Cell Tracking Velocimetry as a tool for defining saturation binding of magnetically conjugated antibodies

Background: Continuous flow immunomagnetic separation is an attractive alternative to current batch mode immunomagnetic separation methods because it is capable of high sorting speeds at mild cell conditions, and grants the operator better control of separation process. The control of the separation is dependent on knowledge of the amount of magnetic label attached to the cell (magnetic labeling intensity), however. Determination of the magnetic labeling is accomplished by measuring cell magnetophoretic mobility using a newly developed technique of Cell Tracking Velocimetry (CTV). Methods: Flow cytometry was used to define the antibody binding characteristics of a fluorescently tagged primary antibody. Subsequently, CTV was used to measure antibody-binding characteristics of a magnetically tagged secondary antibody. Results: The results of this study show that CTV is capable of providing valuable information concerning the cell labeling by magnetically tagged antibodies. It was demonstrated that the magnetically conjugated antibody binding curve exhibits the same exponential increase to saturation characteristics as that seen with the fluorescently tagged antibody. Further, it was shown that the intensity of the secondary magnetic labeling is directly proportional to the intensity of the primary fluorescent label. Conclusions: CTV is an accurate tool for evaluation of magnetically conjugated antibodies. The ability to determine the intensity of magnetic labeling is necessary for the development of continuous flow immunomagnetic separations based on cell magnetophoresis. (C) 2005 Wiley-Liss, Inc.