Identification of AML-specific B-cell clones. (A) Subcloning of miniculture 2K23 yielded an AML-specific clone, producing the antibody AT1413 that binds to AML cell lines (French-American-British classification M0-M5). In all experiments, the recombinant antibody was used. (B) AT1413 also bound to a subset of nonmalignant hematopoietic progenitor cells and to peripheral blood-derived nonmalignant monocytes and granulocytes. AT1413 did not bind to blood-, tonsil-, or thymus-derived mature and immature lymphoid cells, nor did it bind to the tissue cell lines HepG2 (liver), Huh7 (liver), H69 (cholangiocytes), Caco2 (colon), or BJ (foreskin fibroblasts), and primary cultured fibroblasts (normal human adult dermal fibroblasts). (C) Immunohistochemistry of AT1413-biotin with streptavidin-HRP as a secondary antibody confirmed binding to mononuclear cells in tonsil and demonstrated binding to endothelial cells in blood vessels and a punctuate staining pattern of macrophage-type cells in the liver. Biotin immunoreactivity of antibody shown with streptavidin-HRP and the peroxidase substrate diaminobenzidine (DAB). Scale bars, 50 μm. (D) Comparison of AT1413 staining to THP-1 cells (black triangles), granulocytes (white circles), and endothelial cells with FACS analysis. HUVEC, white diamonds; HAEC, white squares; BOEC, blood outgrowth endothelial cells, white triangles. The in-house generated influenza-specific antibody AT1002 was used as a negative control in A-C.