A Novel Aptamer-Based Assay For Capturing Total Erythropoietin (EPO)
| dc.contributor.author | Toh, Saw Yi | |
| dc.date.accessioned | 2019-07-17T02:11:58Z | |
| dc.date.available | 2019-07-17T02:11:58Z | |
| dc.date.issued | 2015-10 | |
| dc.description.abstract | Recombinant human erythropoietin (EPO) abuse is rampant among athletes, which raises the need to monitor its usage to ensure „fair play‟ in sports and for the sake of the athlete‟s health. The current method approved by WADA for the detection of EPO abuse among athletes is the isoelectric focusing (IEF) double-immunoblotting, which requires concentrated form of EPO for a clear-cut result. Current method of concentrating EPO such as ultrafiltration is expensive and non-specific while a more specific strategy such as lectin-based purification leads to the purification of other glycoproteins. In addition, antibody-based purification is also expensive and possibly imparts irreversible denaturation of the target protein, suggesting the „adoption‟ of a more amenable molecular recognition element. Aptamer, owing to its vast array of advantages over the antibodies, can be a potential agent for capturing and concentrating EPO prior to IEF-double immunoblotting. In this study, REPORA-6 aptamer, the previously isolated EPO-specific RNA aptamer, was successfully used in the aptamer-based EPO capture assay. In this assay, REPORA-6 RNA aptamer was functionalized with biotin prior to the immobilization on the streptavidin-coated magnetic beads. Epoetin-α was used as the target protein and was added onto the streptavidin-coated magnetic beads immobilized with REPORA-6 RNA aptamer . The target protein was eluted using 0.1 M of biotin and the lowest amount of the EPO captured/eluted was estimated using dot blot assay. In parallel with this, Reverse Transcriptase-PCR (RT-PCR) was carried out to check for the integrity of the RNA aptamer in this assay. This capture assay developed is able to capture and elute as low as 1.93 nM of EPO. For the EPO protein spiked into urine and serum, the lowest amount of EPO captured and eluted were 48.25 nM and 193 nM, respectively. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/8515 | |
| dc.language.iso | en | en_US |
| dc.publisher | Universiti Sains Malaysia | en_US |
| dc.subject | Recombinant human erythropoietin abuse | en_US |
| dc.subject | is rampant among athletes | en_US |
| dc.title | A Novel Aptamer-Based Assay For Capturing Total Erythropoietin (EPO) | en_US |
| dc.type | Thesis | en_US |
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