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

众所周知，肿瘤细胞中低聚糖的结构存在于糖蛋白中，细胞膜经常发生突变。结果发现，肝癌患者血清糖蛋白α-FetoProtein中寡糖的结构发生了变化。因此，我们打算建立一种简单的方法来制备针对诱变糖蛋白寡糖的单抗。本研究利用微生物内切糖苷酶(Endo-β-N-乙酰氨基葡萄糖苷酶)的转糖基化活性，合成了以糖蛋白寡糖为免疫原的新型糖偶联物(烷基糖苷)，并制备了抗天冬酰胺连接的双烯复合型低聚糖的单抗。结果：1.从蛋黄中制备了大量含有双烯复合型低聚糖的糖肽，作为木糖的供体，与烷基葡萄糖苷结合。我们合成了烷基糖苷作为天冬酰胺连接的双烯复合型…的免疫原更多的寡糖利用毛霉内切糖苷酶(Endo-M)的糖基化活性。将蛋黄糖肽和正辛基-β-D-葡萄糖苷与Endo-M(p H6.0)在37℃下孵育12小时，用高效液相色谱法分离转糖基化产物。产物经质谱学鉴定为烷基糖苷，具有复合型低聚糖。我们用烷基糖苷免疫小鼠。免疫原经小鼠腹膜腔内注射4次。最后一次免疫后，取脾细胞与骨髓瘤细胞混合，进行融合。3周后，杂交瘤细胞生长，并用人血清转铁蛋白糖肽和蛋黄糖肽进行了杂交瘤细胞株的筛选，杂交瘤细胞产生抗体。结果获得了5株产生抗体的杂交瘤细胞株。利用这些杂交瘤细胞，采用有限稀释法进行克隆。然后，我们获得了三个杂交瘤细胞克隆，这些杂交瘤细胞产生了抗双烯复合型寡糖的抗体。我们还按照上述相同的方法制备了杂交瘤细胞株，该杂交瘤细胞产生去唾液酸双烯复合型低聚糖。较少

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