­­­Successful Application of Whole Cell Panning for Isolation of PhageAntibody Fragments Specific to Differentiated Gastric Cancer Cells

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Abstract
Purpose: Generation of antibodies which potentially discriminate between malignant andhealthy cells is an important prerequisite for early diagnosis and treatment of gastric cancer(GC). Comparative analysis of cell surface protein landscape will provide an experimental basisfor biomarker discovery, which is essential for targeted molecular therapies. This study aimedto isolate phage-displayed antibody fragments recognizing cell surface proteins, which weredifferentially expressed between two closely related GC cell lines, namely AGS and MKN-45.Methods: We selected and screened a semisynthetic phage-scFv library on AGS, MKN-45, andNIH-3T3 cell lines by utilizing a tailored selection scheme that was designed to isolate phagescFvsthat not only recognize the differentiated AGS cells but also distinguish them from NIH-3T3 fibroblasts and the poorly differentiated MKN-45 cells.Results: After four rounds of subtractive whole cell panning, 14 unique clones were identifiedby ELISA screening and nucleotide sequencing. For further characterization, we focused on fourphage-scFvs with strong signals in screening, and their specificity was confirmed by cell-basedELISA. Furthermore, the selected phage-scFvs were able to specifically stain AGS cells with38.74% (H1), 11.04% (D11), 76.93% (G11), and 69.03% (D1) in flow cytometry analysis whichsupported the ability of theses phage scFvs in distinguishing AGS from MKN-45 and NIH-3T3cells.Conclusion: Combined with other proteomic techniques, these phage-scFvs can be applied tomembrane proteome analysis and, subsequently, identification of novel tumor-related antigensmediating proliferation and differentiation of cells. Furthermore, such antibody fragments canbe exploited for diagnostic purposes as well as targeted drug delivery of GC.

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