TITLE: SARS-CoV-2 live virus neutralization assay
AUTHORS: Ria   Lassauniere, Anders  Frische

[DESCRIPTION]
Virus neutralization assays provide a means to quantitate functional antibody responses that block virus infection. These assays are instrumental in defining vaccine and therapeutic antibody potency, immune evasion by viral variants, and post-infection immunity. This protocol describe a means by which to measure neutralizing antibody titers in a live virus microneutralization assay specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 microneutralization assay is based on the influenza virus microneutralization assay described by WHO. In this assay, SARS-CoV-2 clinical isolates are pre-incubated with serial diluted antibody and added to Vero E6 cells. Replicating virus is quantitated by enzyme-linked immunosorbent assay (ELISA) targeting the SARS-CoV-2 nucleocapsid protein and the 50% virus inhibition titer calculated. The assay has been systematically optimized at critical test parameters that include virus titration, assay linearity, number of cells, viral dose, incubation period post-inoculation, and normalization methods.

This protocol has three parts:
Part I: Determination of the 50% tissue culture infectious dose per milliliter (TCID50/mL).
Part II: ELISA.
Part III: Virus microneutralization assay.

[BEFORE_START]
The assay requires several steps where liquids are added to and aspirated from live cell monolayers in a 96-well tissue culture plate. Ideally, optimize handling technique to minimize cell loss at these steps before running the full protocol. Cell loss will reduce assay signal and introduce variance.

[GUIDELINES]
No specific guidelines.

[STEPS]
SECTION: Seeding Vero E6 cells into 96-well plates
1. Seeding Vero E6 cells (from T75 flask)
 

SECTION: Seeding Vero E6 cells into 96-well plates
1.1. Verify that Vero E6 cells growing in maintenance media are actively growing (monolayer 70-95% confluent).
 

SECTION: Seeding Vero E6 cells into 96-well plates
1.2. Remove the maintenance media from the flask with a serological pipette and discard.
SECTION: Seeding Vero E6 cells into 96-well plates
1.3. Add 5 mL of phosphate buffered saline (PBS) to the cell monolayer and tilt the flask to cover and wash the whole monolayer. Remove the PBS with a serological pipette.
SECTION: Seeding Vero E6 cells into 96-well plates
1.4. Add 2 mL 10X TrypLE dissociation media to the flask. Tilt the flask to ensure all cells are covered, and remove the dissociation media with a pipette.
 

SECTION: Seeding Vero E6 cells into 96-well plates
1.5. Place the flask horizontally in a 37 °C, 5% CO2 incubator and incubate for 10 minutes.
SECTION: Seeding Vero E6 cells into 96-well plates
1.6. Add 10 mL maintenance media to the flask and gently wash cells from the flask surface. Pipette up and down to mix the single cell suspension.
SECTION: Seeding Vero E6 cells into 96-well plates
1.7. Determine the cell number. Combine 10 μL of the resuspended cells with 10 μL Trypan Blue. Count the cells on a hemocytometer.
 

SECTION: Seeding Vero E6 cells into 96-well plates
1.8. Adjust the cell concentration to 1 × 105 cells/mL with maintenance media and transfer 100 μL to each well in a 96 well plate (10 000 cells per well) using a multichannel pipette.
 

SECTION: Seeding Vero E6 cells into 96-well plates
1.9. Incubate the 96-well plate containing the cells overnight under standard tissue culture conditions (37 °C, 5% CO2) for use the following day.
SECTION: SARS-CoV-2 virus titration
2. SARS-CoV-2 virus titration
 

SECTION: SARS-CoV-2 virus titration
2.1. Thaw a vial of each virus stock just prior to use. Dilute 10 μL virus in 90 μL virus diluent (1:10 dilution).
 

SECTION: SARS-CoV-2 virus titration
2.2. In a sterile 96-well dilution plate, prepare a ½ log (3.16-fold) serial dilution of the viruses in virus diluent (illustrated in the accompanying figure).


SECTION: SARS-CoV-2 virus titration
2.3. Add 158 μL of virus diluent to column 1 and 120 μL of virus diluent to columns 2 to 12.
SECTION: SARS-CoV-2 virus titration
2.4. For each virus stock to be titrated, allocate four rows on the 96-well plate for quadruplicate measurements. Add 17.6 μL of the 1:10 diluted virus to column 1, rows A to D (virus 1) and repeat for virus 2 for rows E to H.
 

SECTION: SARS-CoV-2 virus titration
2.5. Using a multichannel pipette, mix column 1 thoroughly and transfer 55 μL from column 1 to column 2. Discard tips.
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2.6. Using clean tips, mix column 2 thoroughly and transfer 55 μL from column 2 to column 3. Discard tips.
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2.7. Continue with serial dilution to column 11 using clean tips between columns as described in step 2.6. In the final column with virus (column 11), mix thoroughly and discard 55 μL into a disinfectant (e.g. Virkon or sodium hypochlorite).
 

SECTION: SARS-CoV-2 virus titration
2.8. Place plate(s) at 37 °C, 5%CO2 for 1 hour.
 

SECTION: SARS-CoV-2 virus titration
2.9. Remove the 96-well tissue culture plate seeded with 10 000 Vero E6 cells per well the day before from the 37°C, 5%CO2 incubator.
SECTION: SARS-CoV-2 virus titration
2.10. Aspirate the cell culture media from the cell monolayer.
 

SECTION: SARS-CoV-2 virus titration
2.11. Gently wash the cell monolayer twice with 100 μL sterile PBS. Remove the PBS with a pipette.
 

SECTION: SARS-CoV-2 virus titration
2.12. Add 100 μL of serial diluted virus to the Vero E6 cell monolayer in the same order as in the dilution plate.
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2.13. Incubate for 96 hours at 37°C, 5%CO2. Thereafter, the cells are fixed to the plate for the ELISA (described in the Section 3 and 4 of this protocol).
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2.14. Using the optical density values from the ELISA, calculate the 50% tissue culture infectious dose using the method described by Reed and Muench (1938), see attached file.
SECTION: Fixation of SARS-CoV-2 infected cells
3. Fixation of cells
 

SECTION: Fixation of SARS-CoV-2 infected cells
3.1. Prepare fixative and pre-cool at -20°C until just before use.
 

SECTION: Fixation of SARS-CoV-2 infected cells
3.2. Remove the 96-well tissue culture plate with SARS-CoV-2 cells from the 37°C, 5%CO2 incubator.
SECTION: Fixation of SARS-CoV-2 infected cells
3.3. Remove the medium from all the cells in the tissue culture plate.
SECTION: Fixation of SARS-CoV-2 infected cells
3.4. Gently wash the wells with 200 μL PBS.
SECTION: Fixation of SARS-CoV-2 infected cells
3.5. Remove the PBS and add 100 μL cold fixative to each well.
SECTION: Fixation of SARS-CoV-2 infected cells
3.6. Cover the plate with a lid and incubate at room temperature for 10 minutes.
SECTION: Fixation of SARS-CoV-2 infected cells
3.7. Remove fixative with a pipette or by flicking onto stacked paper towel and let the plate air dry (approximately 10-20 minutes).
SECTION: Fixation of SARS-CoV-2 infected cells
3.8. Proceed with ELISA directly (step 4 in this protocol) or add 50 µL 50% glycerol, cover with an adhesive plate seal and store at -20°C until ELISA can be done.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4. Anti-SARS-CoV-2 nucleocapsid ELISA
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.1. Dilute the primary antibody (Cat # bsm-41414; Bioss USA; SARS-CoV-2 nucleocapsid mouse mAb, clone 7E1B) 1:4000 in dilution buffer.
 

SECTION: Enzyme linked immunosorbent assay (ELISA)
4.2. Add 250 μL wash buffer per well to the 96-well plate with fixed cells and soak for 30 seconds at room temperature.
 

SECTION: Enzyme linked immunosorbent assay (ELISA)
4.3. Flick wash buffer out over a sink.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.4. Repeat wash steps twice [repeat steps (4.2 and 4.3) × 2]. After the last wash, gently tap the 96-well plate upside-down on stacked tissue paper to remove excess wash buffer.

SECTION: Enzyme linked immunosorbent assay (ELISA)
4.5. Add 100 μL of the diluted primary antibody to each well.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.6. Cover the plate(s) with a plate sealer and incubate for 5 minutes on shaking incubator (300 rpm) at room temperature and then for 1 hour at 37°C.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.7. Dilute the horse radish peroxidase (HRP)-conjugated secondary antibody (Cat. # A16078; Invitrogen; goat anti-mouse IgG/HRP highly cross-adsorbed) 1:10000 in dilution buffer.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.8. Wash the plate(s) three times with wash buffer as before [repeat steps (4.2 and 4.3) × 3]. After the last wash, gently tap the 96-well plate upside-down on stacked tissue paper to remove excess wash buffer.

SECTION: Enzyme linked immunosorbent assay (ELISA)
4.9. Add 100 μL of the diluted HRP-conjugated secondary antibody to each well.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.10. Cover the plate(s) with a plate sealer and incubate for 5 minutes on shaking incubator (300 rpm) at room temperature and then for 1 hour at 37°C.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.11. Wash the plate(s) five times with wash buffer as before [repeat steps (4.2 and 4.3) × 5].
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.12. Add 250 μL distilled water per well to the 96-well plate and flick out over the sink without an incubation. Repeat twice. After final wash with distilled water, gently tap the plate upside-down on stacked tissue paper to remove excess water.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.13. Add 100 μL 3,3',5,5'-tetramethylbenzidine (TMB; Cat. # 4380; Kementec; TMB One) substrate to each well. Do not cover plate.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.14. Incubate for 15-30 minutes in the dark at room temperature. Monitor colour development.
SECTION: Enzyme linked immunosorbent assay (ELISA)
4.15. Add 100 μL stop solution to all wells.
 

SECTION: Enzyme linked immunosorbent assay (ELISA)
4.16. Read the absorbance of the wells at 450 nm (OD450) with 620 nm as reference.
SECTION: SARS-CoV-2 virus neutralization
5. SARS-CoV-2 virus neutralization
 

SECTION: SARS-CoV-2 virus neutralization
5.1. Prepare sterile 96-well flat bottom plates with 10 000 cells per well as described under Section 1 ‘Seeding Vero E6 cells (from T75 flask)’.
SECTION: SARS-CoV-2 virus neutralization
5.2. Incubate cells overnight at 37°C, 5%CO2.
SECTION: SARS-CoV-2 virus neutralization
5.3. Thaw a vial of virus and place in the biosafety cabinet, mix thoroughly and dilute to 300× TCID50 per 60 μL. Approximately 6 mL/plate is needed. Store on ice or +4°C.
 

SECTION: SARS-CoV-2 virus neutralization
5.4. Add virus diluent to a sterile dilution plate as follows (layout on next page):
 a. 108 μL to row A, wells 1-10
    b. 60 μL to rows B-H, wells 1-10
    c. 60 μL to wells A11 – D11 (virus control)
    d. 60 μL to wells B12 – H12 (virus back titration)
 e. 120 μL to wells E11 – H11 (cells control)
SECTION: SARS-CoV-2 virus neutralization
5.5. Do the serum dilution in a separate sterile 96-well plate
 a. 12 μL serum to the appropriate wells in row A (1:10 serum dilution)
 b. Do a 2-fold serial dilution. Sequentially transfer 60 μL from row A through to row H. Mix thoroughly by pipetting.    Discard 60 μL from row H.
 c. 60 μL diluted serum remains in each well.
SECTION: SARS-CoV-2 virus neutralization
5.6. Do the virus back titration.
a. In column 12, add 120 μL virus to well A12
b. Do a 2-fold serial dilution of virus - transfer 60 μL from one row to the next, start row A through to row 12. Change tips between dilutions.
c. Discard 60 μL from well H12
d. Add 60 μL virus diluent to all wells in column 12 (final volume 120 μL).
 

SECTION: SARS-CoV-2 virus neutralization
5.7. Add 60 μL diluted virus (300× TCID50) to the following wells:
a. All wells in rows A-H, wells 1-10 (test wells)
b. Wells A11 – D11 (virus control)

SECTION: SARS-CoV-2 virus neutralization
5.8. Place plate(s) at 37 °C 5% CO2 for 1 hour.
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5.9. Approximately 45-50 minutes into the incubation period (step 5.8), remove the 96-well tissue culture plate(s) seeded with 10 000 Vero E6 cells the day before from the 37°C, 5%CO2 incubator.

SECTION: SARS-CoV-2 virus neutralization
5.10. Aspirate the cell culture media from the cell monolayer. Do not allow cells to dry out.
 

SECTION: SARS-CoV-2 virus neutralization
5.11. Gently wash the cell monolayer twice with 100 μL sterile PBS. Remove the PBS with a pipette.
 

SECTION: SARS-CoV-2 virus neutralization
5.12. Add 100 μL serum/virus mix to the Vero E6 cell monolayer.
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5.13. Incubate for 22-24 hours at 37°C, 5%CO2.
 

SECTION: SARS-CoV-2 virus neutralization
5.14. After the incubation period, fix the cells as described in Section 3 'Fixation of SARS-CoV-2 infected cells'
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5.15. Perform the ELISA as described in Section 4 'Enzyme linked immunosorbent assay (ELISA)'
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
6. Quality control
Perform the following quality control checks to determine successful execution of the neutralization assay. If the criteria below are not met, the assay should be repeated or optimized by the specific performing laboratory.
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
6.1. The average OD values for the virus controls must be equal to or above 1.00.
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
6.2. The signal-to-noise ratio calculated for the average OD values of the quadruplicate virus controls divided by the average OD values of the quadruplicate cell controls should preferably be above 6.
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
6.3. Determine if the virus test dose (300× TCID50) is acceptable in the virus back titration. In most cases, the test dose of virus is acceptable if the back titration is below or equal to the 50% cut-off value in the 5-6 wells containing the lowest dilutions of test virus.
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
6.4. Confirm that non-specific inhibition is not present on the plate using the negative serum control. The OD450 of the negative serum control for all dilutions should be similar to that observed for the virus control wells.
 

SECTION: Quality control, neutralizing antibody titer calculations, and normalization
6.5. Assess inter-run variability using the positive control. The serum positive control should give titres within +-1.5-fold of the average values obtained in previous tests.
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
7. Determine the serum 50% neutralization titers as follows:
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
7.1. Calculate the optical density value cut-off representing 50% virus infection that represents inhibition of 50% virus infection in the neutralization assay i.e. 50% neutralization.
  

SECTION: Quality control, neutralizing antibody titer calculations, and normalization
7.2. All wells containing diluted serum with an OD value below or equal to the 50% cut-off value are positive for neutralization activity. The reciprocal serum dilution corresponding to that well is the 50% neutralization antibody titre for that serum sample. Serum dilutions are: well A 1:10; well B 1:20; well C 1:40; well D 1:80; well E 1:160; well F 1:320; well G 1:640; and well H 1:1280.

Alternatively, fit a four parameter logistic regression line over all the dilutions for a single sample and calculate the exact 50% neutralization titer as the intercept of the curve with the 50% cut-off OD value.
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
8. Normalize 50% neutralization titers to minimize inter-assay variation
This method can only be applied to exact titers calculated from four parameter logistic regression curves fitted over all serum dilution data points. It requires an exact titer for the same positive control used in all neutralization assays. This exact positive control titer is obtained by running single-use aliquots of the positive control in the neutralization assay on 8-15 different days with optimal cell passage numbers between 8 and 20. The average positive control titer is referred to here as the nominal value.
SECTION: Quality control, neutralizing antibody titer calculations, and normalization
8.1. Use the following equation to determine the normalized 50% neutralization titer for each sample:
  

x is the log10 transformed titer of the sample
Pos is the log10titer of the positive control included on the same assay plate as the sample
Posnom is the average log10 titer of the positive control determined from 8-15 runs denoted as the nominal value of the positive control material.

The second fraction calculates the bias of the result from the positive control according to the nominal “expected” value. Thus, if the positive control in a given run has a titer identical to the nominal value, samples will not be corrected. The first fraction describes the relative size of the titer of the sample compared to the maximum titer in a normal assay (here 1:1280). This weighs each titer according to size. Range is adjusted according to the minimum positive value (log10 10 = 1) to exclude normalization for positive samples at the minimum titer of 10, which is the lower limit of quantification. This exclusion is designed to diminish influence of normalization on the qualitative judgement of borderline samples. The correction increases with titer and is greatest at maximal titer (1280).