Description
IgGs from goat anti-Human immunoserum were immunopurified and labeled with XL665. PAb Anti Human IgG-XL665 may be used to detect the binding of unlabeled human specific antibody on a target molecule or on Ig fusion proteins.
This reagent can be used in both biochemical and cellular formats to study a wide variety of interactions: protein/protein, protein/peptide, protein/DNA, protein/RNA, protein/carbohydrate, protein/small molecule, receptor/ligand.
HTRF can detect a broad range of affinity constants ranging from picomolar to low millimolar.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
In an HTRF interaction assay, one partner is labeled (directly or indirectly) with the donor, and the other with the acceptor (again, directly or indirectly). The intensity of the signal is proportional to the binding of the 2 partners. In the example shown here: PAb Anti Human IgG-XL665 binds to the Human Fc fused tagged partner A while partner B* binds to a specific Ab labeled with an HTRF donor. *partner B can also be biotinylated, tagged, Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with donor for the detection.
Assay protocol
The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a Human Fc fused-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add PAb Anti Human IgG-XL665 (5 µL) and anti-partner B labeled with donor (5 µL), incubate and read. *partner B can also be biotinylated, tagged, Fc fused or directly labeled. In these cases, use the corresponding HTRF reagent (anti-Tag, anti species, protA, Streptavidin) labeled with donor for the detection.
Build HTRF interaction assays for your specific application
Reagents are sold by the number of tests (20 µL reactions). Antibodies conjugated to XL665 or d2 are supplied on the basis of 20 ng 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). The active moiety is defined as the active part of a conjugate (e.g. antibody).
How do the number of tests relate to active moiety?
The average conjugate quantity per well reflects overall biological material content. Using the active moiety amount is generally preferred to the quantity of total conjugate. For Cryptate and d2 conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible. This is not the case for XL665 labeled entities for which the quantity of total conjugate will vary depending on the final molar ratio of the XL665 conjugate, however, the amount of active moiety, provided by Revvity, is constant and based on the number of tests ordered.
Species Antibody Species Specificity Active Moiety 5,000 tests Europium conjugates XL665 conjugates Human immunoglobulins PAb Goat Human Fc Igs 10 µg 100 µg
Recommended quantities of Cryptate and XL665 conjugates
Cryptate conjugates must not be excessive in order to prevent reader saturation and an unacceptable level of background. In most cases, a cryptate concentration of 1 to 5nM is appropriate, and will generate 20,000 to 80,000 cps at 620 nm depending on the HTRF compatible reader used. 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 tagged molecule at an assay concentration of 20nM, the concentration of anti-Tag-XL665 should be equimolar or higher.
CD28/CD86 binding assay
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. HTRF 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 antibody, followed by binding of streptavidin-Eu cryptate. 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.
