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 (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.
Becton Dickinson Immunocytometry Systems, San Jose, California 95131-1807, USA. kdavis@bdis.com
K A
KA
Davis
B
B
Abrams
S B
SB
Iyer
R A
RA
Hoffman
J E
JE
Bishop
D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D015704:CD4 Antigens / Q000032:analysis* / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry* / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008863:Microspheres; D010799:Phycoerythrin; D011994:Recombinant Proteins / Q000276:immunology; D012015:Reference Standards; D013048:Specimen Handling; D016707:Tissue Fixation
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D000939:Epitopes; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D010799:Phycoerythrin