Identification of novel tumor-related antigens and autoantibodies will lead to early diagnosis of cancer and the development of more effective immunotherapies. The purpose of this study was to identify novel tumor antigens from the gastric cancer cell lines MkN-1, MkN-45 and KATOIII, and their related autoantibodies in sera of patients with gastric cancer using a proteomics-based approach. Proteins from the gastric cancer cell lines (MkN-1, MkN-45 and KATOIII) were separated by two-dimensional polyacrylamide gel electrophoresis, followed by Western blotting and antibody reaction with sera from patients with gastric cancer, healthy individuals and patients with other cancers. Positive spots were excised from Coomassie blue stained gels and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Sera from patients with gastric cancer yielded multiple spots, one of which was identified as the 78 kDa glucose-regulated protein (GRP78) by MALDI-TOF/TOF MS. Western blots against recombinant GRP78 showed reactivity in sera from 17/60 (28.3%) patients with gastric cancer and 0/20 (0.0%) of healthy individuals. Autoantibodies against GRP78 were found in 4/15 (26.7%) and 3/15 (20.0%) patients with esophageal and colon cancer, respectively. We identified for the first time an autoantibody against GRP78 in gastric cancer patients. The proteomic approach implemented in this study offers a powerful tool for identifying novel serum markers that may display clinical usefulness in cancer.

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