Isolation and characterization of nerve growth factor (NGF) excreted from cultured eukaryotic cells

Since the discovery in 1954, NGF has been isolated from snake venoms and various tissues and organs of different animals. Recently, Lipps (2000b) reported the isolation of NGF from the human body fluids, saliva, serum, and urine. This investigation reports the isolation of NGF excreted by various types of cells from diverse origin, in serum free medium, proving that its presence is not restricted to neural cells. The established cell lines used were Chang's liver and neuroblastoma of human origin, Vero monkey origin, PC12 rat, and SP/2 mouse origin. The fully grown monolayers of the cells were maintained in serum free medium for 48 hours to excrete NGF in the medium and the cell free medium was concentrated. NGF from cell free concentrated medium for each cell line was isolated by high pressure liquid chromatography (HPLC) and was identified as described by Lipps (1998). The HPLC profiles for cell free concentrate from different types were observed to be similar. The NGF fraction was eluted last in the neutral pH (Fig. 1). The identified fraction for NGF from each cell line was further purified, which resolved into a single peak. The purified NGF was used to study the biological and immunological properties. The biological activities of NGFs from cell free medium were minuscule in comparison to the cobra venom derived NGF. The molecular weights of NGFs from cell free medium for all cell lines were identical, 36.0 kDa. Anti-human NGF reacted strongly immunologically with NGFs from human origin cells and poorly with rat and mouse. PC12 NGF antibody reacted immunologically only with PC12 NGF.