The long term objectives of this research are to understand the structural features of myotoxins that are required for generation of the cellular and biochemical responses that they produce and to generate a series of monoclonal antibodies that will neutralize the toxin actions by binding to selected antigenic determinants on the toxin. An important step in this study will be to develop a tissue culture system that can be used as a tool for assay of myotoxin and chemically modified forms of the toxin. This will obviate much of the need for intact animals and histological preparation of tissues that is common in studies of myotoxin. Chemical modification of several amino acids, each occurring only once in the toxin sequence, will produce well defined modified toxins that will be tested for myotixic activity. The tissue culture system will be used to test the hypothesis that myotoxins stimulate endogenous membrane phospholipase activities which in turn leads to an alteration of membrane structure and function. Radiolabelled toxin will be used to assess toxin-cell membrane binding interactions. Hybridoma technology will be used to fuse spleen cells of myotoxin immunized mice with NS-1 myeloma cells. The resultant hybridoma cell cultures will be screened by ELISA to select cell lines for cloning. Cloned cell lines will be used to produce monoclonal antibodies which will be tested for ability to neutralize the effects of myotoxin on the cell system. The monoclonal antibodies will also be used to explore the number and locations of antigenic sites in the myotoxin sequence.
