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 resulting signal is proportional to the binding of the 2 partners. In the example shown here, MAb Anti FLAG-Eu cryptate binds to the FLAG tagged partner A, while partner B* binds to a specific Ab labeled with an HTRF acceptor. Partner B* can also be biotinylated, tagged, or Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with an acceptor for the detection.

The example on the right describes the protocol using a 20 µL final assay volume for the detection of an interaction between a FLAG-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add MAb Anti FLAG-Eu cryptate (5 µL) and anti-partner B labeled with acceptor (5 µL), then 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 an acceptor for the detection.

The average conjugate quantity per well reflects the overall biological material content. Use of 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.