Isolation and characterization of monoclonal antibodies directed against novel components of macrophage phagosomes

In order to identify novel proteins associated with various stages of macro phage phagocytosis, we have generated monoclonal antibodies that recognize phagosomes, Purified Fc receptor-mediated phagosomes, isolated by feeding I gG-conjugated magnetic beads to LPS-primed murine peritoneal macrophages, w ere used as the immunogen, An immunofluorescence screen was used to isolate and single-cell clone similar to 150 monoclonal antibodies that recognize mouse macrophage phagosomes as well as labeling other cellular components i n patterns which are frequently distinct from those observed with previousl y characterized phagosome-associated proteins, Predominant morphologicall c ategories (in addition to phagosome labeling) include staining of one or mo re of the following: cytoskeletal patterns, vesicular patterns and plasma m embrane localization. In this paper, we describe the antibody screen, preli minary characterization of the antibodies and our identification of the ant igens for three representative monoclonal antibodies. These antibodies iden tify a plasma membrane associated receptor (Mac-1, a subunit of the complem ent receptor), an actin binding protein (coronin-2) and a vesicular protein (amphiphysin II). Some of the antibodies recognize many cell types, wherea s other antibodies are apparently macrophage specific as assessed by flow c ytometry and histology. Remarkably, several of the antibodies crossreact wi th the phagocytic slime mold, Dictyostelium discoideum, recognizing phagoso mes and other cellular elements as assessed by immunofluorescence and immun oblots. These results indicate that macrophage phagocytosis has both conser ved ancestral features and unique specialized aspects associated with the r ole of these phagocytes in immunity.