Assessment of a positive selection technique using an avidin column to isolate human peripheral blood T cell subsets.
The current studies were designed to assess a new technique for positively selecting human T cells from whole peripheral blood mononuclear cells using the minimal amount of monoclonal antibody required to bind the T cell to an avidin column indirectly via a biotin-conjugated secondary antibody. Positive selection of T cells has previously been avoided because the saturating amounts of antibodies required for other isolation procedures can lead to aberrant results in assays of T cell activation and function. The avidin column technique for obtaining purified T cell subsets was compared to a multi-step procedure that included negative selection panning. The positive selection technique was easily performed within 4 h whereas the negative selection technique required a minimum of 12 h to complete. The avidin column technique proved to be a rapid and simple method for isolating T cell subsets of high purity and normal functional capabilities. Since minimal amounts of monoclonal antibodies were used for the purification protocol, no consistent inhibitory or stimulatory effect of the residual antibody was noted in assays of activation and proliferation of positively selected T cells compared to T cells isolated by negative selection panning.
Davis
L S
LS
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8884.
McIlraith
M J
MJ
Paecheo
T
T
Becker
B
B
Adix
L M
LM
Thomas
R
R
Wacholtz
M C
MC
Lipsky
P E
PE
D000328:Adult; D000941:Antigens / Q000276:immunology; D001360:Avidin; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002118:Calcium / Q000097:blood; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000096:biosynthesis; D005455:Fluorescent Antibody Technique; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000502:physiology; D008934:Mitogens / Q000276:immunology
D000941:Antigens; D007376:Interleukin-2; D008934:Mitogens; D001360:Avidin; D004247:DNA; D002118:Calcium