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| Assay Type | Inhibition-TR-FRET |
| Analyte | Human IgG, Human IgG Fc protein, Anti-human CD64 antibody |
| Format | 100T/500T |
| Reactivity | Human |
| Regulatory Status | RUO |
| Sensitivity | IC50=5.276nM |
| Standard Curve Range | 0.244 nM-1000 nM |
| Assay Time | 1 hr |
| Suitable Sample Type | For the binding of IgG Fc region to the human CD64 |
| Sample volume | 10 μL |
Interaction sites for FcγRs are indicated to be located to the hinge proximal region of IgG-Fc via their extracellular domains (ECDs). Within IgG, the fragment antigen binding (Fab) region can exert direct effects through binding interactions with antigen. Meanwhile, the fragment crystallizable (Fc) region interacts with FcγRs to mediate indirect effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and these interactions are important for the efficacy and safety of therapeutic antibodies. One of the major functions of Human FcγRI is to activate myeloid cells to phagocytose IgG1 and IgG-bound target cells via ADCP.
This kit is designed to facilitate the ADCC and ADCP functional performance evaluation of antibody drug candidates, and also high-throughput screening of anti-human CD64 antibodies. It can also be used as a universal detection tool to identify the ability of antibody drugs to bind to human CD64.
It is for research use only.
2. Find the expiration date on the outside packaging and do not use reagents past their expiration date.
3. The opened kit should be stored per components table. The shelf life is 30 days from the date of opening.
| ID | Components | Size |
| FRT03-C01 | Human Fc gamma RI / CD64 Protein Europium-chelate | 0.6 μg/100 tests 3 μg/500 tests |
| FRT03-C02 | FA labeled human IgG antibody | 2.4 μg/100 tests 12 μg/500 tests |
| FRT03-C03 | Human IgG standard | 100 μg/100 tests 500 μg/500 tests |
| FRT03-C04 | Sample Dilution Buffer | 10 mL/100tests & 500tests |
| FRT03-C05 | Detection Buffer | 10 mL/100tests & 500tests |
Inhibition Assay of interaction of Human CD64 and Human IgG1 antibody by Human IgG standard in a homogeneous (no wash) TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) competition assay, with a typical IC50 of 5.276 nM (QC tested).
The kit has been used to detect different subclasses of Human IgG Whole and Fc fragment proteins (Human IgG1, Human IgG2, Human IgG3 and Human IgG4), which exhibit different IC50 results as expected. As shown in the figure, human CD64 is a high affinity receptor that binds to human IgG1, IgG3 and IgG4 with nanomolar affinity, while it has not been shown to bind to human IgG2.
The kit has been used to detect different subclasses of mouse IgG (mouse IgG1, mouse IgG2a and mouse IgG2b), which exhibit different IC50 results as expected. As shown in the figure, human CD64 binds to mouse IgG2a with a high affinity, but not mouse IgG1 and mouse IgG2b.
The kit has been used to detect four FDA approved antibody drugs with different affinities binding to human CD64. Bevacizumab, Toripalimab, and Efgartigimod alfa bind to human CD64 with the nanomolar affinity. The Fc of Eculizumab has been modified into the human IgG2 hinge region and human IgG4 CH2-CH3 region, so it doesn’t bind to human CD64.
Verify potential matrix effects by adding different levels of DEME, RPMI1640, FBS and HSA to the Sample Diluted buffer.
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