Barnaby Abrams

Determination of CD4 antigen density on cells: Role of antibody valency, avidity, clones, and conjugation†

The number of R-phycoerythrin (R-PE)-conjugated antibodies bound to a cell can be quantitated on a flow cytometer by using beads with known numbers of attached R-PE molecules (QuantiBRITE PE). Using these reference beads, we have observed that a number of factors affect the accuracy of the quantitation and conclusions about epitope density. These factors include valence of antibody binding, the use of antibody fragments (Fabs) versus intact monoclonal antibodies (mAbs), fixation, the purity of the conjugate (i.e., percentage of 1:1 ratios), dissociation rate, the use of washed versus unwashed preparations, and the location of epitope on target antigen. We used CD4 on T cells as a model to explore these challenges in detail. We conclude that CD4+ T cells bind approximately 49,000 CD4 (Leu 3a) antibody molecules, that this binding is bivalent, and therefore that there are approximately 98,000 CD4 antigen molecules on the surface of these cells.

Quantum Dots in 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.