UBC Theses and Dissertations
Cell separations by immunoaffinity partition Stocks, Susan Jill
The partition of cells in an aqueous polymer two-phase system composed of dextran T500, polyethylene glycol 8000 and buffer was studied. The effect of various immunoaffinity ligands on erythrocytes and lymphocytes was examined. Separations of rabbit and human erythrocytes were achieved using a combination of monoclonal mouse anti-NN glycophorin IgG with trypan blue-derivatized sheep anti-mouse F[sub c] fragment IgG as well as with a polyacrylamide-derivatized rabbit anti-human erythrocyte IgG (PAA-rochrbc). The PAA-rahrbc was able to completely resolve the two erythrocyte species in a countercurrent distribution (CCD) of 20 transfers. The lymphocyte separation problem was of two sub-lines of a transformed mouse lymphocyte, MBL-2(4.1) and MBL-2(2.6), which differ in the surface densities of an antigen recognized by a rat monoclonal IgG, YE1.48.10. Binding studies showed that at saturation MBL-2(4.1) bound 2.4 x 10⁶ molecules of YE1.48.10. per cell whereas MBL-2(2.6) bound 8 x 10⁵ molecules of YE1.48.10. per cell. This represented an extremely stringent separation problem compared to previous immunoaffinity erythrocyte separations. Attempts to separate the cells by immunoaffinity partition using the following combinations of ligands were not sufficiently successful to achieve useful separations: (a.) PEG 1900-derivatized YE1.48.10. (b.) PEG 1900-derivatized RG7/11.1, a mouse monoclonal IgG specific for rat F[sub c] fragment, and YE1.48.10. (c.) biotin-derivatized YE1.48.10. and PEG-derivatized avidin. However polyacrylamide grafted onto YE1.48.10. produced an effective immunoaffinity ligand, separating MBL-2(4.1) and MBL-2(2.6) on the basis of their antigenic differences. A separation was achieved in 60 CCD transfers.
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