HTRF toolbox reagents include secondary murine, sheep, rabbit, and human antibodies, as well as immunoglobulin binding proteins. These reagents are useful in developing new assays for which directly labeled assay components are not available or which involve chimeric proteins. Most of our anti-immunoglobulin reagents are available conjugated to either Europium-cryptate or XL665, for maximum flexibility.
Anti-Ig HTRF reagents have been strictly selected based on their specificity. Polyclonal antibodies (PAb) to murine, rabbit, and human immunoglobulins (IgGs) are absorbed against most human and animal serum proteins to minimize inter-species cross-reactivity.
| Motif | Active Moiety 5,000 tests | |
|---|---|---|
| Europium conjugate | XL665 conjugate | |
| Protein A | 3.5 µg | 50 µg |
| Species | Antibody | Species | Specificity | Active Moiety 5,000 tests |
|
|---|---|---|---|---|---|
| Europium conjugates |
XL665 conjugates |
||||
| Mouse immunoglobulins | PAb | Rabbit | Mouse Igs | 7.5 µg | 100 µg |
| Rabbit immunoglobulins | PAb | Goat | Rabbit Igs | 7.5 µg | 100 µg |
| Human immunoglobulins | PAb | Goat | Human Fc Igs | 10 µg | 100 µg |
HTRF toolbox reagents provide an added level of flexibility to assay development, and are therefore commonly used when a ready-to-use assay kit is not available.
Reagents are sold by the number of tests (384-well, 20 µl reactions). Antibodies conjugated to XL665 or d2 are supplied on the basis of 20ng of antibody per well. The amount of active moiety per vial is also provided (as well as the number of tracers per vial - see product description sheet). In practice, the amount of active moiety is generally preferred to that of total conjugate as a basis for calculating assay development.
Most assays can be run within the nanomolar range. However, as a tracer, Eu3+ cryptate conjugates must not be excessive in order to prevent reader saturation and an unacceptable level of background. In most cases, a Eu3+ cryptate concentration of 4 to 5nM is appropriate, and will typically generate 620nm fluorescent signals of approximately 20,000 to 80,000 cps depending on the HTRF compatible reader used. As an example, for an antibody conjugated to Eu3+ cryptate with a molar ratio of 5 cryptates/Ab, the recommended value would be close to 1nM of antibody per well. The XL665 conjugate must match its assay counterpart as closely as possible in order for the maximum number of biomolecules to be tagged with the XL665 acceptor. Thus, to detect a GST-tagged molecule at an assay concentration of 20nM, the concentration of anti-GST-XL665 should be equimolar or higher. The actual amount will depend on the assay configuration.
Custom labeling and assay development services are also available to help meet your research needs.
CD28 provides a major costimulatory signal for CD4-positive T cells. Ligation with its natural ligands CD80 (B7-1) and CD86 (B7-2) leads to signals during activation that are required for the production of interleukin-2. This process has been implicated in the regulation of T-cell anergy and programmed cell death. The HTRF anti-immunoglobulin reagents were used to develop an assay for antagonists of CD28 and CD86 binding. Both molecules were respectively tagged with human and rat IgG Fc fragments, and detected using the XL665-labled anti-human antibody. CD86 was expressed as a fusion protein with a rat Ig domain which is recognized by a biotinylated sheep anti-rat antibody, followed by binding of europium-labeled streptavidin. Results of the binding assay were published by Mellor et al.
Data for the CD28-CD86 binding assay is published in: Mellor et al. Development of a CD28/CD86 (B7-2) binding assay for high throughput screening by homogenous time-resolved fluorescence. J Biomol Screening. 1998; 3: 91-99.
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| PAb Anti Human IgG-d2 - 100 | 61HFCDAA | - | |
| PAb Anti Human IgG-K - 5,000 tests | 61HFCKLA | - | |
| PAb Anti Human IgG-K - 20,000 tests | 61HFCKLB | - | |
| PAb Anti Human IgG-XL665 - 5,000 tests | 61HFCXLA | - | |
| PAb Anti Human IgG-XL665 - 20,000 tests | 61HFCXLB | - | |
| PAb Anti Mouse-d2 - 100 | 61PAMDAA | ||
| PAb Anti Mouse-d2 - 400 | 61PAMDAB | - | |
| PAb Anti Mouse IgG-K - 5,000 tests | 61PAMKLA | ||
| PAb Anti Mouse IgG-K - 20,000 tests | 61PAMKLB | ||
| PAb Anti Mouse IgG-XL665 - 5,000 tests | 61PAMXLA | ||
| PAb Anti Mouse IgG-XL665 - 20,000 tests | 61PAMXLB | ||
| PAb Anti Rabbit IgG-K - 5,000 tests | 61PARKLA | ||
| PAb Anti Rabbit IgG-K - 20,000 tests | 61PARKLB | ||
| PAb Anti Rabbit IgG-XL665 - 5,000 tests | 61PARXLA | - | |
| PAb Anti Rabbit IgG-XL666 - 20,000 tests | 61PARXLB | - | |
| Protein A-K - 5,000 tests | 61PRAKLA | - | |
| Protein A-K - 20,000 tests | 61PRAKLB | - | |
| Protein A-XL665 - 5,000 tests | 61PRAXLA | - | |
| Protein A-XL665 - 20,000 tests | 61PRAXLB | - |
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| Streptavidin-XL665 - 5,000 tests | 610SAXLA | ||
| Streptavidin-XL665 - 20,000 tests | 610SAXLB | ||
| Streptavidin-XL665 - 3 mg | 610SAXLG | - | |
| Streptavidin-XLent! - 5,000 tests | 611SAXLA | ||
| Streptavidin-XLent! - 20,000 tests | 611SAXLB |
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