Could Oligonucleotide Aptamer Probe Replace Antibody for Diagnosis?
Zihua Zeng, Peng Zhang, Youli Zu. The Methodist Hospital, Houston, TX
Background: Aptamers are small molecule ligands composed of short single-stranded oligonucleotides. The high sensitivity and specificity to their targets make aptamer an excellent candidate as a diagnostic probe. In addition, aptamer probes are easily generated through chemical synthesis. However, their potential clinical value has not yet been fully explored. In this study, we tested the aptamer probes for cell immunophenotyping, tissue immunohistochemical (IHC) stain, and blood circulating tumor cell detection.
Design: For immunophenotyping a CD30-specific aptamer was synthesized and conjugated with fluorochrome. Cultured lymphoma cells were stained with aptamer probe with antibodies and analyzed by flow cytometry. For IHC study the biotinylated aptamers were used. Formalin-fixed and paraffin-embedded lymphoma tissues were stained with aptamer probes and HRP-streptavidin reporter. For circulating tumor cell detection a novel assay system was developed by conjugating aptamer probe with both fluorochrome and quencher molecule. Normally, quencher molecule interacts with fluorochrome in the same aptamer sequence and renders it inactive.
Results: 1) Flow cytometry showed that the synthetic CD30 aptamer specifically stained anaplastic large cell lymphoma and Hodgkin lymphoma cells, but not control lymphoma cells that do not express CD30 [figure1A]. Cell staining patterns of aptamer probe were identical to CD30 antibody; 2) Tissue IHC stains showed that aptamer probe specifically recognized CD30+ lymphoma cells, but did not react to background cells in tumor sites [figure1B]. The aptamer probe could efficiently stain tissues within shorter time than standard antibody; 3) To detect circulating tumor cells a drop of blood from lymphoma patients was simply mixed with our new assay system, in which the aptamer-mediated cell binding and subsequent intracellular internalization resulted in endosomal degradation of aptamer sequence. Separation of fluorochrome from quencher molecule present in each aptamer probe activated fluorescent signals exclusively within lymphoma cells. This one-step assay could detect single tumor cell among thousands to millions of normal blood cells with no background [figure1C].
Conclusions: Our proof-of-concept studies demonstrated that the synthetic aptamer probe could replace antibody for disease diagnosis.
Tuesday, March 20, 2012 2:00 PM
Platform Session: Section E, Tuesday Afternoon