whitepaper

Application note: Fc-Receptor binding in ADCC assays utilising LANCE Ultra Technology – characterisation of hIGs and FcγRIIIa

In this application note, PerkinElmer demonstrates the ease and utility of using LANCE® Ultra, a homogeneous assay format, for determining binding profiles of therapeutic monoclonal antibodies to FcγRs.

Fc Receptors are cell-surface proteins found on a wide variety of cell types and are involved in some of the actions of the adaptive immune system. Specifically, Fc-Gamma Receptors (FcγRs) are members of the immunoglobin superfamily and play a critical role in the function of therapeutic antibodies.

FcγRIIIa is one of two forms of FcγRs and is a low/intermediate affinity receptor for polyvalent immune-complexed IgG. It is involved in phagocytosis, secretion of enzymes and inflammatory mediators, antibody-dependent cellular cytotoxicity (ADCC), mast cell degranulation and clearance of immune complexes. In humans, a single polymorphism creates two isoforms (high and low binding) that when homozygous, may influence susceptibility to immune diseases or response to therapeutic IgG antibodies. As such, FcγRIIIa is considered to be a therapeutic target.

LANCE and LANCE (Lanthanide chelate excite) Ultra are our TR-FRET (time-resolved fluorescence resonance energy transfer), homogeneous (no wash) technologies. One protein of interest is labelled with a donor fluorophore (a LANCE Europium chelate) and the second protein is labelled with an acceptor fluorophore (ULight™ dye). Upon excitation at 320 or 340 nm, energy can be transferred from the donor Europium chelate to the acceptor fluorophore if sufficiently close for FRET (~10 nm). This results in the emission of light at 665 nm

Here we use the LANCE Ultra TR-FRET homogeneous technology to take a closer look at the binding of human IgGFc fragment to human FcγRIIIa. We also show that LANCE Ultra can be used to study how other antibodies bind to FcγRIIIa via a competition assay.

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