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Among various solid tumours, gastric cancer (GC) is one of the leading causes of cancer‐related deaths worldwide. Expansion into the peritoneal cavity, which results from dissemination of diffuse cancer cells, is the main cause of mortality in gastric adenocarcinoma patients. Therefore, investigation of putative biomarkers involved in metastasis is prerequisite for GC management. In an effort to discover potential tumour markers associated with peritoneal metastasis of GC, a semi‐synthetic human scFv library (Tomlinson I) was used to isolate novel antibody fragments recognizing MKN‐45, a poorly differentiated diffuse gastric adenocarcinoma cell line. Four rounds of subtractive selection each consisting of extensive pre‐absorption of phage library with NIH‐3T3 murine embryonic fibroblasts and AGS (a well‐differentiated intestinal gastric adenocarcinoma) cell line were carried out prior to positive selection on MKN …