Preparative Electrophoretic Separation of Antibody Forming Cells

Zeiller, K.; Pascher, G.; Hannig, Kurt

doi:10.1080/00327487208061450

Cited by 32 publications

(13 citation statements)

References 16 publications

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“…For example, T-responder cells of individual blood donors were purified within two hours until the 'output stream cells' were found not to respond to Concanavalin A unless monocytes or dendritic cells were re-added [74]. Antibody forming cells, which had already been stimulated in a body prior to drawing blood or lymph node dissection, were enriched several fold [75,76]. Kidney cells were subdivided into fractions which showed various physiological characteristics [77] and the pituitary cells secreting growth hormones were enriched [78].…”

Section: Suspension Media For Electrophoresismentioning

confidence: 99%

Electrophoresis of cells and the biological relevance of surface charge

2002

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Recent developments in electrophoresis of cells are reviewed. Problems and progress in automation and miniaturization of analytical electrophoresis instruments as well as in the interpretation of experimentally determined electrophoretic mobility (EPM) data are summarized: In recent times, the EPM determination techniques not only became more reliable and faster, but also more knowledge could be gained about the cell surface electrical properties, the structure of the glycocalyx as well as its influence on the cell peripheral regions and microenvironment by applying cell electrophoresis. In addition, ways are shown to solve discrepancies between physical requirements of a preparative cell electrophoresis procedure and the quantities of ions, which have to be dissolved in cell suspension media. As the modern machines allow the purification of untagged cells suspended in more cell friendly and physiological media, they are likely to be valuable tools in several useful practical applications in clinical transplantation, gene therapy and treatment of disease states.

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Section: Suspension Media For Electrophoresismentioning

confidence: 99%

Electrophoresis of cells and the biological relevance of surface charge

2002

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“…Protected amino acid derivatives were obtained from Bachem. The binding buffer for cell binding experiments was a low ionic strength (0.015 M) buffer made isotonic to the cells by the addition of glucose and sucrose (13). The composition of this buffer was: 0.015 M triethanolamine, 0.24 M glycine, 0.009 M sucrose, 0.025 M glucose, 0.004 M potassium acetate, and 0.0003 M calcium chloride.…”

Section: Methodsmentioning

confidence: 99%

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Sharma

Jiang

Hadley

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone ( The search for a molecular marker for melanoma cancer cells is pivotal for the development of modalities for the early detection and management of melanoma. The identification of a specific universal antigen associated with melanoma has remained elusive. Usually, good antibody candidates specific for a particular melanoma specimen fail to recognize other tumor variants from different patients and sometimes even from the same individual (1, 2). The occurrence of melanotic as well as amelanotic tumors further illustrates the great degree of melanoma variance and the problem of unambiguous detection. A group of acidic cytosolic proteins, commonly referred to as S-100 proteins, have been claimed to be a useful marker for melanoma (3, 4). An immunoassay based on this protein has been used to screen formalin fixed melanoma tumors in vitro, stressing its importance as a good diagnostic tool for melanoma, especially in case of metastatic lymph node melanoma. Unfortunately, a large number of normal tissues also contain S-100 proteins in substantial amounts (5), making it a rather complex issue in distinguishing the truly positive from the truly negative specimens. Furthermore, the cytosolic localization of these proteins suggests that antibody-directed targeting of modalities of therapeutic relevance might not be feasible. For this purpose, the identification of specific cell membrane markers is better suited because of their rather facile accessibility by cell-specific targeting agents.We recently described the development of melanotropin conjugates that could detect the presence of melanotropin receptors on a number of mouse and human melanoma cell lines, melanotic as well as amelanotic, that we investigated (6, 7). These conjugates were composed of multiple copies of both a potent melanotropin analog and a fluorophore that were conjugated to a biologically inert macromolecule in a pendant fashion. The multivalency of the ligand molecule in these conjugates afforded specific binding between the conjugate and the melanotropin receptors, presumably by establishing simultaneous interactions with a number of melanocytestimulating hormone (MSH) receptors on the cell membrane. In addition, these macromolecular composites also allowed visualization of certain phenomena, like capping and internalization, that are associated with certain ligand-receptor binding.Here we describe another class of multivalent melanotropic ligands that can detect the presence or absence of specific MSH binding sites on melanoma cells in a very simple and straightforward manner. These methods provide simple alternatives to the use of fluorescence techniques previously described by and recently by us (6, 7). The specificity of these newer solid-phase reagents for MSH-binding sites is addressed and established. MATERIALS AND METHODSN, N-Dimethylaminopyridine was obtained from Aldrich. DTT, mercaptoethanol, 4-( p-...

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“…Dead cells are preferentially removed from the fractions of interest because they tend to acquire higher negative charge (Sherbet, 1978), whereas the antigen-positive cells are collected in the region of low EPM. In our experiments the vitality of the separated lymphocytes has not yet been verified in functional assays, but the use of electrophoretically separated cells in such tests is well documented (Zeiller et al, 1971;Zeiller et al, 1976;Sherbet, 1978), and antibody treatment is known not to interfere with most lymphocyte functions.…”

Section: Discussionmentioning

confidence: 79%

“…A reduction in surface charge density has been observed in lymphocytes from various mammals after treatment with ALS or anti-Ig antisera (Bert et al, 1969;Phondke et al, 1970;Bert et al, 1971;Bona et al, 1972;Kaplan and Uzgiris, 1976). However, an increase in EPM (Von Boehmer, 1974;Zeiller et al, 1976), or a lack of any change (Bert, 1969;Wioland et al 1976;Zeiller et al, 1976) has also been described. In all of these studies antibody-induced redistribution of surface antigens has to be considered since incubations at room temperature or 37°C were involved.…”

Section: Discussionmentioning

confidence: 99%

“…Free-flow electrophoresis has proven to be a powerful method for the separation of T and B cells, and of lymphocytes in various stages of differentiation and activation, for mouse and rat (Zeiller et al, 1971;Zeiller et al, 1976; for review see : Sherbet, 1978;Pretlow and Pretlow, 1979). Attempts to separate human lymphocytes, however, have been unsatisfactory (Stein et al, 1973;H/iyry et al, 1975;Ault et al, 1976;Chollet et al, 1978).…”

Section: Introductionmentioning

confidence: 99%

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Antigen-Specific Electrophoretic Cell Separation (ASECS): Isolation of human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies

Hansen

Hannig

1982

Journal of Immunological Methods

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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.

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Preparative Electrophoretic Separation of Antibody Forming Cells

Cited by 32 publications

References 16 publications

Electrophoresis of cells and the biological relevance of surface charge

Electrophoresis of cells and the biological relevance of surface charge

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Antigen-Specific Electrophoretic Cell Separation (ASECS): Isolation of human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies

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