Fluorescent Aptamer Tests for Active and Latent Leishmania

Operational Technologies Corporation (OpTech) proposes to couple the sensitivity of fluorescence-based assays with the high affinity and selectivity of DNA aptamers as replacements for antibodies to detect Leishmania infections. The primary target for detection of active cutaneous Leishmania species will be the common 11kD kinetoplastid membrane protein (KMP-11). During Phase I, OpTech will make FRET (fluorescence resonance energy transfer) DNA aptamers to the entire KMP-11 protein and several Leishmania-specific antigens provided by the Army. Latent infections may be detectable by anti-KMP-11 antibodies and possibly infection-related cytokines or markers via a competitive FRET assay employing lower affinity fluorophore-labeled aptamers and quencher-labeled KMP-11, which OpTech will attempt to demonstrate in Phase I. Lower affinity aptamers can be produced by fewer cycles of SELEX (Systematic Evolution of Ligands by EXponential enrichment) and by controlling buffer stringency. If successful, OpTech will optimize and adapt each of these assays for use in field kits to detect Leishmania in both blood samples and captured sand flies by use of a commercially available handheld, battery-operated fluorometer. Final assays will be freeze-dried in sealed plastic cuvettes or in OpTech's proprietary magnetic bead-based microfluidic plastic cartridge, which is currently under development.