Introduction

Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses that belong to the family Coronaviridae. Coronaviruses are known to cause diseases with a spectrum of severity in humans and other animals1,2. In December 2019, a new coronavirus emerged that was identified as a member of the Betacoronavirus genus. It was subsequently named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is the causative agent of coronavirus disease 2019 (COVID-19). The spread of this virus led to a global pandemic affecting millions of people3. The search for an effective vaccine for COVID-19 is a global priority. Immunological assays, such as those for neutralizing antibodies, are important for providing information on correlates of protection for vaccine efficacy and data to support regulatory submissions for vaccine licensure.

Development of the protocol

The initial procedure developed was the plaque reduction neutralization test (PRNT), which measures neutralizing antibodies by in vitro virus neutralization. PRNT is considered the gold standard for assessing humoral correlates of protection for many viral diseases4,5,6. In the absence of phase III efficacy data, neutralizing antibody results have a key role in identifying optimal COVID-19 vaccine candidates and will be important in the regulatory licensure process. It is not yet known whether circulating neutralizing antibody levels can be considered a sole measure of protective immunity against COVID-19, but recent successful treatment of severely ill patients with convalescent plasma and monoclonal antibody therapeutics containing high levels of neutralizing antibodies suggest an important role for these antibodies7,8,9,10. However, PRNT has several limitations that make it unsuitable for large-scale studies of serum samples, such as phase III human clinical vaccine trials. PRNT is technically demanding, has very low throughput, is difficult to automate and, for SARS-CoV-2, has a long turnaround time due to the time taken for the virus to form visible plaques. In addition, the analysis of the count data is typically performed using Excel spreadsheet calculations to determine neutralization titers (e.g., Kärber formula11) or using free statistical packages such as R to perform Probit regression. These analysis solutions represent obstacles for assay validation, which may lead to difficulties in using the data as part of a regulatory submission. For these reasons, of the three assays presented in this article, only the microneutralization assay (MNA) and pseudotyped virus neutralization assay (PNA) (not the PRNT) have been qualified, as only data produced by these assays and analyzed by the validated version of the SoftMax Pro software are likely to be acceptable to regulatory agencies. The qualification results for the MNA and PNA can be found in Extended Data Figs. 1 and 2, respectively.

To improve on the limitations of the PRNT, an alternative assay called the focus-reduction neutralization test (more commonly known as an MNA) has been developed for other viruses12,13,14. This assay is performed in 96-well plates throughout and uses immunostaining to visualize infected foci, which can be counted using computer-controlled imagers (Fig. 1), substantially increasing assay throughput compared with the manual counting performed in PRNT. A further development of MNA is to replace the hazard group 3 wild-type virus with a hazard group 2 pseudotyped virus to perform a PNA. Pseudotyped viruses consist of nonreplicating genetically modified virions that comprise the structural and enzymatic core of one virus, such as vesicular stomatitis virus (VSV), while bearing the surface proteins of another virus, such as the SARS-CoV-2 spike, and encode a quantifiable reporter gene such as firefly luciferase15. In the PNA described in this protocol, the pseudotyped virus consists of a VSV that has been engineered to bear the SARS-CoV-2 spike glycoprotein on its surface and replace its G protein gene with a luciferase reporter. The last 19 amino acids of the spike protein have been deleted, which roughly corresponds to the cytoplasmic tail.

Fig. 1: General overview of the MNA described in Procedure 2.
figure 1

Serum samples are diluted in a 96-well plate (Stage I), before wild-type SARS-CoV-2 virus is added to the diluted serum at BSL3 and neutralization allowed to occur (Stage II). The neutralized virus is then transferred onto Vero E6 cells (Stage III), allowed to adsorb, overlaid with viscous medium and incubated for 24 h (Stage IV). Plates are then fixed (Stage V), virus infected foci immunostained (Stage VI) and foci counted (Stage VII). Well counts are then analyzed with SoftMax Pro to determine median neutralization titers of the reference and test sera (Stage VIII).

In this protocol, we describe procedures for performance of the classical PRNT, a newly developed MNA and a PNA for the measurement of SARS-CoV-2 neutralizing antibodies. The National Institute for Biological Standards and Control (NIBSC) has made available the 1st WHO International Standard for anti-SARS-CoV-2 antibody (20/136)16. In combination with this international antibody standard, we expect these assays to play a pivotal role in the development and licensure of urgently needed COVID-19 vaccines.

Applications

The MNA and PRNT described in this protocol have been applied to the testing of serum from participants in randomized controlled clinical trials for the ChAdOx1 nCoV-19 (AstraZeneca; Oxford University) and Ad26.COV2.S (Janssen) COVID-19 vaccines17,18,19. They have also been applied to the testing of serum from animal models20,21 and vaccine immunogenicity studies22. The wild-type assays may also be used with little adaptation to assess the in vitro efficacy of antiviral drugs23. All three assays permit the detection and measurement of titers of SARS-CoV-2 neutralizing monoclonal antibodies24,25 and could help to elucidate the structural basis of neutralization26. These assays form a critical readout for the development and licensure of current and future COVID-19 vaccines and, moving forward, will be a key element in demonstrating the bioequivalence of future vaccines.

Comparisons between the assays

The PRNT requires 5 d of incubation to develop countable plaques, whereas, in the MNA and PNA, plates are fixed after only 24 h incubation; this considerably shortens the turnaround time of these assays. The MNA and PNA are conducted in 96-well plate format throughout, whereas the PRNT is performed in a mixture of 96-well (for the neutralization stage) and 24-well (for the plaque assay) plates. This increases the number of samples that can be tested per operator on any given testing occasion in the MNA and PNA relative to the PRNT. The use of 96-well plates throughout the assay also facilitates increased automation of the washing, staining and reading stages of the assay. Automation is a key factor in reducing operator error and increasing assay precision.

In the MNA, immunostained foci are counted using a computerized counting system and, in the PNA, luminescence of the luciferase reporter is measured with a plate reader; whereas, in the PRNT, operators perform manual plaque counting. This improves accuracy and objectivity of the reading as well as increasing throughput. In the MNA and PNA, count and luminescence measurements, respectively, are analyzed automatically using curve-fitting software, which allows the majority of test samples to have a median neutralizing dose (ND50) assigned to them. In addition, the curve-fitting method provides a confidence interval around any result derived from the curve. The software used is GxP compliant, which lends itself to validation in accordance with the Good Clinical Practice (GCP) regulations required by regulatory authorities. In contrast, the most common methods for performing this determination for PRNT and some PNA assays27 are use of the Spearman–Kärber or Reed–Muench formulae, both of which require a full neutralization response (0–100%) and neither of which easily provide confidence intervals. These traditional calculations are generally carried out by hand or with the aid of a spreadsheet, representing additional challenges for assay validation to meet the requirements of regulators.

As wild-type virus contains the complete SARS-CoV-2 replicative machinery, both PRNT and MNA can be adapted to assess the efficacy of antiviral compounds, whereas the PNA is appropriate only for measuring neutralization activity of serum or monoclonal antibody-based therapeutics.

Limitations

Limitations of the PRNT are that it is slow to perform, has low throughput and is technically demanding. In addition, both the wild-type MNA and PRNT require access to Biosafety Level 3 (BSL3) facilities and suitably trained staff, whereas the PNA can be conducted in a BSL2 facility. MNA and PNA correlate well with the ‘gold standard’ PRNT when tested with a panel of convalescent human serum; live virus PRNT with MNA (Pearson r = 0.963; P < 0.001) and PRNT with PNA (Pearson r = 0.862; P < 0.001) (Extended Data Figs. 3 and 4). During qualification, assay performance is assessed and documented for key performance metrics, e.g., specificity, linearity, bias, precision and limits of quantification. Both the MNA and the PNA have been qualified and will be utilized in regulatory submissions. However, as the pseudovirus used in PNA contains only the SARS-CoV-2 spike antigen and not the full complement of viral antigens, there is a risk that the PNA might fail to correlate well with the wild-type assays for some vaccine candidates, particularly those containing non-spike subunits of SARS-CoV-2. It is therefore crucial that, for each novel vaccine candidate, serum responses are correlated between MNA and PNA before transferring serum antibody assessments over to the PNA.

A potential limitation of all neutralization assays utilizing wild-type virus relates to the testing of plasma instead of serum derived from clotted whole blood, as plasma is often derived from whole blood that has been collected into tubes containing heparin as an anticoagulant. There have been reports that heparin is an effective inhibitor of SARS-CoV-2 entry into cells, and therefore there is a risk that this sample type might give rise to erroneous results from these assays23,28. Other common anticoagulants have not been reported to exhibit nonspecific antiviral activity.

A final limitation of the PNA is that the assay is appropriate only for measuring neutralization activity of serum or monoclonal antibody-based therapeutics, whereas both wild-type virus assays can be adapted to assess the efficacy of other antiviral compounds. This is because the pseudovirus does not contain the coronavirus replicative machinery that such compounds are directed against.

Alternative methods

All measurements of neutralizing antibody titers are generally performed by PRNT, some form of the MNA, or assays using pseudotyped viruses (e.g., the PNA). PRNT tests generally follow the procedure described here with only minor changes, mainly to the source of the wild-type virus, the semi-solid overlay used (agarose, methylcellulose or AviCell) and the method of analysis. Of these, only the method of analysis is worthy of note. As discussed earlier, methods such as Reed–Muench and Spearman–Karber are often still used to estimate median neutralization doses. In the past, this was due to their ease of calculation; however, curve-fitting methods have now been demonstrated to provide more accurate results29, and their computation no longer presents a barrier. The primary advantage of PRNT over other methods is the ability to detect more subtle changes, such as those to plaque morphology, in response to a serum sample, antibody or therapeutic, as well as a reduction in total plaque number. For example, antibody enhancement may cause plaques to appear larger than normal, or certain types of incomplete neutralization may cause a reduction in plaque size.

Large increases in scale and sample throughput can be achieved by replacing the PRNT with an MNA. In addition, as MNA is performed in a 96-well format, the assays are more amenable to automation of several assay steps. The two main types of MNA differ in the method of readout; some assays (such as those presented here) are read by counting immunostained foci (spots), whereas others are read on an ELISA reader measuring a colorimetric dye such as o-phenylenediamine30. The advantage of foci counting is that microbial contamination of samples or other mishandling causing cell monolayer damage can be seen during the plate quality control stages of the assay; whereas a colorimetric assay lacks this opportunity to reject the well or sample containing the error, leading to an aberrant absorbance reading. The advantages of the colorimetric methods are that they are generally faster to read, require less expensive equipment to perform measurements, and setting up the curve-fitting software is more straightforward. In the MNA presented here, the primary focus detection antibody is directed at the SARS-CoV-2 spike receptor binding domain, which is presented on the surface of infected cells; whereas, in other assays, the primary antibody is directed against the viral nucleocapsid30, which requires the staining procedure to have an additional cell-permeabilization step that increases assay time. The advantage of using a primary antibody against nucleocapsid is that, if one is working with virus variants with many mutations in the spike protein, it is conceivable that the anti-spike antibodies may no longer bind to the spike; however, this is less likely to occur to the nucleocapsid, as it is not under the same immune pressure in the host.

All wild-type assays suffer from the limitation of requiring BSL3 facilities and highly trained staff to perform them. PNAs allow neutralization assays to be performed in a standard BSL2 laboratory. These assays retain the advantages of the MNA, of being more scalable and having higher throughput than the PRNT. The readout of the PNA is luminance, which has the same advantages and disadvantages discussed for colorimetric methods in the MNA above. Median neutralizing doses can be determined using in-house Excel calculations (e.g., Reed–Muench method) or curve-fitting functions in off-the-shelf statistical packages such as GraphPad Prism27,31, but these approaches are unlikely to be acceptable to vaccine licensing agencies. It is preferable, as presented here, to perform calculations within a GxP validated version of curve-fitting software such as SoftMax Pro.

Overview of the procedures

For all neutralization assays described here, the serum must first be heat-treated to inactivate complement using the method described in Box 1. For both wild-type virus assays, PRNT (Procedure 1) and MNA (Procedure 2), a stock of virus needs to be produced as described in Box 2. For the PRNT, the wild-type virus stock is subjected to a rough sighting to estimate the dilution at which to prepare the virus, followed by the fine sighting (Box 3). Once a given virus stock has been sighted into the assay in this manner, samples can be analyzed by the PRNT by following the instructions in Procedure 1. A similar process is used to test a new virus stock in the MNA but can be more simply performed by the semifine sighting (Box 4), which includes regression analysis to determine the working dose for the assay. Once the virus has been sighted into the MNA, samples can be analyzed by following the instructions in Procedure 2. Resighting of the PRNT or MNA would only be required when a new stock of wild-type virus is produced or if the virus-only control (VOC) counts were outside the specified range for the assay.

For the PNA (Procedure 3), stocks of pseudovirus should be produced by following the procedure in Box 5. New virus stocks should be subjected to the sighting procedure (Box 6). Once the optimal pseudotyped virus dilution for a given stock is known, Procedure 3 can be started. Resighting would be required in the event of a new batch of pseudotyped virus being produced or if the VOCs (pseudotyped virus control; PC) luminescence measurements were outside the specified range.

Experimental design

To perform the procedures described in this protocol, commercial individual and/or pooled human serum containing SARS-CoV-2 neutralizing antibodies are required. These have been available from NIBSC since December 202016. To perform the PRNT and MNA assays, implementers will need an appropriate wild-type SARS-CoV-2 strain or isolate and an operational Advisory Committee on Dangerous Pathogens containment level 3 laboratory (BSL3). To perform the PNA, implementers will need to obtain stocks of the pseudotyped virus or have the necessary expertise to generate these stocks.

For all procedures described here, experience in working with cell culture would be a distinct advantage. For all manipulations involving wild-type SARS-CoV-2 in the PRNT and MNA, operators must be competent in performing standard virological techniques at BSL3. For the PNA, implementers would need the molecular biological expertise to generate the pseudotyped virus stocks if they cannot obtain them commercially.

Materials

Biological materials

  • Serum samples (either from clinical trial participants or patients for testing)

    Caution

    Appropriate national laws and institutional regulatory board ethical guidelines must be followed and informed consent obtained from patients for the use of human or animal serum in these protocols.

  • Wild-type SARS-CoV-2 virus (Victoria/1/2020), supplied to us by the Docherty Institute, then grown and stored at <−60 °C in single-use aliquots

    Critical

    In our laboratory, a typical virus stock at ~1 × 107 pfu/ml is typically sufficient to set up 50 96-well plates, which would facilitate the testing of ~300 samples.

  • Vero E6 cells (ECACC, cat. no. 85020206)

  • Heat-inactivated fetal calf serum (Merck, cat. no. F4135-500ML)

    Critical

    All fetal calf serum used for cell cultivation, virus propagation and assay performance must be heat-inactivated to ensure that no active complement is present to interfere with these assays.

Reagents

Reagents required for all assays

  • PBS (Severn Biotech, cat. no. 207410, or equivalent)

  • Trypan blue solution, 0.4% (Gibco, cat. no. 15250061)

  • l-Glutamine solution, 200 mM (Gibco, cat. no. A2916801)

PRNT/MNA-specific reagents

  • Minimal Essential Medium (MEM; Gibco, cat. no. 21090-022)

  • Dulbecco’s PBS (DPBS; Gibco, cat. no. 14190169)

  • Trypsin-EDTA, 0.25% (Gibco, cat. no. 25200056)

  • 10× MEM (Gibco, cat. no. 11430030)

  • Sodium bicarbonate solution, (Gibco, cat no. 25080094)

  • HEPES buffer solution, 1 M (Gibco, cat. no. 15630056)

  • Nonessential amino acids (NEAA), 100× (Gibco, cat. no. 11140035)

  • Antibiotic-antimycotic solution, 100× (Gibco, cat. no. 15240062)

  • Medium-viscosity carboxymethylcellulose (CMC; Merck, cat. no. C4888)

  • Formalin solution, 40% (wt/vol) (e.g., Sigma, cat. no. F8775)

PRNT-specific reagents

  • Crystal violet 0.2% (wt/vol) in 20% (vol/vol) ethanol solution, prepared from 1% stock (Sigma, cat. no. V5265)

MNA-specific reagents

  • Hydrogen peroxide solution 30% (vol/vol) (Merck, cat. no. H1009)

  • Anti-spike rabbit polyclonal antibody (Sino Biologicals, cat. no. 40592-T62)

    Critical

    The MNA assay has been developed and optimized for this primary detection antibody. Other antibodies would require optimization of the immunostaining stage of the procedure.

  • Anti-rabbit IgG goat–horseradish peroxidase conjugate (Invitrogen, cat. no. G21234)

    Critical

    The MNA assay has been developed and optimized for this secondary conjugate. Other antibodies would require optimization of the immunostaining stage of the procedure.

  • Substrate, TrueBlue (SeraCare KPL, cat. no. 5510-0050)

    Critical

    The MNA assay has been developed and optimized for this substrate. Other substrates would require optimization of the immunostaining stage of the procedure.

PNA-specific reagents

  • Dulbecco’s Modified Eagle Medium (DMEM; Gibco, cat. no. 11960-085)

  • ONE-GLO EX Luciferase assay system (Promega, cat. no. E8110)

  • ESF-SFM medium (Expression Systems, cat. no. 98-001)

  • FreeStyle MAX reagent (Thermo Fisher Scientific, cat. no. 16447100)

  • Accutase (Gibco, cat. no. A11105-01)

  • Opti-Pro SFM medium (Thermo Fisher Scientific, cat. no. 12309050)

  • Pseudotyped ΔG-luciferase (G*ΔG-luciferase) rVSV (Kerafast, cat. no. EH1020-PM)

  • pSF-CMV-Amp CMV promoter plasmid (Sigma, cat. no. OGS2) containing the SARS-CoV-2 spike ΔCT encoding gene (ΔCT = deletion the last 19 amino acids of the spike glycoprotein, corresponds to the cytoplasmic tail); the plasmid will need to be generated (not available from the authors or deposited in a bank)

  • 293-ES cells (Expression Systems, cat. no. 94-007F)

Equipment

Equipment required for all assays

  • Sterile 96-well, low-absorption, V-bottomed lidded plates (Thermo Fisher Scientific, cat. no. 249935)

  • Sterile vent/close T175 and T25 cell culture flasks (Thermo Fisher Scientific, cat. nos. 159920 and 156340)

  • Disposable counting chamber, C-chip (NanoEntek, cat. no. DHC-N01)

  • Laboratory single and multichannel pipettes (e.g., Rainin LTS, cat. nos. L-1000XLS+, L-200XLS+, L-20XLS+ and L12-300XLS+)

  • Class II microbiological safety cabinet (e.g., Contained Air Solutions, cat. no. BioMAT 2)

  • Laboratory incubators capable of 37 °C (with additional CO2 control for PNA; e.g., Eppendorf, cat. no. 6734000012)

  • Laboratory refrigerator (2–8 °C; e.g., Fisher Scientific, cat. no. 11317941) and freezer (−15 to −30 °C; e.g., Fisher Scientific, cat. no. 15588229)

  • Laboratory ultralow freezer (≤−60 °C; e.g., Fisher Scientific, cat. no. 15830620)

  • Orbital 96-well plate shakers (e.g., VWR, cat. no. 444-9143 with rack system VWR, cat. no. 444-0519)

  • Inverted phase-contrast microscope (e.g., Leica microsystems, cat. no. 11526216, with accessories, cat. nos. 13613532, 11526118, 11526111, 11506271, 11506272)

  • Microcentrifuge (e.g., Thermo Fisher Scientific, cat. no. 75002410)

  • 96-well plate washer with cell-based assay capability (BioTek, cat. no. 405 TSUS)

  • SoftMax Pro Software version 7.0.3 or greater (Molecular Devices, cat. no. SMP7X GXP SINGLE COMP or SMP7X GXP SERVER)

  • Laboratory water bath (e.g. Fisher Scientific, cat. no. 12718898)

PRNT/MNA-specific equipment

  • Class III microbiological safety cabinet (MSCIII) or suitable equivalent (e.g., flexible film isolator) (e.g., Contained Air Solutions, built to order; contact supplier directly)

  • Plastic boxes with airtight, leakproof, four-sided lockable lid (LocnLock 5.5 L, cat. no. HPL836)

  • ImmunoSpot S6 Ultra-V analyser with BioSpot counting module (Cellular Technologies Europe, contact supplier directly) (only required for MNA)

  • Sterile 96-well, tissue culture treated, flat-bottomed lidded plates (Thermo Fisher Scientific, cat. no. 161093) (only required for MNA)

PNA-specific equipment

  • SpectraMax i3x plate reader (Molecular Device, contact supplier directly)

  • Vacuum pump system (e.g., Fisher Scientific, cat. no. 12911151)

  • 0.22 μm filtration unit (Sigma, cat. no. SCGPU05RE)

  • Sterile 96-well, polypropylene, U-bottomed lidded plates (Thermo Fisher Scientific, cat. no. 267334/264623)

  • Sterile 96-well flat-bottom white plates (Corning, cat. no. 3917)

Reagent setup

PRNT/MNA-specific reagent setup

PRNT/MNA maintenance medium (10% FCS)

To one 500 ml bottle of MEM, add 5 ml of l-glutamine, 5 ml of nonessential amino acids, 13 ml of HEPES buffer (1M) and 50 ml of heat-inactivated FCS. Can be aseptically made in advance and stored at 2–8 °C for up to 2 months.

PRNT/MNA assay diluent (1% FCS)

To one 500 ml bottle of MEM, add 5 ml of l-glutamine, 5 ml of nonessential amino acids, 13 ml of HEPES buffer (1M), 5 ml of heat-inactivated FCS and 5 ml of 100× antibiotic-antimycotic solution. Can be aseptically made in advance and stored at 2–8 °C for up to 2 months.

PRNT/MNA overlay medium

Prepare 2% (for MNA) or 3% (for PRNT) (wt/vol) CMC solution in water and sterilize by autoclaving. Can be aseptically made in advance and stored at ambient temperature for up to 2 months.

Critical

CMC forms clumps easily when attempting to dissolve it in water. To successfully make this reagent, heat the water to ~50 °C in a microwave and add the CMC powder very gradually with regular shaking.

In a class II safety cabinet, aseptically prepare double-strength medium by mixing 142 ml of sterile bottled water with 50 ml of 10× MEM, 15 ml of 7.5% (wt/vol) sodium bicarbonate solution, 5 ml of l-glutamine solution, 5 ml of nonessential amino acids, 13 ml of 1 M HEPES and 20 ml of FCS. Can be made aseptically in advance and stored at 2–8 °C for up to 2 months.

On the day of the assay, mix the CMC solution/paste 1:1 with the double-strength medium, add antibiotic-antimycotic solution to a final concentration of 1× and mix thoroughly by shaking. Warm to 37 °C after preparation, and use within 10 h.

PRNT/MNA fixative

Add 200 ml formalin (40% (wt/vol) formaldehyde solution) to 800 ml of PBS.

Caution

Formalin is a hazardous chemical, and this procedure must be performed in a fume hood or other safety cabinet ducted to atmosphere; wear protective gloves when handling formalin and the prepared fixative.

PNA-specific reagent setup

PNA maintenance medium/assay diluent

To one 1,000 ml bottle of DMEM, add 10.9 ml of l-glutamine, 10.9 ml sodium pyruvate, 10.9 ml of MEM nonessential amino acids and 54.5 ml of heat-inactivated FCS. Can be aseptically made in advance and stored at 2–8 °C for up to 1 month.

Procedure

Procedure 1: PRNT

Timing 1 week

Critical

Before starting this assay, it is essential to inactivate components of the complement system by following the sample-preparation procedure in Box 1. Virus stock solutions must also be prepared in advance by following the procedure in Box 2. When using a new virus stock for the first time, wild-type virus rough and fine sighting experiments must be performed (Box 3).

Critical

All steps in this procedure must be performed aseptically in a class II or class III (where indicated) safety cabinet to ensure the sterility of the assay.

Stage I: serum dilution

Timing 1–2 h

  1. 1

    Day 0: seed 24-well tissue culture plates with 3 × 105 Vero E6 cells/ml (500 μl/well) in PRNT/MNA maintenance medium.

    Critical step

    Maintain cultures of Vero E6 cells such that they are in logarithmic growth (slightly subconfluent) when they are trypsinized, diluted and seeded into plates. Also ensure that the cells are homogeneous and well suspended.

    Critical step

    To ensure even distribution of the cells, rock the 24-well plates at least five times from front to back and side to side to ensure even distribution of the cells, and do not swirl, which can cause the cells to cluster in the middle of the well.

  2. 2

    Stack and seal the plates into a lid-lock plastic box containing several layers of paper towel that have been moistened with ~100 ml of sterile water (for a 5.5 L box). Incubate the plates at 37 °C for a minimum of 20 h before use in the assay.

  3. 3

    Day 1: remove heat-inactivated serum from the freezer, and thaw at ambient temperature. Record any unusual observations of the test samples, e.g., hemolysis or lipemia. These observations can be added to the report along with ND50 measurements.

    Critical step

    Check that the cells seeded on day 0 are ≥85% confluent and are forming an even monolayer before starting this step.

  4. 4

    Dispense PRNT/MNA assay diluent into a 96-well V-bottom plate as follows: into wells C1 to H1 and A7 to H7, dispense 135 μl/well; into wells A6 and B6, dispense 150 μl; and into all other wells, dispense 75 μl. The layout of each 96-well plate (Fig. 2a) corresponds to four 24-well plates (Fig. 2b for the control wells, and three of Fig. 2c for the samples).

    Fig. 2: Typical plate layout of the PRNT.
    figure 2

    a, Due to the plaque assay element of the assay, each 96-well neutralization plate requires four 24-well cell plates. b, The first 24-well cell plate contains the VOC and reference wells. c, The remaining three 24-well cell plates contain samples 1 to 6. VOC contains no serum and is used to determine the foci count (100% unneutralized) for the analysis. NVC contains no serum or virus and is used to ensure that cell monolayers are of good quality and that no virus cross-contamination has occurred during assay setup.

    Critical step

    It is not recommended to exceed two 96-well plates, as more than this can affect the VOC reaching acceptability criteria owing to the additional manipulation times. However, only one control plate per assay is required (i.e., a maximum of 14 samples per control plates).

  5. 5

    Predilute any serum in PRNT/MNA assay diluent that has already been determined to have a neutralizing titer of greater than 1/640. The reference serum is also likely to need predilution to ensure that the 50% neutralization dose falls within the range of the assay, depending upon the source.

    Critical step

    The first dilution into the plate always gives a 1/20 final dilution, so this must be borne in mind when performing any predilution; e.g., if a dilution series starting at 1/640 is required, perform a predilution of 1/32 (given the additional 1/20 performed in Step 6, the first dilution will be 1/640).

  6. 6

    To the first two wells of each sample position (i.e., for sample 1, wells E1 and F1), pipette 15 μl/well of the sample serum (prediluted serum).

    Critical step

    This dilution is a 1/10 of serum in medium, but later in the procedure (prior to neutralization), an equal volume of virus is added to each well, doubling this initial dilution to 1/20.

  7. 7

    Continue to add test serum to the remaining positions on the plate.

  8. 8

    Add the (prediluted) reference serum to wells C1 and D1.

  9. 9

    With a multichannel pipette set to 75 μl, double-dilute the serum (six to eight mixes at each column) across the plate, discarding the final 75 μl at the end of each row.

    Critical step

    At the end of this step, all wells should contain 75 μl except for the two no-virus control (NVC) wells A6 and B6, which should contain 150 μl.

  10. 10

    Take the diluted serum in the neutralization plates to the containment level 3 laboratory.

Stage II: virus addition and neutralization

Timing 1.5 h

Caution

Stages II to V involve the use of live SARS-CoV-2 virus and must be performed within a class III safety cabinet within a BSL3 facility by trained and competent operators.

  1. 11

    Thaw a single-use vial of virus using hand heat, and dilute it to the working dilution determined previously (Box 3). Dilute the virus in PRNT/MNA assay; allow for a volume of 9 ml of diluted virus per plate.

  2. 12

    Using a multichannel pipette, add 75 μl/well of the diluted virus to each well of the V-well neutralization plate except for the NVC wells (A6 and B6).

  3. 13

    Stack and seal the plates into a lid-lock plastic box containing several layers of paper towel that have been moistened with ~100 ml of sterile water (for a 5.5 L box). Incubate the plates at 37 °C for 1 h (1.5 h max) to allow neutralization to occur.

    Caution

    Ensure that the sealed box is externally decontaminated before removing it from the safety cabinet or flexible film isolator.

Stage III: virus adsorption

Timing 1.5 h

  1. 14

    In the cell culture laboratory, take the 24-well plate containing the Vero E6 cells into a class II safety cabinet. Using the cell-capable plate washer, run a cell wash program, which should remove 250 μl of the maintenance medium from the cells. Then perform two 500 μl washes with DPBS, leaving the second wash in the wells.

    Critical step

    To prevent the cells from drying out during the assay, a residual volume of ~250 μl is always left on the cells. At the end of this step, the wells of the cell plate each contain ~750 μl of DPBS.

  2. 15

    Transfer the washed cell plates to the containment level 3 laboratory, label them to match the numbering used on the neutralization plates and place them in the safety cabinet.

  3. 16

    Take the box containing the neutralization plates back into the safety cabinet.

  4. 17

    Using a stripette, remove ~500 μl of DPBS from all wells of the cell plate.

    Critical step

    To avoid the risk of cell monolayers drying out in the high airflow of the class III safety cabinet, perform Steps 17 and 18 one plate at a time.

  5. 18

    Transfer 100 μl of the neutralized virus–antibody mixture from the neutralization plate into the cell plate. This is best performed one row at a time, working from left to right (i.e., strongest serum dilution to weakest, which equates to fewest plaques to most plaques) and changing tips after each row to avoid sample-to-sample contamination.

  6. 19

    Rock the plates at least five times from front to back and side to side.

    Critical step

    To ensure even distribution of the virus, rock the 24-well plate, and do not swirl, which can cause the virus to cluster in the middle of the well.

  7. 20

    Stack and seal the plates into a lid-lock plastic box containing several layers of paper towel that have been moistened with ~100 ml of sterile water (for a 5.5 L box). Incubate the plates at 37 °C for 1 h (1.5 h max) to allow adsorption to occur.

    Caution

    Ensure that the sealed box is externally decontaminated before removing it from the safety cabinet or flexible film isolator.

Stage IV: addition of overlay medium and incubation

Timing 25 min to 1 h

  1. 21

    Take the box containing the adsorption plates back into the safety cabinet.

  2. 22

    Without removing the inoculum, gently add 500 μl of warmed overlay medium to each well of the plate. This is best performed using a stripette owing to the high viscosity of the overlay medium.

  3. 23

    Stack and seal the plates into a lid-lock plastic box containing several layers of paper towel that have been moistened with ~100 ml of sterile water (for a 5.5 L box). Incubate the plates at 37 °C for 5 d to allow plaques to form.

    Caution

    Ensure that the sealed box is externally decontaminated before removing it from the safety cabinet or flexible film isolator.

Stage V: fixing and inactivation of virus

Timing ~12 h

  1. 24

    Take the boxes containing the plates into the MSCIII, and add 500 μl of PRNT/MNA fixative to each well of the assay plates.

  2. 25

    Fumigate the MSCIII overnight with formaldehyde vapor, or make the plates safe for transferring out of the containment level 3 laboratory according to local safety protocols.

    Critical step

    The addition of formalin to the wells inactivates the SARS-CoV-2 virus as well as chemically fixing the cell monolayers. Fumigation should ensure that no residual virus on lids or between wells remains viable. This critical step is an important safety check because, from this point on, the plates are considered noninfectious.

    Caution

    To ensure a good seal of the box, it is good practice to lightly grease the rubber or silicone lid gasket with silicone/vacuum grease after each use.

Stage VI: staining, counting and scoring of plaque counts

Timing ~1 d

  1. 26

    Remove fixed medium from all wells, and collect into a formaldehyde waste receptacle before removing from the MSCIII.

    Caution

    Formaldehyde is a dangerous chemical. Inactivate waste formaldehyde according to local protocols before disposal.

  2. 27

    Wash plates by submerging in copious amounts of tap water to remove traces of formaldehyde.

  3. 28

    Add 0.2% (wt/vol) crystal violet stain to all wells. The volume is not critical but must be sufficient to cover the monolayer (~250 µl). Stain for 5–15 min following addition to the last well.

  4. 29

    Aspirate or pour off the crystal violet solution, and wash plates twice with copious amounts of tap water.

    Caution

    Crystal violet solution is toxic to the aquatic environment, so waste must be collected and disposed of according to local protocols.

  5. 30

    Tap plates on absorbent material to remove excess liquid, and allow to air dry before counting.

  6. 31

    Manually count plaques using a white light box. Use a nonpermanent marker to place a single dot on recognized plaques to prevent double-counting. Record the counts on the plate next to each well. Do not use a microscope, and count only macroscopically visible plaques. If multiple plaques are close together but can be clearly distinguished, these can be counted as separate plaques. If a large plaque does not have internal borders (suggesting multiple plaques growing closely together), this should be counted as a single plaque.

  7. 32

    Collate the counts into a spreadsheet for analysis.

  8. 33

    Ask a second trained operator to check the spreadsheet against information originally recorded on the assay plates to ensure that all the data have been correctly captured.

  9. 34

    In Microsoft Excel, generate a new spreadsheet and limit the plaque counts to the average of the VOC counts (Fig. 3a).

    Fig. 3: Protocol for calculating the ND50 for serum by PRNT assay using Probit analysis in R.
    figure 3

    a, Plaque counts should be limited to the average of the VOC counts. b, Counts should be arranged in a new worksheet, as shown, and saved as a .csv file. c, Following analysis using the R script supplied, a graph is generated and automatically saved as an .EMF file. d, An example of a good result with a full curve and tight confidence intervals. e, An example of a graph generated for a nonneutralizing sample with a poor curve.

  10. 35

    In a new sheet within Microsoft Excel, generate four column headers, starting in cell A1, labeled as conc, success, failure and VOC. In the ‘conc’ column, input the reciprocal of the dilutions used for the sample being analyzed (e.g., 20, 40, 80, 160, 320, 640). In the ‘failure’ column, input the capped plaque counts for one sample alongside the corresponding dilution. In the ‘VOC’ column, add the average VOC count. To calculate the ‘success’ rate, subtract the plaque count (failure) from the VOC (Fig. 3b).

  11. 36

    Save this worksheet as a .csv file with the sample ID.

  12. 37

    Open the freely available software package ‘R’, and paste in the code supplied in the supplementary files (Supplementary Software 1). When prompted, select the .csv file to be analyzed. R will then generate a graph with the appropriate scale and calculate the median neutralizing dose (ND50) with confidence intervals. The graph will be automatically saved as an .EMF file in the original folder with the same name (Fig. 3c–e).

  13. 38

    Check that the data fulfill the requirements of the PRNT assay suitability criteria presented in Table 1.

    Table 1 System suitability criteria (criteria 1–3) and test sample criteria (criteria 4–9) for the PRNT

Procedure 2: microneutralization assay (MNA)

Timing 3 d

Critical

Before starting this assay, it is essential to inactivate components of the complement system by following the sample-preparation procedure in Box 1. Virus stock solutions must also be prepared in advance by following the procedure in Box 2. When using a new virus stock for the first time, wild-type virus semifine sighting experiments must be performed (Box 4).

Critical

All steps in this procedure must be performed aseptically in a class II or class III (where indicated) safety cabinet to ensure the sterility of the assay.

Stage I: serum dilution

Timing 1–2.5 h

  1. 1

    Day 0: seed 96-well tissue culture plates with 2.5 × 105 cells/ml (100 μl/well) in PRNT/MNA maintenance medium.

    Critical step

    Maintain cultures of Vero E6 cells such that they are in logarithmic growth (slightly subconfluent) when they are trypsinized, diluted and seeded into plates. Also ensure that the cells are homogeneous and well suspended.

  2. 2

    Stack and seal the plates into a lid-lock plastic box containing several layers of paper towel that have been moistened with ~100 ml of sterile water (for a 5.5 L box). Incubate the plates at 37 °C for a minimum of 20 h before use in the assay.

  3. 3

    Day 1: remove heat-inactivated serum from the freezer, and thaw at ambient temperature, recording any unusual observations of the test samples, e.g., hemolysis or lipemia. These observations can be added to the report along with ND50 measurements.

    Critical step

    Check that the cells seeded on day 0 are ≥85% confluent and are forming an even monolayer before starting this step.

  4. 4

    Dispense PRNT/MNA assay diluent into a 96-well V-bottom plate as follows: into wells A1 to H1 and A7 to F7, dispense 135 μl/well; into wells G12 and H12, dispense 150 μl; and into all other wells, dispense 75 μl. An example plate layout for testing of one reference and six sera over six dilutions per sample can be seen in Fig. 4a.

    Fig. 4: Typical plate layout of the MNA and the PNA.
    figure 4

    a, MNA. b, PNA. In this example layout, the reference serum (R1) is prediluted 1/6.25 such that the initial in-plate dilution of 1/250 is achieved. Test sera (S1 to S6) are all tested with no predilution. In the PNA, a second reference (R2) is run without predilution. VOC and PC contain no test serum and are used to determine the foci count and maximum luminance (100% unneutralized) for the analysis, respectively. NVC and cell control (CC) contain no serum or virus and are used to ensure that cell monolayers are of good quality and that no virus cross-contamination has occurred during assay setup.

  5. 5

    Predilute any serum in PRNT/MNA assay diluent that has already been determined to have a neutralizing titer of greater than 1/640. The reference serum is also likely to need predilution to fit within the range of the assay, depending upon the source.

    Critical step

    The first dilution into the plate always gives a 1/20 final dilution, so this must be borne in mind when performing any predilution; e.g., if a dilution series starting at 1/640 is required, perform a predilution of 1/32 (given the additional 1/20 performed in Step 6, the first dilution will be 1/640).

  6. 6

    To the first two wells of each sample position (i.e., for sample 1, wells C1 and D1), pipette 15 μl/well of the sample serum (prediluted serum).

    Critical step

    This dilution is a 1/10 of serum in medium, but later in the procedure (prior to neutralization), an equal volume of virus is added to each well, doubling this initial dilution to 1/20.

  7. 7

    Continue to add test serum to the remaining positions on the plate.

  8. 8

    Add the (prediluted) reference serum to wells A1 and B1.

  9. 9

    With a multichannel pipette set to 75 μl, double-dilute the serum (six to eight mixes at each column) across the plate, discarding the final 75 μl at the end of each row.

    Critical step

    At the end of this step, all wells should contain 75 μl except for the two NVC wells G12 and H12, which should contain 150 μl.

  10. 10

    Take the diluted serum in the neutralization plates to the containment level 3 laboratory.

Stage II: virus addition and neutralization

Timing 1.5 h

Caution

Stages II to V involve the use of live SARS-CoV-2 virus and must be performed within a class III safety cabinet within a BSL3 facility by trained and competent operators.

  1. 11

    Thaw a single-use vial of virus using hand heat, and dilute it to the working dilution determined previously (Box 4). Dilute the virus in PRNT/MNA assay diluent; allow for a volume of 9 ml of diluted virus per plate.

  2. 12

    Using a multichannel pipette, add 75 μl/well of the diluted virus to each well of the V-well (neutralization) plate except for the NVC wells (G12 and H12).

  3. 13

    Stack and seal the plates into a lid-lock plastic box containing several layers of paper towel that have been moistened with ~100 ml of sterile water (for a 5.5 L box). Incubate the plates at 37 °C for 1 h (1.5 h max) to allow neutralization to occur.

    Caution

    Ensure that the sealed box is externally decontaminated before removing it from the safety cabinet or flexible film isolator.

Stage III: virus adsorption

Timing 1.5 h

  1. 14

    In the cell culture laboratory, take the 96-well plate containing the Vero E6 cells into a class II safety cabinet. Using the cell-capable plate washer, run the cell wash program, which should remove 65 μl of the maintenance medium from the cells. Then perform two 100 μl washes with DPBS, leaving the second wash in the wells.

    Critical step

    To prevent the cells from drying out during the assay, a residual volume of ~35 μl is always left on the cells. At the end of this step, the wells of the cell plate each contain ~135 μl of DPBS.

  2. 15

    Transfer the washed cell plates to the containment level 3 laboratory, label them to match the numbering used on the neutralization plates and place them in the safety cabinet.

  3. 16

    Take the box containing the neutralization plates back into the safety cabinet.

  4. 17

    Using a multichannel pipette, remove 100 μl of DPBS from all wells of the cell plate.

    Critical step

    To avoid the risk of cell monolayers drying out in the high airflow of the class III safety cabinet, perform Steps 17 and 18 one plate at a time.

  5. 18

    Transfer 100 μl of the neutralized virus–antibody mixture from the neutralization plate into the cell plate. This is best performed two rows at a time, changing tips after each pair of rows to avoid sample-to-sample contamination.

  6. 19

    Stack and seal the plates into the lid-lock plastic box prepared in Step 13. Incubate the plates at 37 °C for 1 h (1.5 h max) to allow adsorption to occur.

    Caution

    Ensure that the sealed box is externally decontaminated before removing it from the safety cabinet or flexible film isolator.

Stage IV: removal of inoculum and addition of overlay medium

Timing 25 min to 1 h

  1. 20

    Take the box containing the adsorption plates back into the safety cabinet.

  2. 21

    Remove 100 μl of virus–antibody mixture from all wells of the plate. This is best performed two rows at a time, changing tips after each pair of rows to avoid sample-to-sample contamination.

    Critical step

    To avoid the risk of cell monolayers drying out in the high airflow of the class III safety cabinet, perform Steps 21 and 22 one plate at a time.

  3. 22

    Add 100 μl of warmed overlay medium to each well of the plate. This is best performed using the reverse pipetting method owing to the high viscosity of the overlay medium.

  4. 23

    Stack and seal the plate(s) into the lid-lock plastic box prepared in Step 13. Incubate the plates at 37 °C for 22–24 h to allow viral foci to form.

    Caution

    Ensure that the sealed box is externally decontaminated before removing it from the safety cabinet or flexible film isolator.

Stage V: fixing and inactivation of virus

Timing ~12 h

  1. 24

    Take the boxes containing the plates into the MSCIII, and add 100 μl of PRNT/MNA fixative to each well of the assay plates.

  2. 25

    Fumigate the MSCIII overnight with formaldehyde vapor according to local safety protocols.

    Critical step

    The addition of formalin to the wells inactivates the SARS-CoV-2 virus, as well as chemically fixing the cell monolayers. Fumigation should ensure that no residual virus on lids or between wells remains viable. This critical step is an important safety check because, from this point on, the plates are considered noninfectious.

    Caution

    To ensure a good seal of the box, it is good practice to lightly grease the rubber or silicone lid gasket with silicone/vacuum grease after each use.

Stage VI: immunostaining of virus-specific foci

Timing ~1 d

  1. 26

    Remove fixed medium from all wells using a multichannel pipette and collect into a formaldehyde waste receptacle before removing from the MSCIII.

    Caution

    Formaldehyde is a dangerous chemical. Inactivate waste formaldehyde according to local protocols before disposal.

  2. 27

    Using a cell-capable plate washer, remove residual fixed medium by performing six 300 μl washes with distilled water.

    Critical step

    Ensure that the plate washer has been set up such that the aspiration and dispense jets do not come into contact with the fixed cell monolayer and that all aspiration and dispensing speeds are set to the minimum setting.

  3. 28

    Prepare hydrogen peroxide at 0.3% (wt/vol) in water from the 30% (wt/vol) stock, and add 100 μl/well to each plate. Allow 11 ml of diluted hydrogen peroxide per plate. Incubate on a plate shaker at ambient temperature for 20–25 min.

    Critical step

    This step removes any residual endogeneous peroxidase activity from the cells which, if not performed, can give rise to background ‘spots’ on the plates. Hydrogen peroxide has a very short lifespan when diluted in water, so make up this solution immediately before use.

    Caution

    Hydrogen peroxide is harmful, so operators must wear gloves, laboratory coats and laboratory eyewear for this step.

  4. 29

    Perform two 300 μl washes with PBS using the plate washer.

  5. 30

    Add 100 μl/well of diluted anti-SARS-CoV-2 antibody (1/2,000) to all wells on the microplates. Allow 11 ml of diluted antibody per plate. Incubate on a plate shaker at ambient temperature for 55–65 min.

  6. 31

    Perform two 300 μl washes with PBS using the plate washer.

  7. 32

    Add 100 μl/well of diluted anti-rabbit IgG conjugate (1/4,000) to all wells on the microplates. Allow 11 ml of diluted antibody per plate. Incubate on a plate shaker at ambient temperature for 55–65 min.

  8. 33

    Perform two 300 μl washes with PBS using the plate washer.

  9. 34

    Allow 11 ml of substrate per plate; prefilter the required amount of TrueBlue substrate using a syringe-mounted 0.45 μm filter. Add 100 μl of filtered substrate to all wells on the microplates. Incubate on a plate shaker at ambient temperature for 10 (±2) min.

    Critical step

    If processing a large number of plates, it is better to batch them into groups of four or five plates to avoid leaving the substrate on the plates for too long.

  10. 35

    Perform four 300 μl washes with water using the plate washer.

    Critical step

    It is important to perform this step with water and not PBS, as the stained foci will rapidly fade if PBS is used.

  11. 36

    Invert the plates and tap them on a paper towel before drying them (generally overnight), face up without lids attached, in a class II safety cabinet or laminar flow hood.

Stage VII: automatic foci counting and neutralization dose determination

Timing ~1 d

  1. 37

    Wipe the plate bases with 70% (vol/vol) isopropanol solution, and load the plate in the correct orientation into the ImmunoSpot analyzer (there is a marking on the plate loading arm adjacent to well A1).

  2. 38

    Open the BioSpot software to scan and count the stained foci using the parameters in Table 2.

    Table 2 BioSpot counting parameters for the MNA
  3. 39

    Paste the counted data from the Excel spreadsheet file produced by the ImmunoSpot analyzer into the SoftMax Pro protocol file.

    Critical step

    The protocol has been developed to fit the focus count data using a four-parameter logistic regression with upper and lower constraints (0–100% of the mean VOC counts).

  4. 40

    Check that the data in SoftMax Pro fulfill the requirements of the MNA assay suitability criteria presented in Table 3. Any plates or samples that do not meet these criteria will require retesting.

    Table 3 System suitability criteria (criteria 1–7) and test sample criteria (criteria 8 and 9) for the MNA

Procedure 3: PNA

Timing 2 d

Critical

Before starting this assay, it is essential to inactivate components of the complement system by following the sample-preparation procedure in Box 1. Pseudovirus stock solutions must also be prepared in advance by following the procedure in Box 5. When using a new pseudovirus stock for the first time, sighting experiments must be performed (Box 6).

Caution

Perform all steps of this procedure within a class II microbiological safety cabinet.

Stage I: plate setup and serum dilution

Timing 2 d

  1. 1

    Day 0: seed white flat-bottomed 96-well plates with 2.0 × 105 cells/ml (100 μl/well) of Vero E6 cells in PNA maintenance medium and incubate overnight at 37 °C/5% CO2. At the same time, seed one clear plate exactly as for the white plates in parallel; this is used to assess the quality of the cell monolayer in the next step.

    Critical step

    Ensure that the assay is performed in white cell culture plates, as these are critical to assess the luminescence per well while avoiding cross-talk that could occur using transparent plates. Multiple types of plates were evaluated during development, but white cell culture plates were demonstrated to provide the best signal-to-noise ratio.

  2. 2

    Day 1: ensure that the cells seeded on day 0 are >80% confluent, using the cells seeded into the clear plate.

  3. 3

    Thaw a vial of pseudotyped virus on ice, and keep on ice until ready for dilution.

  4. 4

    Using a multichannel pipette, add 30 μl of PNA assay diluent to rows B to H of a round-bottomed 96-well plate (the neutralization plate) and 54 μl to the wells of row A.

  5. 5

    Predilute any serum in PNA assay diluent that has already been determined to have a neutralizing titer of greater than 1/1,280. The reference serum is also likely to need predilution to get it into the range of the assay, depending upon the source.

    Caution

    The first dilution into the plate always gives a 1/20 final dilution, so this must be borne in mind when performing any predilution; e.g., if a dilution series starting at 1/1,280 is required, perform a predilution of 1/64 (given the additional 1/20 performed in Step 7, the first dilution will be 1/1,280).

  6. 6

    Add 6 μl of test/reference serum or prediluted test/reference, in duplicate, to the wells in row A of the microplate (Fig. 4b).

  7. 7

    With a multichannel pipette set to 30 μl, double-dilute the serum (6 –8× mixes at each row) down the plate, discarding the final 30 μl after mixing into row G.

    Critical step

    At the end of this step, all wells of the neutralization plate should contain 30 μl.

Stage II: pseudotyped virus addition and neutralization

Timing ~1.5 h

  1. 8

    Perform gentle up-and-down mixing of the thawed pseudotyped virus stock solution before preparing the working dilution.

    Critical step

    Do not vortex the pseudovirus to avoid damaging the virions.

  2. 9

    Prepare sufficient volume (~3.5 mL/plate) of pseudotyped virus working dilution in PNA assay diluent using the dilution ratio predicted in Stage I to yield a target of 150,000 RLUs/well.

  3. 10

    Into the neutralization plate, add 30 μl of diluted virus to all wells of rows A to G and H5 to H12 (PC). To rows H1 to H4, add 30 μl of PNA assay diluent (cell controls).

  4. 11

    Replace the lid and incubate the plate(s) for 55–65 min at 37 °C/5% CO2.

Stage III: cell monolayer infection and incubation

Timing 18–22 h

  1. 12

    Using a vacuum pump or multichannel pipette, remove the cell growth medium from the flat-bottomed cell seeded plates (cell plate) from Step 1.

    Critical step

    This needs to be performed carefully to avoid scratching or otherwise disturbing the cell monolayers.

  2. 13

    Using a multichannel pipette, transfer 50 μl of the serum/pseudotyped virus mixtures from the neutralization plate into the cell plate.

  3. 14

    Replace the lid and incubate the cell plates for 18–22 h at 37 °C/5% CO2

Stage IV: cell lysis, luminescence measurement and determination of neutralization titers

Timing 18–22 h

  1. 15

    Thaw and equilibrate to ambient temperature sufficient ONE-Glo EX luciferase assay reagent for all tested plates; allow at least 5.5 ml of diluted luciferase reagent per plate.

    Critical step

    The luciferase assay reagent is light sensitive, so take measures to avoid light exposure and wrap tubes in aluminium foil.

  2. 16

    Dilute luciferase assay 1/2 in PNA assay diluent.

  3. 17

    Remove the cell plates from the incubator, and use a vacuum pump or multichannel pipette to remove the cell growth medium.

  4. 18

    Add 50 μl/well of diluted luciferase reagent to all wells of the plate. Incubate the plates for 3 min at room temperature (20–25 °C) with shaking set at 600 rpm.

    Critical step

    Avoid air bubbles; reverse pipetting the luciferase reagent into the plate is a good way to achieve this.

  5. 19

    Switch on the SpectraMax i3x plate reader and launch SoftMax Pro software (Table 4).

    Table 4 Spectramax i3X configuration parameters for the PNA
  6. 20

    Read the luminescence of each plate; the SoftMax Pro protocol determines midpoints of each curve using 4PL regression and outputs each sample’s median neutralizing titer (NT50). Check that the data in SoftMax Pro fulfill the requirements of the PNA assay suitability criteria listed in Table 5. Any plates or samples that do not meet these criteria will require retesting.

Timing

  • Box 1, sample preparation for all assays: 1 h

  • Box 2, production of wild-type virus stocks for PRNT and MNA: 5 d

  • Box 3, wild-type virus rough and fine sighting experiments for PRNT: 2 weeks

  • Box 4, wild-type virus semifine sighting and regression experiments for MNA: 3 d

  • Procedure 1, PRNT: 1 week

  • Stage I, serum dilution: 1–2 h

  • Stage II, virus addition and neutralization: 1.5 h

  • Stage III, virus adsorption: 1.5 h

  • Stage IV, addition of overlay medium and incubation: 25 min to 1 h (followed by 5 d incubation)

  • Stage V, fixing and inactivation of virus: ~12 h

  • Stage VI, staining, counting and scoring of plaque counts: ~1 d

  • Procedure 2, MNA: 3 d

  • Stage I, serum dilution: 1–2.5 h

  • Stage II, virus addition and neutralization: 1.5 h

  • Stage III, virus adsorption: 1.5 h

  • Stage IV, removal of inoculum and addition of overlay medium: 25 min to 1 h

  • Stage V, fixing and inactivation of virus: ~12 h

  • Stage VI, immunostaining of virus-specific foci: ~1 d

  • Stage VII, automatic foci counting and neutralization dose determination: ~1 d

  • Box 5, creation and production of pseudotyped virus for PNA: 2 d

  • Box 6, pseudotyped virus dose sighting experiment for PNA assays: 2 d

  • Procedure 3, PNA: 3 d

  • Stage I, plate setup and serum dilution: 2 d

  • Stage II, pseudotyped virus addition and neutralization: ~1.5 h

  • Stage III, cell monolayer infection and incubation: 18–22 h

  • Stage IV, cell lysis, luminescence measurement and determination of neutralization titers: ~1.5 h

Anticipated results

PRNT

Following the PRNT procedure, a wild-type virus stock requiring dilutions in the range of 1/10 should have been generated. A single 1 ml vial of this stock should be sufficient to set up four assay plates (one control plate and three sample plates) with an expected VOC of 70 pfu per well. The protocol should yield neutralization curves similar to those shown in Fig. 3d. Care should be taken with a new reference serum to determine a starting dilution that generates as full a curve as possible (a response of 0–100% of VOC).

It is good practice to track critical parameters such as VOC counts and reference ND50 using trending worksheets, which can give an indication of where changes have occurred in assay performance and provide assurance that assays are in control and operating consistently. It is convenient to use spreadsheet software such as Microsoft Excel to collect and monitor these trending data. An example of a nonneutralizing and highly neutralizing serum are given in Fig. 3e and the effects on the graphs demonstrated. Common problems seen after staining the plates can be found in Fig. 7, and a troubleshooting guide can be found in Table 6.

Table 5 System suitability criteria (criteria 1–4) and test sample criteria (criteria 5 and 6) for the PNA
Table 6 Troubleshooting for the PRNT

MNA

Following the MNA procedure, a wild-type virus stock requiring dilutions in the range of 1/500 or greater should have been generated. A single 1 ml vial of this stock should be sufficient to set up ~60 assay plates with an expected VOC of 100–250 countable foci per well. This protocol should yield neutralization curves similar to those shown in Fig. 5b. Implementers should take care with a new reference serum to determine a starting dilution that generates as full a curve as possible (a response of 0–100% of VOC); an example of a full curve can be seen in Fig. 5b. For clinical trial material, the EC50 generated by the software is reported as the median neutralization dose (ND50). The SoftMax protocol has been designed to validate the foci count data and the results of the regression and indicate whether results are reportable or not. Negative sera are reported as less than the lower limit of quantification (<LLOQ), and an example curve can be seen in Extended Data Fig. 6a. Strongly positive sera are reported as greater than the upper limit of quantification (>ULOQ) and may be retested at a higher initial starting dilution to determine the ND50; an example of such a strongly neutralizing serum can be seen in Extended Data Fig. 6b. Common problems seen after imaging the plates can be found in Fig. 7, and a troubleshooting guide can be found in Table 7.

Fig. 5: Example data generated by the SoftMax Pro protocol.
figure 5

a, The raw foci counts are shown in the Values column, and the percentage neutralization (of the VOC average foci count) is shown in the MeanFinal column. b, An example four-parameter regression output from the SoftMax Pro protocol. The top panel shows the sample regression curve (green) against the reference curve (red). Neutralization doses (ND50) are shown in the column EC50; reference = 3,884 and sample = 204.1.

Table 7 Troubleshooting for the MNA

It is good practice to track critical parameters using trending worksheets, which can give an indication of where changes have occurred in assay performance and provide assurance that assays are in control and operating consistently. Useful parameters to track include VOC counts, reference ND50 and measures of operator performance such as average square residuals of the 4PL fits. It is convenient to use spreadsheet software such as Microsoft Excel to collect, graphically summarize and monitor these trending data.

PNA

Following the PNA procedure, a pseudotyped VSV-spike stock requiring dilutions in the range of 1/50 should have been generated. A single 1 ml vial of this stock should be sufficient to set up ~15 assay plates (60 samples) with an expected VOC of 150,000 RLUs per well. This range was calculated based on the results of runs performed by multiple analysts over several days on a single instrument. However, if additional instruments or critical reagents are to be used, these will need to be bridged. It should be noted that the range of RLUs should be amended only if it can be shown that this does not alter the titers obtained. The PNA protocol should yield neutralization curves similar to those shown in Fig. 6. Care should be taken with a new reference serum to determine a starting dilution that generates as full a curve as possible (a response of 0–100% of the VOC).

Fig. 6: Typical neutralization curves expected from the PNA.
figure 6

Poorly neutralizing sera (yellow) do not cross the dotted line corresponding to 50% of the luminance value of the pseudotyped virus control. Sera in the test range (blue and orange) all cross the 50% neutralization and yield reportable titers. Highly neutralizing sera (green) do not cross the 50% neutralization and must be retested at a higher initial dilution.

Fig. 7: Examples of potential issues that can occur during the PRNT and MNA assays.
figure 7

ad, PRNT. ei, MNA. a, Typical PRNT plaque image from a successful experiment. b, Drying of monolayers during assay setup can result in ‘half-moon’ or patchy staining. c, When monolayers are overconfluent, cells can accumulate in the middle of the wells and detach during assay setup. d, If CMC overlay is not viscous enough, plaques can have a large or comet-shaped morphology. e, Typical MNA foci image from a successful experiment. f, Enlarged MNA foci caused by excessive incubation or insufficiently viscous overlay. gh, Tears in the monolayer caused by ‘rough’ pipetting (g) or incorrect washer settings (h). i, Foreign material obscuring well.

It is good practice to track critical parameters using trending worksheets, which can give an indication of where changes have occurred in assay performance and provide assurance that assays are in control and operating consistently. An example of a nonneutralizing and highly neutralizing serum is given in Fig. 6 and the effects on the graphs demonstrated. A troubleshooting guide for this assay can be found in Table 8.

Table 8 Troubleshooting for the PNA

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.