Monoclonal antibody analysis of complex biological systems. Combination of cell hybridization and immunoadsorbents in a novel cascade procedure and its application to the macrophage cell surface.

To simplify the construction of monoclonal antibody (MAb) libraries to complex biological systems, the use of a cascade procedure has been investigated. The procedure continually restricts MAb production to those antigens previously so unidentified. Murine macrophage membranes were detergent-solubilized and purified by Lens culinaris lectin affinity chromatography. Prior to immunization of rats, the previously recognized heat-stable antigen and common leukocyte antigen were removed with MAb immunoadsorbents. Analysis of serum antibody concentrations to common leukocyte antigen, heat-stable antigen, and Mac-1 (a previously recognized antigen which served as a positive control for nondepleted antigens) demonstrated the efficacy of the procedure. The ratios of anti-Mac-1 to anticommon leukocyte antigen and anti-Mac-1 to anti-heatstable antigen antibodies were over 100-fold higher when purified, MAb immunoadsorbent-depleted material rather than whole peritoneal exudate cells were used for immunization. P3-NSI/l-Ag4-1 y-chain nonsecretor myeloma cells were fused with spleen cells from rats immunized to immunodepleted antigen and hybrid culture supernatants were screened for immunoprecipitation of ’‘‘1 (lactoperoxidase)-labeled material. The common leukocyte antigen was among the most immunodominant antigens on whole peritoneal exudate cells. However, immunization with immunoadsorbentdepleted antigen did not elicit any hybrid cultures precipitating the 200,000 Mr common leukocyte antigen antigen. Eighteen cultures immunoprecipitated four different types of macrophage cell surface antigens. Clones identifying previously undescribed macrophage surface polypeptides of 32,000 M, (Mac-2) and 110,000 M, (Mac-3) have been isolated.