Human, Mouse & Rabbit Fc assay kits

HTRF Fc kits for IgG and any Fc tagged protein quantification

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.

Assay Principle

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.

Product Specifications

  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.

Product Performance

Case study: Whole human IgG and Fc Fragment Detection

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.

Media Compatibility

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).

Assay Miniaturization

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).

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human Fc kit - 500 tests62HFCPEG
-
Human Fc kit - 10,000 tests62HFCPEH
-
Mouse Fc kit - 500 tests6FMIGPEG
-
Mouse Fc kit - 10,000 tests6FMIGPEH
Rabbit Fc kit - 500 tests6FRIGPEG
Rabbit Fc kit - 10,000 tests6FRIGPEH
-

Companion products

DescriptionCat. noProduct insertMSDS
Diluent 1 - 20 ml62DL1DDD
Human IgG kit standard62HFCCDA
-

ドキュメント