Immunohistochemical studies with conventional antisera have been done to examine muscle fiber types and their alterations but conflicting results were obtained. We plan to investigate the properties of fiber types using monoclonal antibodies (MAb's) to myosin isozymes produced by the technique of Kohler and Milstein (1975). For this purpose antibodies to heavy and light chains of slow myosins will be produced using appropriate myosins from both avian and mammalian sources as immunogens. (Some interesting monoclonals have already been generated against the myosin of anterior latissimus dorsi muscle of the chicken). The specificities and binding affinities of the MAb's with respect to various myosin isomorphs will be compared by radioimmune assay and immunofluoresence. Location of the antigenic determinants in heavy chain (and its subfragments) and light chains will be determined by antibody binding assays and ultrastructural immunocytochemistry. MAb's against light and heavy chains of fast myosins have already been produced and are being characterized in Dr. Fischman's laboratory; they will be available for the present study. Using the 2 sets of well characterized antibodies fiber type analysis of chicken and mammalian muscles will be done on frozen sections by immunofluorescence. It is hoped that classification of fiber types and sub-types will be established on the basis of myosin isozyme distribution. Surgical denervation of chicken and rat muscles will be done at both neonatal and adult stages. The differential susceptibility to denervation of fast and slow fibers in respect to both differentiation and maintenance of their myosin isozymes will be evaluated. These studies are directly relevant to neurogenic diseases of muscle. Because of the cross-reactivity of the antibodies with human muscle (several of our MAb's give strong immunofluorescence with biopsied human samples), the monoclonals generated in this project should also be valuable in fiber typing of normal and diseased human muscles.
