The favoured methodology for homogeneous assays is still the use of labelled substrates or indirect signalling to produce fluorescence indicating when the desired reaction occurs. Assay performance depends critically on selecting the labels most suited to the target, and optimising the assay for parameters such as sensitivity, specificity and throughput. This article examines a three-step assay development process for assays using patented technology, homogeneous timeresolved fluorescence (HTRF®), (see Note) a time-resolved fluorescence resonance energy transfer (TR-FRET) technology based on a non-radiative transfer of energy between two compatible fluorophores. The europium cryptate (Eu-K) donor confers a long-lived fluorescence to the acceptor during the FRET process.

MULTIPLE-STEP PROCESS

The first step in developing a new assay is the ‘paper study’. The starting point is a detailed description of the target species and the activity that the assay will detect, whether receptor-ligand binding; protein-protein or protein-DNA interaction; protein cleavage or modification events involving kinases or ubiquitin; or cell-secreted markers such as cytokines and hormones.

This is an important step for a user who has chosen to ask the assay reagent vendor to develop the new assay, because it requires handover of what may commercially be highly sensitive materials such as proprietary drug leads. Clearly it is essential to have a relationship of trust, backed by a comprehensive confidentiality agreement including a non-disclosure agreement (NDA) and biological material transfer agreement (MTA). Cautious customers will examine the partner vendor’s track record before forming such a relationship.

With confidentiality secured, next the user sets out the desired performance specifications for the finished assay. These will include the reference method against which the assay will be calibrated; the nature of the sample, whether serum, plasma, supernatant or cell-based; sensitivity to the presence of organic solvents like DMSO; detection limits; dynamic range (the spread of target concentration over which the assay produces valid, preferably linear, results); the required Z-factor (a measure of separation between maximum and minimum controls in an assay, accounting for the assay’s variability); whether the assay will activate or inhibit the target’s activity; and whether it will be endpoint-based or equilibrium-based.

The paper study can then go ahead. A literature search will reveal any existing assay formats and the availability of suitably specific and stable reagents such as antibodies. Reagent molecules can be labelled using direct labelling of the target species with donor (cryptate) or acceptor (d2 or XL) fluorophores, in which case the detection step visualises the target itself. Alternatively, the target species can be tagged with biotin or 2,4-dinitrophenyl (DNP), or indeed both. This tag is later detected by fluorophore-labelled streptavidin or by anti-DNP antibody labelled with donor or acceptor fluorophore (indirect labelling). Eu-K-based donors are able to transfer energy to the acceptor over a relatively long distance (~9nm), so indirect labelling of this kind can be used successfully to detect protein-protein interactions.