Combination Use of CD4 Aptamer Probe and Antibodies for Multi-Color Flow Cytometry Immunophenotyping Analysis
JT O'Hare, P Zhang, C Chang, Y Zu. The Methodist Hospital, Houston, TX
Background: In the last decade, a new class of small molecule ligands composed of synthetic oligonucleotides known as 'aptamers' have emerged. Studies have shown that aptamers can bind specifically to their targets with significant high affinity. In contrast to protein antibodies, the aptamers are easier to produce with much less cost. However, potential clinical value of the aptamers has not been fully addressed yet. In this study, we adapted a previously reported CD4-specific aptamer and validated its use in flow cytometry immunophenotyping of human lymphocytes in combination with antibodies for multi-color staining.
Design: The reported sequence of a 41-mer RNA-based CD4 aptamer was used and labeled with fluorochrome Cy5 for tracking purposes. To validate specific cell binding of the synthetic CD4 aptamer probe, cultured CD4-positive and CD4-negative human lymphoma cell lines were utilized and cell binding was quantified by flow cytometry analysis. In addition, multi-color cell staining by combination of the CD4 aptamer and antibodies for CD3, CD8, and CD45 were tested. Moreover, immunophenotyping of mononucleated cells in peripheral blood samples were also performed using the combination probes of CD4 aptamer and antibodies.
Results: 1) The synthetic CD4 aptamer probe specifically bound to cultured CD4-positive lymphoma cells with a nearly identical pattern to that detected by CD4 antibody, which is a “gold standard” for immunophenotyping analysis; 2) multi-color cell staining could be achieved by combination of the CD4 aptamer probe and antibodies; and 3) the CD4 aptamer probe could be used for immunophenotyping analysis of mixed mononucleated cells in a whole blood specimen.
Conclusions: Our findings demonstrate that the synthetic CD4 aptamer probe can specifically bind to intact human lymphocytes in whole blood samples, indicating the potential clinical use for flow cytometry immunophenotyping analysis with or without antibodies.
Wednesday, March 24, 2010 1:00 PM
Poster Session VI # 203, Wednesday Afternoon