Table 1.
Comparison of micro-neutralisation tests.
Fig 1.
SARS-CoV-2 flow based micro-neutralisation assay workflow.
1. Heat inactivated (30 mins at 56°C) human plasma samples are serially diluted (8-point, half-log from 1:20) in infection medium, in 96-well deep well plates. 2. Viral neutralisation is carried in standard 96-well plates, where each antibody dilution is co-incubated with WT SARS-CoV-2 or a Variant of Concern (VOC) for 60 mins at 37°C. 3. Infection of confluent Vero-E6 or Vero-E6/TMPRSS2 cells with antibody-virus mixture is carried out in 96-well plates for 18h at 37°C. Each plate can test one plasma sample against three SARS-CoV-2 variants in parallel, in duplicates, including controls (virus alone and infection medium alone) or three samples with only one variant. 4. Cells are trypsinised, fixed (4% formaldehyde), permeabilised and stained for intracellular SARS-CoV-2 NP. Infection is measured using Flow Cytometry, first gating the live, single cell population, and then NP negative and positive populations. 5. Flow cytometry data is analysed to determine the percentage of infected (NP+) cells per well and then the percentage of inhibition can be determined by normalising each dilution to the positive and negative controls. NT50 can be determined from the inhibition curve, as the dilution factor resulting in 50% inhibition of infection. Created with BioRender.com.
Fig 2.
Validation of Flow-Based Micro-NT using the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin and Secondary Standards, (A-B) SARS-CoV-2 (WT-B) was neutralised for 1 hour with an 8-point, half-log serial dilution of WHO SARS-CoV-2 low, medium or high Titre IgG. (A) The antibody-virus mixture was then plated onto Vero E6 cells for 90 mins before overlay (1% CMC) was added and incubated for 96-hours. Plaques were quantified and the inhibition of infection is presented as a percentage, relative to the virus-only control wells. Graph shows the mean and standard deviation of technical duplicates. (B) In parallel, the virus-antibody mixture was added onto Vero E6 cells for 18h before cells were trypsinised, fixed and permeabilised, and stained for the SARS-CoV-2 NP. Percentage of infected cells was determined by flow cytometry analysis. Inhibition of infection is presented as percentage, relative to the virus-only control wells. Graph shows the mean and standard deviation of technical duplicates. (C) Table shows the NT50 values obtained for the three WHO international standards on PRNT and Micro-NT, as well as the NT50 value provided by WHO for each sample in IUs. (D and E) 12 secondary standards were tested in technical duplicates using PRNT and Micro-NT against WT-B.1.177.18 on Vero E6/TMPRSS2 cells. NT50s were determined for each sample (D) and the correlation was plotted using non-linear regression (log-log line) with GraphPad Prism (E).
Table 2.
Reproducibility of Micro-NT 6 secondary standards were run against WT-B.1.177.18 SARS-CoV-2 on Vero E6/TMPRSS2 cells, five times per plate (Row 1–5), on five independent days (Day 1–5).
The NT50s determined for each row are provided, as well the mean across all 25 measurements. The standard deviation (S) and co-efficient of variation (%CV) is provided for both the repeatability test (R) and the intermediate-precision (inter-assay) test (RW). The mean repeatability and intermediate precision is provided which an average of the 6 measurements.
Fig 3.
Dynamic range of flow cytometry-based micro-NT.
We analysed the NT50s of 201 COVID-19 naïve or convalescent plasma samples, using Micro-NT, against SARS-CoV-2 (WT-B) on Vero E6 for 18h. (A-D) Graphs show neutralising capacity of 3 convalescent plasma samples (P1-P3) across 8-point, half-log serial dilution, representative of samples with 50% Neutralisation Titres (NT50) within a similar range. The percentage of viral inhibition of each dilution was calculated relative to the virus only control. (E) Plasma from COVID-19 naïve individuals showed little or no neutralisation capacity against SARS-CoV-2. (F) Graph shows the full range of NT50 values obtained across the cohort of COVID-19 convalescent plasma samples, measured using Micro-NT.
Fig 4.
Neutralisation capacity of secondary convalescent and vaccinated standards against SARS-CoV-2 variants of concern measured using micro-NT.
Here neutralisation capacity of WT.B-177.18, Beta, and Omicron BA.5 was measured using Micro-NT on Vero E6/TMPRSS2 cells, against six secondary standards. The assays were performed in technical duplicates. (A) NT50 values obtained for each standard against the three SARS-CoV-2 variants. (B) Graph shows the reduction in neutralisation capacity of Beta and Omicron VOCs across each standard tested. Lines represent paired samples.