NEUTRALIZATION ASSAY FOR L2 SERA
Patricia Day and Susana Pang
John Schiller’s group
Lab of Cellular Oncology, NCI/NIH
Citation: Clin Vaccine Immunol. 2012 Jul;19(7):1075-82. A human papillomavirus (HPV) in vitro neutralization assay that recapitulates the in vitro process of infection provides a sensitive measure of HPV L2 infection-inhibiting antibodies.
Day PM, Pang YY, Kines RC, Thompson CD, Lowy DR, Schiller JT.
Typical schedule:
Friday:
split CHO-Δfurin cells 1x106cells/17 mls in T75 (enough for 3x 96 plates)
Monday:
plate MCF10A cells 2x104/well 96 well plate 100 µl volume/well (plate in am)
split pgsa-745 cells to be ready on Wednesday
Tuesday:
make ECM from 10A cells: wash cells with PBS 3x
add 50 µl pre-warmed lysis buffer/well
incubate 5 minutes, confirm lysis microscopically
gently add 100 µl PBS (add to side walls)
remove 100 µl liquid
gently add 100 µl PBS
remove 100 µl liquid
gently add 100 µl PBS
remove all liquid
rinse wells 2x with 100 µl PBS
leave plate in PBS until ready for next step
harvest supernatant from CHO-Δfurin cells
pellet cells and decant sup to new tube
make dilutions of virus in L2 assay medium calculate to add 50 µl/well virus (see notes following)
remove PBS from plate
add 70 µl/well of Δfurin sup with Heparin (add heparin to final concentration of 5µg/ml, note that after adding 50µl of virus, the final volume will be 120µl)
add virus dilution, leave one row with no virus for background control
incubate at 37°C overnight
Wednesday:
remove medium/virus mix
wash with PBS 2X. leave on PBS until ready for next step
make antibody titrations in L2 assay media, need 100µl/well
remove last wash and add antibody (or media for infection controls)
incubate at 37°C 6 hours
harvest pgsa-745 cells with 0.05% trypsin
pellet cells and resuspend in L2 assay media
do not remove antibody
add pgsa-745 cells 50 µl/well at concentration of 1.6x105/ml (8x103/well)
incubate 37°C 2 days
Friday
rinse with PBS, add 50 µl/well .05% trypsin, add 150 µl/well FACS buffer
read GFP fluorescence on flow cytometer
Notes:
•Can store 10A ECM at -80°C w/ and w/o 100 µl PBS or 4°C with 100 µl PBS, 0.1% NaN3
for at least 2 months.
•CHO-Δfurin supernatant is good for at least 3 days up to 7 days.
MCF10A cells (ATCC cat# CRL-10317)
media: DMEM/F12
5% horse serum (Sigma Cat# H1138, Lot# 11G413)
glutamine
pen/strep
500 µg/ml hydrocortisone
10 µg/ml insulin
20 ng/ml EGF
CHO-Δfurin cells (NCI Cell Repository)
& pgsa-745 cells (ATCC cat# CRL-2242)
media: DMEM
10% FBS
200 µM proline
1% Pen Strep
L2 assay medium
media: DMEM
10% FBS
200 µM proline
1% Antibiotic/Antimycotic
H-4784 heparin (Sigma)
Lysis buffer: PBS
0.5% v/v Triton x-100
20 mM NH4OH
store at 4oC
FACS buffer: PBS
2% FBS
0.1% NaN3
Pseudovirus: make “ripcord” method pseudovirus with packaged fwB plasmid
titer pseudovirus in assay prior to testing neutralization
the starting concentration should give ~60% infection
at present we assay 3 dilutions of virus (3-fold dilutions starting from 60% infection)
for our HPV16 stock the starting concentration is 13 ng L1/well
typical plate setup
row A: media/cells only
row B: virus/no ab/cells
rows C-H: virus/serial dilution of antibody/cells
column 1: no virus/ab dilutions/cells
columns 2-12: virus dilutions or different viruses
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