The 14-3-3 protein as the antigen for lung cancer-associated human monoclonal antibody AE6F4

Human monoclonal antibody (MAb) AE6F4, which had been shown potentially useful for the immunocytological detection of lung cancer cells in sputum, was characterized for its antigen(s). Of the three MAb-reacting materials found in A549 cells by the immunoblotting analysis, the cytoplasmic 31-kDa protein extractable with phosphate-buffered saline was evidenced as the most plausible antigen by its highest content and outstanding affinity to the MAb AE6F4-derivatized Sepharose 4B column. This 31-kDa protein was identified by the amino acid sequence analysis of the CNBr-cleaved fragment as the 14-3-3 family of proteins, the members of which are known to play important physiological roles such as in the regulation of neurotransmitter levels and intracellular signal transduction. The purified 14-3-3 protein from bovine brain showed a comparable MAb-reacting activity to that of the 31-kDa protein from A549 cells in the enzyme-linked immunosorbent assay (ELISA). The significant reactivity of bovine 14-3-3 protein by MAb AE6F4, shown by the cross inhibition of antibody binding to the coated 31-kDa antigen in ELISA as well as by the inhibition of immunostaining with lung cancer tissues, consistently demonstrated that the antigen(s) recognized by the MAb was involved in the 14-3-3 protein family. It was found that the expression of the 14-3-3 protein was significantly enhanced in lung cancer tissues compared with the neighboring normal part of the lung as examined by the immunoblotting method. These results implicated that some member(s) of the 14-3-3 protein family can be the tumor marker(s), providing a rational basis for the immunocytological diagnosis of lung cancer with this human MAb.