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 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 |
背景（Background）
Human Fc gamma RIIIA / CD16a (V176) Binding Kit (TR-FRET) can detect the binding of IgG Fc region to the human Fc gamma RIIIA / CD16a (V176) 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 RIIIA / CD16a (V176) receptor. It is used as a universal detection tool to identifying the ability of antibody drugs to bind to Fc gamma RIIIA / CD16a (V176).
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 |
| 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 |
原理（Assay Principles）
Human Fc gamma RIIIA / CD16a (V176) binding kit (TR-FRET) is based on TR-FRET technology (Time-Resolved Fluorescence Resonance Energy Transfer). Use the mixture of biotinylated human Fc gamma RIIIA / CD16a (V176) 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 RIIIA / CD16a (V176) 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 RIIIA / CD16a (V176) binding components, the donor and acceptor are in close proximity because of the binding of human Fc gamma RIIIA / CD16a (V176) 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 RIIIA / CD16a (V176) 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 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.
背景（Background）：Fc gamma RIIIA / CD16a
CD16 is a low affinity Fc receptor, and has been identified as Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies. CD16 encoded by two different highly homologous genes in a cell type-specific manner.CD16 is found on the surface of natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages.
CD16a antigen is also known as Low affinity immunoglobulin gamma Fc region receptor III-A, Fc-gamma RIII-alpha. CD16b is a low-affinity, GPI-linked receptor expressed by neutrophils and eosinophils, whereas CD16a is an intermediate affinity polypeptide-anchored transmembrane glycoprotein expressed natural killer cells, macrophages, subpopulation of T-cells, immature thymocytes and placentaltrophoblasts.CD16a is involved in phagocytosis, secretion of enzymes and inflammatory mediators, antibodydependent cytotoxicity and clearance of immune complexes. Aberrant expression or mutations of CD16a is implicated in susceptibility to recurrent
