Elisa-Like Format for Comparing DNA Capture Elements (Aptamers) to Antibody in Diagnostic Efficacy

摘要

DNA Capture Elements (DCEs), or aptamers, are small pieces of artificial DNA (30-60 base pairs) that are selected by increasing stringency for binding to ligand targets varying from pure proteins, lipids, carbohydrates to whole microbes. Due to this stringency of selection, they are expected to have binding constants in the nanomolar range. However, the real test of their efficacy is based on performance in a standard immunoassay-like format. For this purpose, the microtiter, heterologous phase, ELISAlike sandwich assay test was chosen. The antigen of choice was the standard antigen used in commercially available agglutination tests for Francisella tularensis (tularemia bacterium). In addition, data were collected using the antigen available from the DoD Biodefense Critical Reagents Program and whole vaccine strain Francisella tularensis from the Centers for Disease Control and Prevention (CDC). Microtiter plates were coated with 500 pmol of polyclonal capture DCEs (25 total distinct DCE sequences). This minimal amount was able to detect as little as 250 ng of the antigen. The binding was detected using DCE/horseradish peroxidase conjugate indicator and a colorimetric and spectrophotometric ABTS test.