膜杰作
Star Staining
优势与特点（FAB）
- Cost effective - Sufficient quantity at a lower price, accounting for dilution and pipetting losses.
- Comprehensive validation - Validated with various antibody subtypes and antibody drugs.
- Simple and fast operation - No complicated washing steps, significantly reducing time.
- High batch consistency - Strict control over raw materials and finished product quality, ensuring a stable supply.
- Accurate and reliable results - High sensitivity with minimal matrix effects.
- High throughput capability - Supports 500 tests, ideal for high-throughput screening.
- Fast completion - Results in just 1 hour.
产品参数（Product Specifications）
| 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 |
产品概述（Product Overview）
Human Fc gamma RI / CD64 binding Kit (TR-FRET) is based on a homogeneous (no wash) competition TR-FRET technology (Time-Resolved Fluorescence Resonance Energy Transfer) to measure the interaction between human CD64 and antibody drug candidates or CD64 inhibitors. It is designed to facilitate the facilitate the ADCC and ADCP functional performance evaluation of antibody drug candidates, high-throughput screening of CD64 inhibitors within 0.5-1 hours. It can also be used as a universal detection tool to identify the ability of antibody drugs to bind to human CD64.
存储（Storage）
组分（Materials Provided）
| ID | Components | Size |
| FRT03-C01 | Human Fc gamma RI / CD64 Protein Europium-chelate | 100 tests/500 tests |
| FRT03-C02 | FA labeled human IgG antibody | 100 tests/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.
- In the absence of human Fc gamma RI/CD64 binding components, the donor and acceptor are in close proximity due to the binding of human Fc gamma RI/CD64 and FA-labeled Human IgG1 antibody. Upon excitation with a specific light source, the donor emits a 620 nm signal, which is absorbed by the acceptor, resulting in a 665 nm emission.
- In the presence of human Fc gamma RI/CD64 binding components, they disrupt the donor-acceptor interaction, preventing 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.
