Identification and immunolocalization by monoclonal antibody of NSP-5, a surface polypeptide of neural cells

A monoclonal antibody, termed anti-NSP-5 (anti-Neural cell Surface Protein-5) was obtained from an hybridoma generated by fusing rat myeloma cells with splenocytes of a rat immunized with membranes from the cerebella of weaver mutant mice. This antibody reacted with the surface membrane of a subset of neurones in cultures from cerebella and dorsal root ganglia. In both culture systems, only tetanus toxin-positive cells were stained by the antibody. In sections of adult cerebellum a punctate pattern of staining was seen in the molecular layer, the Purkinje cell layer and the upper part of the granule cell layer. The white matter was strongly positive whereas granule cell and Purkinje cell bodies were clearly negative. In sections from adult dorsal root ganglia anti-NSP-5 labeled most sensory neurones including their axones in the dorsal roots. The expression of the antigen was developmentally regulated. It could not be detected in cerebellar cultures prepared from animals younger than 7 days, in good agreement with the data obtained on tissue sections. Similarly, the antigen could not be detected by immunoblotting in neonatal spinal cord, but a NSP-5-reactive band was present at postnatal day 7. The antibody bound a polypeptide of around MW 180 000 in extracts prepared from adult mouse spinal cord or cerebellum. When purified by immunoaffinity chromatography the antigen co-eluted with numerous strongly associated polypeptides. Upon subcellular fractionation most of it remained associated with a Triton-X100 insoluble fraction thus co-distributing with the cytoskeleton.