No abstract

We have used recombinant human CD4 presented on beads as an affinity matrix to screen a 2'-F-pyrimidine-containing RNA library with a complexity of approximately 10(14) molecules. Affinity-selected aptamers bind recombinant CD4 with low nanomolar equilibrium dissociation constants. These high-affinity aptamers conjugated to different fluorophores such as fluorescein and phycoerythrin were used to stain cells, expressing human CD4 on cell surface, for analysis by flow cytometry. Aptamers, conjugated to fluorophores, stained mouse T cells that express human CD4 on the surface, but not the control mouse T cells lacking human CD4. The control cells, however, do express mouse CD4 whose extracellular domain has 55% sequence identity to the human form. These human CD4-specific aptamers selectively stained CD4(+) T cells in a preparation of human peripheral blood mononuclear cells. These results and others suggest that aptamers are emerging as a versatile class of molecules that can be used for various diagnostic applications performed under different formats or platforms.

To investigate the feasibility of using oligonucleotides in flow cytometry we describe a model system consisting of human neutrophil elastase (HNE) coated on 3.3 micro beads and a high affinity DNA ligand for HNE isolated by in vitro selection (SELEX). In this system the fluoresceinated DNA ligand was equally effective as an anti- HNE antibody in detecting HNE on beads. The location on and the chemistry of attachment of fluorescein to the DNA ligand is critical for the sensitivity of detection. DNA constructs in which fluorescein was conjugated via an ethylene glycol tether to either the 5'-end or near the 3'-end gave much higher signals than did probes with fluorescein directly conjugated to either end. Second-step staining with strepavidin-conjugated phycoerythrin was accomplished using a biotinylated DNA ligand in the initial staining of HNE beads. These data suggest that instead of, or in addition to, antibodies high affinity oligonucleotide probes can be useful in diagnostic applications based on flow cytometry.

The development of new fluorophores has experienced a tremendous advance over the last two decades. The unique photophysical properties of quantum dots (QDs), such as their large Stokes shifts and exceptional brightness, make them attractive probes in flow cytometry applications. In this chapter, the spectral overlap and the fluorescence intensity of a single Qdot nanocrystal species (Qdot-655) was investigated in the context of a panel containing conventional fluorophores. Certain compensation issues remain because of the unique absorption characteristics of QDs. To demonstrate the potential of QDs for multicolor flow cytometry, human lymphocytes were surface stained with an eight-color panel where one of its standard violet laser reagents, CD4 AmCyan, was substituted with the CD4 Qdot-655 conjugate.