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 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.
Comella
K
K
Department of Chemical Engineering, The Ohio State University, Columbus, Ohio 43210, USA.
Nakamura
M
M
Melnik
K
K
Chosy
J
J
Zborowski
M
M
Cooper
M A
MA
Fehniger
T A
TA
Caligiuri
M A
MA
Chalmers
J J
JJ
D000937:Antigen-Antibody Reactions / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology*; D019002:CD56 Antigen / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods*; D007694:Killer Cells, Natural / Q000166:cytology*; D010799:Phycoerythrin / Q000276:immunology
D019002:CD56 Antigen; D007074:Immunoglobulin G; D010799:Phycoerythrin