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Preparation of Monoclonal Antibody against Mutagenic Sugar Chain Which Specifically Occurs in Hepatoma Cells and Its Evaluation for Diagnostics

肝癌细胞特异糖链突变单克隆抗体的制备及其诊断评价

基本信息

批准号：
10556079
负责人：
KUMAGAI Hidehiko
金额：
$ 7.55万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

It is known that the structure of oligosaccharide existed in glycoproteins and cell membrane frequently mutate in cancer cells. It was found that the structure of oligosaccharide in glycoprotein α-fetoprotein from serum was varied in hepatoma patients. Therefore, we intended to develop a simple method to prepare the monoclonal antibody specific for the mutagenic oligosaccharide of glycoprotein. In this study, we synthesized the novel glycoconjugate (alkylglycoside) having oligosaccharide of glycoprotein as immunogen using transglycosylation activity of microbial endoglycosidase (endo-β-N-acetylglucosaminidase), and prepared the monoclonal antibody against asparagine-linked bianntenary complex-type of oligosaccharide.1. We prepared a large amount of glycopeptide containing bianntenary complex-type of oligosaccharide from hen egg yolk as the donor of ligosaccharide for binding to alkylgucoside.2. We synthesized alkylglycoside as the immunogen of asparagine-linked bianntenary complex-type … More of oligosaccharide using transglycosylation activity of Mucor endoglycosidase (Endo-M). We incubated egg yolk glycopeptide and n-octyl-β-D-glucoside with Endo-M, pH6.0, at 37℃ for 12hr, and isolated the transglycosylation product by HPLC. The product was confirmed as the alkylglycoside having complex-type of oligosaccharide by mass spectrometry.3. We immunized mice by the alkylglycoside. The immunogen was injected to mice intraperitonealy four times. After last booster, spleen cells were harvested and mixed with myeloma cells, and fused. Three weeks later, hybridomas grew and the screening of hybridomas producing antibody against oligosaccharide was carried out by ELISA method using human serum tranferrin glycopeptide and egg yolk glycopetide, both of which contained bianntenary complex-type of oligosaccharide. As the result, five hybridoma cell lines were obtained as the producer of antibody. Using these hybridomas, cloning was carried out by limiting dilution method. Then, we obtained three hybridoma clones which produced antibody against bianntenary complex-type of oligosaccharide.4. We also prepared the hybridoma producing asialo-bianntenary complex-type of oligo-saccharide according to the same method described above. Less

已知癌细胞中糖蛋白和细胞膜上的寡糖结构经常发生突变。发现肝癌患者血清甲胎蛋白糖蛋白中的寡糖结构存在差异。因此，我们拟建立一种简便的方法制备特异性抗糖蛋白致突变寡糖的单克隆抗体。本研究利用微生物内切糖苷酶（endo-β-N-acetylglucosaminidase）的转糖基化活性，合成了以糖蛋白寡糖为免疫原的新型糖缀合物（烷基糖苷），并制备了抗天冬酰胺连接的二元复合型寡糖的单克隆抗体.从鸡蛋黄中制备了大量的含双元复合型寡糖的糖肽，作为寡糖的供体，与烷基葡糖苷结合.本文合成了天冬酰胺联双元复合物型免疫原烷基糖苷 ...更多信息利用毛霉内切糖苷酶（Endo-M）的转糖基化活性对低聚糖进行纯化。将蛋黄糖肽和正辛基-β-D-葡萄糖苷与Endo-M（pH6.0）在37℃孵育12小时，用HPLC分离转糖基化产物。产物经质谱鉴定为具有复合型寡糖的烷基糖苷.用烷基糖苷免疫小鼠。免疫原经腹腔注射小鼠4次。最后一次加强后，收获脾细胞并与骨髓瘤细胞混合，并融合。3周后，杂交瘤生长，并使用人血清转铁蛋白糖肽和蛋黄糖肽（两者均含有二元复合型寡糖）通过ELISA方法筛选产生抗寡糖抗体的杂交瘤。结果，获得了5株杂交瘤细胞系作为抗体生产者。利用这些杂交瘤，通过有限稀释法进行克隆。获得了3株抗双元复合型寡糖抗体的杂交瘤细胞株.我们还按照上述相同的方法制备了产生脱唾液酸双元复合型低聚糖的杂交瘤。少