Jul 02, 2025
HPV pseudovirus neutralization assay: Antibody Titration
This protocol is a draft, published without a DOI.
- joseph Carter1
- 1Fred Hutchinson Cancer Center
Protocol Citation: joseph Carter 2025. HPV pseudovirus neutralization assay: Antibody Titration. protocols.io https://protocols.io/view/hpv-pseudovirus-neutralization-assay-antibody-titr-g4dmbys47
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 30, 2025
Last Modified: July 02, 2025
Protocol Integer ID: 221325
Keywords: quantitative measure of hpv, hpvl1l2 pseudovirus, antibody titration this method, pseudovirus infection by half, antibody titration, pseudovirus infection, hpv, dilution of antibody, specific antibody potency, concentration of antibody, antibody, monoclonal antibody, assay
Funders Acknowledgements:
Denise A Galloway
Grant ID: R01 AI038382
Abstract
This method will provide a quantitative measure of HPV specific antibody potency. It can be used with any HPVL1L2 pseudovirus that express well in 293TT cells (pSHELL plasmids) that have the pYSEP plasmid encapsidated. If a monoclonal antibody is used with a known concentration then the result with be an IC50 in picomoles. If concentration of antibodies in not known (serum, or crude preparations of monoclonals) the IC50s with be in fold dilution (the dilution of antibody needed to reduce pseudovirus infection by half).
Guidelines
Like most cell culture assays there can be a fair amount of variation. To limit variation split the cells about 1:3 the day before every assay. The cells should not be confluent on the day the assay is setup. If you can't work on the weekend: cut back cells on Mondays and Thursdays, setup assays on Tuesdays and Fridays and harvest assays on Mondays and Fridays.
This protocol uses a 1:3 dilution of antibodies.
To get an accurate estimation of IC50s having several data points on the curve near the IC50 is required
Ideally, use smaller dilutions steps (1:2 or 1:3) but this will limit the concentration range
One way to extend the range while keeping smaller dilution steps is to titrate down the plate in the first column (column 2) before titrating across the plate. I have found that doing 3 1:3 dilutions down the plate and then 1:3 dilutions across gives an broad range of dilutions in small steps. If you use this approach be sure to calculate the antibody concentration/dilution for each well correctly (each row will be offset)
IC50s should be towards the center of the titration curve - If it is too far to the right repeat using 10 lower conc of antibody. If IC50s are too far left use 10X higher conc antibody and also consider also going to 2 fold serial dilutions
The variation within an experiment is generally small (CV < 10%) but expect modest levels of variation between experiments ~ 20%
Materials
Dulbecco's modified eagle's medium high glucose Thermofisher Gibco 11965092
100X Non-essential amino acids Thermofisher 11140050
100X GlutaMAX Thermofisher 35050061
Trypsin Thermofisher 15090046
Hygromysin B Thermofisher 10687010
PBS: Sterile 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO47H2O, 1.4 mM KH2PO4 pH ~7.3
Fetal Bovine serum
10 cm and 15 cm tissue culture dishes
Polystyrene, Flat bottom, Sterile, Cell culture treated 96 well plates Corning Life Science Product Number 3997
Polypropylene round bottomed 96 well plates
Polystyrene, Flat bottom, non-sterile 96 well plates. Fisherbrand 21-377-203. Part of Thermofisher
Adhesive tops for 96 well plate
Developer: 0.1M CO3, 10mM MgCl2 pH9.5. [7.32 g NaHCO3, 1.37 g Na2CO3, 2 g MgCl2 6H2O, pH 9.5 per liter]
Prior to use add Phosphatase substrate – 4.3 g/ ml.
Phosphatase substrate: 4-Nitrophenyl phosphate disodium salt hexahydrate, pNPP disodium salt hexahydrate, Millipore Sigma P4744-5G
Synergy H4 plate reader (BioTek, Winooski, VT)
Tissue culture hood
Centrifuge
Temperature controlled incubator with 5% CO2
Pipettor: For 1 - 10 microliter, 20 - 200 microliter, 1 mL. and for serological pipettes.
Pipet tips sterile, and serologic pipets, sterile and plugged: 5mL, 10mL and 25 mL.
Sterile cell culture reservoirs
Disposable nitrile gloves
Ice tray
Data handling software: Microsoft EXCEL and GraphPad Prism version 10.3.1
Troubleshooting
Before start
Have pseudovirus stocks at a known concentration
Have sub-confluent 293TT cells that were split the day previous
Make a template in EXCEL that will calculate the dilution of cells, pseudovirus and antibodies when the concentrations are input. The template is also used to calculate the concentration of antibodies after serial dilutions and to keep track of which antibodies are used.
HPV pseudovirus neutralization assay: Ab titration
Prepare the cells:
Remove 293TT cells from plate by rinsing a plate (10 cm or 15 cm) once with PBS. Remove PBS by suction
Add 1 mL Trypsin and return to incubator for 5 min
Add 10 mL of complete media and count the cells.
Complete media is DMEM with 10% FBS + nonessential amino acids, Glutamax and penstrep (antibiotics)
Plate the cells:
Calculate the volume of cells = number of plates x 6ml + 10%
In a 50 mL conical tube: Resuspend 293TT cells at 3 x 105/mL in complete media with hygromysin B 0.4 mg/mL. Vortex briefly and transfer cell to reservoir
Pipet 100 uL into the inner wells of a 96 well plate (60 wells total) using a multichannel pipettor. Mix the cells between plates. It is best to use multichannel pipettor for this and the next few steps if possible because it will be faster and not allow the cells to settle out.
Fill the outer wells with 200 uL PBS
Put into tissue culture incubator and wait at least 2 hours to allow the cells to adhere.
Antibody dilutions:
Using polypropylene 96 well plates, add 100 uL of complete media (no Hygro) to the inner 60 wells
In the first column (2) add 50 uL of culture media containing 6X the final concentration of antibody (when added to the 100 uL in the well the Ab conc is now 2X the final)
Serially dilute across the plate but stop at row 10 and discard the 50 uL from those wells so that all wells now have 100 uL
Prepare pseudovirus:
Calculate the volume of pseudovirus needed: = number of wells X 100 uL + 10% For example 1 plate is 60 wells - 6000 uL + 600 uL = 6.6 mL
Pipet that volume of complete media into an appropriate sized conical tube
Add Hygro to 2X the normal concentration (0.8 mg/mL)
Thaw a frozen tube of pseudovirus and dilute to 4X the target concentration
For example if the final dilution is 1:4000 make a 1:1000 dilution
Vortex briefly and transfer to a cell culture reservoir
In a separate tube dilute hygro (0.8 mg/mL) in complete media - about 350 uL for each plate
Add pseudovirus to antibodies:
Add 100 uL diluted pseudovirus to all except three wells of column 11 of the plates with diluted antibodies
(At this step the antibody is at 1X the final conc and pseudovirus is at 2X the final conc)
For each plate I use one set of tips on the multichannel pipettor but carefully add liquid moving right to left without touching the liquid
Add 100 uL of the diluted hygro without pseudovirus to the other 3 well in column 11
Seal the plates with adhesive and leave on ice for 1 hour
Add pseudovirus/antibodies to cells:
After 1 hr transfer 100 uL from each of the wells of the polypropylene plate to the cell culture plate
Return to the incubator and wait for three days
Prepare developer:
Calculate the amount of developer needed = 6 mL/plate + 10%
weigh out phosphatase substrate ~ 4.3 X vol needed
Add the volume of developer buffer that results in exactly 4.3 mg/mL
Vortex to mix thoroughly and transfer to a cell culture reservoir
Harvest supernatant:
When the plates are ready to harvest (day 3) carefully remove 30 uL from each well and transfer to an flat bottom ELISA plate
Save the cell culture plates in the incubator until the data are collected and you are satisfied with results
Add 100 uL from of developer containing substrate to all wells
Spin in centrifuge for 1 min at 300 xg - this is to remove any bubbles
Incubate in the dark for 30 min00:00:00
Read Plate:
On a plate reader - Read at OD = 405
If many of the well are over the limit of the reader you can repeat the last 2 steps using another 30 uL from the cells. Read the plate multiple times so that all of the values are within the limit of the reader. This is not ideal and indicates that the concentration of pseudovirus is higher than anticipated
Export the raw data to EXCEL for analysis
Convert OD values to percent neutralization:
To get robust data there should be a large difference between the background (ideally less than 500) and 100% reactivity (ideally OD > 1500) [if this difference can't be reached the errors will be higher than anticipated]
Calculate background = the average OD value of the 3 wells in column 11 with no pseudovirus
Calculate 100% reactivity = The average OD value of the 3 wells in column 11 with pseudovirus without antibody
Percent reactivity for each well = 100 X (OD - background) / (Reactivity-background)
Percent neutralization = 100 - percent reactivity
Calculate the IC50 values:
If IC50 reported as ambiguous this indicates one or more values were outliers or there were too few informative data points (not enough data points close to IC50)
Inspect the data to see if there are outliers and eliminate
Recalculate IC50 values
If result is still ambiguous - Repeat the experiment adjusting the concentration of Abs
Ambiguous will also be reported if there is no neutralization activity.
Transfer the % neutralization values to the Y-columns in Prism X-Y
In the X-column enter the log dilution or log concentration of antibody corresponding for each well
Under analysis using non-linear regression choose Sigmoidal 5PL (or 4PL) X is log concentration
You will find the IC50 values in the analysis tab
Protocol references
D. V. Pastrana, C. B. Buck, Y. Y. Pang, C. D. Thompson, P. E. Castle, P. C. FitzGerald, S. J. Kjaer, D. R. Lowy and J. T. Schiller. Virology 2004 Vol. 321 Issue 2 Pages 205-216. Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18
J. J. Carter, G. C. Wipf, M. M. Madeleine, S. M. Schwartz, L. A. Koutsky and D. A. Galloway. J Virol 2006 Vol. 80 Issue 10 Pages 4664-72 Identification of human papillomavirus type 16 L1 surface loops required for neutralization by human sera
Acknowledgements
This assay was developed in John Schillers laboratory and uses cells and pseudovirus created there by Chris Buck and Diane Pastrana among others.