Evidence of vimentin/cardiolipin binding. (A) Vimentin/cardiolipin complexes were resuspended in a buffer containing 20mM Tris-HCl, pH 7.5; 0.15M NaCl; 1mM EDTA; 0.02% NaN₃; and 10mM NaF. The mixtures were immunoprecipitated with polyclonal antivimentin. The immunoprecipitates were subjected to phospholipid extraction and analyzed by monodimensional HPTLC analysis and then stained by exposure to iodide vapors. As a negative control, immunoprecipitation was performed with an irrelevant goat IgG. (B) Vimentin/cardiolipin complexes were resuspended in a buffer containing 20mM Tris-HCl, pH 7.5; 0.15M NaCl; 1mM EDTA; 0.02% NaN₃; and 10mM NaF. The unextracted precipitates were separated by SDS-PAGE and probed with the antivimentin Ab. As a negative control, immunoprecipitation was performed with an irrelevant goat IgG. (C) Scalar doses of vimentin in 0.05μM NaHCO₃ buffer, pH 9.5, from 10 μg/mL to 1.25 μg/mL, were incubated with 50 μg/mL of cardiolipin in methanol. After washes with PBS-T and blocking in PBS containing 3% BSA, plates were incubated with goat polyclonal antibodies against vimentin. (D) Sera of SN-APS patients with detectable levels of antivimentin/cardiolipin antibodies (diluted 1:100, 1:200, 1:400, 1:800, 1:1600) were tested by the use of ELISA with vimentin/cardiolipin complex. As a control of binding specificity sera were previously adsorbed with vimentin/cardiolipin (incubation, 2:1 vol/vol, with 3 mg/mL cardiolipin micelles containing 3 μg/mL vimentin for 1 hour at 37°C and then overnight at 4°C. The mixture was centrifuged at 27 000g for 15 minutes at 4°C). The supernatants were kept as adsorbed sera and tested by ELISA.