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Your Position: Start > Kits > FcRn (FCGRT & B2M) > FRT-01

Human FcRn binding Kit (TR-FRET)

For research use only.

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    Product Details
    Assay TypeInhibition-TR-FRET
    AnalyteFcRn binding component
    Format100T, 500T, 10000T
    ReactivityHuman
    Regulatory StatusRUO
    SensitivityIC50=20.14μg/mL
    Standard Curve Range0.24 μg/mL-1000 μg/mL
    Assay Time1 hr
    Suitable Sample TypeFor the binding of IgG Fc region to the human FcRn receptor
    Sample volume10 μL
  • Background
    FcRn (neonatal Fc receptor for IgG) 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) 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, short detection time and easy to use.

  • 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.

  • 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.

    Materials Provided
    IDComponentsSize
    FRT01-C01Human FCRN&B2M Heterodimer Protein Europium-chelate2.7 μg/100 tests
    13.5 μg/500 tests
    270 μg/10000 tests
    FRT01-C02FA labeled human IgG antibody2.4 μg/100 tests
    12 μg/500 tests
    240 μg/10000 tests
    FRT01-C03Human IgG standard200 μg/100 tests
    1 mg/500 tests
    20 mg/10000tests
    FRT01-C04Sample Dilution Buffer10 mL/100tests & 500tests
    100 mL/10000tests
    FRT01-C05Detection Buffer10 mL/100tests & 500tests
    100 mL/10000tests
  • 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:
    1) 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.
    2) Add FA labeled human IgG antibody (Acceptor) and incubate at room temperature for more than 0.5 hour.
    3) Use the TR-FRET module of a microplate reader to read the fluorescence signal at 665nm and 620nm. 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.
    - 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.

    Assay Principles

Bioactivity-TR-FRET Please refer to DS document for the assay protocol.
 FcRn (FCGRT & B2M) TR-FRET

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

The half-lives of monoclonal antibodies currently in clinical use generally correlate with measurements of the affinity of their Fc for FcRn. The kit has been used to detect three FDA approved antibody drugs with different affinities binding to FcRn. The result is consistent with SPR test results. In the same experiment, the lower the IC50, the stronger binding affinity between IgG antibodies and FcRn, and the longer half-life of the antibody drug. Bevacizumab and Toripalimab have consistent binding affinity with FcRn and have a half-life of about 20 days. The Fc of Eculizumab has been modified to bind relatively weakly to FcRn, resulting in a relatively short half-life of approximately 11 days.

 FcRn (FCGRT & B2M) TR-FRET

The kit has been used to detect the binding activity between Human FcRn and Human IgG standard (FRT-C03) under different pH. The affinity between Human FcRn and Human IgG standard (FRT-C03) decreases with increasing pH.

 FcRn (FCGRT & B2M) TR-FRET

The kit has been used to detect different human IgG isoforms of Human IgG1, Human IgG2, Human IgG3, Human IgG4, with different affinities binding to FcRn.

 FcRn (FCGRT & B2M) TR-FRET

The kit has been used to detect the Efgartigimod drug and Human IgG1 Fc (C103S, M135Y, S137T, T139E, H316K, N317F), His Tag (Cat.No. IG1-H52H8) that inhibit FcRn.

  • Clinical and Translational Updates

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Drug Development Status

  • Number of Launched Drugs:3 Details
  • Number of Drugs in Clinical Trials:3 Details
  • Latest Research Phase:Approved

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