膜杰作
Star Staining
产品描述（Product Details）
| 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 |
背景（Background）
Human FcγRI (CD64) is a 70kDa transmembrane glycoprotein, a member of the Ig superfamily, with three C2 structures in the extracellular region. Human FcγRI mainly binds to human monomer IgG1/ IgG3 and mouse IgG2a/IgG3, the binding affinity with human IgG4 is clearly reduced. In contrast, Human FcγRI has no binding to human IgG2.
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.
应用说明（Application）
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.
存储（Storage）
1. Unopened kit should be stored at 2℃-8℃ upon receiving.
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.
组分（Materials Provided）
| 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 |
原理（Assay Principles）
This Human Fc gamma RI / CD64 binding kit (TR-FRET) is based on TR-FRET technology (Time-Resolved Fluorescence Resonance Energy Transfer). Use the mixture of biotinylated human Fc gamma RI / CD64 and Europium-chelate labeled streptavidin as the donor, FA labeled Human IgG1 antibody as the acceptor.
Your experiment will include 3 simple steps:
1) Mix the sample or Human IgG standard in the kit with Human Fc gamma RI / CD64 Protein Europium-chelate (Donor) and incubate at room temperature for 0.5 hours.
2) Add FA labeled human IgG antibody (Acceptor) and incubate at room temperature for at least 0.5 hours.
3) Use the TR-FRET module of a microplate reader to read the fluorescence signal at 665nm and 620nm. Calculate the Ratio based on the formula Ratio = "Signal 665 nm" /" Signal 620 nm" × 10^4. The Ratio value is negatively correlated with the antibody content in the sample.
— When the sample does not contain human Fc gamma RI / CD64 binding components, the donor and acceptor are in close proximity because of the binding of human Fc gamma RI / CD64 and FA labeled Human IgG1 antibody. The 620nm signal emitted by the donor under specific light source excitation is received by the acceptor, emitting a 665nm signal.
— When the sample contains human Fc gamma RI / CD64 binding components, the components inhibit the binding between the donor and acceptor and thereby prevents FRET from occurring.
活性（Bioactivity）-TR-FRET Please refer to DS document for the assay protocol.
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.
背景（Background）：Fc gamma RI / CD64
Receptors that recognize the Fc portion of IgG are divided into three groups designated Fc gamma RI, RII, and RIII, also known respectively as CD64, CD32, and CD16. Fc gamma RI binds IgG with high affinity and functions during early immune responses. Fc gamma RII and RIII are low affinity receptors that recognize IgG as aggregates surrounding multivalent antigens during late immune responses.
High affinity immunoglobulin gamma Fc receptor I is also known as FCGR1A, FCG1, FCGR1, CD64 and IGFR1, is a type of integral membrane glycoprotein that binds monomeric IgG-type antibodies with high affinity, which belongs to the immunoglobulin superfamily or FCGR1 family. FCGR1A / CD64 contains 3 Ig-like C2-type (immunoglobulin-like) domains. CD64 is constitutively found on only macrophages and monocytes, but treatment of polymorphonuclear leukocytes with cytokines like IFNγ and G-CSF can induce CD64 expression on these cells.
