Target identification of AML-specific antibodies: U5 snRNP200 complex. (A) Immunoblotting of THP-1 immunoprecipitates (IP) with AT1223, AT1331, and AT1002 with rabbit snRNP200–, PRPF8–, and EFTUD2– and mouse GAPDH–specific antibodies. (B) Jurkat and THP-1 cells were stained with a commercially available rabbit anti-PRPF8 (Abcam), -snRNP200 (Bethyl Labs), and -EFTUD2 (Abcam) antibodies on the membrane (left panels) and intracellularly (right panels). Polyclonal rabbit antihepatitis core antigen (HBcAg) was used as negative control (gray-filled histograms). (A-B) Depicted is 1 representative experiment of at least 3 independent experiments (Table 2). (C) Seven AML-specific antibodies recognize the U5 snRNP200 complex, as tested in ELISA. Depicted are means of 3 independent experiments and standard deviations. Flag-tagged hepatitis C–specific protein (H77/E1/E2-flag) was used as a negative control. SnRNP200, commercially available rabbit anti-snRNP200; snRNP200-flag, flag-tagged snRNP200 lysate; AT1002, influenza-specific antibody. *P < .003. (D) Screening of 20 cells per well cultures of 5 allogeneic HSCT recipients with lasting anti-AML responses (AML), 3 patients with multiple myeloma (MM) in long-time remission after allogeneic HSCT, and 5 healthy individuals for antibodies binding to snRNP200 (ELISA). In total, 23.040 B cells per patient were screened. NS, not significant. (E) Immunoblotting of THP-1 (human AML) and WEHI-3B (mouse AML) immunoprecipitates with AT1331 and AT1002 with rabbit snRNP200– and mouse GAPDH–specific antibodies. Specific U5 snRNP200 complex stain is seen for both human and mouse AML cells. (Right panel) Binding of AT1331 to WEHI-3B cells.