Development of a Fluorescent Enzyme-Linked DNA Aptamer-Magnetic Bead Sandwich Assay and Portable Fluorometer for Sensitive and Rapid Leishmania Detection in Sandflies
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A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (∼100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.
KeywordsAmplex Red Aptamer Assay Leishmania Magnetic bead Portable fluorometer Resazurin Resorufin SELEX
Funding was provided by Phase 2 SBIR Contract No. W81XWH-10-C-0179. The authors are grateful to Texas State University (San Marcos, TX) and its faculty (Profs. Joseph Koke, Dana Garcia and Shannon Weigum) for advice and guidance related to confocal fluorescence microscopy. Additionally, the authors acknowledge the technical assistance of Alexander Carr at Texas State University for culture of Leishmania promastigotes. Finally, the authors express gratitude to Dr. Edgar Rowton of the Walter Reed Army Institute of Research (WRAIR) for guidance on culturing of Leishmania promastigotes and assistance in obtaining infected and uninfected sandflies.
- 4.Sundar S, Rai M (2002) Laboratory diagnosis of visceral leishmaniasis. Clin Diag Lab Immunol 9:951–958Google Scholar
- 5.Souza AP, Soto M, Costa JM, Boaventura VS, de Oliveira CI, Cristal JR, Barral-Netto M, Barral A (2013) Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins. PLoS One 8(6):e66110. doi: 10.1371/journal.pone.0066110 PubMedCentralPubMedCrossRefGoogle Scholar
- 6.Moreno EC, Gonçalves AV, Chaves AV, Melo MN, Lambertucci JR, Andrade AS, Negrão-Corrêa D, de Figueiredo Antunes CM, Carneiro M (2009) Inaccuracy of enzyme-linked immunosorbent assay using soluble and recombinant antigens to detect asymptomatic infection by Leishmania infantum. PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0000536, 3e536PubMedCentralPubMedGoogle Scholar
- 10.Wei B, Li F, Yang H, Yu L, Zhao K, Zhou R, Hu Y (2012) Magnetic beads-based enzymatic spectrofluorometric assay for rapid and sensitive detection of antibody against ApxIVA of Actinobacillus pleuropneumoniae. Biosens Bioelectron 35:390–393. doi: 10.1016/j.bios.2012.03.027 PubMedCrossRefGoogle Scholar
- 30.Bruno JG, Carrillo MP, Phillips T (2007) Effects of immobilization chemistry on enzyme-linked aptamer assays for Leishmania surface antigens. J Clin Ligand Assay 30:37–43Google Scholar
- 31.Gonzalez VM, Martin ME, Moreno M (2013) Aptamers targeting protozoan parasites. In: Bruno JG (ed) Biomedical applications of aptamers. Nova, New York, pp 73–88Google Scholar
- 38.Burns JM, Shreffler WG, Benson DR, Ghalib HW, Badaro R, Reed SG (1993) Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral Leishmaniasis. Proc Natl Acad Sci U S A 90:775–779PubMedCentralPubMedCrossRefGoogle Scholar
- 39.Burchardt ER, Kroll W, Gehrmann M, Schroder W (2009) Monoclonal antibody and assay for detecting PIIINP. U.S. Patent No. 7,541,149Google Scholar
- 40.Bruno JG, Carrillo MP, Phillips T, Edge A (2011) Discrimination of recombinant from natural human growth hormone using DNA aptamers. J Biomolec Techn 22:27–36Google Scholar
- 42.Holzer TR, McMaster WR, Forney JD (2006) Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana. Mol Biochem Parasitol 146:198–218PubMedCrossRefGoogle Scholar