ADCD was assessed using a flow cytometry-based complement-fixing assay.97 (link) Briefly, 10 × 106 HUT78 cells were pulsed with 2 μg of recombinant HIV-1 gp120 from strain CN54 (Acro Biosystems; catalog #GP0-V182E6) for 20 minutes at 37°C. Unbound gp120 was removed by washing the cells twice with 1% PBS-BSA buffer. Bulk IgG samples from study participants were added to the antigen-pulsed cells and incubated for another 30 minutes at 37°C. Freshly resuspended lyophilized guinea pig complement (Cedarlane Labs; catalog #CL4051), diluted 1:20 with veronal buffer 0.1% gelatin with calcium and magnesium (Boston BioProducts; catalog #IBB-300), was added to the cells for 2 hours at 37°C. After washing with 1X PBS, the cells were assessed for complement deposition by staining with goat anti-guinea pig C3-FITC (MP Biomedicals; catalog #0855385). After fixing, the cells were analyzed by flow cytometry, and ADCD was reported as the mean fluorescence intensity (MFI) of FITC-positive cells.
Protocol detail
Flow Cytometry-Based ADCD Assay
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Antigen
Assay
Buffer
Bulk
Calcium
Cells
Fitc
Flow cytometry
Fluorescence
Gelatin
Goat
Gp120
Guinea pig
Hiv 2
Magnesium
Strain
Corresponding Organization : The Wistar Institute
Other organizations : Cornell University, Johns Hopkins University, Albert Einstein College of Medicine, Emory University, SUNY Downstate Health Sciences University, University of Miami, University of Pittsburgh, University of North Carolina at Chapel Hill, University of California, Los Angeles, Georgetown University Medical Center, Georgetown University, University of California, San Francisco, University of Alabama at Birmingham, Northwestern University, The Lundquist Institute, Rush University, University of Pennsylvania
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Variable analysis
independent variables
- Amount of recombinant HIV-1 gp120 from strain CN54 (2 μg)
- Complement deposition on antigen-pulsed cells, measured as mean fluorescence intensity (MFI) of FITC-positive cells
- Number of HUT78 cells (10 × 10^6)
- Incubation time of cells with gp120 (20 minutes)
- Incubation time of cells with participant IgG samples (30 minutes)
- Incubation time of cells with guinea pig complement (2 hours)
- Concentration of guinea pig complement (diluted 1:20 with veronal buffer)
- Freshly resuspended lyophilized guinea pig complement
- Not explicitly mentioned
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