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A systematic approach for the determination of epitope specificities of
monoclonal antibodies to a complex antigen system is described. After initial
screening to identify antigen-binding monoclonal antibodies, one or more of the
clones are isolated by limiting dilution cloning, grown in ascites, and the
resulting antibodies secreted into the ascitic fluid are affinity purified on
Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from
other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice
were immunized with either purified carcinoembryonic antigen (CEA) or the
CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma
cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant
binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned,
resulting in 33 different clones. The antibodies produced by the different
cloned hybrids and the remaining uncloned hybrids recognized a total of five
different epitopes on CEA. All of the epitopes reside on the protein moiety of
the molecule as determined by antibody binding to deglycosylated CEA. The
monoclonal antibodies with five different epitope specificities were reacted
with tissue sections of normal and cancerous tissues and with peripheral blood
smears. Each of the five monoclonal antibodies reacted with tissue sections from
colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic
polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted
with normal liver tissue. Granulocytes in peripheral blood smears bound three
antibodies strongly and one antibody weakly, and one antibody was not bound. One
monoclonal antibody that reacted with normal liver tissue was not bound by
granulocytes. The ability of these five monoclonal antibodies to differentially
detect three different CEA-related antigens in normal and malignant tissues may
have clinical utility.
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