermocycler are required.
• Concordant with PRNT.
• Sub-femtogram/ml detection, sufficiently sensitive to quantify neutralizing antibodies from dried blood spots (DBS).
• High precision, low lab-to-lab variability.
• Amenable to high-throughput screening through liquid handling automation.
• BSL-1 facilities.
• Multiple variant test kits available (WT, BA4/5, BQ1.1, XBB.1.5).
• 6 hour assay time (+1.5hr if starting from DBS).

Disadvantages:

• Doesn’t assess neutralization by NAbs that bind epitopes outside the RBD (but still highly concordant with PRNT)
• Does not measure impact on replication.

References:

1. Rocha, V.P.C.; Quadros, H.C.; Fernandes, A.M.S.; Gonçalves, L.P.; Badaró, R.J.d.S.; Soares, M.B.P.; Machado, B.A.S. An Overview of the Conventional and Novel Methods Employed for SARS-CoV-2 Neutralizing Antibody Measurement. Viruses2023, 15, 1504. https://doi.org/10.3390/v15071504

2. Bewley, K.R., Coombes, N.S., Gagnon, L. et al.Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays. Nat Protoc 16, 3114–3140 (2021). https://doi.org/10.1038/s41596-021-00536-y

3. Liu, K. T., Han, Y. J., Wu, G. H., Huang, K. A., & Huang, P. N. (2022). Overview of Neutralization Assays and International Standard for Detecting SARS-CoV-2 Neutralizing Antibody. Viruses, 14(7), 1560. https://doi.org/10.3390/v14071560