HTRF® Technology - Toolbox Reagent Usage

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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. Cisbio Bioassays would like to take the opportunity to explain how toolbox reagents are provided and concentrations to be used.

Information provided about toolbox reagents:
You will notice that the ordering information for all HTRF® toolbox reagents includes the number of tests (384-well, 20 µl reactions) and the average quantities of active moiety per vial. The active moiety is defined as the active part of a conjugate (e.g. antibody, streptavidin).

How do the number of tests relate to active moiety?
The average conjugate quantity per well is information that reflects overall biological material content. For Eu3+ Cryptate, biotin, and DNP conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible.

For XL665 labeled entities, however, the amount of active moiety will not equal the amount of total conjugate. In practice, using the active moiety amount is generally preferred to the quantity of total conjugate as a basis for calculating reagent usage per well during assay development. The quantity of total conjugate supplied by Cisbio Bioassays will vary depending on the final molar ratio of the XL665 conjugate. The amount of active moiety, however, is constant and based on the number of tests ordered.

Recommended quantities of Eu3+ Cryptate and XL665 conjugates:
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 40,000cps on a RUBYstar, the HTRF® reference reader. 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.