Monoclonal antibodies to epitopes on different regions of the 200 000 dalton neurofilament protein. Probes for the geometry of the filament.
Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.
Liem
R K
RK
Chin
S S
SS
Moraru
E
E
Wang
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002531:Cerebellum / Q000648:ultrastructure; D002918:Chymotrypsin; D003102:Colloids; D003599:Cytoskeleton / Q000032:analysis* / Q000648:ultrastructure; D000939:Epitopes / Q000276:immunology; D006046:Gold; D007118:Immunoassay; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000276:immunology*; D008970:Molecular Weight; D016900:Neurofilament Proteins; D051381:Rats
D000911:Antibodies, Monoclonal; D003102:Colloids; D000939:Epitopes; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D006046:Gold; D002918:Chymotrypsin