Cell separation based on the reversible interaction between calmodulin and a calmodulin-binding peptide.
A cell separation system based on the calcium-dependent interaction of calmodulin (CM) with a calmodulin-binding peptide (CBP) has been developed. The prototype of this system utilizes an indirect method to label the target cell population. Cells are first labeled with a primary monoclonal antibody directed to a specific cell surface antigen, then with a secondary affinity reagent, consisting of a polyclonal goat anti-mouse IgG (GAM-IgG) that has been cross-linked to a CBP derived from the sequence of the rabbit skeletal muscle myosin light chain kinase. In the presence of Ca2+, the CBP on the cells labeled with GAM-IgG-CBP binds to biotinylated calmodulin (CM-Biotin) with high affinity. The target cells are then captured with a solid-phase streptavidin. The unbound non-target cells are washed away and the immobilized target cells are released by chelating Ca2+ with EGTA. The specificity of the GAM-IgG-CBP and CM-Biotin and the feasibility of using this system to separate cells was demonstrated using the KG-1 human acute myelogenous leukemia cell line. KG-1 cells were fractionated on the basis of cell surface expression of HLA-DR. The cell selection reagents and the cell separation process did not affect KG-1 cell viability while cells selected by this procedure were 90% pure with a yield of 75%. This cell separation system also was used for rare cell isolation from normal human peripheral blood mononuclear cells. T cells expressing the Vbeta5 T cell receptor, which represent < 5% of the unfractionated cells, were isolated with 89% viability, 72% purity, 80% yield, and retained the ability to respond to activation signals as measured by blast transformation. The results from this study show that a cell selection system based on the reversible interaction between CM and a CBP can be applied to gently and efficiently isolate cells from a heterogeneous starting population that are free of the solid matrix without exposure to the stresses of mechanical or enzymatic release.
Colinas
R J
RJ
The Wadsworth Center, New York State Department of Health, Albany 12201-0509, USA. colinas@wadsworth.org
Walsh
A C
AC
D001710:Biotin; D017252:CD3 Complex / Q000302:isolation & purification; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D018914:Cells, Immobilized; D006684:HLA-DR Antigens / Q000302:isolation & purification; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000276:immunology; D008213:Lymphocyte Activation; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000302:isolation & purification; D019809:Streptavidin; D014407:Tumor Cells, Cultured
D017252:CD3 Complex; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D006684:HLA-DR Antigens; D010446:Peptide Fragments; D016693:Receptors, Antigen, T-Cell, alpha-beta; D001710:Biotin; D019809:Streptavidin; D009219:Myosin-Light-Chain Kinase