We are continuing our efforts to characterize and purify mediators elaborated by human cell lines, e.g., GCT. Our efforts in the current year are aimed at large scale immunization of animals with purified human GM-CSF preparations, possibly using acrylamide gels as added immunogens or alternative coupling methods. We have used several techniques to scale up the purification sequence while minimizing losses. Currently, we have begun to explore the use of hydrophobic HPLC separations and are comparing overall yields between reverse phase supports C4 and C8 and the new phenyl-TSK columns, which may provide similar resolution but avoid the use of solvents such as acetonitrile. Our CSF preparations of highest purity still are able to stimulate erythroid-enhancing activity and mixed Eo-CSF and GM-CSF. We are also investigating factors such as cell concentration, cell cycle, and chemicals such as dimethylsulfoxide on the responsiveness of the leukemia cell-line, HL-60, to CSF. We believe that with careful control of such parameters this population can be used to perform CSF assays using tritiated thymidine incorporation and cytochemical staining techniques, for instance. The availability of such tools will assist us in selecting suitable target cell conditions for study of CSF binding to its receptors, as well as screening for relevant antibodies, polyclonal or monoclonal. (M)
