The goal of this project is to generate monoclonal antibodies to different cytoskeletal proteins and use these antibodies as tissue-specific reagents in diagnostic surgical pathology. The cytoskeleton (particularly the intermediate filaments) of different cells is composed of different proteins, and antibodies capable of recognizing specific proteins are therefore useful as tissue-specific reagents. We intend to generate a panel of such hybridomas capable of recognizing different cells and use them to accurately determine the cellular nature of poorly differentiated neoplasms. Cytoskeleton from different normal and malignant human cells such as lymphocytes, lymphomas, melanomas, different carcinomas, different sarcomas, and neural tumors will be used as immunogens. Antibodies will be selected for their ability to react specifically with one cell type and not with others. Antigens recognized by the different antibodies will be determined by Western blot experiments. The different antibodies will be used to study the cell biology of the different cytoskeletal proteins. We will localize the different intermediate filament proteins in different cells by immunoelectron microscopy, asking if the different proteins are found in similar filaments or if they polymerize into homogeneous filaments. The protein composition of tonofilament bundles and desmosomes will also be investigated. Attempts at sequencing fragments of the different intermediate filament proteins will be undertaken by fragmenting the different proteins with proteases or cyanogen bromide and isolating fragments that retain the ability to react with the different antibodies. The amino acid sequence of these fragments will be determined. These studies should identify subtle differences in intermediate filament proteins, which then can be used to generate additional antibodies with greater specificity. (4)
