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Antigen‐specific electrophoretic cell separation for immunological investigations
Author(s) -
Hansen Ernil,
Wustrow Thomas P. U.,
Hannig Kurt
Publication year - 1989
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150100819
Subject(s) - antigen , polyclonal antibodies , antibody , lymphocyte , microbiology and biotechnology , monoclonal antibody , biology , in vitro , cell , immunology , chemistry , biochemistry
Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non‐capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen‐positive and antigen‐negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence‐labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen‐positive cells in the fractions of electrophoresis. Carrier‐free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti‐IgM‐antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate‐labelled anti‐IgG resulted in the large‐scale separation of highly pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen‐specific induction and regulation of antibody synthesis in vitro . These separated lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell sub‐populations. We studied the influence of the anesthetic thiopental on separated, human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B‐Cells, T‐helper and T‐suppressor cells were equally affected and showed the same dose response. The electrophoretic separation of cells after reaction with antibodies is applicable to a wide range of human lymphocyte subpopulations defined by specific antibodies. Their gentle isolation in large quantities is a powerful tool for investigations of the immune system.