Enzyme-Linked Oligonucleotide Assays (ELONA)
ELISA are a popular method of quantifying the amounts of a specific product in a given sample. This may include protein, metabolite or cellular changes and once developed, are very high-throughput and capable of generating large amounts of data. Numerous types of ELISA exist including:
- Direct ELISA – target is immobilised and an antibody conjugated to a fluorophore acts as a reporter
- Indirect ELISA – target is immobilised but assay requires a primary antibody and separate reporter.
- Sandwich ELISA – target is immobilised via monoclonal antibody and detected by separate polyclonal antibody. Can be direct or indirect.
The development of ELISA can be a time-consuming process and is heavily dependent on the availability of sometimes multiple, 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.
Incorporating aptamers in ELISA
Aptamers can easily be incorporated into existing ELISA platforms with ease. 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 existing high-quality monoclonal antibodies.
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.