title: Rapid positive selection of CD34+ cells using magnetic microspheres coated with monoclonal antibody QBEND/10 linked via a cleavable disulphide bond.
abstract: Positive selection of CD34+ cells has applications in diagnostic pathology, in peripheral blood and bone marrow transplantation, and in studies on the function and regulation of primitive haemopoietic stem cells. Antibody-coated magnetic microspheres (dynabeads) can be used to isolate these cells by positive selection procedures. However, the advantages of using dynabeads in some positive selection protocols are compromised by the retention of the beads on the cells. We present a protocol which allows the rapid chemical release of the beads from positively sorted cells. The murine immunoglobulin (Ig) G1 CD34 antibody, QBEND/10, was immobilised onto dynabeads as part of a three-layered immune complex: QBEND/10 was attached to F(ab')2 anti-mouse immunoglobulin antibody fragments, which were immunologically bound to a mouse IgG1 myeloma protein. The myeloma protein covalently bonded the triplex to the beads. Thus, disulphide bonds in the hinge region of the F(ab')2 could be reduced with 10 microM dithiothreitol and CD34+ cells released within 20 min. Purified cells can be re-phenotyped by multiple markers and subsets identified. Purity of 97%, recovery of > 50%, and viability over 90% of the CD34+ cells was readily achieved. Furthermore, granulocyte-macrophage colony-forming cells were retained in the positive fraction. This methodology can be used to purify other cell types, including T and B lymphocytes.
authors.affiliation: Leukaemia Research Fund Centre, Institute of Cancer Research, London, UK.
authors.forename: P G
authors.initials: PG
authors.lastname: Grimsley
authors.forename: T A
authors.initials: TA
authors.lastname: Amos
authors.forename: M Y
authors.initials: MY
authors.lastname: Gordon
authors.forename: M F
authors.initials: MF
authors.lastname: Greaves
mesh_terms: D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D001402:B-Lymphocytes / Q000166:cytology; D002469:Cell Separation / Q000379:methods*; D004220:Disulfides; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D066298:In Vitro Techniques; D008099:Liver / Q000196:embryology; D008280:Magnetics; D008863:Microspheres; D013601:T-Lymphocytes / Q000166:cytology
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018952:Antigens, CD34; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments