Choice of ligand and other reagents

Depending on the design of the assay, there may be a range of possible ligands. Immunological assays typically offer a panel of antibodies that may be used as ligands or detecting molecules. On the other hand, assays based for example on the binding of analyte to a specific cell surface receptor are limited in their choice of detecting molecule.

In general, ligand and other reagents are selected on the basis of known interaction properties, but the suitability of the selection always needs to be confirmed in practice. If details of the interaction properties are not known in advance, selection is done entirely through practical tests.

Practical approaches to reagent selection

To confirm the suitability of a selected reagent, perform measurements using samples with known analyte concentrations that cover at least the intended range of the assay. Construct a calibration curve for the assay as a tool for deciding whether the reagent choice is acceptable.

Measuring a calibration curve with known analyte samples presupposes that assay conditions have been established, including conditions for surface preparation and regeneration. The process of selecting and optimizing reagents is necessarily iterative, since all steps in the assay set-up and execution need to be confirmed before the assay as a whole can be approved. A reagent that is acceptable in terms of interaction properties may for instance be discarded on the grounds of difficulties in regeneration. Be prepared to review your choice of ligand or other reagents on the basis of results from later steps in the assay development procedure.

Choice of ligand and other reagents for surface competition assay

The ligand in surface competition assays is analogous to that in direct binding assays and the choice of molecule is subject to the same considerations. In addition, it is necessary that the analyte and competing molecule each bind to the ligand to the exclusion of the other, so that a true competition situation is achieved. The relative affinities of analyte and competing molecule will determine to some extent the range of analyte concentrations that can be measured.

The detecting molecule for surface competition assays should be larger than the analyte, so that the observed response derives almost exclusively from binding of the detecting molecule. Response that derives from binding of analyte will result in apparently incomplete inhibition of the response at high analyte concentrations, reducing the range of measured response and potentially impairing the assay performance.

Choice of ligand and other reagents for inhibition assay

The ligand for inhibition (solution competition) assays is the same as the analyte or an analogue thereof. The choice of ligand is thus given, with a certain degree of freedom in finding or synthesizing analogues that are suitable for immobilization on the surface.

Demands on the detecting molecule for inhibition assays are broadly similar to the demands on ligand for direct binding assays. The interaction between detecting molecule and analyte should be moderately fast, so that incubation times of sample with detecting molecule and contact times of injection over the surface are kept reasonably short in the interests of assay throughput.

Choice of ligand and other reagents for direct binding assay

Choice of ligand

For direct binding assays, base the choice of ligand on the following considerations:

The ligand should if possible be available in a purified state. If partially purified ligand preparations are used, additional control experiments will be necessary to establish the specificity of the assay. High levels of ligand immobilization should be possible to achieve.
The interaction of ligand with analyte should show selectivity characteristics that are compatible with the demands of the assay. Assay selectivity can be improved in some cases by the use of enhancement reagents that bind specifically to analyte on the surface.
The binding of analyte to ligand should be fairly rapid so that analysis cycle times can be kept short. In addition, slow dissociation rates help to improve assay robustness when the response is measured after the end of the sample injection. In practice this means that the ligand should have a high affinity for the analyte. The affinity of ligand for analyte may be a determining factor in the operating range of the assay: a higher affinity interaction can often be used to measure lower analyte concentrations.
It must be possible to regenerate the ligand efficiently without loss of ligand activity. If a compromise is necessary between regeneration characteristics and affinity for the analyte, regeneration characteristics are more important than high affinity.

Enhancement reagent

The enhancement reagent used in sandwich assay formats must be able to bind to the analyte at a site independent of the ligand binding site. Antibodies are most commonly used as enhancement reagents, but in principle any macromolecule that can bind to analyte independently of ligand can be used.

Enhancement reagents intended primarily to amplify the analyte response must be larger molecules than the analyte or (less commonly) must bind to multiple sites on the analyte. Reagents used to enhance specificity, on the other hand, do not necessarily have to be larger than the analyte provided that a confidently measurable signal is obtained.

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