Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry

Cytometry
O SiimanM Forman

Abstract

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. 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. 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...Continue Reading

References

Aug 1, 1990·The Journal of Clinical Investigation·T W HuizingaA E von dem Borne
Jan 5, 2000·Journal of Immunological Methods·S F van EedenJ C Hogg
Jan 5, 2000·Journal of Immunological Methods·T W KuijpersD Roos
Jan 7, 2000·The Journal of Immunology : Official Journal of the American Association of Immunologists·A L KindzelskiiH R Petty

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