Enzyme Linked Oligonucleotide Assays (ELONA)
The development of an ELISA can be time-consuming and heavily dependent on the availability of high-quality antibodies with validation in an ELISA platform. This is complicated further by the requirement for a reporter molecule, limiting the choice of antibodies and introducing more chance of error in reporting due to non-specific binding. Using aptamers can be beneficial through Enzyme-Linked Oligonucleotide Assays (ELONA)
Aptamers can be easily incorporated into existing ELISA platforms. The resulting assays are known as ELONA and benefit from:
- Increased sensitivity– using the principle of counter-selection, aptamers can be designed to not recognise closely-related compounds
- Wider range of outputs – aptamers can be conjugated to multiple reporter molecules including fluorophores, biotin and quencher molecules.
- Easier to develop – aptamer selection identifies multiple aptamers capable of binding to different epitopes on the target’s surface, allowing a pair to be easily determined.
- Works with existing technology – can be used by all existing plate reader models and ELONA can be designed using high-quality monoclonal antibodies.
Our approach to small molecule aptamer selection gives us affinity reagents that are already suited to an ELISA-like format. The aptamers are labelled, so may be used in a ‘gain of signal’ format and do not require the use of a ‘pair’; greatly simplifying assay development.
Small molecule binding aptamers can be used in a range of ‘ELISA-like’ formats. They can be labelled with fluorophores to allow direct reading or with traditional moieties (such as biotin) to facilitate more sensitive assays.
Aptamers can be selected to function in a range of matrices.
An area of significant growth for Lateral Flow Device use is for ‘point-of-care’ diagnostic tests in healthcare. Aptamers show numerous advantages over antibodies, which may present issues when trying to develop a rapid assay kit. The aspects where aptamers are superior include:
- Temperature control requirements: High stability of aptamers at high temperatures and ability to refold allow application in low resource settings.
- Restrictions to conditions: Aptamers show more resistance to different chemical buffers such as large ranges of pH, ion strength, and organic reagents.
- Batch-to-batch variations: Aptamers can be replicated by simple chemical synthesis with very high fidelity once the sequence of the aptamers is known.
- High cost: Aptamers show cost advantages over antibodies as aptamers are produced entirely synthetically.
- Reagent immobilisation:Aptamers are much smaller than antibodies therefore more can be immobilised on the same GNP or NC membrane surface.
- Target limitation: Nearly any target can be used including low molecular weight substances for aptamer selection in unlimited conditions.
Example of in-house developed indirect ELONA. Three different human proteins were biotinylated and adhered to a 96-well plate. Aptamers raised against these proteins were conjugated to biotin and incubated with the target in varying concentrations for X hour. A reporter molecule (streptavidin-HRP conjugate) was added with a commercially available reagent and the respective colour change measured using a plate reader.