Monoclonal antibodies as probes for exposed determinants on desmin intermediate filaments

The intermediate filaments are a group of cytoskeletal filaments the structural analysis of which is under active investigation in many laboratories (Steinert et al., 1985). The use of antibodies to probe the surfaces of organelles and protein filaments promises to provide important information about the molecular assembly of these structures (Pool et al., 1983). In the current study, monoclonal antibodies to chicken gizzard desmin have been used to decorate synthetic intermediate filaments, and the effectiveness of the use of gold-labelled secondary antibodies (Probert et al., 198 1; De Mey, 19x3) for visualization of the primary antibody has been evaluated. Monoclonal antibodies were generated and screened against purified desmin by standard techniques (Kohler & Milstein, 1075). Four anti-desmin monoclonal antibodies were obtained and isotyped as IgG1 kappa (IA10, 187 and 1A3) and IgM kappa (3C6). Desmin fragments were prepared by chemical and enzymic cleavage of the intact molecule and their cross-reactivities towards each of the monoclonal antibodies were determined after initial electrophoretic separation of the fragments, followed by electrotransfer to nitrocellulose or polyvinylidene difluoride (Millipore) membranes (Towbin et al., 1979). Binding of antibody to the desmin fragments was detected by the use of peroxidase-labelled goat anti-mouse antibody and the use of 4-chloro1 -naphthol as substrate for the peroxidase (Hawkes et al., 1982 j. For immuno electron microscopy. synthctic dcsmin filaments were made by the procedure of Rueger et ul. (1981) and the antibodies to be used were concentrated in the same filament buffer. Desmin filaments were applied to Formvarcoated 300-mesh copper grids, and then subsequently treated with primary antibody by flotation of the grid on a droplet of antibody solution. An alternative procedure involved interaction of filament with antibodies before application to the grid to maximize binding of antibody to filament. In studies using goat anti-mouse secondary antibodies which contained 5 or 20 nm diameter gold particles, it was found that saturation of all the pre-attached primary antibody did not occur. Furthermore, in the case of the antibody containing 20 nm gold particles, significant levels of non-specific binding of the secondary antibody t o the desmin filaments was found to occur in the absence of the primary antibody. These results therefore indicate that the goldlabelled secondary antibody technique is not useful in studies of isolated desmin filaments, despite its utility in immunolocalization studies in sectioned tissues (DoerrSchott & Lichte, 1986). Studies in which the binding of primary antibody t o the desmin filament has been visualized without the use of a labelled secondary antibody have also been performed (Fig.1). Antibodies 1AlO and 1A3 decorated the filaments with a periodicity of 54 nm. These antibodies have been shown to recognize an epitope in the C-terminal third of the desrnin molecule (residues 325-463). Antibody 1B7, which recognizes an epitope within the large NTCB fragment (residues 1-324) which consists of the N-terminal globular region and the central coil region, also decorated the filaments with a periodicity of 54 nm. Antibody 3C6, which