Grimsley P G, Amos T A, Gordon M Y, Greaves M F
Leukaemia Research Fund Centre, Institute of Cancer Research, London, UK.
Leukemia. 1993 Jun;7(6):898-908.
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.
CD34+细胞的阳性选择在诊断病理学、外周血和骨髓移植以及原始造血干细胞的功能与调控研究中具有应用价值。抗体包被的磁性微球（磁珠）可用于通过阳性选择程序分离这些细胞。然而，在某些阳性选择方案中使用磁珠的优势因磁珠在细胞上的残留而受到影响。我们提出了一种方案，可使磁珠从阳性分选的细胞中快速化学释放。将鼠免疫球蛋白（Ig）G1 CD34抗体QBEND/10固定在磁珠上，形成三层免疫复合物的一部分：QBEND/10与F(ab')2抗鼠免疫球蛋白抗体片段相连，后者通过免疫反应与鼠IgG1骨髓瘤蛋白结合。骨髓瘤蛋白将三联体共价连接到磁珠上。因此，F(ab')2铰链区的二硫键可用10微摩尔二硫苏糖醇还原，CD34+细胞在20分钟内即可释放。纯化后的细胞可用多种标志物进行重新表型分析，并鉴定出亚群。很容易实现CD34+细胞97%的纯度、>50%的回收率以及超过90%的活力。此外，粒细胞-巨噬细胞集落形成细胞保留在阳性组分中。该方法可用于纯化其他细胞类型，包括T淋巴细胞和B淋巴细胞。
