Quantitative Evaluation of Neurite Outgrowth in Cultures of Human Foetal Brain and Dorsal Root Ganglion Cells Using an Enzyme‐Linked Immunoadsorbent Assay for Human Neurofilament Protein

Abstract

An enzyme-linked immunoadsorbent assay has been developed to evaluate comparative levels of neurofilament protein in developing primary cultures of human foetal dorsal root ganglion and brain tissue. The quantitative parameters of the assay, relating linearity of response with varying levels of neurofilament protein, were verified by comparing the relative binding of human species-specific (BF10) and cross-species-reactive (RT97) monoclonal antibodies to mixtures of human and baboon spinal cord homogenates that had been passively adsorbed onto microtitre wells. In human neural cultures, the localisation of neurofilament protein to growing neurites was determined by indirect immunofluorescence staining with anti-neurofilament antibodies and, using the immunoadsorbent assay, a time-dependent increase in the level of neurofilament protein was detected that correlated with the morphological time course of neurite development. In the case of dorsal root ganglion cells over 6 days in vitro, a seven- to ninefold greater increase in neurofilament protein levels was observed in cultures treated with nerve growth factor when compared with control unstimulated preparations. The quantitative responsiveness of dorsal root ganglion neurones to nerve growth factor detected by the neurofilament assay indicates its potential usefulness in the identification and analysis of neurotrophic and neurotoxic factors or cellular interactions operating in vitro.