Antigen-specific electrophoretic cell separation (ASECS): isolation by human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies.
The electrophoretic mobility of human lymphocytes can be reduced by incubation with surface antigen specific antibodies under non-capping conditions. This renders subpopulations of human peripheral blood lymphocytes accessible to separation by free-flow electrophoresis. After reaction of lymphocyte preparations with anti-IgM antibody and a fluorescent second antibody, B lymphocytes showed a considerable shift in position in preparative cell electrophoresis and could be separated with high yield, purity and vitality. Similarly, a T cell subpopulation reactive with the monoclonal antibody T811 could be isolated, even though only small amounts of this antibody were bound, by using a double-sandwich method. Non-specific antibody uptake via Fc-receptors did not contribute to the observed shift of antibody-labelled cells to lower electrophoretic mobility. Flow cytometric analysis showed that cells were separated according to their antigen density. Thus cell electrophoresis can be used to separate antibody-labelled cells. With a flow rate of 100,000 cells/sec this method has a much higher separation capacity than fluorescence-activated cell sorting. The described method should be applicable to the separation of a wide range of cell populations for which specific antibodies are available.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001402:B-Lymphocytes; D002465:Cell Movement; D002469:Cell Separation / Q000379:methods*; D004586:Electrophoresis / Q000379:methods; D000939:Epitopes; D005434:Flow Cytometry; D006041:Goats; D006801:Humans; D007075:Immunoglobulin M; D051379:Mice; D011817:Rabbits; D051381:Rats; D013601:T-Lymphocytes
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007075:Immunoglobulin M