David S. Secher

Abstract We have previously described the derivation of M1/70, a hybrid myeloma line secreting monoclonal rat anti‐mouse cell surface antibody (Springer, T., Galfre, G., Secher, D. S. and Milstein, C, Eur. J. Immunol. 1978. 8 : 539). We have now investigated the cellular distribution of this antigen using a 125 I‐labeled anti‐rat IgG indirect binding assay, the fluorescence‐activated cell sorter, autoradiography and precipitation of cell surface molecules. Screening with a tumor cell panel showed strong reactivity with a macrophage‐like line but no reactivity with B or T lymphoma lines. In normal tissues, M1/70 antigen was found to be present in small amounts on spleen and exudate granulocytes and a subpopulation of bone marrow cells, in moderate amounts on spleen and blood monocytes and expressed in much larger amounts on spleen histiocytes and peritoneal exudate macrophages. In contrast, M1/70 antigen was found to be absent from erythroid and lymphoid cells. M1/70 antibody precipitated two polypeptides of 190 000 and 105 000 mol. wt. which were present in much greater amounts on peritoneal exudate macrophages than on spleen cells. The expression on phagocytes of two other antigens identified by monoclonal antibodies M1/69 and M1/9.3 was also examined. Monocytes and granulocytes expressed large amounts of M1/69 and low amounts of M1/70 antigen, while in peritoneal exudate macrophages this pattern was dramatically reversed. M1/70 thus defines a differentiation antigen on mononuclear phagocytes and granulocytes, the expression of which is specifically increased during monocyte maturation. This antibody is the first to be described which recognizes a discrete molecule specific to phagocytes.

Abstract Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125 I‐labeled anti‐rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125 I‐labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross‐inhibition of binding of different monoclonal antibodies. It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat‐stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125 I‐labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105000. Five IgG‐secreting clones identify the fourth antigen, a heat‐stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross‐inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.