Drug Discovery and Bioprocess often require accurate quantification technologies. These HTRF Fc kits enable rapid quantification of human, mouse and rabbit IgG, and any proteins containing a Fc region. The assays are compatible with natural IgG as well as recombinant antibodies. They are based on a competitive format and are reliable over a wide concentration range, avoiding the need for sample dilution.
The HTRF human and mouse Fc assays are competitive immunoassays for the measurement of IgGs or any Fc-tagged proteins in a range of biological materials. The assays use a Eu3+-Cryptate conjugated antibody specific to Fc IgGs, and XL665 (human and rabbit) or d2 (mouse) coupled IgGs. These homogeneous assays enable precise assessment of IgGs, and require only a single 2-hour incubation following the addition of detection reagent.
The human Fc assay principle is shown here as an example.
| Human Fc | Mouse Fc | Rabbit Fc | |
|---|---|---|---|
| Detection limit: | 5.5 ng/mL | 6.8 ng/mL | 5 ng/mL |
| Dynamic range: | 5.5 to 4,000 ng/mL | 0.8 – 400,000 ng/mL | 4.9 – 80,000 ng/mL |
| Incubation time: | 2h 30 to ON | 2h at RT | 2h at RT |
| EC50: | 226 ng/mL | 138 ng/mL | 178 ng/mL |
Total human IgG standard curve drawn up using the protocol detailed in product insert.
Human Fc chimeric antibodies as well as proteins containing a human Fc region can be detected using the HTRF human Fc assay. As shown in Figure 1, the affinity depends on the nature of each Fc chimera and is at its maximum for whole IgG. This means that the assessment of the concentration can be made on a relative basis using the human IgG calibrator. Absolute concentration can be approached if the calibrator is made with the same protein as that being assessed.
Figure 1: Various concentrations of four different human Fc chimeras were tested using the Total human IgG assay, following standard assay protocols.
Figure 2 demonstrates the compatibility of the HTRF human Fc assay with different media, even in the presence of 10% FCS. No impact on assay performance was seen. Unlike other immunoassays involving streptavidin-biotin detection, this assay is insensitive to the presence of culture medium, whose quenching effects can be compensated for by our patented 665/620 ratio.
Figure 2: IgG standard curve was drawn up using IgG in phosphate buffer, culture media and RPMI containing 10% FCS+/- 4.4% PSGHT (Penicillin, Streptamycin, Glutamine, Hypoxanthine, Thymidine).
The HTRF human Fc assay was shown to be fully adapted to the screening of clone libraries producing human Fc-tagged proteins. As an example, figure 3 shows that sample size can be reduced to as low as 10 µL without affecting assay performance, simply by resizing each addition volume proportionally.
| 96 half-well (100µL) | 96w LV (40µL) | 384w LV (20 µL) | |
|---|---|---|---|
| Sample | 50µL | 20µL | 10µL |
| XL665 conjugate | 25µL | 10µL | 5µL |
| Cryptate conjugate | 25µL | 10µL | 5µL |
Figure 3: IgG standard curve was drawn up using an Fc-tagged protein. Standard assay conditions were followed. Final assay volumes ranged from 50 to 12.5µl. The plate format used for each assay volume is indicated in the legend (standard 96-well, 96- half well or standard 384-well plate).
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| Human Fc kit - 500 tests | 62HFCPEG |
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| Human Fc kit - 10,000 tests | 62HFCPEH |
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| Mouse Fc kit - 500 tests | 6FMIGPEG |
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| Mouse Fc kit - 10,000 tests | 6FMIGPEH | ||
| Rabbit Fc kit - 500 tests | 6FRIGPEG | ||
| Rabbit Fc kit - 10,000 tests | 6FRIGPEH |
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Accelarate throughput without compromising on perforance: HTRF® reagents for biotherapeutics find_in_pageCisbio Bioassays, Codolet, France . Cisbio US, Bedford, MA, USA - Cisbio Bioassays, Codolet, France . Cisbio US, Bedford, MA, USA |
Automated Human Total IgG HTRF Assay: A Comparison with ELISAWhalley D - MRC Technology Centre for Therapeutics Discovery, London, UK. |
Truly homogeneous functional assays
FRET (Fluorescence Resonance Energy Transfer) is based on the transfer of energy between two fluorophores – a donor and an acceptor – when in close proximity. Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and by detecting the level of energy transfer.
