ADCP was measured as previously described (65 (link)). Briefly, gp120 CM235 (NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 CM235 gp120 Recombinant Protein, ARP-12816, contributed by NIAID, DAIDS) was biotinylated at a biotin-to-protein ratio of 50 according to the manufacturer’s instructions (Thermo Fisher Scientific) and incubated with yellow-green streptavidin fluorescent beads (Molecular Probes) for 2 hours at 37°C. Ten microliters of a 100-fold dilution of beads-protein was incubated for 2 hours at 37°C with 100 μL of 200-fold diluted plasma samples before addition of Tamm-Horsfall protein 1 (THP-1) cells (20,000/well; MilliporeSigma). After an 18-hour incubation at 37oC, the cells were fixed with 4% formaldehyde solution (Tousimis), and fluorescence was evaluated on an LSR II (BD Biosciences). The phagocytic score was calculated by multiplying the percentage of bead-positive cells by the geo MFI of the bead-positive cells and dividing by 104.
Mitchell J.L., Pollara J., Dietze K., Edwards R.W., Nohara J., N’guessan K.F., Zemil M., Buranapraditkun S., Takata H., Li Y., Muir R., Kroon E., Pinyakorn S., Jha S., Manasnayakorn S., Chottanapund S., Thantiworasit P., Prueksakaew P., Ratnaratorn N., Nuntapinit B., Fox L., Tovanabutra S., Paquin-Proulx D., Wieczorek L., Polonis V.R., Maldarelli F., Haddad E.K., Phanuphak P., Sacdalan C.P., Rolland M., Phanuphak N., Ananworanich J., Vasan S., Ferrari G, & Trautmann L. (2022). Anti-HIV antibody development up to 1 year after antiretroviral therapy initiation in acute HIV infection. The Journal of Clinical Investigation, 132(1), e150937.
Other organizations :
Oregon Health & Science University, Walter Reed Army Institute of Research, Henry M. Jackson Foundation, Duke Medical Center, Chulalongkorn University, Drexel University, HIV Netherlands Australia Thailand Research Collaboration, Bamrasnaradura Infectious Diseases Institute, Armed Forces Research Institute of Medical Science, National Institute of Allergy and Infectious Diseases, National Cancer Institute, Amsterdam Institute for Global Health and Development, University of Amsterdam
Biotinylation ratio of gp120 CM235 (50 biotin-to-protein ratio)
dependent variables
Phagocytic score (calculated by multiplying the percentage of bead-positive cells by the geo MFI of the bead-positive cells and dividing by 10^4)
control variables
Incubation time of beads-protein with plasma samples (2 hours at 37°C)
Incubation time of beads-protein-plasma with THP-1 cells (18 hours at 37°C)
Concentration of THP-1 cells (20,000 cells per well)
positive controls
None specified
negative controls
None specified
Annotations
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