This paper reports results first disclosed at the Fourth Annual Clinical Cytometry and Imaging Symposium, March 13–15, 1997, Rockville, Maryland.
Determination of CD4 antigen density on cells: Role of antibody valency, avidity, clones, and conjugation†
Article first published online: 6 JAN 1999
Copyright © 1998 Wiley-Liss, Inc.
Special Issue: Quantitative Fluorescence Cytometry: An Emerging Consensus
Volume 33, Issue 2, pages 197–205, 1 October 1998
How to Cite
Davis, K. A., Abrams, B., Iyer, S. B., Hoffman, R. A. and Bishop, J. E. (1998), Determination of CD4 antigen density on cells: Role of antibody valency, avidity, clones, and conjugation. Cytometry, 33: 197–205. doi: 10.1002/(SICI)1097-0320(19981001)33:2<197::AID-CYTO14>3.0.CO;2-P
- Issue published online: 6 JAN 1999
- Article first published online: 6 JAN 1999
- Manuscript Accepted: 16 JUN 1998
- Manuscript Received: 5 JUN 1998
- flow cytometry;
- QuantiBRITE method;
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 (Fab's) versus intact monoclonal antibodies (mAb's), 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. Cytometry 33:197–205, 1998. © 1998 Wiley-Liss, Inc.