膜杰作
Star Staining
产品描述（Product Details）
| Assay Type | Inhibition-TR-FRET |
| Analyte | FcRn binding component |
| Format | 100T, 500T, 10000T |
| Reactivity | Human |
| Regulatory Status | RUO |
| Sensitivity | IC50=20.14μg/mL |
| Standard Curve Range | 0.24 μg/mL-1000 μg/mL |
| Assay Time | 1 hr |
| Suitable Sample Type | For the binding of IgG Fc region to the human FcRn receptor |
| Sample volume | 10 μL |
背景（Background）
FcRn is a heterodimer comprising a β2-microglobulin (β2m) light chain and a major histocompatibility complex class I-like heavy chain. It is widely accepted that the interaction of IgGs with the Fc FcRn plays a critical role in regulating IgG homeostasis in vivo. FcRn interacts with the CH2-CH3 portion of the Fc domain of IgGs in a tightly regulated pH-dependent manner with high affinity binding occurring at an acidic (pH 6.0) and weak to no binding interactions as the pH is raised to neutral (pH 7.4). FcRn is responsible for the extended serum half-life of IgG and also serum albumin, and for the transport across endothelial and epithelial barriers, increasing the overall bioavailability of IgG and serum albumin. The binding affinity between FcRn and IgG are commonly used to characterize the metabolic levels of antibody drugs.
The Human FcRn Binding Kit (TR-FRET) takes advantage of binding of Europium-chelate labeled FcRn (donor) and FA labeled Human IgG1 antibody (acceptor) in a homogeneous (no wash) TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) competition assay to measure the interaction between human FcRn and antibody drug candidates or FcRn inhibitors. It is designed to facilitate the half-life evaluation of antibody drug candidates, and also high-throughput screening of FcRn inhibitors within 0.5-1 hours. It is highly sensitive, has a short detection time and easy to use.
应用说明（Application）
This kit is designed to facilitate the half-life evaluation of antibody drug candidates, and also high-throughput screening of FcRn inhibitors. It can also be used as a universal detection tool to identifying the ability of antibody drugs to bind to FcRn.
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 |
| FRT01-C01 | Human FCRN&B2M Heterodimer Protein Europium-chelate | 2.7 μg/100 tests
13.5 μg/500 tests 270 μg/10000 tests |
| FRT01-C02 | FA labeled human IgG antibody | 2.4 μg/100 tests
12 μg/500 tests 240 μg/10000 tests |
| FRT01-C03 | Human IgG standard | 200 μg/100 tests
1 mg/500 tests 20 mg/10000tests |
| FRT01-C04 | Sample Dilution Buffer | 10 mL/100tests & 500tests
100 mL/10000tests |
| FRT01-C05 | Detection Buffer | 10 mL/100tests & 500tests
100 mL/10000tests |
原理（Assay Principles）
This Human FcRn binding Kit (TR-FRET) is based on TR-FRET technology (Time-Resolved Fluorescence Resonance Energy Transfer). Use the mixture of biotinylated FcRn and Europium-chelate labeled streptavidin as the donor, FA labeled Human IgG1 antibody as the acceptor.
Your experiment will include 3 simple steps:
a) Mix the sample or Human IgG standard in the kit with Human FCRN&B2M Heterodimer Protein Europium-chelate (Donor) and incubate at room temperature for 0.5 hours.
b) Add FA labeled human IgG antibody (Acceptor) and incubate at room temperature for at least 0.5 hours.
c) 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 FcRn binding components, the donor and acceptor are in close proximity because of the binding of FcRn 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 FcRn 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 FcRn 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 20.14 μg/mL (QC tested).
The kit has been used to detect different subclasses of Human IgG Fc proteins, which exhibit different IC50 results as expected.
The kit has been used to detect different subclasses of Human IgG, which exhibit different IC50 results as expected.
The kit has been used to detect the binding activity between Human FcRn and Human IgG standard under different pH. The IC50 shows a significant increase with increasing pH, which corelates with a decreased binding between Human FcRn and Human IgG standard.
The half-lives of these 3 monoclonal antibodies currently in clinical use generally correlate with the binding affinity to FcRn. The kit has been used to detect 3 FDA approved antibody drugs of different binding affinity to FcRn, and the IC50 trends are consistent with affinity constant from SPR as well as the actual in vivo half-life published.
The kit is suitable for the detection of FcRn inhibitors. It shows that both Efgartigimod and its biosimilar, Human IgG1 Fc (C103S, M135Y, S137T, T139E, H316K, N317F) His Tag (Cat. No. IG1-H52H8) exhibit good inhibitory activity in this TR-FRET competition assay.
