Immunocytochemical detection of the growth‐associated protein B‐50 by newly characterized monoclonal antibodies in human brain and muscle

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Abstract
The growth‐associated protein B‐50 also termed GAP‐43, F1, pp46, P‐57 and neuromodulin is a nervous tissuespecific protein kinase C (PKC) substrate that is considered to play a major role in neurite formation, regeneration, and neuroplasticity. We describe the isolation of seven mouse monoclonal antibodies (Mabs) directed against B‐50. The Mabs are produced against the bovine B‐50, selected by ELISA for cross‐reactivity with its human counterpart, and evaluated on Western blots in comparison with the well‐characterized affinity‐purified rabbit polyclonal antibodies to rat‐B‐50. The Western blots show that the Mabs NM1, NM4, and NM6 recognize specifically the B‐50 of bovine, human, and rat brain extract and the purified PKC phosphorylated and unphosphorylated rat B‐50 isoforms. The Mabs NM2 and NM3 cross‐react with bovine B‐50 immunoreactive c‐kinase substrate (BICKS), a protein sharing a 17 amino acid sequence homology with B‐50. Two Mabs are useful for the detection of B‐50 immunoreactivity in formalin‐fixed human and rat brain tissues. In human specimen of the hippocampus, a characteristic neuropil distribution of B‐50 is detected by the Mabs. In human muscle, Mabs reveal B‐50 in nerve bundles and in axons at motor end plates. Thus, these Mabs are useful in investigating the function and localization of the B‐50 protein.