Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system

• Published in: Cytometry (New York, N.Y.) Vol. 45; no. 4; pp. 285 - 293
• Main Authors: Comella, K., Nakamura, M., Melnik, K., Chosy, J., Zborowski, M., Cooper, M.A., Fehniger, T.A., Caligiuri, M.A., Chalmers, J.J.
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 01.12.2001
• Subjects: Antigen-Antibody Reactions - immunology; Binding Sites, Antibody - immunology; CD56 Antigen - immunology; cell tracking velocimetry; Dose-Response Relationship, Immunologic; Flow Cytometry - methods; Humans; Immunoglobulin G - blood; immunomagnetic label; Immunomagnetic Separation - instrumentation; Immunomagnetic Separation - methods; Killer Cells, Natural - cytology; magnetic cell separation; MiniMACS separation column; natural killer cells; Phycoerythrin - immunology
• ISSN: 0196-4763; 1097-0320
• DOI: 10.1002/1097-0320(20011201)45:4<285::AID-CYTO10018>3.0.CO;2-W

Background The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. Methods Specifically, the degree of saturation of antibody binding sites using a two‐step antibody sandwich was quantified. The quantification of the first step, a primary anti‐CD56‐PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti‐PE‐microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. Results From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. Conclusions As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process. Cytometry 45:285–293, 2001. © 2001 Wiley‐Liss, Inc.