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| Assay Type | Inhibition-TR-FRET |
| Analyte | Human IgG, Human IgG Fc protein, Anti-human CD16a antibody |
| Format | 100T/500T |
| Reactivity | Human |
| Regulatory Status | RUO |
| Sensitivity | IC50=390.4nM |
| Standard Curve Range | 2.4414 nM-10000 nM |
| Assay Time | 1 hr |
| Suitable Sample Type | For the binding of IgG Fc region to the human CD16a |
| Sample volume | 10 μL |
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 |
| FRT07-C01 | Human Fc gamma RIIIA / CD16a (V176) Protein Europium-chelate | 100 tests/500 tests |
| FRT07-C02 | FA labeled human IgG antibody | 100 tests/500 tests |
| FRT07-C03 | Human IgG Standard | 400 μg/100 tests 2000 μg/500 tests |
| FRT07-C04 | Sample Dilution Buffer | 10 mL/100tests & 500tests |
| FRT07-C05 | Detection Buffer | 10 mL/100tests & 500tests |
Inhibition Assay of interaction of Europium-chelate labeled human Fc gamma RIIIA / CD16a (V176) and FA labeled human lgG by Human IgG standard in a homogeneous (no wash) TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) competition assay, with a typical IC50 of 387.2 nM (QC tested).
The kit has been used to detect different subclasses of Human IgG (Human IgG1, Human IgG2, Human IgG3 and Human IgG4), which exhibit different IC50 results as expected. As shown in the figure, human CD16a (V176) binds to human IgG1, IgG2, IgG3 and IgG4 with low affinity, and IgG1 and IgG3 show the higher affinity than IgG2 and IgG4.
The kit has been used to detect different subclasses of mouse IgG, which exhibit different IC50 results as expected. The figure shows that human CD16a (V176) has very weak or no binding to mouse IgG1, mouse IgG2a, and mouse IgG2b as observed.
The kit has been used to detect four FDA approved antibody drugs with different affinities binding to human CD16a (V176). Bevacizumab and Efgartigimod alfa bind to human CD16a (V176) with the nanomolar affinity from 300nM to 700nM. Toripalimab doesn’t bind to human CD16a (V176). 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 CD16a (V176).
Verify potential matrix effects by adding different levels of DEME, RPMI1640, FBS and HSA to the Sample Diluted buffer.
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