The objective of this project is to generate a library of well-characterized, antihuman skeletal myosin monoclonal antibodies to be used as sensitive and specific reagents to ascertain and characterize the diversity of such isoforms and investigate their modification under physiologic and pathologic conditions. The McAbs will be generated using human skeletal myosin light chains as immunogen to BALB/C mice. Their specificity will be characterized as to species specificity, heavy versus light chain specificity, subfragment and macromolecular location of recognized epitope, as well as to native isozyme specificity. This will be done with the use of one and two dimensional electrophoresis as well as electrophoresis of myosin under nondissociating conditions coupled with Western blotting to which McAbs will be reacted and displayed using a second radioactively labeled antibody and developed autoradiographically. Macromolecular epitope localization will be done with immuno-electron microscopy. In addition, the myosin isoform-specific McAbs generated, will be used in immuno-affinity chromatography to isolate and purify human skeletal myosin isoenzymes for use in future work aimed at correlating the heterogeneity of human myosins with functional properties and ultimately with clinical manifestations of neuromuscular disease. As these McAbs are generated and characterized, they will begin to be applied to survey the normal diversity of myosin isoforms in human skeletal muscle and also used to examine pathological specimens for abberations or abnormalities in the distribution or structure of myosin in neuromuscular disease.
