人肝癌组织中DSA强结合的γ-谷氨酰基转移酶单克隆抗体的制备

Preparation of monoclonal antibody against gamma-glutamyltransferase strongly bound to Datura stramonium (DSA) lectin in primary hepatocellular carcinoma tissue

目的制备与欧蔓陀罗凝集素(DSA)强结合的γ-谷氨酰基转移酶(γGT)的单克隆抗体(mAb),并对抗体进行鉴定。方法以纯化的人肝癌组织DSA强结合的γGT为抗原,免疫Balb/c小鼠,取小鼠脾细胞与小鼠骨髓瘤(SP2/0)细胞融合,经ELISA筛选和有限稀释法获得稳定分泌mAb的细胞株。结果获得5株分泌抗DSA强结合γGT的mAb细胞株,其中3株分泌的mAb具有较高的特异性;ELISA法测定小鼠腹水mAb的效价≥625000;亚类鉴定mAb为小鼠IgG1。结论成功制备了抗DSA强结合γGT的单克隆抗体,为进一步建立免疫学检测方法奠定了基础。

Objective To prepare a monoclonal antibody （mAb） against gamma-glutamyltransferase （GGT） strongly bound to Datura stramonium （DSA） lectin in primary hepatocellular carcinoma tissue, and to identify the properties of the monoclonal antibody. Methods The fraction of gamma-glutamyltransferase strongly bound to DSA was purified from human hepatocellular carcinoma, and then was used to immunize BALB/c mice. The mouse splenic cells were then fused with mouse myeloma cells. Stable cell clones were screened by enzyme-linked immunosorbent assay （ELISA） and limiting dilution. The immunoglobulin subtype of the mAb was identified by specific reagents. The specificity and bioaffinity of the mAb were identified by ELISA. Results Five cell lines producing monoclonal antibody which can specifically recognize gamma-glutamyltransferase strongly bound to DSA were acquired. The specificity of mAb was relatively high. The titre of the mAbs from hybridoma cell line in ascites was higher than 1 ： 625000 in ELISA test. The immunoglobulin subtype of mAb was identified as mouse IgG1. Conclusion The bybridoma cell lines producing mAb against gamma-glutamyltransferase strong binding to DSA were successfully prepared, and on this basis the corresponding immunoassay will be established.