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 CD32a antibody |
| Format | 100T/500T |
| Reactivity | Human |
| Regulatory Status | RUO |
| Sensitivity | IC50=816.6nM |
| 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 CD32a |
| Sample volume | 10 μL |
背景（Background）
Human Fc gamma RIIA / CD32a (R167) Binding Kit (TR-FRET) can detect the binding of IgG Fc region to the human Fc gamma RIIA / CD32a (R167) receptor in homogeneous system within 0.5-1 hours. it is high sensitive, short detection time and easy to use.
应用说明（Application）
The kit is developed for the binding of IgG Fc region to the human Fc gamma RIIA / CD32a (R167) receptor. It is used as a universal detection tool to identifying the ability of antibody drugs to bind to Fc gamma RIIA / CD32a (R167).
It is for research use only.
重构方法（Reconstitution）
Please see Certificate of Analysis for details of reconstitution instruction and specific concentration.
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 |
| FRT05-C01 | Human Fc gamma RIIA / CD32a (R167) Protein Europium-chelate | 100 tests/500 tests |
| FRT05-C02 | FA labeled human IgG antibody | 100 tests/500 tests |
| FRT05-C03 | Human IgG Standard | 400 μg/100 tests
2000 μg/500 tests |
| FRT05-C04 | Sample Dilution Buffer | 10 mL/100tests & 500tests |
| FRT05-C05 | Detection Buffer | 10 mL/100tests & 500tests |
原理（Assay Principles）
Human Fc gamma RIIA / CD32a (R167) binding kit (TR-FRET) is based on TR-FRET technology (Time-Resolved Fluorescence Resonance Energy Transfer). Use the mixture of biotinylated human Fc gamma RIIA / CD32a (R167) 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 RIIA / CD32a (R167) 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 = × . The Ratio value is negatively correlated with the antibody content in the sample.
- When the sample does not contain human Fc gamma RIIA / CD32a (R167) binding components, the donor and acceptor are in close proximity because of the binding of human Fc gamma RIIA / CD32a (R167) 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 RIIA / CD32a (R167) 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 Europium-chelate labeled human Fc gamma RIIA / CD32a (R167) 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 913 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 following figure, human CD32a (R167) binds to human IgG1, IgG2, IgG3 and IgG4 with low affinity.
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 CD32a (R167) binds to mouse IgG1, mouse IgG2a, and mouse IgG2b with low affinity as observed, and mouse IgG1 show the higher affinity than mouse IgG2a or mouse IgG2b.
The kit has been used to detect four FDA approved antibody drugs with different affinities binding to human CD32a (R167). Bevacizumab, Toripalimab, Eculizumab and Efgartigimod alfa bind to human CD32a (R167) with the low affinity from 1 μM to 10 μM.
Verify potential matrix effects by adding different levels of DEME, RPMI1640, FBS and HSA to the Sample Diluted buffer.
背景（Background）：Fc gamma RIIA / CD32a
Receptors for the Fc region of IgG (Fc γ R) are members of the Ig superfamily that function in the activation or inhibition of immune responses. Three classes of human Fc γ Rs: RI (CD64), RII (CD32), and RIII (CD16), which generate multiple isoforms, are recognized.
There are three genes for human Fcγ RII /CD32 (A, B, and C) and one for mouse Fcγ RII B (CD32B). CD32 is a low affinity receptor for IgG. The activating isoform, CD32A, is expressed on monocytes, neutrophils, platelets and dendritic cells. CD32A is expressed on many immune cell types (macrophage, neutrophil, eosinophils, platelets, dendritic cells and Langerhan cells), where inhibitory ITIMbearing receptors may also be coexpressed and coengaged by specific ligands. CD32A delivers an activating signal upon ligand binding, and results in the initiation of inflammatory responses including cytolysis, phagocytosis, degranulation and cytokine production. The responses can be modulated by signals from the coexpressed inhibitory receptors such as CD32B
