Table 2 In vitro assays used to assess Fc-dependent antibody effector functions

Binding assays

Antibody–FcR binding

Surface plasmon resonance

FcRs are immobilized on a biosensor and antibody binding is measured

173,174

ELISA

Recombinant FcRs are coated onto plates and antibodies are serially diluted to measure binding

174

Flow cytometry/FACS

Fluorescent antibodies are incubated with cells expressing FcRs. Median fluorescence intensity is measured to assess antibody binding

174

Fc-array with microspheres

Antigen-coated microspheres are incubated with serum. Microsphere–antibody complexes are subsequently incubated with fluorescent FcRs to assess median fluorescence intensity

175

Reporter-based assays

FcR-mediated transcriptional activation

ADCC and ADCP reporter bioassays

Engagement of FcR on effector cells activates intracellular signalling resulting in transcriptional activation of reporter gene expression

176

Functional assays

ADCC

Fluorescence reduction

Effector cell killing of fluorescent target cells results in a reduction in the size of the target cell population as measured by flow cytometry

177

Rapid fluorescent ADCC

Target cells are dual-stained with CFSE (viability) and PKH-26 (membrane dye). Incubation with effector cells results in target cell killing, as measured by the CFSE-negative population within the PKH-26-positive gate as measured by flow cytometry

173,178

Chromium release assay

Target cells are labelled with ⁵¹Cr and co-incubated with effector cells. ⁵¹Cr is released into the supernatant upon cell killing and radioactivity is measured

179

Effector cell activation

Target cells are co-incubated with effector cells. Flow cytometry is used to assess the percentage of effector cells positive for IFNγ, CCL4 and CD107a

173,180

ADCP

Fluorescence uptake

Uptake of antigen-coated beads by effector cells is assessed by flow cytometry. Both median fluorescence intensity and the percentage of bead-positive effector cells can be measured

180

Fluorescent dye-labelled target cells are co-incubated with effector cells. Flow cytometry is used to assess the percentage of fluorescent dye-positive effector cells

181

ADCD

Bead-based assays

Antibodies are incubated with antigen-coated beads and subsequently incubated with complement. C3 deposition on beads can then be assessed as a measure of median fluorescence intensity

182

Virus-based assays

Virions are incubated with complement, resulting in viral membrane disruption. Viral proteins are conjugated to streptavidin-coated beads and fluorescence intensity is determined by flow cytometry

183

Live/dead staining

Antigen-expressing target cells are incubated with serum and cells are stained with a viability marker to assess membrane integrity. Flow cytometry is used to measure the magnitude of cell killing

183

Antibody-dependent cell-mediated virus inhibition

ELISA

Infected target cells are incubated with antibodies and effector cells. An ELISA is performed on the supernatant to measure viral antigen

174,184