{"id":5984,"date":"2024-08-19T15:45:27","date_gmt":"2024-08-19T19:45:27","guid":{"rendered":"https:\/\/neoaptamers.com\/?p=5984"},"modified":"2024-08-19T15:45:27","modified_gmt":"2024-08-19T19:45:27","slug":"enzyme-linked-oligonucleotide-assay","status":"publish","type":"post","link":"https:\/\/neoaptamers.com\/es\/enzyme-linked-oligonucleotide-assay\/","title":{"rendered":"The Enzyme-Linked Oligonucleotide Assay. The aptamer ELISA."},"content":{"rendered":"

The Enzyme-Linked Immunosorbent Assay (ELISA) was invented by Swedish scientists Eva Engvall and Peter Perlmann in 1971. This groundbreaking diagnostic method revolutionized medicine by using antibodies to detect the presence of specific hormones or viruses, providing results much faster than traditional methods. The Enzyme-Linked Immunosorbent Assay (ELISA) has been developed into a variety of tests\u00a0from pregnancy tests, infectious disease detection, allergy testing, hormone level measurements, food industry applications to COVID.<\/p>\n

The global market<\/a> for ELISA based assays was estimated at $2.52B in 2023 but what has happed with the sister assays, the aptamer based ELISA or ELONA? Why are they no FDA or CE mark approved tests for Enzyme-linked Oligonucleotide Assay?<\/p>\n

We spend a lot of time talking, working and developing cutting edge sophisticated diagnostic assays based on microfluidics, electrochemistry and fluorescent switches and lateral flow assays. It may make sense to pick some of the low hanging fruit, like the ELONA, prior to reaching out on limbs for the ones that are more difficult to capture.<\/p>\n

In a lateral flow assay, the target is forced to interact with the immobilized aptamer, but then it is subject to constant washing as more solution passes over. The ELONA assay is attractive compared to lateral flow because it is possible to allow much more time for target\/aptamer binding, and because it is possible to amplify the colour signal. In an ELONA, there is no loading of a capture zone, but it is possible to allow 30 minutes to an hour for complexes to form.<\/p>\n

The key to any diagnostic approach is to maximize the signal to noise ratio. We have achieved this with ELONA applications by functionalizing gold nanoparticles with both aptamers that bind to the target and oligos that are labeled with a biotin for the capture of streptavidin\/horseradish peroxidase (Strep\/HRP) conjugates.<\/p>\n

\"Gold<\/p>\n

First, we conjugate a capture aptamer to streptavidin with a biotin on the aptamer and a long enough spacer such that the aptamer is able to rise above the surface of the protein. This is passively immobilized to the surface of a microtiter well. The target in a sample is added, along with the dual functionalized gold nanoparticles described earlier.<\/p>\n

Unbound functionalized GNPs and target are removed with a wash step. Streptavidin\/HRP is added, unbound Strep\/HRP is removed with an additional wash step. Then, colour is developed with the addition of TMB.<\/p>\n

An example of results obtained at NVB with this approach is provided below.<\/p>\n

\"graph<\/p>\n

With this approach we were able to detect the presence of 1 fmole of target (confidential protein) from the absence of the protein with a t-test significance of 0.014 across three replications.<\/p>\n

There are two keys to our success with this application:<\/p>\n