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A Lentiviral Pseudotype ELLA for the Measurement of Antibodies Against Influenza Neuraminidase   

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Original research article

A brief version of this protocol appeared in:
The Journal of Neuroscience
Feb 2017

Abstract

This protocol describes the rapid and safe production of lentiviral pseudotypes characterized by a lentiviral core containing a reporter, in conjunction with avian influenza haemagglutinin (HA) and human neuraminidase (NA) glycoproteins on the surface. Production is optimized with Endofectin LentiTM transfection reagent in 6-well plate format. These pseudotyped viruses can be employed for serological assays of surface glycoproteins HA and NA. They can be efficiently used to perform the ELLA (Enzyme-linked lectin assay) to measure NA inhibiting antibodies in lieu of using reassortant virus or Triton X-100 inactivated wild-type virus as source of antigen, which may require higher biosafety levels.

Keywords: ELLA, PV, OD, Assay, Neuraminidase, Antibody, Neutralisation

Background

The production of influenza virus pseudotypes has been extensively described previously (Nefkens et al., 2007; Temperton et al., 2007; Carnell et al., 2015). The need for a safe and rapid system to evaluate antibodies targeting the NA via the ELLA assay, avoiding the employment of reassortant mismatched virus or wild-type virus, has been met by producing influenza NA bearing pseudotypes (Prevato et al., 2015). A recent study (Biuso et al., 2017) confirmed that the co-expression of HA with NA improves the release of newly formed pseudotyped lentiviruses. Here we report a simple, widely applicable and optimized protocol for PV production by using the Endofectin LentiTM transfection reagent in 6-well plate format, and the methodology to perform an ELLA assay with the resulting influenza pseudotypes. While PV production for HA-based assays has made use of the lentiviral genome containing a reporter gene, the ELLA assay utilizes solely the surface NA glycoprotein, rendering a PV-incorporated reporter irrelevant to this protocol. The original ELLA assay from 1990 was recently improved (Couzens et al., 2014). This assay enables the detection of exposed galactose residues resulting from the enzymatic action of NA on sialic acids present on the fetuin substrate. This assay allows the measurement of NA inhibiting antibodies, through detection of a drop in enzymatic NA activity. The ELLA overcomes the limits of the cumbersome thiobarbituric acid assay (TBA) that employs hazardous materials, allowing for large-scale screening of serum samples. The basis of the assay is simple, 96-well plates are coated in the carbohydrate fetuin, which is then exposed to NA through NA bearing PV. The NA enzyme cleaves terminal sialic acid residues from the fetuin, exposing galactose that is then bound by the peanut agglutinin from Arachis hypogeal, conjugated to horseradish peroxidase (PNA-HRPO). This reagent then forms the basis for colorimetric reading of NA activity by a spectrophotometer. This activity can then be knocked down using an inhibitor (such as antibodies found in human sera) in subsequent assays. The described protocol combines the ability to screen a large number of sera through the ELLA assay, with a simple and safe lentiviral pseudotype production protocol. NA targeting antibodies are typically neglected in current influenza vaccines (compared to HA targeting antibodies), despite being shown to limit influenza symptoms and transmission (Marcelin et al., 2011 and 2012; Wohlbold and Krammer, 2014).

Materials and Reagents

  1. Multi Guard Barrier pipette tips 1-20 and 1-200 μl (Sorenson BioScience, catalog number: 30550T )
  2. NuncTM Cell-Culture Treated Multi dishes (6-well) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  3. Microcentrifuge tube 1.5 ml
  4. Sterile syringes (10 ml), generic
  5. Millex-HA 0.45 μm filters (Merck, catalog number: SLHAM33SS )
  6. 96-well microtiter plates Maxi Sorp surface (Thermo Fisher Scientific, NuncTM, catalog number: 439454 )
  7. 96-well microtiter plates round-bottom, for dilutions (Thermo Fisher Scientific, NuncTM, catalog number: 267245 )
  8. Reservoirs (Fisher Scientific, catalog number: 11543412 )
  9. Aluminum foil (Household item)
  10. Falcon tube (15 ml, any supplier)
  11. HEK 293T/17 cells (ATCC, catalog number: CRL-11268 )
  12. Plasmids
    1. Glycoprotein expression plasmids: phCMV-H11 and pI.18-NA
    2. Lentiviral vector expressing firefly luciferase: pCSFLW
    3. Second-generation lentiviral packaging construct plasmid: p8.91 (expresses gag, pol and rev)
    Note: Information on the plasmids above can be found in Temperton et al. (2007), Carnell et al. (2015) and Biuso et al. (2017). Plasmids available from Viral Pseudotype Unit, University of Kent: n.temperton@kent.ac.uk.
  13. Dulbecco’s modified Eagle medium (DMEM) (PAN-Biotech, catalog number: P04-04510 ) supplemented with 10% foetal bovine serum (FBS) (PAN-Biotech, catalog number: P40-37500 ) and 1% penicillin/streptomycin (P/S) (PAN-Biotech, catalog number: P06-07100 )
  14. Gibco Reduced Serum media Opti-MEM® (Thermo Fisher Scientific, catalog number: 31985047 )
  15. Endofectin LentiTM (Tebu-bio, catalog number: EFL1001-01 , EFL1001-02 )
  16. Phosphate-buffered saline (DPBS) for cell culture (Thermo Fisher Scientific, catalog number: 14040133 )
  17. Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  18. Internal negative and positive serum controls [e.g., from the U.S Food and Drug Administration (FDA), the National Institute for Biological Standards and Control (NIBSC) or Consortium for the standardization of influenza seroepidemiology (CONSISE)]
  19. Coating buffer (KPL, catalog number: 50-84-01 )
  20. Fetuin (Sigma-Aldrich, catalog number: F3385 )
  21. BSA (Sigma-Aldrich, catalog number: A8327 )
  22. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  23. Lectin from Arachis hypogaea (peanut)-Peroxidase (PNA-HRPO) (Sigma-Aldrich, catalog number: L7759-1MG )
  24. DPBS-T (Sigma-Aldrich, catalog number: P3563-10PAK )
  25. o-Phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich, catalog number: P8287 )
  26. 1 N Sulfuric acid (e.g., Fisher Scientific, Fisher ChemicalTM, catalog number: VL3171000 )
  27. Citrate Buffer (Sigma-Aldrich, catalog number: P4922 )

Equipment

  1. Class II biosafety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM Class II )
  2. Chemical hood
  3. Cell culture Incubator
  4. Water bath or heat block
  5. Plate sealer
  6. -80 °C freezer (e.g., Thermo Fisher Scientific, model: TSX ULT )
  7. Electronic serological pipette (e.g., Gilson, model: MACROMAN® )
  8. Pipettes (Gilson, models: PIPETMAN® Classic P2, P20, P200 and P1000, catalog numbers: F144801 , F123600 , F123601 and F123602 )
  9. Multichannel pipettes (e.g., Gilson, model: PIPETMAN® L Multichanel, 8 or 12 channels)
  10. Plate centrifuge (ELMI, model: SkySpinTM CM-6MT )
  11. Spectrophotometer for ELISA plate reading (e.g., Tecan Trading, model: SunriseTM )
  12. Inverted microscope (e.g., OLYMPUS, model: IX53 )

Procedure

  1. Plasmid transfection to produce Influenza viral pseudotypes
    Note: Class II biosafety cabinet is required to avoid any contamination.
    Timeline: Transfection–24 h.

    Day 1
    1. Seed 4 x 105 HEK 293T/17 cells into each well of a 6-well plate. The right confluence for transfection is 70-90% the day after seeding. An example is shown in Figure 1.


      Figure 1. An example of ready to transfect HEK293/17 cells (shown at x100 magnification), at around 70-90% confluence the day after subculture/seeding

    Day 2
    1. Pre-warm DMEM (10% FBS, 1% Penicillin/Streptomycin) and Opti-MEM® to 37 °C using a water bath or alternative.
    2. Label two sterile 1.5 ml microcentrifuge tubes (Tube 1 and Tube 2) for every well of a 6-well plate.
    3. Add 100 μl of Opti-MEM® to each Tube 1.
    4. Then add the following plasmids to Tube 1:
      0.5 μg of phCMV-H11 (HA from A/duck/Memphis/546/1974 (H11N9))
      1 μg pI.18-NA (NA from A/California/07/2009 (H1N1) or A/Texas/50/2012 (H3N2))
      0.75 μg pCSFLW
      0.5 μg of p8.91
      Note: If transfecting multiple identical wells, DNA mix can be scaled up accordingly.
    5. Add 100 μl of Opti-MEM® and 3 μl of Endofectin LentiTM for each microgram of plasmid DNA, to every Tube 2 (in this case add 8.25 μl of EndofectinLentiTM).
    6. Mix tube by inversion or by pipetting, then incubate at room temperature for 5 min (a thorough mix will help the combination of DNA with EndofectinLentiTM).
    7. Transfer the Opti-MEM®/Endofectin LentiTM mix from Tube 2 to every Tube 1 and incubate at room temperature for 25 min. Gently flick the tube every 5 min.
    8. During the incubation, take out pre-seeded 6-well plates from the incubator and remove the medium with an electronic serological pipette. Change the medium by slowly adding 2 ml of fresh DMEM/10% FBS/1% P/S on the side of each well to avoid detaching the cells, after removing the spent medium.
    9. After 25 min incubation, pipette the DNA/Opti-MEM®/Endofectin LentiTM solution onto each well of confluent HEK293T/17 cells monolayer dropwise. Swirl the 6-well plate(s) gently to ensure even dispersal of transfection mix.
    10. Incubate plates at 37 °C, 5% CO2 overnight.

    Day 3
    1. Pre-warm DMEM (10% FBS, 1% Penicillin/Streptomycin) to 37 °C using a water bath, or alternative.
    2. Check the 6-well plates under an inverted microscope. The confluence should be no greater than 90%. If cells are over confluent the yield will be suboptimal.
    3. Gently remove the medium from each well and substitute with 2 ml of fresh DMEM (10% FBS, 1% Penicillin/Streptomycin). Replenish the media with 2 ml of fresh DMEM, taking care not to disturb the cell monolayer.
    4. Incubate the plates at 37 °C, 5% CO2 for 48 h.

    Day 5
    1. The supernatant containing the viral pseudotypes is harvested using a sterile syringe and subsequently passed through a Millex-HA 0.45 μm filter.
    2. Aliquot the supernatant as required and store at -80 °C until downstream use.
      Note: The best condition for harvesting should be assessed after several attempts at PV production. The NA incorporation into PV can be accessed through western blot, and activity can be measured directly using the ELLA.

  2. Influenza viral pseudotype titration using ELLA
    The Influenza viral pseudotypes contain both HA and NA and can be employed in different assays, such as pseudotype based microneutralization (pMN, Temperton et al., 2007) or ELLA (Prevato et al., 2015). In this section of the protocol, we will describe how the ELLA assay is carried out within the Viral Pseudotype Unit (VPU), according to the CONSISE consensus protocol (Couzens et al., 2014).
    Before starting the titration by ELLA, Maxi Sorp plates must be coated with fetuin.
    Briefly:
    1. Coat Maxi Sorp plates by dispensing 100 μl/well of a 1x Coating buffer with 25 μg/ml of fetuin.
      Note: 10x concentrate coating buffer should be dissolved in deionized H2O.
    2. Seal the Maxi Sorp plates using a plate sealer and incubate them at between 2 and 8 °C for at least 48 h.
    3. Prepare a 1 N solution of sulfuric acid.

    Day 1
    1. Prepare the Sample Diluent: DPBS, 1% BSA, 0.5% Tween 20.
      Note: Sample diluent is used as a blank and to dilute the viral antigen across the plate.
    2. Prepare the wash buffer by dissolving one packet of DPBS-T (DPBS pH 7.4 with Tween 20) in one liter of deionized water.
    3. Remove an aliquot of pseudotyped virus (PV) from the -80 °C freezer and keep this on ice inside a laminar flow cabinet.
    4. Prepare the materials to be used in the laminar flow cabinet:
      1. 96-well dilution plates (one for every ELLA plate required)
      2. Pipette tips (1-20 μl/20-200 μl/100-1,000 μl)
      3. Mono-channel pipettes of different sizes (P20/P200/P1000) and multichannel pipettes (either 8 or 12 channels, P200)
      4. Waste disposal
      5. Reservoirs
      6. Fresh sample diluent
    5. Add 120 μl of sample diluent from Column 1 to Column 12 to the dilution plate.
    6. Add 120 μl of viral pseudotype (H11N1, or H11N2) to the wells in Column 1 for a 1:2 starting dilution.
    7. Perform a two-fold dilution from Column 1 to Column 11 (e.g., move 120 μl from Column 1 to Column 2, mix, and then move 120 μl from Column 2 to Column 3, etc.).
    8. Take coated Maxi Sorp plates out of the fridge (2-8 °C), remove the plate sealer and remove the Coating buffer by aspirator pipette (or plate washer).
    9. Wash the plates three times with wash buffer.
    10. Bring the coated plates into the biosafety cabinet.
    11. Add 50 μl of Sample Diluent to each well.
    12. Transfer 50 μl of diluted virus to each well of the coated Maxi Sorp plate from the dilution plate.
    13. Seal the plate and incubate overnight (16-20 h) at 37 °C, 5% CO2.

    Day 2
    1. Prepare the Conjugate Diluent with DPBS, 1% BSA.
    2. Prepare the materials to be used in the laminar flow hood:
      1. Tips of different volumes (1-20 μl/20-200 μl/100-1,000 μl)
      2. Mono-channel pipettes of different sizes (P20/P200/P1000) and multichannel pipettes (either 8 or 12 channels, P200 μl)
      3. Waste disposal
      4. Reservoirs
      5. Fresh Conjugate diluent
    3. Take out the plates from the incubator, remove the plate sealer and wash six times with DPBS-T wash buffer, as reported in Steps B8-B9, Day 1.
    4. Bring the coated plates into the biosafety cabinet.
    5. Prepare the PNA-HRPO solution by dissolving 1 mg of PNA-HRPO in 1 ml of Conjugate Diluent. Aliquot this accordingly and store them at -20 °C.
    6. Complete the Conjugate Diluent by adding PNA-HRPO to the sample conjugate at desired PNA-HRPO ratio.
      Note: Usually a dilution of 1:1,000 is sufficient. However, this can be optimized based on results to prevent waste of PNA-HRPO. If a plateau is reached at 1:1,000 dilution for the first few data points, the dilution can be increased accordingly.
    7. Add 100 μl/well of the Conjugate Diluent containing PNA-HRPO to all wells of 96 well plates.
    8. Cover the plates with aluminum foil (to avoid HRPO exposure to light) then incubate them for 2 h at room temperature.
    9. Immediately before the incubation ends, prepare the citrate buffer by adding one tablet of citrate buffer to 100 ml of deionized water.
    10. Remove the plates from the aluminum foil and wash them 3 times as reported in Steps B8-B9, Day 1.
    11. Bring the coated plates into the biosafety cabinet.
    12. Prepare the substrate solution by adding 1 capsule of OPD to 20 ml of citrate buffer (cover the Falcon tube with aluminum foil to avoid exposure to light).
    13. Add 100 μl of OPD substrate to every well of the 96 wells ELLA plates.
      Note: 10 ml of substrate solution is usually needed for every plate, meaning that 50 ml of citrate buffer is sufficient for 5 plates.
    14. Cover the plates with aluminum foil (to avoid exposure to light) then incubate them for 10 min at room temperature.
    15. Switch on the spectrophotometer and set up the following parameters:
      1. Wavelength: OD490
      2. Reading speed: 0.1 sec
    16. Stop the reaction by adding 100 μl/well of 1 N sulfuric acid per well. This will prevent saturation of the wells.
    17. Insert the plate into the spectrophotometer and read.
    18. Export results as an Excel file.
      1. Calculate the average of the blank values (background).
      2. Subtract the blank average from all the values from Column 1 to Column 11.
    19. Open Prism GraphPad (in this case version 5 was used):
      1. Open "New tables and graphs" and go on "XY", then select “Points and connecting lines” and enter 2 replicates values in side by side column and press create.
      2. Copy and transpose the results from the Excel file on the Y-axis.
      3. Convert dilution by Log2 to linearize the results and insert them onto the X-axis.
      4. Normalize the data by using the highest OD490 value as 100% activity threshold, and the average of the blank as a 0% threshold (Figure 2A).
      5. Perform non-linear regression to obtain a sigmoidal curve (non-linear regression curve fit, and select Variable slop) (Figure 2C) to obtain the final titration curve (Figure 2B).
      6. In order to evaluate the 90% of the maximum OD490 as reported by Couzens and colleagues (Couzens et al., 2014), simply calculate the average of the highest OD for every replicate and insert it at the bottom of data section and normalize as described above.
      7. Analyze by choosing the non-linear regression and select lines and straight lines from the list. Tick "interpolate unknowns from the standard curve and choose 90% from the list" (Figure 2C).
      8. Reconvert the log2 number found to find the right dilution that corresponds to 90% of the maximum OD. Record this result as viral dilution factor to analyze sera through ELLA assay.


        Figure 2. Evaluation of ELLA titre and its working dilution through Prism GraphPad. Results transposed are normalized (A) to identify a maximum and a minimum, then a curve is created by performing non-linear regression (B). The 90% of the average of the maximum OD from every replicate is then added to the data, normalized and successively identified on the curve by interpolating with a straight line. The result found [red circle on the right side of (C)] is reconverted to find the dilution that corresponds to 90% of the maximum signal.

  3. Anti-neuraminidase antibody detection by ELLA assay
    Timeline: 4 days (including the coating)
    Once that the viral dilution factor has been calculated (in order to dilute the virus to 90% of the maximum signal obtained in the titration), it is possible to perform the ELLA assay. The protocol described by Couzens (Couzens et al., 2014) has been improved.
    Before starting the titration by ELLA, plates need to be coated with fetuin.
    Briefly:
    1. Coat plate by dispensing 100 μl/well of 1x Coating buffer and 25 μg/ml of fetuin.
    2. Seal the plates and incubate them at 2-8 °C for at least 48 h.
    3. Prepare 1 N of sulfuric acid.

    Day 1
    1. Prepare the Sample Diluent: DPBS, 1% BSA, 0.5% Tween 20.
    2. Prepare the wash buffer by dissolving one pack of DPBS-T (DPBS pH 7.4 with Tween 20) for every liter of distilled deionized water.
    3. Take the sera out of the freezer, thaw them slowly on ice, leave at room temperature and successively heat inactivate them at 56 °C for 30 min, by using a water bath.
    4. Take out a viral aliquot from the freezer (-80 °C) and keep it on ice under the laminar flow.
    5. Prepare the materials to be used into the laminar flow:
      1. Dilution plates (one for every viral strain)
      2. Tips of different volumes (1-20 μl/20-200 μl/100-1,000 μl)
      3. Mono-channel pipettes of different sizes (P20/P200/P1000) and multichannel pipettes (either 8 or 12 channels, P200 μl)
      4. Waste disposal
      5. Reservoir
      6. Fresh sample diluent
    6. Add 120 μl of sample diluent from Column 3 up to Column 11.
    7. Add 216 μl of sample diluent on Column 2 and add 24 μl of each serum (1:10 dilution).
      Note: Initial serum dilution is up to the user. It could start with 240 μl of pure serum or with 120 μl of serum and 120 μl of sample diluent.
    8. Perform a serial two-fold dilution by taking 120 μl from Column 2 up to Column 11. Discard tips.
    9. Take coated plates out of the fridge (2-8 °C), remove the plate sealer and remove the Coating buffer by aspirator pipette (or plate washer), or remove by hand inverting the plate on a big reservoir.
    10. Wash the plates three times with wash buffer and empty the plate as reported in the previous step.
    11. Remove all the drops on the top of the wells by applying some clean paper, then bring the coated plates into the biosafety cabinet.
    12. Transfer 50 μl of diluted serum from the dilution to the coated plate. Each row (serum) allow for a duplicate test.
    13. Calculate the amount of viral antigen needed for each plate (~5 ml). Divide this number by the titer that corresponds to 90% of the overall signal (calculated as described in Step C7, Day 1) and dilute in sample diluent.
      Example:
      10 plates = ~50 ml of viral solution. If 90% of the overall signal corresponds to a dilution step of 25, we thus do 50 ml/25 = 2 ml of viral solution (original thawed aliquot stock) in 48 ml of sample diluent
    14. Add 50 μl of antigen solution to each plate from Column 2 to Column 11.
    15. Add 100 μl of antigen solution to Column 1 of each plate (it serves as viral control VC).
    16. Add 100 μl of sample dilution to Column 12 of each plate (it serves as blank).
    17. Seal the plate and incubate overnight (16-20 h) within the incubator (37 °C, 5% CO2).

    Day 2
    1. Prepare the conjugate diluent with DPBS, 1% BSA.
    2. Prepare the materials to be used in the laminar flow:
      1. Tips for different volumes (1-20 μl/20-200 μl/100-1,000 μl)
      2. Mono-channel pipettes of different sizes (P20/P200/P1000) and multichannel pipettes (either 8 or 12 channels, P200 μl)
      3. Waste disposal
      4. Reservoirs
      5. Fresh conjugate diluent
    3. Take out the plates from the incubator, remove the plate sealer and wash six times with DPBS-T wash buffer, as reported in Steps C9-C11, Day 1.
    4. Remove the remaining liquid from the top of the wells by applying some clean paper, then bring the coated plates into the biosafety cabinet.
    5. Thaw an aliquot of PNA-HRPO.
    6. Complete the conjugate diluent by adding PNA-HRPO to the sample conjugate.
      Note: Usually the best dilution is 1:1,000, but a proper setup should be established for every virus. A range from 1:500 to 1:2,000 usually allows for the best results.
    7. Add 100 μl/well of the conjugate diluent containing PNA-HRPO to each well of 96-well plates.
    8. Cover the plates with an aluminum foil (to avoid HRPO to be exposed to light) then incubate them for 2 h at room temperature.
    9. Immediately before the incubation ends, prepare the citrate buffer by adding one tablet of citrate buffer to 100 ml of deionized water.
    10. Remove the aluminum from the plates and wash them 3 times as reported in Steps C6-C7, Day 1.
    11. Remove all the drops on the top of the wells by applying some clean paper, then bring the coated plates into the biosafety cabinet.
    12. Prepare the substrate solution by adding 1 pill of OPD to 20 ml of citrate buffer (cover the falcon with aluminum foil to avoid direct exposure to light).
    13. Add 100 μl of OPD substrate to each well of a 96 wells plate.
      Note: 10 ml of substrate solutions are usually needed for every plate, meaning that 100 ml of citrate buffer ensure the reaction for 5 plates.
    14. Cover the plates with an aluminum foil (to avoid reaction to the exposed to light) then incubate them for 10 min at room temperature.
    15. Switch on the spectrophotometer and set up the following parameters:
      1. OD490
      2. Reading speed 0.1 sec
    16. Stop the reaction by adding 100 μl/well of 1 N sulfuric acid.
    17. Insert the plate into the spectrophotometer and read.
    18. Export results to Excel file.
      1. Calculate the average of blank (background noise).
      2. Subtract the blank from all of the values, Column 1 to Column 11.
    19. Open Prism GraphPad (in this case version 5 was used):
      1. Open Tables and Columns.
      2. Select “Points and connecting lines” and enter 2 replicates values side by side column, then press create.
      3. Copy and transpose the results from the Excel file on Y-axis.
      4. Convert dilution by Log2 to linearize the results and insert them on X-axis (Figure 3A).
      5. Normalize the curve using the highest OD490 value as the maximum value (100%) and the average of the blank as the lowest value (0%) (Figure 3B).
      6. Analyze using non-linear regression to obtain a sigmoid curve (non-linear regression curve fit, and select Variable slope) (Figure 3C).
      7. Check for the section “Data” for IC50 values (Figure 3D).
      8. See Figure 4 for elaboration on the data and requirements for generation of antibody titer.


        Figure 3. Analysis of the results (anti-neuraminidase antibody detection through ELLA) with Prism GraphPad. Insert OD values on the Y-axis and Log2 dilution on the X-axis (A), normalize results (B), analyse through ‘non-linear regression, curve fit, variable slope’ (C), then check and record the IC50 outcomes, highlighted in red circles, for “Normalize OD Data” (D).


        Figure 4. Elaboration of data. Raw data obtained by spectrophotometer are pasted in Excel (A), then the cut-off value is obtained by the formula . A representation is shown in (B), where bold numbers are higher than the cut-off. The highest results which remain higher than the cut-off represent the titre.

Notes

As with most manually performed serological assays, variation in results for the same experiment can be observed between members of the same laboratory, depending on user experience. To reduce variation, it is advisable to ensure lab members performing the ELLA assay have similar training and expertise in regards to use of multichannel pipettes and other standard lab techniques (pipetting, mixing of reagents). As there are multiple time-sensitive steps in this protocol, it is essential to keep to these timings between experiments. Two experiments carried out under even the smallest difference in timings may lead to different results. For any focused problem-solving queries, please contact the senior author: n.temperton@kent.ac.uk.

Acknowledgments

This work was funded by the Medway School of Pharmacy and VisMederi. The authors declare no conflicts of interest or competing interests.

References

  1. Biuso, F., Carnell, G., Montomoli, E. and Temperton, N. J. (2017). The study of antibody responses to influenza neuraminidase using a lentiviral pseudotype based ELLA. bioRxiv 218800.
  2. Carnell, G. W., Ferrara, F., Grehan, K., Thompson, C. P. and Temperton, N. J. (2015). Pseudotype-based neutralization assays for influenza: a systematic analysis. Front Immunol 6: 161.
  3. Couzens, L., Gao, J., Westgeest, K., Sandbulte, M., Lugovtsev, V., Fouchier, R. and Eichelberger, M. (2014). An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. J Virol Methods 210: 7-14.
  4. Marcelin, G., DuBois, R., Rubrum, A., Russell, C. J., McElhaney, J. E. and Webby, R. J. (2011). A contributing role for anti-neuraminidase antibodies on immunity to pandemic H1N1 2009 influenza A virus. PLoS One 6(10): e26335.
  5. Marcelin, G., Sandbulte, M. R. and Webby, R. J. (2012). Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines. Rev Med Virol 22(4): 267-279.
  6. Nefkens, I., Garcia, J. M., Ling, C. S., Lagarde, N., Nicholls, J., Tang, D. J., Peiris, M., Buchy, P. and Altmeyer, R. (2007). Hemagglutinin pseudotyped lentiviral particles: characterization of a new method for avian H5N1 influenza sero-diagnosis. J Clin Virol 39(1): 27-33.
  7. Prevato, M., Cozzi, R., Pezzicoli, A., Taddei, A. R., Ferlenghi, I., Nandi, A., Montomoli, E., Settembre, E. C., Bertholet, S., Bonci, A. and Legay, F. (2015). An innovative pseudotypes-based enzyme-linked lectin assay for the measurement of functional anti-neuraminidase antibodies. PLoS One 10(8): e0135383.
  8. Temperton, N. J., Hoschler, K., Major, D., Nicolson, C., Manvell, R., Hien, V. M., Ha do, Q., de Jong, M., Zambon, M., Takeuchi, Y. and Weiss, R. A. (2007). A sensitive retroviral pseudotype assay for influenza H5N1-neutralizing antibodies. Influenza Other Respir Viruses 1(3): 105-112.
  9. Wohlbold, T. J. and Krammer, F. (2014). In the shadow of hemagglutinin: a growing interest in influenza viral neuraminidase and its role as a vaccine antigen. Viruses 6(6): 2465-2494.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Biuso, F., Carnell, G., Montomoli, E. and Temperton, N. (2018). A Lentiviral Pseudotype ELLA for the Measurement of Antibodies Against Influenza Neuraminidase. Bio-protocol Bio101: e2936. DOI: 10.21769/BioProtoc.2936.
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