Cytokeratin 8 and 19 as antigens recognized by adenocarcinoma-reactive human monoclonal antibody AE6F4

Article type: Research Article

Authors: Ichikawa, Akira^{a; *} | Tachibana, Hirofumi^b | Kawamoto, Seiji^a | Kamei, Masanori^c | Honjoh, Tsutomu^c | Hashizume, Shuichi^c | Shirahata, Sanetaka^a

Affiliations: [a] Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka 812, Japan | [b] Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan | [c] Morinaga Institute of Biological Science, Yokohama 230, Japan

Correspondence: [*] Correspondence and reprint requests to: Akira Ichikawa, Cellular Regulation Technology Laboratory, Graduate School of Genetic Resources Technology, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan.

Abstract: The human monoclonal antibody (MAb) AE6F4 is secreted by a human-human hybridoma line established from the in vitro immunization of normal human peripheral blood lymphocytes with the human lung adenocarcinoma cell line, A549. This MAb is strongly reactive to lung cancer tissues. In the previous study, the antigens recognized by the MAb AE6F4 were purified from A549 cells and identified as 14-3-3 protein and 31 kDa cytosolic phospholipase A2 (cPLA2). The MAb AE6F4 also binds two kinds of antigens (53 kDa and 40 kDa), which are not related to 14-3-3 protein or 31 kDa cPLA2, in the human breast adenocarcinoma cell line, MCF-7. We purified a 38 kDa antigen, which is a degradation product of 53 kDa antigen from breast adenocarcinoma MCF-7 cells using ion-exchange and hydroxyapatite column chromatography. Two partial amino acid sequences of the purified 38 kDa antigen showed 95–100% homology to human cytokeratin 8 (CK8). Two-dimensional gel electrophoresis and immunoblot analysis of intermediate filament fraction separated from MCF-7 cells demonstrated that the 53 kDa and 40 kDa antigens were CK8 and CK19, respectively. Antigenic determinants on CK8 and CK19 recognized by the MAb AE6F4 were resistant to sodium periodate treatment, although antigenic determinant on 31 kDa antigen (14-3-3 protein and(or) cPLA2) was, sensitive to this treatment. These results suggest that the MAb AE6F4 reacts with both carbohydrate and peptide antigenic determinants.

Keywords: Human monoclonal antibody, adenocarcinoma, cytokeratin 8, cytokeratin 19, cytosolic phospholipase A₂, 14-3-3 protein

DOI: 10.3233/HAB-1997-8405

Journal: Human Antibodies, vol. 8, no. 4, pp. 195-202, 1997