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. 2023 Jul 5;15(7):1504. doi: 10.3390/v15071504

Table 2.

Principle, advantages, and limitations of NAbs detection assays.

Assay Principle Advantages Limitations
Plaque reduction neutralization test (PRNT) Infection of Vero cells with live viruses. The measurement of NAbs’ potency is determined through the quantification of the plaque-forming units (PFU) in specimens containing the antibodies, such as plasma, serum, or blood.

  • Since PRNT uses live viruses, the replication capacity of the virus is considered.

  • Safety concerns regarding the use of live viruses.

  • Laboratorial infrastructure (BSL-3) to use live viruses.

  • Highly qualified human resources to work in the BSL-3 laboratory.

  • PRNT is not compatible with high throughput, time-consuming, laborious.
Pseudovirus neutralization test (pVNT) Production of lentivirus expressing spike in the surface and transduction of HEK cells overexpressing ACE2. HEK cells are transduced to express GFP or luciferase. The redout is based on the reduction of the percentage of GFP positive cells of the decrease in the luciferase bioluminescence.

  • It requires BSL-2.

  • Compatible with high throughput.

  • Can be quickly adapted to produce lentiviral particles expressing spike from VoC.

  • Since pVNT uses non-replicative viral particles, viral replication is not considered in this assay.

  • Evaluates only spike-ACE-2 interaction.

  • Antiretroviral treatment may produce false positive results regarding pseudoviral neutralization.

  • pVNT requires ACE2-expressing cells to measure anti-SARS-CoV-2 NAbs.
Foci reduction neutralization test (FRNT) or microneutralization assay (MNA) FRNT has a similar principle to the PRNT assay. However, the readout is based on a monoclonal antibody conjugated to horseradish peroxidase (HRP) directed against the spike protein. This method uses live viruses. The PFU is quantified by a microanalyzer.

  • Since PRNT uses live viruses, the replication capacity of the virus is considered.

  • Compatible with high throughput.

  • Safety concerns regarding the use of live viruses.

  • Laboratorial infrastructure (BSL-3) to use live viruses.

  • Highly qualified human resources to work in the BSL-3 laboratory.
Surrogate virus neutralization test (sVNT) The method is based on an enzyme linked immunosorbent Assay (ELISA) and measures the RBD-ACE2 interaction. The RBD conjugates with HRP interacts with ACE2 in an ELISA plate in the absence of NAbs

  • sVNT is easy to execute.

  • sVNT presents less safety concern compared to PRNT and pVNT, since it uses recombinant protein.

  • It has a reduced cost compared to cell-based and’ live-virus methods.

  • Can be quickly adapted to produce lentiviral particles ex-pressing spike from VoC.

  • Compatible with high throughput.

  • The assay evaluates RBD-ACE2 interaction.

  • sVNT may lose NAbs binding outside the RBD
Lateral flow immunoassay (LFIA) The assay uses RBD or full-length spike conjugated to a colloidal gold, colorful microsphere or fluorescent beads. NAbs from the sample interact with RBD in the conjugation pad. NAbs-RBD binding will avoid RBD binding to ACE2 coated in the test line. In this case the test line will be less colorful, or less fluorescent depending on the NAbs titer.

  • Easy to execute test.

  • Usually, it takes 10 to 15 min to see the result.

  • The use of conjugated RBD may miss NAbs targeting spike domains outside the RBD

  • It can be performed in laboratories with high or low infrastructure.

  • It can be used in places without laboratorial infrastructure.

  • Can be adapted for point of care tests.

  • Can be adapted for different readouts, such as colloidal gold, microspheres and fluorescence.

  • LFIA is a cell-free platform and a cheap method.

  • The assay can be qualitative or quantitative.

  • It can be adapted to high throughput screenings.

  • It requires high binding affinity between the target protein and its receptor (RBD and ACE2).

  • Most LFIA are qualitative and to become quantitative it must be associated with an equipment or system (like mobile phones) to measure the intensity of the test and control lines.

Biosafety level (BSL); green fluorescent protein (GFP); receptor binding domains (RBD); Angiotensin-converting enzyme 2 (ACE2); horseradish peroxidase (HRP); neutralizing antibodies (NAbs).