Development of a rapid, highly sensitive, non-radioactive assay system for hematopoietic growth factors.

The aim of this study was to develop non-radioactive cell line proliferation assays. The human leukemic cell line TF1 (Kitamura et al., 1989) was used for the determination of the specific biological activity of recombinant human (rhu) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhu Interleukin 3 (IL-3) by a simple and economical fluorometric assay with a sensitivity similar to the measurement of 3H-thymidine uptake. The TF1 cell line responds to rhu IL-3, rhu GM-CSF and to a lesser extent to rhu Erythropoietin (EPO) and mast cell growth factor (MGF), but not to rhu G-CSF. It is dependent upon rhu GM-CSF for survival in culture. For the proliferation assay 1 x 10(4) TF1 cells were incubated with 20 ng - 0.256 pg rhu GM-CSF or rhu IL-3 at 37 degrees C and 5% CO2 in humidified atmosphere. After 48 h the cells were washed twice with PBS and were incubated with 4-Methylumbelliferyl-heptanoate for 60 min. Fluorescence was determined on a Titertek Fluoroskan II (Flow Lab.), and results were given as fluorescence units using a 355 nm excitation filter and a 480 nm emission filter. The developed assay showed an interassay variability lower than 15%. The sensitivity of the proliferation assays in the same range as the thymidine incorporation assays.