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Cell separation based on the reversible interaction between calmodulin and a calmodulin-binding peptide.
- Source :
-
Journal of immunological methods [J Immunol Methods] 1998 Mar 01; Vol. 212 (1), pp. 69-78. - Publication Year :
- 1998
-
Abstract
- 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.
- Subjects :
- Biotin
CD3 Complex isolation & purification
Cells, Immobilized
HLA-DR Antigens isolation & purification
Humans
Leukemia, Myeloid, Acute immunology
Lymphocyte Activation
Myosin-Light-Chain Kinase metabolism
Peptide Fragments metabolism
Protein Binding
Receptors, Antigen, T-Cell, alpha-beta isolation & purification
Streptavidin
Tumor Cells, Cultured
Calmodulin metabolism
Calmodulin-Binding Proteins metabolism
Cell Separation methods
Subjects
Details
- Language :
- English
- ISSN :
- 0022-1759
- Volume :
- 212
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 9671154
- Full Text :
- https://doi.org/10.1016/s0022-1759(98)00009-x