Human MAb Screening Assay
Drug discovery and bioprocess often require the quantification of human IgGs, Fc fragments or tagged proteins. The successful production of human-Fc tagged chimeras and humanized monoclonal antibodies has raised considerable interest in their potential for therapeutic uses. In addition, libraries of humanized monoclonal antibodies are important sources of drug candidates. The introduction of our HTRF®-based Human MAb Assay offers new capabilities for improved throughput, accuracy and reliability in the areas of IgG quantification, screening and quality control.
- Simple and rapid assay protocol
- Highly sensitive & accurate assay
- Amendable to automation
- Easily miniaturized
- Compatible with culture media and additives
- IgG quantification in a variety of biological materials
- Fc-tagged protein quantification
- Screening of humanized antibodies
- Bioprocess quality control
Assay Principle:
The HTRF® Human MAb Assay is a competitive immunoassay for measurement of human IgGs or 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 source. This homogeneous assay enables precise assessment of all human IgG isotypes and requires only a single 2 hour incubation following detection reagent addition.
Product Specifications:
| Detection limit: | 5.5 ng/mL |
| Dynamic range: | 5.5 to 4,000 ng/mL |
| S/B: | 11 |
| Formats: | 96- or 384-well plates |
| Specificity: | subclasses IgG1, IgG2, IgG3 & IgG4 |
Figure 1: Human IgG standard curve performed using protocol outlined in product insert.
Assay Performance:
Whole IgG and Fc Fragment Detection:
Several human Fc chimera with different characteristics were assayed using the HTRF Human MAb assay (figure 2).
All fusion proteins can be detected using this kit, however the affinity depends on the nature of each Fc chimera. The
affinity of the anti-human Fc-cryptate conjugate is higher for complete IgGs than for truncated Fc, but the
displacement curves for whole IgG and Fc fragments are parallel. This means that the assessment of the concentration
can be made on a relative basis with a human IgG calibrator. Absolute concentration can be approached if the
calibrator is made with the same protein as the one being assessed.
Media Compatibility:
Figure 3 demonstrates the compatibility of the HTRF Human MAb 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.
Assay Miniaturization:
The HTRF® Human MAb Screening assay was shown to be fully adapted to the screening of clone libraries producing
human Fc-tagged proteins. The results in figure 4 show that sample size can be reduced to as low as 12.5 µl
without effecting assay performance. Reduction in assay volume must also be accompanied by a change to a reduced
volume plate.
More information on plate recommendations.
Figure 2: Various concentrations of four different human Fc chimeras were tested using the Human MAb assay following standard assay protocols
Figure 3: 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).
Figure 4: 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).
Ordering Info:
Human MAb Screening Reagents:
| Description | Quantity* | Cat no. | Product Insert |
| Human MAb screening kit | 1,000 tests | 62HFCPEB | |
| Human MAb screening kit bulk | 20,000 tests | 62HFCPEC | |
| *based on 20 µl assay volume | |||
For more information or To Place an Order:
U.S. and Canada
1-888-963-4567
Europe and other countries
+33(0)466 796 705
Japan
+81-3-5510-2932
