Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays

Abstract

Virus neutralization assays measure neutralizing antibodies in serum and plasma, and the plaque reduction neutralization test (PRNT) is considered the gold standard for measuring levels of these antibodies for many viral diseases. We have developed procedures for the standard PRNT, microneutralization assay (MNA) and pseudotyped virus neutralization assay (PNA) for severe acute respiratory syndrome coronavirus 2. The MNA offers advantages over the PRNT by reducing assay time, allowing increased throughput and reducing operator workload while remaining dependent upon the use of wild-type virus. This ensures that all severe acute respiratory syndrome coronavirus 2 antigens are present, but Biosafety Level 3 facilities are required. In addition to the advantages of MNA, PNA can be performed with lower biocontainment (Biosafety Level 2 facilities) and allows for further increases in throughput. For each new vaccine, it is critical to ensure good correlation of the neutralizing activity measured using PNA against the PRNT or MNA. These assays have been used in the development and licensure of the ChAdOx1 nCoV-19 (AstraZeneca; Oxford University) and Ad26.COV2.S (Janssen) coronavirus disease 2019 vaccines and are critical for demonstrating bioequivalence of future vaccines.

Safety and immunogenicity of heterologous versus homologous prime-boost schedules with an adenoviral vectored and mRNA COVID-19 vaccine (Com-COV): a single-blind, randomised, non-inferiority trial

“…Serum samples were analysed at Nexelis (Laval, Canada) to determine SARS-CoV-2 anti-spike IgG concentrations by ELISA (reported as ELISA laboratory units [ELU]/mL) and the 50% neutralising antibody titre (NT 50 ) for SARS-CoV-2 pseudotype virus neutralisation assay (PNA), using a vesicular stomatitis virus backbone adapted to bear the SARS-CoV-2 spike protein. 17 The conversion factors to international standard units can be found in the appendix (p 12) . Sera from day 0 were analysed at Porton Down, Public Health England, by electrochemiluminescence immunoassay (Cobas platform, Roche Diagnostics) to determine anti-SARS-CoV-2 nucleocapsid IgG status (reported as negative if below a cutoff index of 1·0).…”

“…Normalised NT 50 for live SARS-CoV-2 virus (lineage Victoria/01/2020) was determined by microneutralisation assay (MNA), also at Porton Down, on day 0 and day 56 samples in the ChAd-primed groups only, due to limited laboratory capacity. 17 IFNγ-secreting T cells specific to whole spike protein epitopes designed based on the Wuhan-Hu-1 sequence (YP_009724390 · 1) were detected using a modified T-SPOT-Discovery test done at Oxford Immunotec (Abingdon, UK) within 32 h of venepuncture, using the addition of T-Cell Xtend reagent to extend peripheral blood mononuclear cell (PBMC) survival. 18 T-cell frequencies were reported as spot forming cells (SFC) per 250 000 PBMCs with a lower limit of detection of one in 250 000 PBMCs, and these results were multiplied by four to express frequencies per million PBMCs.…”

The CEPI centralised laboratory network: supporting COVID-19 vaccine development

“…Given that diabetes-specific distress is "anchored in the day-to-day experience of living with diabetes", 3 it can be assessed and addressed within the diabetes care team, and this approach is what people with diabetes want. 5 Integrating the management of diabetes-specific distress into a basic care plan is practical and inexpensive, 6 with no need for advanced equipment or diagnostic tools. Nor, in most cases, is there need for referral to mental health professionals.…”

“…The wild-type virus neutralisation assay (VNA) and the pseudovirus neutralising assay (PNA) measure virus-specific neutralising antibodies in serum samples. 5 VNA targets the original SARS-CoV-2 Victoria/1/2020 (subsequently renamed BetaCoV/ Australia/VIC01/2020) strain and requires biosafety level 3 facilities, whereas PNA allows determina tion of neutralisation activity in a standard biosafety level 2 laboratory. The ELISAs and the neutralisation assays are standardised to the WHO International Standard for anti-SARS-CoV-2 immunoglobulin developed by the National Institute for Biological Standards and Control, 6 which will allow harmonisation of data produced in laboratories around the globe.…”

A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19

“…At necropsy nasal washes and oropharyngeal swabs and tissue samples (lung, trachea and duodenum) were collected in PBS and stored frozen at -80°C for viral RNA measurement and viral culture. Tissue samples for histopathological examination were xed in 10% buffered formalin at room temperature (see below).A micro-plaque assay57 was used to determine the amount of virus in tissue samples. The animal sample was serially diluted in assay diluent (MEM supplemented with L-glutamine (Life Technologies), nonessential amino acids (Life Technologies), 25mM HEPES (Sigma) and 1x antibiotic/antimycotic) and added to con uent monolayers of Vero E6 cells.…”