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首页> 外文期刊>Immunology: An Official Journal of the British Society for Immunology >Tailoring subtractive cell biopanning to identify diffuse gastric adenocarcinoma‐associated antigens via human scFv antibodies
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Tailoring subtractive cell biopanning to identify diffuse gastric adenocarcinoma‐associated antigens via human scFv antibodies

机译：纵向降解细胞生物丙酮通过人SCFV抗体鉴定弥漫性胃腺癌相关的抗原

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Summary 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.
机译：发明内容在各种实体肿瘤中，胃癌（GC）是全球癌症相关死亡的主要原因之一。膨胀进入腹膜腔，这导致弥漫性癌细胞的传播，是胃腺癌患者死亡率的主要原因。因此，涉及转移的推定生物标志物的调查是GC管理的先决条件。为了发现与GC的腹膜转移相关的潜在肿瘤标志物，使用半合成的人SCFV库（Tomlinson I）分离识别MKN-45的新型抗体片段，这是一种差异差异化的弥漫性胃腺癌细胞系。在MKN-45靶细胞上阳性选择之前，在MKN-45靶细胞上阳性选择之前，进行四轮的减法选择各自由具有NIH-3T3鼠胚胎成纤维细胞和AGS（良好分化的肠胃腺癌）细胞系进行的噬菌体文库的广泛预热。基于ELISA的筛选192个噬菌体显示的SCFV克隆表示21个高亲和力粘合剂，与AGS细胞相比，MKN-45的特异性染色。所选噬菌体-SCFV的分集分析导致五个不同频率的不同序列。 ELISA和流式细胞术的进一步分析验证了三种特异性识别MKN-45细胞的克隆。 SCFV-免疫沉淀蛋白的液相色谱 - 质谱分析导致C-Met，Hsp90α和Hsp90β作为与弥漫性GC相关的候选生物标志物。免疫组织化学揭示了纯化的SCFV来区分弥漫性和肠胃腺癌的能力。占用的孤立的MKN-45特异性SCFV片段及其同源抗原在筛选和管理方面是有益的，以及弥漫性胃腺癌的靶向和治疗。

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