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Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.

By using four distinct monoclonal antibodies to CEA, the molecular profile of which was clarified in our accompanying companion paper, immunohistochemical distribution of the antigenic determinants on both cancerous and noncancerous tissues as well as fetal tissues was studied with the use of the immunoperoxidase method. All of the monoclonal antibodies recognize different antigenic determinants on the tissue section. None of the antibodies stained granulocytes in the peripheral blood or in the normal liver tissues tested. Three of our monoclonal antibodies stained columnar epithelial cells in morphologically normal colonic mucosa; however, monoclonal antibody YK024 did not stain them. This antibody was also found to be unreactive with intestinal metaplasia lesions of the stomach, but reacted with a 16-wk-old fetal stomach as well as with cancerous parts of the colon and of the stomach. Moreover, it was found that this monoclonal antibody mainly reacted with moderately or poorly differentiated adenocarcinoma lesions of the colon and the stomach. Periodic acid treatment in this study, together with trypsin treatment on the antigen as described in our accompanying companion paper, may suggest that this antibody recognizes the carbohydrate antigenic determinant in nature.

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The serologic and molecular characterization of four monoclonal antibodies to human gastric cancer cells and to CEA were described and the immunohistologic patterns of reactions with freshly obtained gastrointestinal tissues were examined. Monoclonal antibody YK004, which was secreted by the hybridoma constructed with the gastric carcinoma cell line KATO III, reacted with an antigenic determinant preferentially expressed on human gastric and colonic carcinoma cells, but it failed to bind any nonmalignant tissues tested except the stomach. Although this antibody reacted with fetal digestive tract tissues, it failed to immunoprecipitate CEA. Monoclonal antibodies YK013 and YK024 prepared in the same manner by immunizing with the gastric carcinoma cell line KATO III reacted with antigenic determinants on CEA, but not on NCA or NCA2. The antigenic profiles recognized by these two antibodies were found to be identical by sequential immunoprecipitation experiments. However, the antigenic determinant recognized by antibody YK013 may be different from that recognized by antibody YK024, since the immunohistologic reactivity patterns in carcinoma tissue sections were clearly different. Monoclonal antibody AS001, which was secreted by the hybridomas constructed with purified CEA as immunogen, immunoprecipitated CEA molecule with a single antigenic structure of 200 Kd, which was different from the antigenic profile detected by two other monoclonal antibodies, YK013 and YK024. The unique distribution of the antigenic determinants recognized by our monoclonal antibodies on cancerous tissues suggests that antibodies might be able to detect the unique determinants of the circulating CEA molecule or CEA-related molecules in the serum of cancer patients.