Skuse D R, Brooks D E
Department of Chemistry, University of British Columbia, Vancouver, Canada.
J Chromatogr. 1988 Nov 18;432:127-35. doi: 10.1016/s0378-4347(00)80639-8.
Cell separation using aqueous polymeric two-phase systems is well established. For separations of cells having similar partition coefficients a multi-step counter-current distribution procedure has been used. As an alternative strategy, we have developed a column-based technique in which the dextran-rich phase of a dextran-polyethylene glycol (PEG) phase system is immobilized onto derivatized chromatography beads. The PEG-rich phase is used as the eluent. A separation of erythrocytes from different species utilizing an affinity ligand was performed. The following behaviour was observed: (i) no elution occurs below a critical affinity ligand concentration; (ii) cell recovery is proportional to affinity ligand concentration; (iii) elution volume is independent of affinity ligand concentration. The above suggest that no multi-step partitioning occurs. However, the separation achieved is considerably better than in a corresponding single-tube separation. A 1:1 mixture of dog and human erythrocytes was separated to a purity of greater than 98% using the column-based technique. The corresponding optimized single-tube separation gave a maximum purity of 84%.
使用水性聚合物双相系统进行细胞分离已得到充分确立。对于具有相似分配系数的细胞分离，已采用多步逆流分配程序。作为一种替代策略，我们开发了一种基于柱的技术，其中葡聚糖 - 聚乙二醇（PEG）相系统中富含葡聚糖的相被固定在衍生化的色谱珠上。富含PEG的相用作洗脱液。利用亲和配体对不同物种的红细胞进行了分离。观察到以下行为：（i）在临界亲和配体浓度以下不发生洗脱；（ii）细胞回收率与亲和配体浓度成正比；（iii）洗脱体积与亲和配体浓度无关。上述情况表明没有发生多步分配。然而，所实现的分离比相应的单管分离要好得多。使用基于柱的技术将犬和人红细胞的1:1混合物分离至纯度大于98％。相应的优化单管分离的最大纯度为84％。
