SARS-CoV-2 Spike Protein Phagocytosis Assay

We measured ADCP as previously described (18 (link)). We incubated biotinylated SARS-CoV-2, SARS-CoV-1, or MERS-CoV S protein with yellow-green neutravidin-fluorescent beads (Molecular Probes-Thermo Fisher Scientific, https://www.thermofisher.com) for 2 h (37°C). We incubated a 100-fold dilution of beads to protein (10 μL) for 2 h at 37°C along with 100 μL of 100-fold diluted plasma before adding THP-1 cells (MilliporeSigma, https://www.sigmaaldrich.com) at 25,000 cells per well. After a 19-h incubation, we fixed cells with 4% formaldehyde solution (Tousimis, https://www.tousimis.com) and evaluated fluorescence on an LSRII (BD Biosciences, https://www.bdbiosciences.com). We calculated the phagocytic score by multiplying the percentage of bead-positive cells by the geometric MFI and dividing by 10⁴.

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Li Y., Merbah M., Wollen-Roberts S., Beckman B., Mdluli T., Swafford I., Mayer S.V., King J., Corbitt C., Currier J.R., Liu H., Esber A., Pinyakorn S., Parikh A., Francisco L.V., Phanuphak N., Maswai J., Owuoth J., Kibuuka H., Iroezindu M., Bahemana E., Vasan S., Ake J.A., Modjarrad K., Gromowski G., Paquin-Proulx D, & Rolland M. (2022). Coronavirus Antibody Responses before COVID-19 Pandemic, Africa and Thailand. Emerging Infectious Diseases, 28(11), 2214-2225.

Publication 2022

yellow-green neutravidin-fluorescent beads THP-1 cells 4% formaldehyde solution

Cells plasma Dilution Fluorescence Formaldehyde solution Green fluorescent protein Mers cov Molecular probes Neutravidin Phagocytic Protein Sars cov 1 Sars cov 2

Corresponding Organization : Makerere University Walter Reed Project

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Variable analysis

independent variables

Incubation of biotinylated SARS-CoV-2, SARS-CoV-1, or MERS-CoV S protein with yellow-green neutravidin-fluorescent beads

dependent variables

Percentage of bead-positive cells
Geometric MFI

control variables

Incubation time (2 h)
Incubation temperature (37°C)
Dilution of beads to protein (100-fold)
Dilution of plasma (100-fold)
Number of THP-1 cells per well (25,000)
Incubation time with THP-1 cells (19 h)
Fixation method (4% formaldehyde solution)
Fluorescence evaluation (LSRII)

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