Phosphorothyoate oligodeoxynucleotides block nonspecific binding of Cy5 conjugates to monocytes

Cy5-based monoclonal antibody conjugates, including those containing PE-Cy5 and Cy5 alone, are used widely for multi-color flow cytometry in both research and clinical applications. One problem with these conjugates is they bind to monocytes and macrophages irrespective of antibody specificity. This effect, which is thought to be due to binding of the Cy5 itself to the human high affinity receptor for IgG (FcγRI, CD64), can complicate interpretion of flow cytometric results. In the present study we demonstrate that phosphorothioate oligodeoxynucleotides (PS-ODN) suppress this nonspecific binding in a manner that is independent of PS-ODN sequence. Binding of FITC-labeled PS-ODN to monocytes was blocked by CD64-specific monoclonal antibodies, suggesting CD64 is a PS-ODN-binding protein. We conclude PS-ODN can be used as an effective, simple and low-priced reagent to prevent nonspecific binding of Cy5-based immunoconjugates to monocytes. This effect appears to be mediated by PS-ODN binding to CD64 thereby blocking the binding of Cy5 to this receptor.