Zusammenfassung
Mit Hilfe eines Plaque Assays wurde die Kinetik der durch Pokeweed Mitogen induzierten Transformation menschlicher B-Lymphozyten in Immunglobulin-sezernierende Zellen untersucht. Als Indikatorzellen dienten Schafserythrozyten, an die Protein A gekoppelt war. Die maximale Stimulation wurde in allen Experimenten nach einer Kulturdauer von 6–8 Tagen erreicht. Nach Optimierung der Versuchsbedingungen erwies sich dieser Plaque Assay als ausgezeichnete Methode zur quantitativen Bestimmung der Anzahl Ig-sezernierender Zellen vitro; er war außerdem gut geeignet, die B-Zellfunktion eines Individuums über mehrere Monate zu verfolgen.
Die Ergebnisse haben gezeigt, daß die durch Pokeweed Mitogen bewirkte B-Zellaktivierung an das Vorhandensein von Helfer-T-Zellen gebunden war. Außerdem konnte mit anderen, typischen T-Zellmitogenen, ebenfalls eine polyklonale B-Zellaktivierung bewirkt werden. — In weiteren Experimenten wurde gezeigt, daß die Ko-Kultivierung von Lymphozyten zweier nicht HLA-identischer Spender nicht zu einer B-Zellaktivierung führt; nach Stimulierung mit Pokeweed Mitogen wurde auch in diesen Kulturen eine Aktivierung gefunden, die dem erwarteten Ausmaß entsprach. Diese Befunde lassen den Schluß zu, daß mit dieser Technik wertvolle Ergebnisse in der experimentellen und klinischen Immunologie gewonnen werden können.
Summary
The kinetics of Pokeweed mitogen-induced transformation of human B-lymphocytes into immunoglobulin-secreting cells were examined in a reverse hemolytic plaque assay using protein A-coated sheep red blood cells as indicator cells. Peak responses occurred consistently after 6–8 days of incubation. After determination of the optimal experimental conditions the RHPA was found to be a reliable tool to estimate ISC in vitro; the technique was also found to be applicable for experiments surveying the B-cell response of an individual over a period of months.
The PWM-induced transformation of B cells was absolutely T-cell-dependent. Other substances known as typical T-cell mitogens were also tested for polyclonal B-cell activation and some of them showed significant responses. Further experiments have shown that co-cultivation of non-HLA identical cells did not increase the number of plaques in unstimulated cultures whereas the addition of PWM leads to the generation of ISC within the expected range. These findings open a wide field of application of the RHPA in experimental and clinical immunology.
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Supported by Deutsche Forschungsgemeinschaft (Ru 215/2)
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Sieber, G., Bochert, G., Enders, B. et al. Characteristics of immunoglobulin secretion in man evaluated by a reverse hemolytic plaque assay. Blut 41, 81–92 (1980). https://doi.org/10.1007/BF01039652
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DOI: https://doi.org/10.1007/BF01039652