An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

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

1. Preparation of Reagents and Starting Material

Note: Refer to the Materials Table to obtain the source of all reagents.

1. Fetuin-coated Plates
   1. Prepare the coating buffer by mixing 10 ml of the 10x coating buffer with 90 ml of deionized H₂O.
   2. Prepare a stock solution of fetuin at 25 mg/ml in 1x coating buffer and store in 500 µl aliquots at -20 °C.
   3. Prepare a working solution of fetuin (25 µg/ml) immediately before coating plates by diluting the stock solution 1,000-fold in 1x coating buffer.
   4. Use a multichannel pipette to dispense 100 µl of the working solution of fetuin into all wells of a 96-well plate with high protein-binding capacity.
   5. Cover each plate with a plate sealer, stack the plates in groups of 10, and wrap them in foil.
   6. Place the plates in a refrigerator (2-8 °C) for at least 18 hr before performing an assay.
      Note: Plates may be coated in advance and stored with the coating solution at 2-8 °C for up to 2 months.
2. Diluents
   1. To prepare the diluent to make virus and sample dilutions (2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, 20 mM CaCl₂, 1% bovine serum albumin (BSA) and 0.5% Tween 20), mix 94.2 ml 1X MES, pH 6.5 with 2 ml CaCl₂ (10 mg/ml), 3.3 ml 30% BSA and 0.5 ml Tween 20.
   2. To prepare the diluent for Peanut agglutinin-horseradish peroxidase conjugate (PNA-HRPO) (MES, pH 6.5 with 20 mM CaCl₂ and 1% BSA), mix 94.7 ml 1x MES, pH 6.5 with 2 ml CaCl₂ (10 mg/ml), and 3.3 ml 30% BSA.
3. Neuraminidase (NA)
   Note: H6Nx virus reassortants (these have HA of the H6 subtype and the NA of interest) are generated. Request vials of inactivated reassortant virus from a collaborator if they are unavailable in-house. When using inactivated virus, confirm that the preparation is suitable for use in the ELLA through demonstration that the inactivation process did not significantly impact NA activity.
   1. Culture a stock of H6Nx virus in embryonated chicken eggs and store 0.5 ml aliquots of neat allantoic fluid at -80 °C.
   2. Use a new aliquot of virus for each assay. Do not freeze and thaw the aliquots.
4. Serum Samples and Controls
   1. Heat-inactivate all sera (test samples, e.g., pre- and post-vaccination sera, and controls, e.g., sample with known NI titer) in a water bath at 56 °C for 45-60 min. Store the sera at -20 to -70 °C before or after the heat treatment. If the samples are to be tested repeatedly, make several aliquots before freezing so that the sample is not repeatedly frozen and thawed.
   2. Use the ELLA to measure the NI titers of human sera available in reasonable quantity (>5 ml) to identify at least one with a low titer and another with a high titer. Store 100 µl aliquots at ≤-20 °C so that the same sample can be used as a control in many assays to track assay performance.
5. Peanut Agglutinin (PNA)-Horseradish Peroxidase (HRPO)
   1. Prepare the PNA-HRPO diluent (MES, pH 6.5 with CaCl2 and 1% BSA).
   2. Prepare a PNA-HRPO stock solution by dissolving 1 mg of PNA-HRPO in 1 ml diluent. Store 20-200 µl aliquots at -20 °C.
   3. Before using a new PNA-HRPO lot, test 1:500, 1:750, 1:1,000, and 1:2000 dilutions of this reagent to identify the amount that results in maximal optical density (OD) with the positive control (virus only) and a background (no virus) that is <10% of the positive signal.
      1. Make quadruplicate virus dilutions as described in section 2.1.
      2. Transfer the dilutions to a fetuin-coated plate as described in section 2.2 and incubate the plate at 37 °C.
      3. Wash the plate 16-18 hr later and add 1:500, 1:750, 1:1,000, and 1:2,000 dilutions of PNA-HRPO (100 µl/well) to duplicate rows of the plate.
      4. Complete the assay as described in steps 2.3.4 to 2.3.9.
      5. Review the data to select the dilution resulting in a maximum signal of>10 times the background.
   4. Immediately before use, prepare the optimal dilution of PNA-HRPO identified in 1.5.3.
6. Prepare a large volume of Wash buffer (0.01 M phosphate-buffered saline (PBS), pH 7.4, 0.05% Tween 20 (PBS-T)). Store at RT.
7. Prepare the peroxidase substrate (o-Phenylenediamine dihydrochloride (OPD)): Note: alternative peroxidase substrates such as 3,3′,5,5′-Tetramethylbenzidine (TMB), may be used
   1. Prepare phosphate-citrate buffer by dissolving 1 capsule in 100 ml dH₂O on the day of assay.
   2. Dissolve 1 OPD tablet (10 mg) in 20 ml of phosphate-citrate buffer immediately before use.
8. Prepare the stop solution (1 N H₂SO₄): add 27.2 ml stock 98% H₂SO₄ to 973 ml dH₂O. Mix and then store at RT.

2. Determination of the Amount of NA to Use in ELLA

1. Prepare Virus Dilutions in a 96-well Plate
   1. Dispense 120 µl sample diluent (section 1.2) into columns 1-11 of the dilution plate.
   2. To column 1, add an additional 96 µl sample diluent.
   3. Thaw and then vortex the virus vial before adding 24 µl to duplicate wells in column 1. This gives a 1:10 dilution of the virus.
   4. Make serial 2-fold dilutions of virus in sample diluent by transferring 120 µl from one well to the next using clean pipette tips for each dilution.
2. Transfer Virus Dilutions to a Fetuin-coated Plate
   1. Wash the fetuin-coated plate 3 times with PBS-T (section 1.6) and then blot each plate onto an absorbent paper towel to remove any excess wash buffer.
   2. Add 50 µl of sample diluent (section 1.2.1) to each well in columns 1 to 11 of the fetuin-coated plate.
   3. Add 100 µl of sample diluent to column 12 (these wells are the negative control).
   4. Transfer 50 µl of diluted virus from columns 1-11 of the dilution plate to the corresponding wells of the fetuin-coated plate.
   5. Cover the plate with a plate sealer and place it in a humidified incubator at 37 °C.
   6. Note when the remaining steps will be performed (16-18 hr later).
3. Complete the NA Determination
   1. When the incubation is complete (16-18 hr), transfer the plate to the bench and remove the plate sealer.
   2. Wash the plate 6 times with PBS-T (section 1.7), then invert and pat on absorbent paper towels to ensure all liquid has been removed from the wells.
   3. Add 100 µl/well PNA-HRPO solution (at the dilution determined in 1.5.3) to all wells and incubate the plate for 2 hr at RT.
   4. Less than 15 min before the incubation ends, prepare the OPD solution as described in section 1.7.
   5. Wash the test plates 3 times to remove the PNA-HRPO and blot dry before adding 100 µl of the OPD substrate to each well.
   6. Incubate the plate for exactly 10 min at RT.
   7. Add 100 µl/well of 1N sulfuric acid to stop the reaction.
   8. Use a plate reader to measure the Optical Density (OD) at 490 nm for 0.1 sec.
   9. Save and export all data files.
4. Select the Virus Dilution that will be used for Serology
   1. Draw a graph that plots OD_490nm values at each virus dilution. Inspect the titration curve to identify the maximum signal (this is usually the signal that corresponds to a plateau at the beginning of the titration curve), the minimum signal (wells that contain no virus in column 12 of the plate provide the background control), and the dilutions of virus that result in OD that is proportional to the input dilution (i.e., linear region of the curve).
   2. Select the virus dilution that gives approximately 90% of the maximum signal and is within the linear range. Confirm that the OD at the selected dilution is at least 10-fold greater than the background signal. Use the selected virus dilution for all assays employing this virus stock.
      Note: Alternatively, measure the NA activity of the virus and use ~15-20 µU NA activity/ml in the ELLA.

3. Enzyme-linked Lectin Assay

Note: Figure 1 shows the setup of the dilution and assay plates.

1. Make Sample Dilutions
   1. Place the heat-inactivated samples on ice.
      ​Note: 8 sera can be diluted in each plate.
   2. For a setup using dilutions starting at 1:10 across the plate, add 120 µl sample diluent to all wells in columns 3-11.
   3. To column 2, add 216 µl of sample diluent and 24 µl of each sample. Mix the sample in the well by pipetting up and down 3 times and then transfer 120 µl to the next column.
   4. After changing pipette tips, mix the contents of the well by pipetting up and down and then transfer 120 µl to the next column.
   5. Repeat step 3.1.4 until the sample has been transferred to column 11 and the remaining 120 µl discarded.
2. Add Samples and Viruses to the Fetuin-coated Plate
   1. Thaw a vial of virus, vortex, and resuspend the virus in the diluent (section 1.2) at the dilution selected in step 2.4.2. Prepare at least 5 ml of virus for each assay plate. Keep the diluted virus on ice until the plates are washed and serum samples are added.
   2. Decide on the number of fetuin-coated plates needed for the assay (generally apply 4 sera per plate). Wash the fetuin-coated plate 3 times with PBS-T, invert each plate, and blot onto an absorbent paper towel to remove excess wash buffer.
   3. Use a multichannel pipette to transfer 50 µl of each serum control or sample dilution from the dilution plate into duplicate wells in columns 2-11.
   4. Add 50 µl of diluted virus to all wells except for the negative control (column 12).
   5. Add 50 µl of sample diluent to wells in column 1 and add 100 µl of sample diluent to column 12.
   6. Cover the wells with a plate sealer and then mix by gently tapping the sides of the plate or placing them on a plate shaker at moderate speed for 10 sec.
   7. Place the plate in a humidified incubator at 37 °C for 16-18 hr.
3. Add PNA-HRPO and complete the assay as described in section 2.3

4. Data Analysis

1. Determine the Validity of the Assay Results
   1. Confirm that the background values (no virus) are less than 10% of the positive control (virus and no serum).
   2. Confirm that the titers of control sera run in different assays using the same conditions are within 2-fold of the median titer.
   3. Confirm that OD measurements of control wells are consistent (≤20% different) and that OD measurements of duplicate sample wells are consistent (≤10% different).
   4. Determine the root cause of invalid results and repeat the assay if the criteria listed in 4.1.1, 4.1.2, or 4.1.3 are unmet. Consider the factors presented in Table 1 when trying to troubleshoot.
2. Assign a 50% End-point Titer
   1. For each assay plate, subtract the average background (no antigen added to wells) from all readings.
   2. Calculate the percent inhibition at each serum dilution using the formula: 100 x (OD_{virus only control} - OD_{test sample})/ OD_{virus only control}.
   3. Identify the highest dilution that resulted in at least 50% inhibition of the maximum signal.
   4. Report the reciprocal of this dilution as the 50% end-point titer.
      Note: If 50% inhibition was not achieved at any dilution, the titer is less than the first dilution tested, e.g., <10 when 1:10 is the first dilution tested.
3. Calculate the 50% Inhibitory Concentration (IC₅₀)
   1. Use four parameter logistic regressions to determine the IC₅₀ titer as follows: subtract the average background from all readings and then transfer the results to a program that performs regression analysis.
   2. Use non-linear regression, with the maximum set to the virus-only control and the minimum set to zero, to determine the dilution corresponding to exactly 50% inhibition.
   3. Report the reciprocal of this dilution as the IC₅₀ titer.