MONOCLONAL ANTI-CYTOSKELETON ANTIBODY DERIVED FROM AN ITP PATIENT

A monoclonal anti-platelet IcpI antibody was established by the fusion of splenocytes from Immune Thrombocytopenic purpura (ITP) patient with a human-mouse-hetercmyelcma cell line. The splenic lynphocytes were cultured with platelets and lipopoly saccharide for 9 days prior to fusion. Anti-platelet activity in the hybridcma supernatant was tested by the ELISA technique using plastic adherent platelets as antigen. Out of three hybridomas that demonstrated anti-platelets activity, one (4G9) appeared to be stable producer of IgM, was cloned and served for further characterizations. In immunofluorescence studies it was found that 4G9 stains both platelets and mononuclear cells, exhibiting mainly intracellular pattern. When different cell lines were fixed and stained, specific decoration of cytoskeletal elements was detected on normal and transformed fibroblasts and not in epithelial or carcinoma cell lines. To characterize the antigen reacting with 4G9 antibody, whole platelet lysate was electrophoreased on SDS-PAGE, transferred to nitrocellulose filter and reacted with 4G9 followed by peroxidase anti-Ig staining. Major band of 45 Kd corresponding to act in, and minor band of 16 Kd reacted with 4G9 but not with control antibodies. The anti-actin activity of 4G9 was confirmed by immunoblot using purified rabbit-muscle actin, and by ELISA, using act in coated plates. Whether this autoantibody plays a role in the pathogenesis of ITP, or whether it belongs to the family of natural autoantibodies is under investigation.