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Lumit™ FcγR Binding Immunoassays

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Measure Fcγ Receptor Binding

  • No wash or transfer; simple add-mix-read protocol
  • Results in less than 1 hour
  • Scalable and high throughput; perform assays in 96-well or 384-well plates

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Lumit™ FcγR Binding Immunoassays
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Faster, Simpler Fc Receptor Binding Assays

The Lumit™ FcγR Binding Immunoassays are novel homogeneous (no-wash) competition assays to measure the interaction between human Fc receptors and antibodies or Fc fusion proteins. Lumit™ Immunoassays are based on NanoBiT® complementation to generate a luminescent signal. In the assays, a human IgG1 labeled with LgBiT (Tracer-LgBiT) is used as the tracer. A C-terminal biotinylated human FcγR bound to Streptavidin-SmBiT (FcγR-Biotin-SA-SmBiT) is used as the target. In the absence of an antibody analyte sample, Tracer-LgBiT binds to the FcγR-SmBiT target, resulting in maximum luminescence signal. In analyte samples, unlabeled IgG will compete with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in luminescent signal. These easy-to-use biochemical assays can complement and provide orthogonal data to support results from cell-based functional bioassays.

Fcγ Receptor Function

Fc receptors for IgG (FcγRs) are expressed by a variety of cells of hematopoietic origin and contribute to protective functions of the immune system. These receptors are so named because they possess affinity for the fragment-crystallizable (Fc) region of antibody molecules. Upon binding antibodies attached to diseased cells or pathogens, these receptors mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The various Fcγ receptors differ in their affinity for IgG subclasses, and these interactions are further modified by the IgG glycosylation pattern.

Learn more about the Lumit™ FcγR Binding Immunoassays

Nath, N. et al. (2022) A homogeneous bioluminescent immunoassay for parallel characterization of binding between a panel of antibodies and a family of Fcγ receptors. Sci Rep 12, 12185.

Read the Paper
nath-fcgr-paper

Simple Protocol, Reproducible Results

The Lumit™ FcγR Binding Immunoassays are solution-based homogeneous assays that require no washes or sample transfer steps.

Lumit FcγR Binding Immunoassay workflow diagram. 17985ma

How the Lumit™ FcγR Binding Immunoassays Work

In the absence of an antibody analyte, Tracer-LgBiT binds to the FcγR-SmBiT target, resulting in maximum luminescence signal. In samples containing analyte, nonlabeled IgG will compete with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in the luminescent signal.

Schematic for Lumit FcγR Binding Immunoassays. 17986ma

Assay Performance Data

Lumit™ FcγRI Binding Immunoassay

Lumit FcγRI Binding Immunoassay. Cetuximab, Pembrotizumab, Pantinumumab17987ma

FcγRI binds to IgG in a subclass-specific manner in the Lumit™ Immunoassay with IC50 values reflective of relative IgG affinity (IgG3>IgG1>IgG4>>>IgG2).

Lumit™ FcγRIIa (H131) Binding Immunoassay

Lumit FcγRIIa (H131) Binding Immunoassay: Cetuximab, Pembrotizumab, Pantinumumab. 17988ma

FcγRIIa (H131) binds to IgG in a subclass-specific manner in the Lumit™ Immunoassay with IC50 values reflective of relative IgG affinity (IgG3>IgG1≈IgG2>>>IgG4).

Lumit™ FcγRIIa (R131) Binding Immunoassay

A rightward shift in IC50 is observed following control antibody dilution in the Lumit FcγRIIa (R131) Binding assay. 17989ma

Lumit™ FcγR Binding Immunoassays can be used as potency assays to complement cell-based functional Fc effector activity assays. A rightward shift in IC50 is observed following control antibody dilution in the Lumit™ FcγRIIa (R131) Binding Immunoassay.

Lumit™ FcγRIIIa (V158) Binding Immunoassay

FcγRIIIa (V158) binds to IgG in a subclass-specific manner in the Lumit™ Immunoassay with IC50 values reflective of relative IgG affinity. 17990ma

FcγRIIIa (V158) binds to IgG in a subclass-specific manner in the Lumit™ Immunoassay with IC50 values reflective of relative IgG affinity (IgG1>IgG3>IgG4>IgG2).

Lumit™ FcγRIIIa (F158) Binding Immunoassay

Lumit FcγR Binding Immunoassays can be used to assess antibody glycan status. 17991ma

Lumit™ FcγR Binding Immunoassays can be used to assess antibody glycan status. IC50 shifts in non-fucosylated (Anti-hCD20-hIgG1fut) or non-glycosylated (Anti-hCD20-hIgG1NQ) antibodies can be detected with Lumit™ FcγR Binding Immunoassays.

Kit Contents

  • Tracer-LgBiT
  • FcγR-SmBiT
  • Lumit™ Detection Substrate
  • FcγR Assay Buffer
  • FcγR Control Antibody

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