An improved solid-phase bioassay for evaluation of neurotrophic factors

A bioassay system originally described by other investigators was improved to evaluate the biological activities of neurotrophic factors (NTFs) and is referred to as a solid-phase bioassay (SPB). The principle and an outline of the SPB are as follows: (a) Test samples containing NTFs are applied to polyacrylamide gel electrophoresis (PAGE) of the sodium dodecyl sulfate (SDS) or two-dimensional type and then transferred onto nitrocellulose membranes, (b) neurons are cultured on the protein-blotted membranes, and (c) the distributions of the surviving neurons are estimated following fixation and clarification of the nitrocellulose membranes. The rationale is that neuron survival will be restricted to the migration positions of the NTF(s). We used nerve growth factor (NGF) as a NTF and sympathetic neurons of 10- to 12-day-old chick embryos as NGF-responsive neurons. Neuronal survival was observed in the areas coinciding with the positions of NGF migration on the membranes in both systems following SDS-PAGE and two-dimensional PAGE. These results indicate that the SPB can identify the positions of NGF-like molecules on nitrocellulose membranes. Using this system, we found active entities, with molecular masses of approximately 100-200 kDa, different from NGF in crude extracts of mouse submaxillary glands. The SPB developed is considered to be a useful tool for obtaining information on the physicochemical and/or biological properties of putative NTFs in crude samples.