Evidence that the monoclonal antibodies SMI-31 and SMI-34 recognize different phosphorylation-dependent epitopes of the murine high molecular mass neurofilament subunit

The monoclonal antibodies SMI-31 and SMI-34 react with phosphate-dependent epitopes of the high molecular mass (200 kDa) neurofilament protein (Hphos). Determination of whether or not these monoclonals react with different epitopes would assist in interpretation of post mortem immunocytochemical analyses in neurodegenerative disorders and in normal aging. We therefore examined the relative immunoreactivity of these antibodies against Triton-insoluble (cytoskeleton-associated) and Triton-soluble Hphos variants in NB2a/d1 neuroblastoma and post-natal mouse brain in immunoblot analysis. Densitometric analysis yielded a ‘reactivity ratio’ (soluble Hphos/insoluble Hphos) for each antibody. This ratio was approximately 44% and 87% less for SMI-34 than for SMI-31 in neuroblastoma and brain, respectively. These findings confirm that the SMI-34 epitope is distinct from that recognized by SMI-31, and, in these systems, is preferentially associated with the cytoskeleton.