A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

All influenza viruses should be handled according to appropriate biosafety level requirements (BSL-2 or higher) as defined in the Biosafety on Microbiological and Biomedical Laboratories (BMBL).

1. Preparation of Reagents and Starting Material

1. Prepare Madine Darby Canine Kidney-α2,6-sial transferase (MDCK-SIAT1) cells and sterile cell culture medium
   1. Prepare the MDCK-SIAT1 cell culture medium using 500 mL of Dulbecco's Modified Eagle Medium (DMEM) with high glucose, 10% v/v heat-inactivated fetal bovine serum (FBS), 2 mM L- glutamine, 1 mM sodium pyruvate, 1 mg/mL G418 sulfate (e.g., geneticin), and 100 U/ml penicillin with 100 µg/mL streptomycin (optional). Sterilize by filtration through a 0.2 µM pore-size membrane. G418 sulfate is added to ensure plasmid stability in the MDCK-SIAT1 cells.
   2. Prepare the MDCK-SIAT1 cell line. In a 162 cm²– tissue culture flask(s) containing 30 mL of sterile cell culture media, seed flasks with 2 – 2.5 x 10⁶ MDCK-SIAT1 cells and incubate at 37 °C in 5% CO₂ for 2 days; this will be used for tissue-culture infectious dose (TCID) determination and MN assays.
      NOTE: MDCK-SIAT1 cells were kindly provided by Dr. M. Matrosovich, Marburg, Germany. This cell line can also be obtained commercially (see the Table of Materials).
2. Prepare the sterile virus propagation media using 500 mL of DMEM high glucose, 0.3% bovine serum albumin (BSA) fraction V, 100 U/mL penicillin, 100 µg/mL streptomycin, and 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Sterilize by filtration through a 0.2 µM pore-size membrane.
3. Prepare 0.75% (v/v) guinea pig red blood cells (gpRBCs) in phosphate-buffered saline (PBS) containing 0.01 M PBS, at pH 7.2.
4. Prepare the sterile virus diluent using DMEM high glucose, 1% Bovine Albumin Fraction V (BSA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 20 mM HEPES. Sterilize by filtration with a 0.2 µm pore-size membrane and prepare fresh for each assay.
5. Prepare the cold cell fixative as 80% cold acetone in PBS (0.01 M PBS, pH 7.2).
6. Prepare the wash buffer using PBS (0.01 M PBS pH 7.2) and 0.3% (v/v) tween-20.
7. Prepare the antibody diluent using PBS (0.01 M PBS, pH 7.2), 0.3% (v/v) tween-20, and 5% non-fat dry milk.
8. Use anti-influenza A NP mouse monoclonal antibody clones A1 and A3 pool as the primary antibody. Dilute in the antibody diluent at the optimal concentration as determined by titration.
9. Use goat anti-mouse IgG conjugated to horse radish peroxidase (HRP) as the secondary antibody. Dilute in the antibody diluent at the optimal concentration as determined by titration.
10. Prepare the peroxidase substrate using o-phenylenediamine dihydrochloride (OPD) in 0.05 M phosphate citrate buffer at pH 5. Prepare 0.05 M phosphate citrate buffer by dissolving 1 capsule/100 mL deionized H₂O. Dissolve 1 OPD tablet (10 mg)/20 mL of phosphate citrate buffer immediately before use.
11. Use 0.5 M of sulfuric acid as the OPD stop solution. Add 28 mL of 18 M sulfuric acid stock to 972 mL of deionized H₂O in a chemical hood.
12. Treatment of human and animal sera
    1. Heat-inactivate the human sera used in the MN assays in a water bath at 56 °C for 30 min prior to the assay. Use immediately or if needed, store thawed sera at 4 °C for no more than 24 h; if a longer storage period is needed, store sera frozen at -20 °C or colder.
    2. Treat the animal sera used in the MN assays with Receptor Destroying Enzyme (RDE) and heat inactivate prior to the assay as follows.
       1. Thaw the sera in a 37 ˚C water bath then place on ice. Mix 1 volume of animal serum sample with 3 volumes of RDE. Incubate at 37 ˚C for 18 – 20 h.
       2. Heat inactivate the RDE-treated sera at 56 ˚C for 30 min. Add 6 volumes of PBS, pH 7.2 to each sample for a final pre-dilution of 1:10. If needed, store thawed sera at 4 °C for no more than 24 h; if a longer storage period is needed, store sera frozen at -20 °C or colder.
          NOTE: Animal sera may contain various sialic acid glycans which may bind to the hemagglutinins, or HAs of influenza viruses and inhibit the binding of influenza-specific anti-HA antibodies. Therefore, it is necessary to RDE treat all animal sera prior to the MN assays to remove these non-specific viral HA binders in the serum samples.

2. Passage of MDCK-SIAT1 Cell Culture

NOTE: All cell cultures must be performed in a biological safety cabinet to prevent contamination.

1. Decant the cell culture medium from the cell monolayer in 162 cm² flasks. Trypsinize the cells by rinsing the monolayer with trypsin-ethylenediamine tetraacetic acid (EDTA). Add 5 mL trypsin-EDTA to cover the cell monolayer. Incubate at 37 °C, 5% carbon dioxide, CO₂ until the monolayer detaches (5-10 min). Add 15 mL of MDCK-SIAT1 cell culture media to each flask containing trypsinized cells, and pipette up and down to separate cells.
2. Use a hemocytometer and trypan blue to determine the cell counts and viability. Seed the cells in new 162 cm² tissue culture flasks containing 30 mL of cell culture media with 2-2.5 x 10⁶ cells /flask and incubate at 37 °C with 5% CO₂ for 2 days for use in TCID and MN assays. Seed cells at 4 – 5 x 10⁶ cells/flask and incubate at 37°C with 5% CO₂ for 2 days for use in virus propagation.
   NOTE: The seeding density can be adjusted if shorter or longer culture periods are desired. For virus propagation, cells should reach over 100% confluency. For Tissue Culture Infection Dose (TCID) and MN assays, the cells should be at 75 – 95% confluency (Figure 1).

3. Propagation of A(H3N2) Viruses in MDCK-SIAT1 Cells

NOTE: A(H3N2) virus can be propagated either in 10 – 11 day old embryonated hen's eggs according to standard protocol, or MDCK-SIAT1 cell cultures. MDCK-SIAT1 cells should reach over 100% confluency for virus inoculation. Virus seed stocks can be inoculated with multiple dilutions of inoculum. The inoculum dilutions with the best harvest HA and infectivity can be used for further MN assays.

1. Remove the cell culture medium from the cell monolayer (>100% confluency) in 162 cm² flasks. Wash the monolayer twice with 15 mL of sterile 0.01 M PBS, pH 7.2, and decant.
2. Label 1 flask as control and add 20 mL of virus propagation media. Incubate the flask at 37 °C with 5% CO₂. Leave this flask untouched and use it for comparison after day 1 of virus propagation.
3. Add 10 mL of sterile virus propagation media to the flask(s) and incubate all flask(s) at 37 °C, 5% CO₂. Thaw a vial of influenza virus stock at room temperature then place on ice. Dilute with the virus propagation media to the target dilutions.
   NOTE: Target dilutions can be adjusted based on HA titers of virus stocks. To prepare a 1:1000 diluted inoculum, add 100 µL of virus to 9.9 mL of sterile virus propagation media. Invert gently, remove 1 mL of 1:100 dilution to 9 mL of sterile virus propagation media and invert gently for a 1:1000 diluted inoculum. Place on ice.
4. Remove all flasks except the control flask from the incubator. Remove the virus propagation media from the flask(s) of MDCK-SIAT1 cells and inoculate each flask with 10 mL of diluted virus. Incubate the flasks at 37 °C, 5% CO₂ for 1 h, rotating the flask(s) every 15 min.
5. Remove 5 mL of inoculum from the flask(s) and replace with 15 mL virus propagation media containing 1 µg/mL Tosyl phenylalanyl chloromethyl ketone-treated trypsin (TPCK-trypsin). Incubate the flask(s) at 37 °C, 5% CO₂ for 16-18 h.
6. After the overnight incubation, observe the flask(s) under 100X microscope magnification and look for cytopathic effects (CPE) on the cells. Remove 15-17 mL of virus supernatant from the flask(s), and replace it with 15 – 17 mL of freshly prepared virus propagation media containing 2 µg/mL TPCK-trypsin.
7. Incubate the flasks at 37 °C, 5% CO₂. Monitor the CPE of the monolayer (by comparison with the cell control flask) and check the HA periodically (every 4 h) with 0.75% gpRBC until harvest.
   1. Set up an HA 96-well microtiter plate. Add 50 µL of 0.01 M PBS, pH 7.2 to wells A2 – A12 and B2 - B12 (duplicates), and wells H1 – H12 for control.
   2. Add 100 µL of virus supernatant to A1 and A2, and perform a 2-fold serial dilution from A1 and B1 through A12 and B12. Discard 50 µL after mixing in A12 and B12. Add 50 µL of 0.75% gpRBCs to Rows A, B, and H.
   3. Tap the plate and incubate at room temperature for 1 h.
      1. Determine HA of virus supernatant by tilting the 96-well microtiter plate at a 45° to 60° angle.
      2. Read plates for hemagglutination; the highest dilution of the virus that achieves complete hemagglutination is considered the HA titration endpoint for the specific virus. Record the reciprocal of the highest virus dilution with complete hemagglutination as the HA titer of the virus.
         NOTE: The settled RBCs in row H (controls) should start "running" and form a small teardrop shape due to gravity. Wait till RBCs in control wells finish running, then read the RBC buttons in the virus titration wells. Those that exhibit RBC buttons and "run", do not achieve hemagglutination. Locate the highest dilution of the virus that completely inhibits hemagglutination as the endpoint HA titer of the virus.
8. Harvest the virus when the HA of the virus culture plateaus or the virus culture reaches the target HA (for example, >16 HA unit), but before the cell monolayer starts to display significant CPE.
   1. Centrifuge the virus culture supernatant at 300 x g for 10 min at 4 °C to pellet the cellular debris. Transfer the clarified supernatant containing the virus harvest to a clean tube. Aliquot the supernatant containing the virus harvest into single-use sterile cryogenic vials and freeze immediately at -70 °C or colder.  
      NOTE: The virus stocks used for the MN assays should have high infectious titer and minimal defective particles. The minimum dilution of virus stocks to achieve 100 TCID₅₀/50 µL for MN assay is 1:100.     
      NOTE: The virus stocks used in the MN assays should not be thawed and refrozen.

4. Determination of TCID of the Virus

1. Day 1: Virus titration
   1. Thaw a vial of the virus at room temperature and immediately place it on ice.
   2. Test the virus at two different starting dilutions: 10^-2 and 10^-3. Add 100 µL of virus to 9.9 mL of virus diluent for 10^-2 dilution. Add 1 mL of 10^-2 dilution to 9.0 mL of virus diluent for 10^-3 dilution.
   3. Using two microtiter plates (plate 1 for 10^-2 dilution and plate 2 for 10^-3 dilution), add 100 µL of virus diluent to all wells except column 1 of the 96-well microtiter plate.
   4. Perform ½log₁₀ dilutions (10^-2, 10^-2.5, 10^-3, etc.). Add 146 µL of the virus starting dilution to all wells in column 1 and transfer 46 µL serially from column 1 through column 11 (Figure 2). Change pipette tips between wells. After mixing column 11, discard the tips with the 46 µL dilution.
   5. Use column 12 as the Cell Control (CC); it only contains virus diluent. Incubate for 1 h at 37 °C, 5% CO_2.
2. Day 1: Preparation of MDCK-SIAT1 cells      
   NOTE: The MDCK-SIAT1 cell monolayer should reach 75 – 95% confluency for TCID and MN assays. One 162-cm² flask at 95% confluency should yield enough cells to seed ~ 4 – 5 microtiter plates.
   1. Wash the 75 – 95% confluent monolayer with 20 mL PBS to remove the FBS in the culture media. Trypsinize the cells as follows.
      1. Rinse the monolayer with sterile PBS, and add 7 mL trypsin-EDTA to cover the cell monolayer. Lay the flask flat and incubate at 37 °C, 5% CO₂ until the monolayer detaches (approximately 5 – 10 min). Add 7 mL of virus diluent to each flask containing the trypsinized cells.
   2. Wash the cells with virus diluent to remove the FBS.
      1. Gently pipette up and down to separate the cells. Transfer the cells to a 50-mL conical tube; fill the tube with virus diluent.
      2. Centrifuge at 485 x g for 5 min. Decant the virus diluent, replace it with fresh 50 mL virus diluent, and centrifuge at 485 x g for 5 min. Decant the virus diluent and replace it with fresh virus diluent (10 mL/flask) to resuspend the pellet.
   3. Use a hemocytometer and trypan blue to determine the cell count and viability. Adjust the cell concentration with the virus diluent to 1.5 x 10⁵ cells/mL.
   4. Add 100 µL of diluted MDCK-SIAT1 cells to each well of the microtiter plates (1.5 x 10⁴ cells/well) and cover the plates. Incubate at 37 °C, 5% CO₂ for 18 – 20 h.
3. Day 2: ELISA
   1. Fixation of cells
      1. Remove the medium from the microtiter plates. Wash each well with 200 µL of PBS. Add 300 µL cold 80% acetone to each well and incubate at room temperature for 10 min. Remove the fixative and allow the plates to air dry.
   2. ELISA
      1. Primary antibody addition        
         NOTE: Anti-influenza A NP monoclonal antibody should be used in excess as the primary antibody in ELISA. Determine the optimal antibody dilution for each lot of primary antibodies by performing antibody titrations in MN. Select the primary antibody concentration that is in excess and with the best signal-to-background ratio.
         1. Dilute the anti-influenza A NP monoclonal antibody (primary antibody) to the target concentration in the antibody diluent (e.g. add 30 µL of primary antibody to 30 mL of antibody diluent for a target dilution of 1:1000).
         2. Wash plates 3 times with 300 µL of wash buffer. Add 100 µL diluted primary antibody to each well. Incubate at room temperature for 1 h.
      2. Secondary antibody addition   
         NOTE: Goat anti-mouse immunoglobulin, IgG conjugated to horse radish peroxidase (HRP) should be used in excess as the secondary antibody in ELISA. Determine the optimal antibody dilution for each lot of secondary antibodies by performing antibody titrations. Select the secondary antibody concentration in excess and with the best signal-to-background ratio.
         1. Dilute the goat anti-mouse IgG conjugated to HRP antibody (secondary antibody) to the target concentration in the antibody diluent (e.g. Add 7.5 µL of secondary antibody to 30 mL of antibody diluent for a target 1:4000 dilution).
         2. Wash the plates 3 times with 300 µL wash buffer. Add 100 µL diluted secondary antibody to each well. Incubate at room temperature for 1 h.
      3. Substrate addition and plate reading
         1. Wash the plates 5 times with 300 µL wash buffer and tap on a lint-free wipe.
         2. Add 100 µL of freshly prepared substrate to each well and incubate at room temperature until the color development saturates and the optical density (OD) of cell control wells <0.2.
         3. Add 100 µL of stop solution to all wells. Read the OD of wells at 490 nm using a microplate spectrophotometer.
4. TCID ₅₀ calculation
   1. Calculate the median OD₄₉₀ of the cell controls (column 12).
   2. Consider any test well with an OD₄₉₀ greater than twice the median OD₄₉₀ of the CC wells as "positive"; otherwise, it is considered "negative".
   3. Calculate the TCID₅₀ of the virus using the Reed-Muench method.
      1. Determine the number of positives and negatives at each dilution.
      2. Calculate the "cumulative positive", "Cumulative negative", "Ratio", and "% positive" as illustrated in Table 1.
      3. Calculate the "proportional distance" between the dilution showing >50% positives and the dilution showing <50% positives using the following:
         
         NOTE: The correction factor for ½ log dilution is 0.5. For example, in Table 1: (80 − 50)/(80 − 20) x 0.5 = 0.25
      4. Calculate the virus TCID₅₀ by adding the proportional distance to the dilution showing > 50% positive.
         NOTE: For example, in Table 2, TCID₅₀ is 10^-5+(-0.25) = 10^-5.25. NOTE this is the TCID₅₀ of the virus per 100 µL (or 10^-5.25/100 µL).
   4. Calculate the virus dilution. For MN assays, dilute the virus to 200 TCID₅₀/100 µL (equivalent to 100 TCID₅₀/50 µL per well).
      NOTE: In the example in Table 1, 1 TCID₅₀ is 10^-5.25 in 100 µL, and the dilution to achieve 200 TCID₅₀/100 µL is 1:891 based on the calculations: 200 x 10^-5.25 = 10^-2.95 = 1/10^2.95 = 1/891

5. MN Assay Using MDCK-SIAT1 Cells

1. Day 1: Test and control sera preparation and plate layout
   1. Thaw the sera in a 37 °C water bath and remove immediately after thawing. Aliquot the amount of sera that needs to be tested; a minimum of 10 µL of original sera is needed to test with one virus in a singlet. Test sera in duplicates if possible.
   2. Heat inactivate the human sera for 30 min in a 56 °C water bath as in step 1.12.1. Place sera on ice post heat inactivation, and add the virus diluent to sera to achieve a 1:10 pre-dilution.
   3. RDE treat and pre-dilute animal sera to 1:10 to use for controls per 1.12.2.
      NOTE: Thawed and treated human and animal sera can be stored at 4 °C for no longer than 24 h. Sera should be stored frozen at -20 °C or colder if a longer storage period is needed prior to the assay.
   4. To test with one virus, add 100 µL 1:10 diluted sera to columns A1 to A10 (Figure 3). Add 50 µL of virus diluent to rows B through H, except columns 11 and 12 (Figure 3). Perform a 2-fold serial dilution from rows A through H, and discard the last 50 µL at row H (Figure 3).
      NOTE: When multiple viruses are to be tested, sera can be diluted in titer tubes. The dilution of serum, to determine the serum titer, in well A is 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, well H 1:1,280 (Figure 3).
   5. For the virus control, add 50 µL of virus diluent to wells A12, B12, C12, and D12 (no sera). For the cell control, add 100 µL of virus diluent to wells E12, F12, G12, and H12 (no virus, no sera).
   6. For the control sera (for example, ferret sera controls), add 100 µL of diluted control sera to well A column 11 and add 50 µL of virus diluent to wells B11 – H11. Serial dilute down.
   7. Cover the plates, and incubate at 37 °C, 5% CO₂ until ready for the virus addition.
2. Day 1: Virus addition
   1. Dilute the virus to 100 TCID₅₀/50 µL with virus diluent.
   2. Add 50 µL diluted virus to all wells, except for column 11 on the back titration (BT) plates and the cell control wells E12, F12, G12, and H12 on all plates (Figure 3). For those plates with control sera in column 11, add the virus to column 11.
   3. Back titration (BT)
      1. Include a BT in column 11 of one set of duplicate plates (for example, plates 1A and 1B). Add 50 µL of virus diluent to all wells in column 11. Add 50 µL of the virus at 100 TCID₅₀/50 µL to the first well (A11). Mix by pipetting up and down.
      2. Mix and transfer 50 µL to successive wells to perform 2-fold serial dilutions. Change the pipette tips between wells to avoid virus carry-over. Discard 50 µL from well H11.
      3. Add 50 µL of virus diluent to column 11 to bring the final volume to 100 µL. Tap the plates to mix.
   4. Incubate plates at 37 °C, 5% CO₂ for 1 h.
   5. Perform MDCK-SIAT1 cell addition on day 1 and ELISA on day 2 as described in steps 4.2 and 4.3 (also described in 5.3 and 5.4)
3. Day 1: MDCK-SIAT1 cell addition
   1. Prepare the MDCK-SIAT1 cells as described in step 4.2. Add 100 µL MDCK-SIAT1 cells at 1.5 x 10⁵ cells/mL to each well (1.5 x 10⁴ cells/well). Incubate the plates at 37 °C, 5% CO₂ for 18 – 20 h.
4. Day 2: ELISA
   1. After overnight incubation, on the second day, fix the cells with 80% cold acetone as described in step 4.3.
   2. Primary antibody addition
      1. Wash the plates 3 times with 300 µL wash buffer. Dilute the anti-influenza A NP monoclonal antibody (primary antibody) to the optimal concentration as determined by titration in antibody diluent (e.g. add 30 µL of primary antibody to 30 mL of antibody diluent for a target dilution of 1:1000).    
         NOTE: 100 µL of primary antibody is required per well. ~10 mL is needed per plate.
      2. Add 100 µL diluted primary antibody to each well. Incubate at room temperature for 1 h.
   3. Secondary antibody addition
      1. Wash plates 3 times with 300 µL wash buffer. Dilute the goat anti-mouse IgG conjugated to HRP antibody (secondary antibody) to the target concentration in antibody diluent (e.g. Add 7.5 µL of secondary antibody to 30 mL of antibody diluent for a target 1:4000.)
         NOTE: 100 µL of secondary antibody is required per 96 well. ~10 mL is needed per plate.
      2. Add 100 µL diluted secondary antibody to each well. Incubate at room temperature for 1 h.
   4. Substrate addition and plate reading
      1. Wash the plates 5 times with 300 µL wash buffer and tap on the lint-free wipe.
      2. Add 100 µL of freshly prepared substrate to each well and incubate at room temperature until the virus control wells reach an OD₄₉₀ = 0.8 – 3, with the cell control at a low background OD₄₉₀ <0.2.
      3. Add 100 µL of the stop solution to all wells. Read the OD of wells at 490 nm using a microplate spectrophotometer.
5. Data analysis
   NOTE: The MN calculations are determined for each plate individually.
   1. Determine the neutralizing antibody titer of each serum sample.
      1. Calculate the OD₄₉₀ cut-off of 50% virus neutralization for each plate using the following equation:
         
         NOTE: Here x = 50% of the neutralization cut-off. The reciprocal serum dilution corresponding to the highest dilution with OD₄₉₀ less than 50% of the cut-off (≥50% inhibition) is considered the neutralization antibody titer for that serum sample (Figure 4).
   2. Check that the cell control wells have an OD₄₉₀ <0.2 and the virus control wells have an OD₄₉₀ = 0.8 – 3.
   3. Verify the virus infectivity in each assay (100x TCID₅₀) by virus BT. The acceptable range of BT is 50, 100, or 200 TCID. If using the same cut-off as defined in step 4.4.2, the highest virus dilution in the BT column with OD above the cut-off should be in wells E11, F11, and G11.
      NOTE: The serum positive controls should give titers within 2-fold of the values obtained in the previous tests. The OD₄₉₀ of the negative serum control should be similar to that observed for the virus control.