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: Streptavidin-d2 binds to the biotinylated partner A while partner B* binds to a specific antibody labeled to an HTRF donor.

*partner B can also be tagged, Fc fused. In these cases use for the detection, the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin), labeled with donor.

Assay protocol

The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a biotinylated-tagged partner A and a non-tagged partner B*.

Dispense the 2 partners (10 µL), incubate, add Streptavidin-d2 (5 µL) and anti-partner B labeled with donor (5 µL), incubate and read.

*partner B can also be tagged, Fc fused or directly labeled. In these cases use for the detection, the corresponding HTRF reagent (anti-Tag, anti species, protA, Streptavidin), labeled with donor.

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.