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Tailoring subtractive cell biopanning to identify diffuse gastric adenocarcinoma-associated antigens via human scFv antibodies.
Tailoring subtractive cell biopanning to identify diffuse gastric adenocarcinoma-associated antigens via human scFv antibodies.
- Source :
-
Immunology [Immunology] 2020 Jan; Vol. 159 (1), pp. 96-108. Date of Electronic Publication: 2019 Nov 08. - Publication Year :
- 2020
-
Abstract
- 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-45 target cells. ELISA-based screening of 192 phage-displayed scFv clones indicated 21 high-affinity binders with specific staining of MKN-45 compared with AGS cells. Diversity analysis of the selected phage-scFvs resulted in five distinct sequences with multiple frequency. Further analysis by ELISA and flow cytometry verified three clones that specifically recognized MKN-45 cells. Liquid chromatography-mass spectrometry analysis of the scFv-immunoprecipitated proteins has led to identification of c-Met, HSP90 α and HSP90 β as candidate biomarkers associated with diffuse GC. Immunohistochemistry revealed the capability of purified scFvs to differentiate diffuse and intestinal gastric adenocarcinoma. Taken together, the isolated MKN-45-specific scFv fragments and their cognate antigens would be beneficial in screening and management as well as targeting and therapy of the diffuse gastric adenocarcinoma.<br /> (© 2019 John Wiley & Sons Ltd.)
- Subjects :
- Adenocarcinoma pathology
Animals
Cell Line, Tumor
Chromatography, High Pressure Liquid
Diagnosis, Differential
Humans
Immunohistochemistry
Mice
NIH 3T3 Cells
Predictive Value of Tests
Single-Chain Antibodies genetics
Stomach Neoplasms pathology
Tandem Mass Spectrometry
Adenocarcinoma immunology
Biomarkers, Tumor analysis
Bioprospecting methods
Cell Surface Display Techniques
HSP90 Heat-Shock Proteins analysis
Proto-Oncogene Proteins c-met analysis
Single-Chain Antibodies immunology
Stomach Neoplasms immunology
Subjects
Details
- Language :
- English
- ISSN :
- 1365-2567
- Volume :
- 159
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Immunology
- Publication Type :
- Academic Journal
- Accession number :
- 31596953
- Full Text :
- https://doi.org/10.1111/imm.13129