An Assay to Measure the Neutralizing Antibody Titer Specific for Respiratory Syncytial Virus

Overview

This video demonstrates an assay for measuring neutralizing antibodies against respiratory syncytial virus (RSV). Pre-incubation with serum containing RSV-neutralizing antibodies neutralizes the virus particles, reducing cellular infection and plaque formation. Virus-infected cells are then subjected to fixation, blocking, and fluorophore-conjugated antibody labeling for plaque visualization. This process enables the identification of the highest serum dilution that neutralizes fifty percent of RSV, representing the neutralization titer.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

NOTE: All steps must be performed in a BSL2 hood unless stated differently. Viral titration is required before a plaque-reduction neutralization (PRN) assay to determine the optimal RSV concentration used in the PRN assay. It is recommended to aliquot the virus stocks in a small volume that will be thawed once and used for each NAb assay. Using the same viral stock for all NAb assays performed for all samples from one study is also recommended. Ensure culture media and phosphate-buffered saline (PBS) are warmed at 37 °C before adding them to cell plates.

1. RSV Viral Titration

NOTE: Depending on the number of virus stocks and the number of duplicates, the assay plate can be set up according to Figure 1. Each virus stock should be titrated in triplicate down the assay plate, starting at the highest viral concentration (i.e., 1:10). Serial titrations can be typically 1:10. A549 cell culture and maintenance and RSV culture procedures are done using standard procedures and are not included in this protocol.

1. Seeding plates (Day 1)
   1. Resuspend A549 cells in Dulbecco's Modified Eagles medium (DMEM) + 10% fetal calf serum (FCS) + 1000 IU penicillin/streptomycin (pen/strep) at 4 x 10⁵/mL. Seed 96-well flat-bottom sterile plates with 100 μL/well containing 4 x 10⁴ A459 cells.
   2. Incubate plates overnight at 37 °C, 5% carbon dioxide (CO₂) (cells will be in the log-phase growth).    
      NOTE: Use a new A549 cell vial after 23 passages. It is recommended not to start the experiments when a new cell vial is still at the first three passages.
2. Virus infection (Day 2)
   1. Preparation of virus serial dilution
      1. Rapidly thaw a single frozen vial of respiratory syncytial virus (RSV) in a 37 °C water bath until almost completely thawed and place immediately on ice. Prepare 3 replicates for each RSV virus stock to be assayed, use an initial virus stock dilution of 1:10 with 100 μL of diluted virus/well, and reserve at least one column for negative control.        
         NOTE: Figure 1 shows negative control in triplicates.
      2. Add 100 μL of each diluted virus at 1:10 in triplicate of row A of a 96-well U-bottom sterile plate. Add 90 μL/well of DMEM + 1000 IU pen/strep without FCS to rows B to H.
      3. Perform serial 1:10 dilutions down the plate by transferring 10 μL from row A to row B. Mix solution by pipetting content up and down 5 times and continue the ten-fold dilutions until row H. Discard the final 10 μL so that the final volume in each well is 90 μL.
         NOTE: It is important to use new tips for each dilution.
   2. Virus inoculation to A549 cells
      1. Retrieve A549 cell plate(s) prepared on the previous day. Ensure that A549 cell monolayers in the 96-well plates are ~80% confluent. Discard the media by gently inverting the plate and lightly blotting it on a sterile absorbent paper towel.
      2. Wash all wells with 100 μL of PBS twice; discard excess PBS by gently inverting the plate and lightly blotting on a sterile absorbent paper towel after each wash. Do not allow plates to dry.
      3. Transfer the virus dilutions from the viral titration plate (Figure 1) to corresponding wells on the A549 cell plate. Incubate the plate for 1 h at 37 °C, 5% CO₂. After 1 h incubation, decant supernatants using a pipette (to avoid cross-contamination). Take out 90 μL/well.
      4. Add 100 μL of 1x medium 199 (M199, see Table of Materials) + 1.5% carboxymethylcellulose sodium salt (CMC) low viscosity + 2% FCS containing pen/strep to each well.    
         NOTE: 2x M199 + 4% FCS + 2% pen/strep and 3% CMC solutions must be prepared and stored at 4 °C for future use. Make sure the solution is warmed at 37 °C before adding to plates.
         1. Prepare 2x M199 solution: 1 sachet/500 mL distilled water + 20 mL FCS + 10 mL pen/strep (to make up 2x MI99). Filter the solution using a 0.22 μm filter unit.
         2. For preparation of 3% CMC, add 15 g of CMC slowly into 500 mL of distilled water. Dissolve the CMC in water using a metallic stirrer heater at 50 °C, which takes about 1 hour to prepare. Mix well to make 3% CMC and autoclave the solution.
            NOTE: The CMC solid must be added to the water to be dissolved; adding water to the dry solid produces a "clump" of solid that is very difficult to dissolve.
         3. Prepare 1x M199 + 1.5% CMC low viscosity + 2% FCS containing pen/strep by mixing 1:1 the 2x M199 and 3% CMC prepared in step 1.2.2.4.2.
      5. Incubate plate for 3 days at 37 °C, 5% CO₂.
3. Developing assay plate and analysis (Day 5)
   1. Fixation and developing assay plate
      1. Discard M199 + CMC + 2% FCS containing pen/strep by gently inverting the plate. Add slowly (to avoid disturbing the cell layer) 200 μL of fixation buffer (80% acetone, 20% PBS, stored at -20 °C) to fix cells. Incubate at -20 °C for 20 min.
      2. Discard the fixation buffer and gently blot it on an absorbent paper towel. Leave the plate face down to dry for 10 minutes.       
         NOTE: From this step onwards, the assay can be performed outside the BSL2 hood.
      3. Prepare 5% milk diluent blocking solution in filtered PBS containing 0.05% polysorbate 20 (PBS-polysorbate, see Table of Materials). Calculate the volume needed for blocking for one plate based on 200 /well and 110 wells: 200 μL/well x 110 wells = 22 mL (1.1 mL concentrated milk diluent in 20.9 mL filtered PBS-polysorbate). At this stage, also make up sufficient blocking solution for primary and secondary antibodies. Calculate the volume needed for each antibody solution for one plate based on 50 μL/well and 110 wells: 50 μL/well x 110 wells = 5.5 mL. Therefore, the total volume of milk diluent blocking solution required for a single plate assay is 33 mL.
      4. Add 200 μL/well of blocking solution. Incubate plate at room temperature (RT) for 30 min. Discard the solution by gently inverting the plate and blot on an absorbent paper towel.
      5. Prepare 1:500 Goat X RSV antibody (mAb – primary antibody) diluted in blocking solution. Add 50 μL/well of the mAb solution and incubate at 37 °C for 1 h. Wash the plate 3 times with filtered PBS-polysorbate.
      6. Prepare 1:5,000 Alexa-Fluor donkey anti-goat IgG (secondary antibody) diluted in blocking solution. Add 50 μL/well of the secondary antibody solution and incubate at 37 °C for 1 h. Wash the plate 5 times with filtered PBS-polysorbate.
      7. Read the plate on an automated spot reader using the fluorescein isothiocyanate (FITC) channel and count settings, as shown in Figure 2. Wrap the plate in foil and store it at 4 °C. Calibrate the instrument using an unused 96-well culture plate and inputting count settings before the assay.
         NOTE: Re-scanning within a few days is possible if required. The calibration and count setting can be saved and used for future scanning if the assay uses the same type of plate used for the calibration.
      8. Check the images of wells for artifact plaques or disrupted cell monolayers (Figure 3). Exclude wells with disrupted cell monolayers.
         NOTE: Depending on the size of the artifact plaques, manual counts may need to be performed for those wells, or those wells may need to be discarded from data analysis. Criteria for a valid result include no disrupted cell monolayer or artifact plaques detected in more than one replicate well and no plaque-forming units (PFU) detected in negative wells (wells without added virus).
   2. Determine viral concentrations
      1. Choose the last two dilutions where spots can be counted clearly. Calculate the average number of spots from replicate wells at the same dilution. Calculate the viral titer (PFU per mL) of the stock sample using the formula below:
         PFU/mL = Average number of plaques/(Dilution factor × Volume of diluted virus added to the well)
         NOTE: The virus concentration determined for each RSV stock can now be used in the PRN assay (step 2).

2. RSV Neutralization Assay

1. Seeding plates (Day 1)
   1. Resuspend A549 cells in DMEM + 10% FCS + 1000 IU pen/strep at 4 x 10⁵/mL. Seed 96-well flat-bottom sterile plates with 100 μL/well containing 4 x 10⁴ A459 cells and incubate plates overnight at 37 °C, 5% CO₂ (cells will be in the log-phase growth).
      NOTE: Use a new A549 cell vial after 23 passages. It is not recommended to use A549 cells before passage number 3.
2. Virus and serum preparation (Day 2)
   NOTE: Depending on the sample type, there are required steps for pre-processing samples before the NAb assay. It is recommended to use the RSV international standard sera that is now available upon request from the National Institute for Biological Standards and Controls (NIBSC).
   1. Preparation of serum dilution
      1. Thaw the human RSV reference antiserum (reference serum, REF) and serum test samples at RT. Heat-inactivate serum samples and reference antiserum in a water bath at 56 °C for 30 min before use. Prepare dilutions as per the plate template (Figure 4).
      2. Prepare a 1:100 dilution of the REF. Prepare a minimum of 110 μL of REF diluted in DMEM + pen/strep without FCS per assay plate (e.g., 1.5 μL of REF in a total volume of 150 μL). Add 110 μL of 1:100 diluted REF in well A12 of a 96-well U-bottom sterile plate.
      3. Add 55 μL of DMEM + pen/strep without FCS to wells B12 to H12 in column 12. Perform serial 1:2 dilutions down the plate by transferring 55 μL from row A to row B, mixing solution by pipetting content up and down 5 times and continuing until row H. Discard the final 55 μL of media so that the final volume in each well is 55 μL. Use new tips for each dilution.
      4. Prepare 1:100 dilution of serum test samples. As all serum samples are assayed in triplicate, prepare a minimum volume of 110 μL/well x 3 = 330 μL per serum test sample.
      5. Add 110 μL of each diluted serum test sample (1:100; S1 = serum 1, S2 = serum 2, etc.) to corresponding wells A1 to A9 and also E1 to E9 of the 96-well sterile plate according to Figure 4.
      6. Add 55 μL of DMEM + pen/strep without FCS to all wells labeled X. Perform serial 1:2 dilutions as per step 2.2.1.3 until row D (for samples 1, 2, and 3). Discard the final 55 μL of media so that the final volume in each well is 55 μL. Similarly, perform the serial 1:2 dilutions for samples 4, 5, and 6 for rows E to H.
         NOTE: It is essential to use new tips for each dilution.
      7. Add 55 μL of DMEM + pen/strep without FCS to wells in columns 10 and 11.
   2. Preparation of virus​
      1. Rapidly thaw a single frozen vial of RSV in a 37 °C water bath until almost completely thawed and place immediately on ice.
      2. Dilute virus in DMEM + pen/strep without FCS based on the concentration of the RSV aliquot determined in step 1.3.2. Apply a dilution that gives approximately 200 PFU/well. Prepare a total volume of 5.5 mL (for 100 wells).
   3. Preparation of virus-serum mixture
      1. Add 55 μL of the diluted virus to all wells of columns 1-9 and wells of rows E-H of columns 10 and 11 (positive control) according to the plate template (Figure 4).
      2. Add 55 μL DMEM + pen/strep without FCS to all wells of rows A-D of columns 10 and 11  (negative control). Incubate the plate for 1 h at 37 °C, 5% CO₂.
   4. Virus inoculation to A549 cells
      1. Before the completion of incubation period, retrieve A549 cell plate(s) prepared in step 2.1.1. Ensure that A549 cell monolayers in the 96-well plates are ~80% confluent.
      2. Discard the media by gently inverting the plate and lightly blotting it on a sterile absorbent paper towel. Wash all wells with 100 μL of PBS twice, discard excess PBS by gently inverting the plate and lightly blotting on a sterile absorbent paper towel after each wash. Do not allow plates to dry.
      3. Transfer 100 μL/well of the virus-serum mixture to the corresponding wells on the A549 cell plate following the plate template. Incubate the plate for 1 h at 37 °C, 5% CO₂.
      4. After 1 h, using a pipette, take out 90 μL/well and add 100 μL of prewarmed 1x M199 + 1.5% CMC + 2% FCS + pen/strep. Incubate plate for 3 days at 37 °C, 5% CO₂.
         NOTE: Ensure the solution is warmed at 37 °C before adding to plates.
3. Developing assay plate and analysis (Day 5)​
   1. Develop fixation and assay plate following steps 1.3.1.1 to 1.3.1.8.
   2. Determine the 50% neutralization titer
      1. Determine the 50% neutralization titer by calculating the proportional distance between the reciprocal serum dilutions above and below 50% of the ‘no serum' control wells (positive virus control - column 10, rows E-H).
      2. Count the number of PFU (i.e., plaques) arising from individual viral infections in serum wells and no serum control wells. Calculate the mean number of PFU of the no serum control wells and determine the 50% value.
      3. Identify serum dilutions with counts immediately above and below the 50% value of the no serum control wells. Using a semi-log graph or spreadsheet template, plot the number of PFU on the x-axis (linear scale) and the reciprocal serum dilution on the y-axis (log₁₀ scale). Draw a line between the two points and read the 50% neutralization titers of the test serum samples from this line.

Representative Results

Figure 1: Viral titration plate layout for quantitation of RSV stocks. v = stock of RSV virus (v1, stock of viral batch 1; v2, stock of viral batch 2; v3, stock of viral batch 3), N = negative control well, X = media added. Color coding is only to aid in the visualization of plate layout.

Figure 2: Example count settings used for the spot reader. Screenshot of the parameters typically used for counting virus plaques.

Figure 3: Examples of artifact and disrupted cell monolayers. (A) Artefacts. (B) Disrupted cell monolayers. 

Figure 4: Plate template for RSV PRN assay. V = viral positive control wells, N = negative control wells, X = media added, S = reference antiserum with all dilutions (from 1:100 to 1:12,800) in column 12. Color coding is only to aid in the visualization of plate layout.

Materials

Name	Company	Catalog Number	Comments
Cell line
A549	ATCC	CCL-185	provided by Dr Keith Chappell, University of Queensland
Viral strains
RSV A2	ATCC	VR-1540	lot number 60430286
Reagents
Acetone	Merck	1000142511
Alexa-Fluor donkey anti-goat IgG (stored at 4 °C)	Life Technologies	A11055
CMC sodium salt powder	Sigma-Aldrich	C5678-500G
DMEM (no serum, 3.7 g/L NaHC, P/S) (stored at 4 °C)	Scientific Services – Tissue Culture		MCRI in house supply
Foetal calf Serum (stored in 50ml aliquots at -20 °C)	Interpath	SFBS-F
Goat X RSV antibody	Merck	AB1128
Human polyclonal antiserum to respiratory syncytial virus (RSV) (stored in 45 µL aliquots at -20 °C)	BEI Resources	NR-4022	Free order through BEI Resources upon registration. This serum belong to a panel of human antiserum and immune globulin to RSV (NR-32832)
M199 powder	Life Technologies	31100035
Milk diluent blocking solution (stored at 4 °C)	Australian Biosearch	50-82-01
Penicillin/Streptomycin (stored in 6mL aliquots at -20 °C)	Life Technologies	15140122
s.d.H2O from Milli-Q dispenser	Merck		In-house dispensation
Sterile 1X PBS for culture (stored at 4°C)	Scientific Services – Tissue Culture		MCRI in house supply
Tween 20 polysorbate	Sigma-Aldrich	9005-64-5
General Consumables
Conical Falcon tubes (50 mL)	Invitro Technologies	FAL352070
Filter unit 0.22um (500 mL)	Thermo Fisher	NAL5660020
Sterile Eppendorf tubes (1.5 mL)	Australia PL	AM12400
Sterile flat-bottom plates (96-well with lid)	Interpath	655180
Sterile U-bottom plates (96-well with lid)	Interpath	650180
5ml serological pipette	Sigma-Aldrich	CLS4487-200EA
10 mL serological pipette	Interpath	607180
25 mL serological pipette	Sigma-Aldrich	CLS4251-200EA
Tip Pipette 1-200 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960	Fisher Biotec	TF-200-L-R-S
Tip Pipette 5-20 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960	Fisher Biotec	TF-20-L-R-S
Tip Pipette 100-1000 µLClear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1000	Fisher Biotec	TF-1000-L-R-S
Tip Pipette 1-10 µL Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1001	Fisher Biotec	TXLF-10-L-R-S
Equipments and softwares
ELISpot reader system	AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany
AID ELISpot software version 5.0	AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany
Microsoft Excel 2007