CD16a and variants cellular binding assays

HTRF CD16a and variants binding assays for the cell-based characterization of therapeutic antibodies and for ADCC prediction

The CD16a cellular binding assays are simple and robust tests to monitor binding of the IgG  Fc region to the human CD16a receptor (or FcγIIIRa) on live cells and to select the most potent antibody for use in Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC). The assays can also be used to assess the degree of antibody afucosylation.  Two separate CD16a assays are available to specifically target the V158 or F158 variants. These assays, based on our innovative tag-lite technology, can easily be automated to create a high-throughput solution for predicting ADCC.

The contribution of IgG Fc-mediated effector functions in ADCC has motivated the efforts being made to enhance interactions with Fc gamma receptors (FcγRs) for the development and manufacturing of therapeutic antibodies. In humans, Fc gamma Rs are divided into three groups, known as CD64 (FcγRI), CD32 (FcγRI) and CD16 (FcγRIII). Cisbio Bioassays has developed a cell-based FcR assay platform for selective antibody binding analyses using each type of receptor.

NEW! The  FcR platform has been extended to neonatal Fc-receptor in a biochemical assay.

Assay Theory

Like all Fc gamma receptor binding assays, the CD16a receptor cellular binding assays are competitive assays here based on Tag-lite technology. Each assay uses HEK293 cells expressing a specific CD16a variant conjugated to Lumi4-terbium cryptate, and human IgG1 labeled with d2. 

The FcRn assay, based on a biochemical competition format, uses a recombinant human FcRn receptor labelled with Europium cryptate and d2-labelled human IgG-1 antibody.

For each assay format, the unlabelled antibody to be characterized competes with d2-IgG1 for binding to the receptor, resulting in a decrease in the specific FRET signal. An example of the HTRF cell-based CD16a binding assay is shown below.

Assay Principle

The CD16a receptor binding assays (FcgIIIa V158 or F158) use a one-step dispensing protocol: Lumi4-terbium Cryptate pre-labelled cells are dispensed, followed by the addition of IgG1 labelled with d2 and the sample. After 2hrs incubation at RT, the signal can be read.

Product Performance

Case study: CD16a (FcgIIIa V158)

Specificity for IgG subclasses

The frozen cryptate pre-labeled cells were thawed and plated as indicated in the protocol in a 384 small-volume plate. Increasing concentrations of unlabeled IgGs were incubated in presence of a constant concentration of d2-labeled IgG1. The plate was then read on a PHERAstar microplate reader (BMG Labtech). As previously described, we confirm that the affinity of IgG1 for CD16a is better than that of IgG3 and considerably higher than IgG2 and IgG4 (Bruhns et al., Blood 2009).

Evaluation of a-fucosylation on affinity

To evaluate the impact of IgG a-fucosylation on CD16a affinity, the same IgG with various degrees of fucosylation was tested following the previously-described protocol.
IgG affinity for CD16a and degree of a-fucosylation were very well correlated. These results confirm that a substantial gain of immunoglobulin affinity can be reached when the level of a-fucosylation is higher than 50%.
The CD16a assay can therefore be used for the prediction of a-fucosylation degree and ADCC potential.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
CD16a (FcgRIIIa, V158) - 5 packs 62C16PAG62C16PAE
CD16a (FcgRIIIa, V158) - 10 packs 62C16PAG62C16PAF
CD16a (FcgRIIIa, V158) cellular kit - 100 tests62C16PAG
CD16a (FcgRIIIa, F158) - 5 packs 6FR3APAG6FR3APAE
CD16a (FcgRIIIa, F158) - 10 packs 6FR3APAG6FR3APAF
CD16a (FcgRIIIa, F158) cellular kit - 100 tests6FR3APAG

Companion products

DescriptionCat. noProduct insertMSDS
FcRn kit - Standard63ADK045CDA
FcRn kit - 100 tests63ADK045PEG
CD64 (FcgRI) - 5 packs 6FC64PAG6FC64PAE
CD64 (FcgRI) - 10 packs 6FC64PAG6FC64PAF
CD64 (FcgRI) cellular kit - 100 tests6FC64PAG
CD32b (FcgRIIb) - 5 packs 6FR2BPAG6FR2BPAE
CD32b (FcgRIIb) - 10 packs 6FR2BPAG6FR2BPAF
CD32b (FcgRIIb) cellular kit - 100 tests6FR2BPAG
CD32a (FcgRIIa, H131) - 5 packs 6FRAHPAG6FRAHPAE
CD32a (FcgRIIa, H131) - 10 packs 6FRAHPAG6FRAHPAF
CD32a (FcgRIIa, H131) cellular kit - 100 tests6FRAHPAG
CD32a (FcgRIIa, R131) - 5 packs 6FRARPAG6FRARPAE
CD32a (FcgRIIa, R131) - 10 packs 6FRARPAG6FRARPAF
CD32a (FcgRIIa, R131) cellular kit - 100 tests6FRARPAG