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Human Fc kit

Human Fc kit for IgG and any Fc tagged protein quantification

 

Drug Discovery and Bioprocess often require accurate quantification technologies. Our human Fc kit enables rapid quantification of human IgG and any proteins containing a human Fc region. This assay is compatible with natural human IgG as well as recombinant humanized or chimera antibodies. The assay is based on a competitive format and is reliable over a wide concentration range, avoiding the need for sample dilution.

Features

  • One addition, no washing step
  • Fast: results in 2h
  • Wide dynamic range: 5 to 4,000 ng/mL
  • Low sample volume: < 8µL
  • Easy automation & miniaturization
  • Robust: compatible with culture media

Applications

  • Cell culture screening
  • High-producing clone selection
  • Human IgG quantification - recombinants & chimeras
  • Human Fc tagged protein quantification
  • Production monitoring & Bioprocess quality control

The HTRF human Fc Assay is a competitive immunoassay for the measurement of human IgGs or any Fc-tagged proteins in a range of biological materials. The assay uses a Eu3+-Cryptate conjugated polyclonal antibody specific to human Fc IgGs, and XL665 coupled human IgG. This homogeneous assay enables precise assessment of IgGs, and requires only a single 2 hour incubation following detection reagent addition.

HTRF human Fc assay principle

Detection limit: 5.5 ng/mL
Dynamic range: 5.5 to 4,000 ng/mL
S/B: 11
Formats: 96- or 384-well plates

HTRF human IgG Fc assay principle

Total human IgG standard curve performed using protocol outlined in product insert.

 

Whole 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.

Fc tag protein or IgG titration using HTRF huamn Fc assay

Media Compatibility:

Figure 2 demonstrates the compatibility of the HTRF human Fc assay with media even in the presence of 10% FCS. No effects on assay performance were 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 the patented 665/620 ratio.

Figure 2: IgG standard curve was performed 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.

Figure 3: IgG standard curve was performed 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).

HTRF human Fc assay miniaturization

  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

 

Ordering Info

DescriptionCat. noProduct insertMSDS
Human Fc kit - 1,000 tests62HFCPEB
Human Fc kit - 20,000 tests62HFCPEC

Companion products

DescriptionCat. noProduct insertMSDS
Human IgG calibrator62HFCCDA
-
Human IgG control62HFCTDA
-




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