Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry.
BACKGROUND
Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous.


METHODS
Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand.


RESULTS
Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen.


CONCLUSIONS
The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.
Siiman
O
O
Advanced Technology, Beckman Coulter, Inc., Miami, Florida 33196-2500, USA. olavi.siiman@coulter.com
Burshteyn
A
A
Concepcion
O
O
Forman
M
M
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007118:Immunoassay; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000276:immunology*; D017452:Receptors, IgG / Q000276:immunology*; D012995:Solubility
D017452:Receptors, IgG