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 |
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 |
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 receptor (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) and weak to no binding interactions as the pH is raised to neutral (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) can detect the binding of IgG Fc region to the human FcRn 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 FcRn receptor. It is 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.
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. Firstly, 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 hour. Then, add FA labeled human IgG antibody (Acceptor) and incubate at room temperature for more than 0.5 hour. Use the TR-FRET module of a microplate reader to read the fluorescence signal at 665nm and 620nm. When the sample does not contain FcRn binding component, 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 compete with the binding between the donor and acceptor and thereby prevents FRET from occurring. Calculate 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.
活性(Bioactivity)-TR-FRET Please refer to DS document for the assay protocol.
![FcRn (FCGRT & B2M) TR-FRET](images/tr_fret/FRT-01-TR1.jpg)
Inhibition of Europium-chelate labeled FcRn: FA labeled human IgG antibody binding by Human IgG
Premix serial dilutions of Human IgG standard (Catalog # FRT01-C03) (1:4 serial dilution, from 1000 μg/mL to 0.24 μg/mL (6666.67-1.63 nM)) and Human FCRN&B2M Heterodimer Protein Europium-chelate (Catalog # FRT01-C01) and incubate at room temperature (20℃-25℃) for 0.5 hour. Then add FA labeled human IgG antibody (Catalog # FRT01-C02) and incubate at room temperature (20℃-25℃) for 0.5 hour. Detection was performed with IC50 of 20.14 μg/mL. The assay was performed according to the above-described Datasheet (QC tested).
![FcRn (FCGRT & B2M) TR-FRET](images/tr_fret/FRT-01-TR2.jpg)
The kit has been used to detect different human IgG isoforms of Human IgG1, Human IgG2, Human IgG3, Human IgG4, and modified Human IgG1 Fc (C103S, M135Y, S137T, T139E, H316K, N317F) Protein, His Tag (Cat. No. IG1-H52H8) with different affinities binding to FcRn.