Characterization of antibody binding to three cancer‐related antigens using flow cytometry and cell tracking velocimetry

Abstract

Proper antibody labeling is a fundamental step in the positive selection/isolation of rare cancer cells using immunomagnetic cell separation technology. Using either a two-step or single-step labeling protocol, we examined a combination of six different antibodies specific for three different antigens (epithelial specific antigen, epithelial membrane antigen, and HER-2/Neu) on two different breast cancer cell lines (HCC1954 and MCF-7). When a two-step labeling protocol was used (i.e., anti-surface marker-fluoroscein-isothiocyanate [FITC] [primary Ab], anti-FITC magnetic colloid [secondary Ab]) saturation of the primary antibody was determined using fluorescence intensity measurements from flow cytometry (FCM). The saturation of the secondary antibody (or saturation of a single-step labeling) was determined using magnetophoretic mobility measurements from cell tracking velocimetry (CTV). When the maximum magnetophoretic mobility was the primary objective, our results demonstrate that the quantities necessary for antibody saturation with respect to fluorescence intensity were generally higher than those recommended by the manufacturer. The results demonstrate that magnetophoretic mobility varies depending on the types of cell lines, primary antibodies, and concentration of secondary magnetic colloid-conjugated antibody. It is concluded that saturation studies are a vital preparatory step in any separation method involving antibody labeling, especially those that require the specificity of rare cell detection.

Binding affinities/avidities of antibody–antigen interactions: Quantification and scale‐up implications

“…In addition, it was observed that the magnetophoretic mobility of immunomagnetically labeled cells is a saturation type function of the concentration of the antibody-nanoparticle conjugates used to label the cells (Chosy et al, 2003;Comella et al, 2001). While a saturation type relationship is theoretically expected, based on typical antibody-antigen interactions, the scale-up of immunomagnetic cell separations for clinical applications may require a considerable amount of antibody which can be financially limiting.…”

“…While a saturation type relationship is theoretically expected, based on typical antibody-antigen interactions, the scale-up of immunomagnetic cell separations for clinical applications may require a considerable amount of antibody which can be financially limiting. Significant experimental evidence has demonstrated that the performance of both the commercial MACS systems as well as the flow through technology being developed in our laboratories is a clear function of the magnetophoretic mobility of the labeled cell (Chosy et al, 2003;Comella et al, 2001;McCloskey et al, 2003a).…”

Migration Analysis of Micro-Particles in Liquids Using Microscopically Designed External Fields

“…Particle Tracking Velocimetry (PTV) and magnetophoresis of immunomagnetically labeled cells was combined by Zborowski et al [35][36][37][38][39][40][41] They measured the magnetophoretic velocity of an individual labeled cell under an inhomogeneous magnetic field, and then the magnetophoretic mobility was determined. In their theoretical treatment, the magnetophoretic mobility depended on the number of magnetic labels.…”

“…Antibody Binding Capacity (ABC) was defined as the quantity representing how many magnetic nanoparticles could bind per single cell, and the difference of ABC between individual cells was investigated. 41 The magnetophoretic velocity of a macrophagic cell, which was fed by the magnetic nanoparticle, was investigated and the number of nanoparticles uptaken was determined quantitatively by Wilhelm and co-workers. 42 The number of magnetic particles in the cell was determined by the ferromagnetic resonance as well.…”

Magnetic needles and superparamagnetic cells

“…The nanoparticles are fixed to the cell via the attachment of antibodies to the specific antigens on the cell's surface (Chosy et al 2003). The Stokes viscous drag on the spherical cell is given by…”