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

Background: The magnetic separation of a cell population based on cell surf ace 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 u sing a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluoresc ence intensity measurements using a flow cytometer. The quantification of t he second step, an anti-PE-microbeads antibody reagent, was achieved throug h magnetophoretic mobility measurements using cell tracking velocimetry. Results: From the results of these studies, two different labeling protocol s were used to separate CD56+ cells from human, peripheral blood by a Milte nyi Biotech Mini-MACS cell separation system. The first of these two labeli ng protocols was based on company recommendations, whereas the second was b ased on the results of the saturation studies. The results from these studi es demonstrate that the magnetophoretic mobility is a function of both prim ary and secondary antibody concentrations and that mobility does have an ef fect on the performance of the separation system. Conclusions: As the mobility, increased due to an increase in bound antibod ies, 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 th at these cells are irreversibly retained in the column. These results demon strate the complexity of immunomagnetic cell separation and the need to fur ther optimize the cell separation process. (C) 2001 Wiley-Liss, Inc.