PEG-precipitated ICs from serum of patients with HIV-1-ITP contain talin degradation products, and sera from HIV-1-ITP patients contained anti-talin IgG. (A) Circulating ICs from HIV-1-ITP patients and healthy HIV-1-negative controls were PEG precipitated and analyzed by SDS-PAGE and Coomassie staining or immunoblotting. Talin degradation products in platelet lysate or PEG-ICs were visualized with a murine anti-talin-H mAb, TA205, and a secondary HRP-labeled rabbit anti-mouse IgG F(ab′)2 antibody with minimal cross-reactivity to human serum proteins. No staining was detected with PEG-ICs of healthy controls or with the secondary anti-mouse IgG F(ab′)2 alone. (B) Sera, diluted 1:100 in PBS, from patients with HIV-1-ITP, alloimmune thrombocytopenia (TP), autoimmune thrombocytopenia, and healthy donors were tested for binding to purified talin by ELISA. Bound IgG was detected with an AP-conjugated F(ab′)2 goat antihuman IgG-Fc. Samples with A405 values of more than 2 SD above the mean of alloimmune thrombocytopenia patient values (> 0.7, dashed line) were considered positive. Depicted points are the mean of triplicate determinations.
