Monoclonal antibodies to the heavy neurofilament subunit (NF-H) of Torpedo cholinergic neurons

Abstract

Previous studies from our laboratory suggest that Alzheimer's disease sera contain a repertoire of antibodies to the heavy neurofilament subunit (NF-H) and that a subpopulation of these antibodies bind specifically to epitopes highly enriched in NF-H isolated from the purely cholinergic electromotor neurons of Torpedo. In the present study, we prepared and characterized monoclonal antibodies (MAbs) that bind to epitopes specifically enriched in Torpedo cholinergic neurons. This was performed by a differential enzyme-linked immunosorbent assay (ELISA) in which MAbs were selected that bind to epitopes much more abundant in the NF-H protein of Torpedo cholinergic neurons than in NF-H from the chemically heterogeneous Torpedo spinal cord. This yielded four MAbs, three of which (TC4, TC8, and TC21) were found to be specific to NF-H and one (TC15) that reacts with both NF-H and the medium-size neurofilament subunit NF-M. Dephosphorylation abolishes the binding of MAbs TC4 and TC15 to Torpedo cholinergic NF-H, partially reduces that of MAb TC21 and has no effect on the binding of MAb TC8. This suggests that the antigenic sites specific to Torpedo cholinergic NF-H contain phosphorylated as well as non phosphorylated epitopes. All the MAbs cross-react with rat brain NF-H.