To measure release of endogenous BDNF from neurons, we developed an in vitro model using dissociate cultures of rat primary sensory ganglion cells and a modified ELISA, termed ELISA in situ. Nodose-petrosal ganglion (NPG) neurons were grown in 96 well ELISA plates precoated with Promega Anti-BDNF mAb to capture released BDNF, which we subsequently detected using the antibody sandwich format of the Promega BDNF Emax® ImmunoAssay System. Release was measured in response to either patterned electrical field stimulation or chronic depolarization with elevated extracellular potassium and compared with basal release in the absence of depolarizing stimuli. Incorporation of the coating antibody into the culture system dramatically increased detectability of BDNF in both control and stimulated cultures. The efficacy of the in situ assay appears to be related primarily to rapid capture of released BDNF.
Promega Notes 78, 17–20.
Agnieszka Balkowiec and David M. Katz