An assay for measuring antibody-dependent THP-1 monocyte / cellular phagocytosis (ADCP) was used as previously described.68 (link) Briefly, 1 μm yellow-green fluorescent NeutrAvidin beads were coupled to biotinylated PfRH5 antigen and blocked overnight with PBSA. The beads were then washed twice with PBSA, diluted to 1.8 × 108 beads/mL, and 10 μL beads/well were added to a 96-well round-bottom microplate. Diluted plasma from immunized subjects (10 μL/well) was added to the beads and incubated at 37°C for 2 h to allow the formation of immune complexes. Unbound antibodies were washed off, then 25,000 THP-1 cells/well (ATCC, #TIB-202) were added to the beads in 200 μL THP-1 medium (R-10 + 55 μM β-ME) and incubated overnight at 37°C. Cells were fixed and acquired on an Intellicyt iQue Screener PLUS flow cytometer. The phagocytic score for each sample was calculated as (% bead-positive cells) x (gMFI of bead-positive cells)/(10 x gMFI of first bead-positive peak).
Minassian A.M., Silk S.E., Barrett J.R., Nielsen C.M., Miura K., Diouf A., Loos C., Fallon J.K., Michell A.R., White M.T., Edwards N.J., Poulton I.D., Mitton C.H., Payne R.O., Marks M., Maxwell-Scott H., Querol-Rubiera A., Bisnauthsing K., Batra R., Ogrina T., Brendish N.J., Themistocleous Y., Rawlinson T.A., Ellis K.J., Quinkert D., Baker M., Lopez Ramon R., Ramos Lopez F., Barfod L., Folegatti P.M., Silman D., Datoo M., Taylor I.J., Jin J., Pulido D., Douglas A.D., de Jongh W.A., Smith R., Berrie E., Noe A.R., Diggs C.L., Soisson L.A., Ashfield R., Faust S.N., Goodman A.L., Lawrie A.M., Nugent F.L., Alter G., Long C.A, & Draper S.J. (2021). Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination. Med (New York, N.y.), 2(6), 701-719.e19.
Other organizations :
Vector Oncology (United States), Massachusetts Institute of Technology, Ragon Institute of MGH, MIT and Harvard, Institut Pasteur, Guy's and St Thomas' NHS Foundation Trust, King's College London, University Hospital Southampton NHS Foundation Trust, University of Southampton, Wellcome Trust, ExpreS2ion Biotechnologies (Denmark), United States Agency for International Development
Phagocytic score for each sample, calculated as (% bead-positive cells) x (gMFI of bead-positive cells)/(10 x gMFI of first bead-positive peak)
control variables
1 μm yellow-green fluorescent NeutrAvidin beads coupled to biotinylated PfRH5 antigen and blocked overnight with PBSA
THP-1 cells (ATCC, #TIB-202)
positive controls
Not explicitly mentioned
negative controls
Not explicitly mentioned
Annotations
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