All density separation techniques have the same basic limitation; they can not separate subpopulations of cells with overlapping density distributions such as human lymphocyte subsets. To address this, dense particles have been targeted to cell using monoclonal antibodies with affinity to cell surface antigens and used in discontinous or continous density gradient centrifugation to separate cell populations with similar densities.
Density (isopycnic) gradient separation is one of the most important techniques used to separate different subpopulations of cells. This technique allows cells to sediment to positions according to their density in continuous or discontinous gradients. For example, magnetic dynabeads have been used to separate partially differentiated cells, on the basis of their immunological identity and T and B lymphocytes can be separated from human peripheral blood mononuclear cells using antibody coated dense polystyrene beads and magnetic dynabeads. The basis of density perturbation is that the size and density of the beads that bind to the cell determine to what extent they modify the density of the cell. (Bildirici, J. Immunological Methods, 252 (2001) 57-62).
Levine (US 5,593,848) discloses methods of assaying target cells of specimen samples using particles such as liposomes which are attached to antibodies which are specific to a surface antigen known to occur on the cell. After the sample is incubated with the liposome-antibody, the mixture is then centrifued to separate the various cells types on the basis of the specific gravity of the liposomes which makes the tagged cells into a distinct band in the mixture.
Depletion of B and T cells using Antibody-beads:
Patel (Clinica Chimica Acta 240 (1995) 187-193) discloses using the binding of antibody-coated dense polystyrene beads to increase the density of specific sub-populations of cells. By incubating a total monocnuclear fraction from human peripheral blood together with antibody-coated beads in a lymphocyte separation medium, a depletion of 94.9% of the T cells could be obtained and a depletion of 69.7% of the B cells was also achieved.
Woodside (US20030124719) discloses a method for separating a first population of cells form a second population of cells in a sample by linking dense particles to the first population of cells in the sample, layering the sample over a density separation medium (DSM) having a density at least about 0.001 g/cm3 greater than the mean density of the second population of cells, allowing the cells to settle so that the particle-linked to the first popluation of cells will settle to below the interface between the DSM and the sample and the second population of cells will settle to the interface between the DSM and the sample. The settling of the cells is accelerated by centrifugation.
Van Vlasselaer (US5840502) discloses methods of enriching for a desired cell population from cell sources such as body fluids by using a centrifugation tube tontaining a specific density gradient solution adjusted to the specific density of a desired cell population to enrich for the desired cell from a cell source. In addition, cell type specific binding agents such as antibodies linked to carrier particles to impart different density to the undersired populations can be used. The method can be used as for example to enrich fetal cells from circulating maternal blood or to isolate tumour cells.