肾移植受者血清抗血管内皮细胞特异性抗体的提取和纯化

摘要

Objective To investigate the extraction and purification methods of serum specific endothelial cell antibody of renal transplant recipients with rejection after renal transplantation.Methods Human umbilical vein endothelial cell (HUVEC)was isolated and cultured.The serum samples of the renal transplant recipients with poor renal function after renal transplantation were collected.Specific endothelial cell antibody was screened out with flow cytometry;antibodies against human leukocyte antigen (HLA)and major histocompatibility complex class Ⅰ-related chain A (MICA)were detected by Luminex platform.After the existence of specific endothelial cell antibody in the serum sample was confirmed,specific endothelial cell antibody was absorbed with HUVEC.The cell was washed and then the absorbed antibody was eluted from the cell membrane.Antibody IgG in the eluent was purified and concentrated again with Protein-A /G magnetic beads.Antibody activity in the eluent was detected by flow cytometry and the purified specific endothelial cell antibody (IgG)was identified by SDS-polyacrylamide gel (SDS-PAGE)and Western blot.Results In the serum of 386 renal transplant recipients,the serum samples of 5 renal transplant recipients with serum creatinine (Scr) >400 μmoI /L,negative anti-HLA antibody,negative anti-MICA antibody and median fluorescence intensity (MFI) >16 were selected.Purified specific endothelial cell antibody IgG showed immunoglobulin heavy chain (purity > 95%)by SDS-PAGE gel.Flow cytometry showed that the purified antibody had the feature of rebinding with the surface antigen of vascular endothelial cell.Conclusions The purification method of using human umbilical vein endothelial cell to absorb specific endothelial cell antibody in the serum of renal transplant recipients may obtain good effect.%目的：探讨肾移植术后出现排斥反应的受者血清中抗血管内皮细胞特异性抗体的提取和纯化方法。方法首先分离培养人脐静脉血管内皮细胞（HUVEC）。收集肾移植术后肾功能不良受者的血清样本，应用流式细胞术进行抗血管内皮细胞特异性抗体的筛选；用 Luminex 抗体检测平台检测血清中抗人类白细胞抗原（HLA）和主要组织相容性复合体Ⅰ类相关链 A （MICA）的抗体。在确定血清标本中存在有抗血管内皮细胞特异性抗体后，用 HUVEC 将其吸附。洗涤细胞后再将吸附的抗体从细胞膜上洗脱下来，再用 Protein-A ／G 磁珠再次纯化和浓缩洗脱液中的抗体 IgG。采用流式细胞术检测洗脱液中抗体活性，用 SDS-聚丙烯酰胺凝胶（SDS-PAGE）凝胶和免疫印迹法鉴定纯化的抗血管内皮细胞特异性抗体（IgG）。结果在386例肾移植受者的血清中，选取血清肌酐（Scr ）＞400μmoI ／L、抗 HLA 抗体阴性、抗 MICA 抗体阴性和荧光反应强度（MFI）＞16的5例受者的血清样本，纯化后的抗血管内皮细胞特异性抗体 IgG 在 SDS-PAGE 凝胶显示免疫球蛋白重链（纯度＞95％）。流式细胞术结果显示纯化的抗体具有重新结合血管内皮细胞表面抗原的特性。结论采用人脐静脉血管内皮细胞吸附肾移植受者血清中抗血管内皮细胞特异性抗体的提纯方法可取得较好效果。