Separation of cell mixtures by immunoaffinity cell partitioning: Strategies for low abundance cells

The partitioning of cells in aqueous two-phase systems formed by poly(ethylene glycol) (PEG) and dextran can be changed by incubating the cells with a PEG-modified antibody directed specifically against its surface. We have developed a new approach for immunoaffinity cell partitioning (IACP) in which the antibodies are first reacted with tresylated monomethoxyPEG (TMPEG) in sodium phosphate buffer, pH 7.5, the excess TMPEG is quenched by reaction with bovine serum albumin, and the resulting preparation is used directly for incubation with the cells without any isolation of the monomethoxyPEG (MPEG)-antibody conjugates. We have demonstrated the specificity of this IACP method by showing that MPEG-modified anti-human red blood cell antibody increases the partition of human erythrocytes from the interface to the PEG-rich top phase (up to 100%) but not the partitioning of either neutrophils or HL60 cells. Irrelevant antibodies do not affect the partitioning of red blood cells. The partitioning behaviors of erythrocytes and HL60 cells in mixtures varying from 75 to 10% red blood cells subjected to IACP are similar to those of the pure cell population, i.e., erythrocytes ca. 100% and HL60 cells 3% in top phase. Thus, the population of erythrocytes can be almost completely extracted into the top phase in a single step. The contaminant cells represent only a small percentage (<5% in most of the cases) of the cell mixture recovered in top phase. Both cell populations can be completely separated by countercurrent distribution (CCD). However, in cell mixtures where the erythrocytes represent only 1% of the population, two factors decrease the efficiency of their extraction. First, the partitioning of erythrocytes is reduced to 50% in the top phase and second, even though the separation by CCD of both cell types is theoretically predicted, the low number of erythrocytes in the sample makes them difficult to detect. A combination of bulk extractions and CCD can overcome these problems, allowing the isolation of cells of low abundance in high yield and purity.