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.
The Wadsworth Center, New York State Department of Health, Albany 12201-0509, USA. colinas@wadsworth.org
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Walsh
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